Wave type fiber SPR sensor for rapid and highly sensitive detection of hyperoside

The fiber surface plasmon resonance (SPR) sensor used for the detection of active ingredients in traditional Chinese medicine has the problems of low sensitivity and difficult specific recognition. This paper proposed a wave type fiber SPR sensor, which reduced the mode of transmitted light through a periodic wave structure and caused concentrated and total reflection of the transmitted beam at the interface between the bent peak cladding and the air. A 50 nm gold film was coated on the surface of the cladding in the wave structure area to form the SPR sensing area. By controlling the width and height of the wave structure to control the total reflection angle of the transmitted light, i.e., the SPR incidence angle, the sensitivity of the fiber SPR sensor was effectively improved to 4972 nm/RIU. Furthermore, HSP90AA protein was modified on the gold film of the sensor to achieve specific detection of hyperoside. The longest single detection time was only 3 minutes, and the detection sensitivity was 0.53 nm/(µg/ml), with a detection limit as low as 0.68µg/ml, which is comparable to liquid chromatography. The proposed wave type fiber SPR sensor is fast in production and has high structural mechanical strength, providing a new approach for the rapid, highly sensitive, and specific detection of active ingredients in traditional Chinese medicine.


Introduction
Hyperoside is the highest content flavonoid compound in the traditional Chinese medicine Huangshu Kui, which has anti-inflammatory [1] and anti-tumor effects [2].Recent studies have shown that hyperoside also has potential functions such as inhibiting pancreatic lipase [3] and treating Alzheimer's disease [4].To ensure the therapeutic effect of Huangshu Kui in clinical practice, it is necessary to quickly and sensitively detect the content of the active ingredient hyperoside in Huangshu Kui [5].Traditional methods for detecting active ingredients in traditional Chinese medicine include chromatography, mass spectrometry, electrophoresis, etc.These methods require expensive equipment, complex sample pretreatment, long detection time, and require professional personnel to operate.Therefore, proposing a new method that can achieve rapid, highly sensitive, and specific detection of hyperoside content is of great significance.
SPR technology is an optical sensing technology that is extremely sensitive to changes in refractive index, with the advantages of fast response speed and high sensitivity, which is widely used in the field of biochemical detection [6].At present, SPR based sensor is mainly used to search for therapeutic targets of active ingredients in traditional Chinese medicine [7,8] by using commercialized prism-based SPR sensor devices.Fiber SPR sensor has the advantages of small size and remote sensing capabilities, solving the problem of poor flexibility in prism-based SPR sensor.In recent years, they have received increasing attention in the field of active ingredient detection in traditional Chinese medicine [9,10].The detection of active ingredients in traditional Chinese medicine based on SPR technology essentially converts changes in the concentration of active ingredients into changes in the refractive index of the gold film surface of the sensor.Therefore, the sensor needs to have high refractive index sensing sensitivity and specific recognition ability for active ingredients.The traditional fiber SPR sensor has low sensitivity in refractive index sensing due to the use of multi-mode fiber as the substrate.Multiple transmission modes in the multi-mode fiber jointly excite SPR, and the resonance valley of SPR is generated by the superposition of multiple modes, resulting in the broadening of the resonance valley and a decrease in sensitivity.In addition, the total reflection angle of transmitted light in fiber is difficult to control, and it is impossible to achieve a smaller total reflection angle to excite SPR, which is another reason for its low sensitivity.Using single-mode fiber instead of multi-mode fiber is an effective method to reduce the number of transmitted optical modes.However, to construct SPR sensor using single-mode fiber, the original structure of the fiber needs to be significantly changed [11][12][13], so that the fiber core is exposed on the outside and directly contacts the gold film, which greatly increases the difficulty of fabrication.Meanwhile, the sensor becomes very fragile.Therefore, SPR sensor based on single-mode fiber is not conducive to application in the detection of active ingredients in traditional Chinese medicine.It is necessary to propose a new scheme that can reduce the number of transmitted optical modes in the fiber and control the total reflection angle.
In a bent fiber waveguide, the refractive index of the material on the outer side of the bent part is higher than that on the inner side of the bent part [14].Therefore, the optical field transmitted in the bent fiber tends towards the outer direction of the bent part [15].The greater the degree of bending, the more the mode field shifts, and the more concentrated the optical mode is at the bent structure.If a periodic bending structure can be constructed on a straight fiber, the residual stress after processing will cause a change in the refractive index distribution of the material at the bending structure, which can modulate the mode field offset direction of the light transmitted in the bending area, highly concentrate the light energy in the bending area, and achieve the function of reducing light modes.In addition, by controlling the curvature radius of the bent structure, the total light reflection angle of the bent area can be adjusted.Based on theoretical deduction, a bending structure with an appropriate curvature radius can be processed, which can further reduce the incidence angle of light and greatly improve the sensitivity of the fiber SPR sensor.
This article proposed a wave type fiber SPR sensor, which reduced the number of optical modes in the sensing area and reduced the total reflection angle of transmitted light in the wave area through a continuous and consistent bending structure, achieving a significant increase in sensitivity of the sensor.The fabrication cost of this sensor is relatively low, and wave structured fiber can be made using common multimode fiber only by a fiber fused biconical taper machine.Overexpression of heat shock protein 90α family A members (HSP90AA) will cause massive proliferation of breast cancer in human body [16], while hyperin can specifically bind to HSP90AA protein, inhibit its expression, and further inhibit the proliferation of breast cancer.Therefore, this article modified the HSP90AA protein on the surface of the sensing gold film to achieve specific detection of hyperoside, providing a new approach for rapid, highly sensitive, and specific detection of active ingredients in traditional Chinese medicine.

Sensor structure and principle
The schematic diagram of the wave type fiber SPR sensor structure for high sensitivity and rapid detection of hyperoside is shown in Fig. 1.The sensor body is composed of a periodic wave area fabricated in the middle of step multimode fiber with a core diameter of 105µm.A gold film with a thickness of 50 nm is coated in the wave area to excite SPR.During fabrication, the curvature radius of the wave can be controlled by controlling the spacing length L between the two peaks of the wave structure and the wave height H.The micrograph of the internal beam transmission in the fiber wave structure is shown in Fig. 1(b), which concentrates the energy of the transmitted beam and effectively reduces the number of light modes.The transmission light field shifts towards the upper and lower peaks of each wave, resulting in total reflection with the incident angle of θ.After the fiber wave area is coated with a gold film, the SPR effect is excited with the incident angle of θ.Therefore, the incident angle of the SPR excitation can also be controlled by controlling the curvature radius of the waves (the smaller the SPR incident angle, the higher the sensitivity of the SPR sensor), achieving the effect of sensor sensitization.To specifically recognize hyperoside, HSP90AA protein is modified on the surface of the sensing gold film.The hyperoside molecules that are free around the sensor will be specifically captured by HSP90AA protein, causing a change in the refractive index of the surface of the sensor gold film, thereby causing a shift in the SPR resonance wavelength.The content of hyperoside is directly proportional to the shift in the SPR resonance wavelength, achieving the purpose of SPR resonance wavelength sensing of hyperoside content.
We simulated the beam transmission path of a wave structured fiber SPR sensor using the BEAMPROP module of RSOFT simulation software.The diameter of the fiber cladding and core were set to 125µm and 105µm.As the refractive index of the outer side of the bent fiber is higher than that of the inner side of the bent fiber, a simple refractive index distribution model was established.The refractive index distribution function of the bent fiber was set to y=± 0.01x + 1, with positive and negative representing the refractive index distribution of rightward and leftward bending, respectively.The central refractive indices of the cladding and core were set to 1.440 and 1.446.The refractive index distribution of the fiber core when bent to the right is shown in Fig. 2(a).The L and H of the wave structure were set to 1600µm and 350µm, respectively.According to the curvature radius calculation formula (R = (H2 + (L/2) 2)/2 H), the curvature radius R = 1089µm was obtained.The simulation results are shown in Fig. 2(b).The simulation results were measured, and the total light reflection angle at the peak of the wave structure was θ=77 °.To obtain a smaller total reflection angle θ, a wave structure with a larger H value was simulated.The L and H values of the wave structure were set to 1600µm and 450µm, respectively, with a curvature radius of R = 936µm.The simulation results are shown in Fig. 2(c).The simulation results were measured, and the total light reflection angle at the peak of the wave structure was θ=75 °.The simulation results of the beam path in a straight fiber are shown in Fig. 2(d).Through comparison, it can be seen that the wave structured fiber has the function of concentrating light modes and controlling the total reflection angle of the beam.But as the H value increases, the loss of light beam propagation at the wave structure is also greater.Compared with Fig. 2

Sensor fabrication and parameter optimization
The steps to fabricate the wave type fiber SPR sensor are as follows: (1) Fix both ends of the step index multimode fiber (SI 105/125-/250, YOFC) with the coating stripped in the rotating fixture of the fiber fused biconical taper machine(full-featured research type-2, COUPLER), adjust the left fixture rising at the distance of H, select a round hydrogen oxygen flame head with a diameter of 5 mm, set the hydrogen flow rate to 180SCCM, heat the fiber using the central area of the flame, and the fiber is processed into S type, as shown in Fig. 3 (3) After rotating the right fixture twice, stop and close the hydrogen valve to create a wave type fiber as shown in Fig. 3(c).During fabrication, control the distance H that the left fixture rises and the diameter of the hydrogen oxygen flame head to achieve fiber wave structure with different peak spacing lengths L and wave heights H. (4) Insert the left and right ends of the wave type fiber into the bare fiber adapter of the rotating coating clamping device and fix it.After starting the rotating clamping device, place the wave type fiber in the vacuum chamber of the magnetron sputtering instrument (ETD-650 MS, YLBT), with the wave structure area of the fiber placed directly below the gold target.Meanwhile, turn on the coating machine and film thickness monitoring instrument" and deposit a 50 nm gold film on the wave structure area according to the typical film thickness optimizing parameters of fiber SPR sensor [17].(5) To specifically detect hyperoside, it is necessary to modify the sensing gold film by soaking the wave sensing area of the wave type fiber SPR sensor in a polydopamine (PDA) (B25300, Yuanye biological) solution with PH of 8.5 for 20 minutes, forming a dense PDA film on the surface of the gold film.( 6) Soak the wave shaped fiber SPR sensor in HSP90AA protein solution (ZY66281HuP, Zeye biological) for 2 hours, and use PDA to adsorb a layer of protein film.At this point, the fabrication of a wave shaped fiber SPR sensor that can specifically recognize hyperoside has been completed.According to the aforementioned simulation research, when the wave height H of the fiber wave structure is 350µm and 450µm, both beam transmission mode concentration and SPR angle control can be achieved.To optimize the fabrication parameters, the following tests are conducted.Fabricate wave type fiber SPR sensor with L of 1600µm and H of 350µm and 450µm respectively, as shown in Fig. 4.And the refractive index sensitivity of the two was tested.A glycerol solution with refractive indices of 1.333, 1.345, 1.355, 1.365, 1.375, and 1.385 was prepared using an Abbe refractometer.The sensors were used to detect the solutions sequentially, and the results are shown in Fig. 5.The refractive index sensitivity of the wave type fiber SPR sensor with an H of 450µm has increased by 46% compared to the wave type fiber SPR sensor with an H of 350µm, reaching 4972.31nm/RIU, which is consistent with the previous simulation results.The simulation results show that the SPR incidence angle of the wave type fiber SPR sensor with an H of 350µm is 77 °, and the SPR incidence angle of the wave type fiber SPR sensor with an H of 450µm is 75 °.The smaller the incidence angle of the SPR sensor, the higher the refractive index sensitivity.Therefore, a wave type fiber SPR sensor with L = 1600µm and H = 450µm will be used for the detection experiment of hyperoside.

Experimental preparation
The left end of the wave type fiber SPR sensor was connected to a broadband light source (HL-2000, Ocean Optics), and the right end of it was connected to a spectrometer (USB2000+, Ocean Optics).The spectrum collected by the spectrometer was sent to computer software for processing, as shown in Fig. 6.To conduct detection experiments on different concentrations of hyperoside solutions, different concentrations of hyperoside solutions need to be prepared.10 mg of hyperoside crystals (BP0753, Purifa) were dissolved in 0.5 ml of dimethyl sulfoxide (YS24295, Yaji biological) solution, and then diluted with PBS phosphate buffer (R26180, Yuanye biological) to prepare hyperoside solutions with concentrations of 20µg/ml, 40µg/ml, 60µg/ml, 80µg/ml, and 100µg/ml, respectively.To investigate the specificity of the probe for detecting hyperoside, isoquercetin crystals (BP0793, Purifa) and bicoumarin crystals (BP4979, Purifa) were dissolved and diluted using the same method to prepare isoquercetin and bicoumarin solutions with the same concentration range.Then, equal amounts of hyperoside crystals, isoquercetin crystals, and bicoumarin crystals were taken, mixed and dissolved simultaneously, and then diluted to prepare a mixture of three active ingredients with the same concentration range.Using a wave type fiber SPR sensor with L = 1600µm and H = 450µm, detection experiments were conducted on different concentrations of hyperoside solutions.Firstly, PBS phosphate buffer was added dropwise to the wave sensing area to collect transmission spectra, which were subtracted from the sensor's spectrum in the air to obtain the SPR resonance spectrum at a hyperoside concentration of 0µg/ml.The SPR resonance wavelength at this time was extracted as the reference wavelength.Dropwise add solutions of hyperoside with concentrations of 20µg/ml, 40µg/ml, 60µg/ml, 80µg/ml, and 100µg/ml in the wave sensing area.Collect transmission spectra every 30 seconds, extract the SPR resonance wavelength and subtract it from the reference wavelength to obtain the shift displacement of the resonance wavelength every 30 seconds.After the transmission spectrum stabilizes and does not move, end the test (takes 3 minutes).The relationship between the resonance wavelength shift and time of different concentrations of hyperoside solutions using a wave type fiber SPR sensor is shown in Fig. 7(a), and the 3-minute stable SPR spectra of hyperoside solutions at different concentrations are summarized in Fig. 7(b).It can be seen that when the concentration of hyperoside solution changes in the range of 0-100µg/ml, the SPR resonance wavelength shifts by 53 nm, and the average sensitivity reaches 0.53 nm/(µg/ml).The detection limit is calculated using the formula LOD=δλ/S, where δλ is the resolution of the spectrometer used in the experiment (0.36 nm), S is the average sensitivity, resulting in a detection limit of 0.68µg/ml.To verify the sensitivity of the proposed wave type fiber SPR sensor for the detection of hyperoside, which is higher than that of traditional structured fiber SPR sensor, a traditional multimode straight fiber SPR sensor was prepared using plastic clad fiber.The sensing gold film surface was subjected to the same functional treatment, and the 3-minute stable SPR spectra of various concentrations of hyperoside solutions were summarized in Fig. 7(c).The SPR resonance valley wavelength shifted 23 nm, with an average sensitivity of 0.23 nm/(µg/ml).Which indicates that the sensitivity of the wave type fiber SPR sensor for detecting hyperoside concentration was 2.3 times higher than that of the traditional multimode straight fiber SPR sensor.The relationship curves between the concentration of hyperoside solution detected by two sensors and the SPR resonance valley wavelength are plotted in Fig. 7(d).

Sensing region
To test the specificity of the wave type fiber SPR sensor for detecting hyperoside, solutions of isoquercetin and dicoumarin (the other two active ingredients in the traditional Chinese medicine Huangshu Kui) with concentrations of 20µg/ml, 40µg/ml, 60µg/ml, 80µg/ml, and 100µg/ml were added to the wave sensing area.The detection results (SPR stable spectrum) are shown in Fig. 8(a) and (b), and the sensor is almost unresponsive to these two components.Add mixed solutions of active ingredients with concentrations of 20µg/ml, 40µg/ml, 60µg/ml, 80µg/ml, and 100µg/ml dropwise to the wave sensing area for detection.The results are shown in Fig. 8(c), where the resonance wavelength shifts significantly and the results are consistent with the movement detected by a simple solution of hyperoside.The fitting curves of the above three experimental groups are shown in Fig. 8(d).The experimental results indicate that the wave fiber SPR sensor is specific for detecting hyperoside, and the sensor only responds to solutions containing hyperoside.

Discussion
The proposed wave type fiber SPR sensor achieves high sensitivity and specificity detection of hyperoside.By constructing a periodic bending structure on the fiber, the light transmission mode is concentrated towards the top of the wave, reducing the number of transmitted light modes in the wave structure area.By controlling the height of the wave to control the SPR incidence angle, the sensor is significantly sensitized, and the refractive index sensing sensitivity reaches 4972 nm/RIU.The HSP90AA protein modified outside the gold film can specifically bind to hyperoside, achieving specific detection of hyperoside with a detection limit as low as 0.68µg/ml, which is equivalent to the detection limit of liquid chromatography [14].We have selected several fiber SPR sensors that have been proposed in recent years to reduce transmission light modes and control incident angles.We have compared them in terms of the fiber used, fiber integrity, refractive index sensitivity, and their biochemical detection sensitivity.The results are shown in Table 1.By comparison, we fabricate a wave structured fiber SPR sensor using a fiber fused biconical taper machine.The sensor is manufactured by machine which has good consistency in fabrication.Also, it can be seen that this article has achieved a breakthrough in reducing the transmission mode of light and precise control of SPR incidence angle in multimode fiber.Moreover, the wave type fiber SPR sensor retains the overall integrity of the fiber and has a stable sensor structure, and good long-term stability, which is more conducive to practical applications in biochemical detection.When further developing in the future, it may be considered to replace the type of multimode fiber used, such as step multimode fibers with different core diameters and numerical apertures, or graded multimode fiber can be used for experimental development, due to the unique self-focusing effect of graded multimode fiber, the control effect on the optical path varies after processing into wave structure.In addition, a temperature sensing area can be added behind the wave structure area, combined with temperature compensation algorithms to reduce the impact of temperature on sensor detection performance.

Conclusion
This article proposed a wave type fiber SPR sensor, which processed a continuous and consistent bent structure on a straight fiber, concentrating the optical mode field towards the outer side of the bent structure and reducing the number of optical modes in the wave structure area.By controlling the shape parameters of the wave structure during processing to control the light incidence angle in the wave structure area, the SPR incidence angle was reduced, and the sensor was significantly sensitized.Further modification of HSP90AA protein on the surface of the sensing gold film achieved specific detection of hyperoside, with a single detection time of only 3 minutes.By replacing different proteins, detection of active ingredients in different drugs can also be achieved.This study provides a new approach for the rapid, highly sensitive, and specific detection of active ingredients in traditional Chinese medicine.

Fig. 1 .
Fig. 1.Schematic diagram of the fiber SPR sensor based on wave structure.(a) Microscopic photo of the optical field at the end face of a step multimode fiber, (b) microscopic photo of internal beam transmission in fiber wave structure.

Fig. 2 .
Fig. 2. (a) Distribution of refractive index of fiber bent to the right.The simulation results of the Wave type fiber with (b) L = 1600µm, H = 350µm, (c) L = 1600µm, H = 450µm.(d) The simulation results of the straight fiber.
(a). (2) Set the rotation speed of the right clamp of the fiber taper machine to 12000 r/min and the left clamp to 0 r/min.After igniting the flame for 1 second, start the rotation of the right clamp, as shown in Fig. 3(b).

Fig. 3 .
Fig. 3. Schematic diagram of the fabrication steps of a wave type fiber SPR sensor for detecting hyperoside.(a) S-shaped fiber fabrication, (b) heating and single-sided rotation of S-shaped fiber, (c) Wave type fiber fabrication, (d) coating gold film, (e) growing PDA membrane outside the gold film, (f) PDA adsorbing proteins.

Fig. 6 .
Fig. 6.Experimental setup for detecting active ingredients in traditional Chinese medicine using fiber SPR sensor.

Fig. 7 .
Fig. 7. (a) Wavelength shift and time relationship diagram of wave type fiber SPR sensor, (b) Wave type fiber SPR sensor stabilizes SPR spectrum for 3 minutes, (c) traditional plastic clad fiber SPR sensor stabilizes SPR spectrum for 3 minutes.(d) Fitting curves between resonance wavelengths of two sensors and the concentration of hyperoside.

Fig. 8 .
Fig. 8. Specific testing results on the detection of hyperoside using a wave type fiber SPR sensor.(a) The detection results of isoquercetin, (b) Double coumarin test results, (c) the detection results of a mixture solution of three active ingredients.(d) Fitting curves of concentration and resonance wavelength.