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Abstract
The current temperature-compensated fiber-optic surface plasmon resonance (SPR) biosensors are mainly open-ended outside the sensing structure, and there is a lack of temperature compensation schemes in fiber-optic microfluidic chips. In this paper, we proposed a temperature-compensated optical fiber SPR microfluidic sensor based on micro-nano 3D printing. Through the optical fiber micro-machining technology, the two sensing areas were designed on both sides of the same sensing fiber. The wavelength division multiplexing technology was used to collect the sensing light signals of the two sensing areas at the same time. The specific measurement of berberine and the detection of ambient temperature in the optical fiber SPR biological microfluidic channel were realized, and the temperature compensation matrix relationship was constructed, and then the temperature compensation was realized when measuring berberine biomolecules. Experiments have shown that the temperature sensitivity of the optical fiber SPR microfluidic sensor was 2.18 nm/°C, the sensitivity of the detection of berberine was 0.2646 nm/(µg/ml), the detection limit (LOD) was 0.38 µg/ml, and in a mixed solution showed an excellent specific detection impact.
© 2023 Optica Publishing Group under the terms of the Optica Open Access Publishing Agreement
1. Introduction
The optical fiber SPR microfluidic biochip has the advantages of label-free, high sensitivity [1–3], low sample consumption and fast response [4–6]. However, the measurement results are susceptible to external temperature. At present, the temperature compensation function has not been realized in the optical fiber SPR microfluidic biochip. If the temperature compensation function is realized in the fiber microfluidic biosensor, the anti-temperature crosstalk ability can be improved and the error can be reduced while maintaining the advantage of low sample consumption rate.
In recent years, two schemes have been proposed to realize temperature-compensated fiber-optic SPR biosensors. The first plan merely constructs one SPR sensing region on the sensing probe. The modified sensing area first detects biomolecules and records the resonance wavelength shift; then the sensing area is placed in distilled water, and the refractive index of distilled water at different temperatures changes with temperature to realize temperature detection and SPR resonance wavelengths are recorded at different temperatures. Finally, the temperature compensation matrix of resonance wavelength shift, sensing biomolecular sensitivity and temperature sensitivity are constructed [7,8]. However, the temperature compensation of this sensing structure requires multiple measurements alone, and the process is cumbersome.
The second scheme is to construct two SPR sensing regions at different positions in the axial direction of the sensing fiber. The temperature sensing region covers the temperature-sensitive material while the biomolecular sensing region is created by modifying one sensing region with recognition molecules [9–12]. Xue Zhou et al. put forward a dual-channel optical fiber SPR sensor based on a heterogeneous core construction. The first channel was coated with a temperature-sensitive material to sense temperature, and the second channel surface was functionalized for detecting DNA, which can simultaneously detect the sensitivity of the two substances and construct a temperature compensation matrix [13]. Xuegang Li et al. proposed a dual surface plasmon resonance combining fiber Bragg grating (FBG) and fiber SPR to simultaneously realize three-parameter detection of DNA sequence, temperature and pH value. A temperature compensation matrix was constructed based on the dependence of SPR resonance wavelength on DNA sequence, temperature and pH [14]. Bin Li et al. proposed to plate two kinds of sensing metal films on the core of the coreless fiber to form two sensing zones for the detection of temperature and glucose, respectively. A function was created to implement temperature compensation based on the relationship between temperature, glucose solution concentration, and resonance wavelength [15]. However, the biomolecular sensing area of this type of sensor has a certain lateral distance from the temperature sensing area. If it is in an uneven temperature environment, it is easy to have errors. At present, the proposed temperature-compensated fiber-optic SPR sensing structure is open-ended, which is not easy to integrate with microfluidic chips, and the sample consumption is large during measurement.
Based on this, this paper proposed a temperature-compensated optical fiber SPR microfluidic sensor based on 3D micro-nano printing. The 3D micro-nano printing device was used as the liquid inlet and outlet of the optical fiber microfluidic channel, and the SPR biosensing area and temperature sensing area were constructed on the upper and lower sides of the sensing fiber at the same position. The upper sensing area was modified with a targeted protein for the detection of berberine, and the lower sensing area was used as a temperature sensing area. The optical signals of the two sensing areas were collected simultaneously by wavelength division multiplexing technology. The concentration of berberine and ambient temperature were detected in the upper and lower sensing areas of the optical fiber SPR microfluidic sensor, respectively. The sensitivity matrix of the resonance wavelength shift and the sensor detection of berberine and temperature was constructed, and the optical fiber microfluidic sensor was able to perform temperature compensation.
2. Design and fabrication of microfluidic sensor
2.1 Design of microfluidic sensor
In the paper, we constructed the structure of the temperature compensated optical fiber SPR microfluidic sensor based on 3D printing (Fig. 1). The sensing fiber was a plastic cladding multimode fiber, and the sensing area was a flat-bottom groove area etched on the plastic cladding multimode fiber. The SPR sensing area is formed by rotating and plating a sensing gold film in the flat bottom groove area of the optical fiber. The flat-bottom groove area of the optical fiber was covered with a capillary fiber with grooves on the side. The 3D micro-nano printed tees were respectively sleeved at both ends of the capillary fiber. The hoses of the microfluidic pump and the waste liquid pool were respectively connected to the upper end of the tee to form the inlet and outlet of the test sample. After sealing the voids at various places, the optical fiber sensing microfluidic channel was formed, and the capillary fiber side hole groove was filled with the temperature-sensitive material PDMS.
Fig. 1. Microfluidic sensor structure (a) Photo of sensor not plugged into microflow hose (b)Photo of sensor with microflow hose.
The microfluidic sensor's left side received the injection of the light source. The SPR phenomenon occurs when the transmitted light is in contact with the gold film in the sensing region of the D-type fiber. Among them, the upper side plane of the D-type fiber was the solution channel to be measured, which contacted with the solution to be measured to generate a SPR resonance valley. The lower side arc surface of the D-type fiber was the temperature compensation sensing area, which was coated by PDMS to generate another SPR resonance valley. Because the refractive index of PDMS was higher than that of the solution to be measured, the refractive index sensing resonance valley of the upper side of the D-type fiber appeared in the short wavelength region in the spectral range and the temperature sensing resonance valley of the lower side of the D-type fiber appeared in the long wavelength region, which was realized in a same SPR spectrum acquisition. The D-type fiber, capillary fiber and 3D precision printed three-way tube jointly constructed the microfluidic channel of the liquid to be tested, and the high-throughput flow of the liquid to be tested was controlled by the microfluidic pump.
2.2 Sensor temperature compensation theory
The sensor designed in this paper used wavelength division multiplexing technology to simultaneously detect two SPR sensing regions, in which the SPR spectrum of the biosensor region and the SPR spectrum of the temperature compensation region will not interfere with each other. The sensor used the movement of the resonance valley in the biosensing area to demodulate the corresponding berberine concentration, and the increase in temperature will increase the refractive index of the liquid, causing the resonance valley to blue shift. The measured resonance valley movement was smaller than the accurate value, and the demodulated liquid concentration was low. At the same time, the temperature compensation matrix was constructed by using the characteristic that the resonance valley of the temperature compensation zone was only sensitive to temperature. Seen as formula (2), it eliminated the offset error caused by temperature change on the corresponding resonance valley of the biosensing zone, and then accurately compensated the concentration of the liquid in the biosensing zone.
In the formula, $\Delta {\lambda _1}$ is the amount of movement of the resonance wavelength corresponding to the biosensing area, $\Delta {\lambda _2}$ is the amount of movement of the resonance wavelength corresponding to the temperature compensation area, $\Delta n$ is the concentration of the drug solution, $\Delta T$ is the amount of external temperature change, ${S_{spr - 1}}$ is the sensitivity of the biosensing area to detect berberine, ${S_{spr - 2}}$ is the sensitivity of the biosensing area to temperature, ${S_{spr - 3}}$ is the sensitivity of the temperature compensation area to temperature.
2.3 Fabrication of microfluidic sensor
We used a multimode fiber with a plastic coating and a 125 µm core diameter (HP125/193-37/245, Beijing Scitlion Technology). The coating layer and plastic cladding were mechanically peeled from the right side of the multimode fiber with plastic cladding to a length of 10 cm, allowing the core to seep out. Then the core area was fixed on the fiber clamping mobile platform and placed directly below the CO2 laser (MC-E-B, GD Hans Yueming Laser). The laser scanning region was marked, and the processing settings were set to 3 processing times, 80% power, and 50 KHz frequency. A D-type fiber region was created by processing a flat-bottomed groove area that was 3 cm long and 65 µm deep on a 125 µm fiber core, as illustrated in Fig. 2(a). The D-type fiber region was placed into the fiber rotary coating clamping device of a magnetron sputtering instrument (ETD-650 MS, YLBT), and then a gold film with a thickness of 50 nm was rotatingly plated, as shown in Fig. 2(b). We took a section of capillary fiber (MX130/200, XYAT) with a hollow diameter of 130 µm and an outer diameter of 200 µm. After the coating layer was removed, it was placed directly below the CO2 laser to draw the laser scanning area, and the processing parameters were adjusted (40% power, 50 KHz frequency, and 3 processing times). A groove of 1.5 cm in length and 50 µm in depth was machined on the capillary fiber to leak out the inner wall of the capillary, as shown in Fig. 2(c). The etched capillary fiber with a groove was cut to a length of 2.8 cm, and the cut capillary fiber was inserted into the D-type sensing area from the right side of the plastic cladding multimode fiber, as shown in Fig. 2(d). The left 3D micro-nano printed tee had following characteristics including the overall diameter of the left tee as 2 mm, an opening at the upper end, a sealing at the lower end and the left and right sides with circular openings with a diameter of 260 µm. The right 3D micro-nano printed tee had the following features, including an opening at the top end, a sealing at the lower end, and circular holes with diameters of 210 µm and 135 µm on the left and right sides, respectively. The overall diameter of the tee tube on the right side was 2 mm. The 3D micro/nano printed tee tubes were placed on both sides of the capillary and rotated so that the arc surface of the D-type fiber was in contact with the groove of the capillary fiber, as shown in Fig. 2(e), we used sealant to close the space between the tee tube and the core of the plastic-clad multimode fiber and the capillary fiber. The microfluidic catheter was connected to the opened upper end of the three-way tube (BMF Precision Tech Inc), and finally, the capillary groove area, which was in contact with the sensing gold layer on the arc surface of the D-type fiber, was coated with polydimethylsiloxane (PDMS, aladdin), which has a refractive index of 1.39, and solidified, as shown in Fig. 2(f).
2.4 Microfluidic sensor test device
The microfluidic sensor test system was constructed after the temperature compensation fiber microfluidic sensor based on micro and nano 3D printing was created, as shown in Fig. 3. The microflow sensing probe is located in the middle of the temperature test platform (P-20, Fastelectronics Company). The three-way tube on the microflow sensor is connected to the microflow pump (LSP01-1A, longer pump, Set flow is 0.08 µl/min) and the waste liquid tank, respectively. A wide-spectrum light source (HL-2000, Ocean Optics) and a spectrometer (USB2000+, Ocean Optics) were coupled to the left and right ends of the microflow sensing probe, respectively. The spectrometer captured the sensing fiber's spectrum and sent it to the computer for analysis.
2.5 Refractive index test of microfluidic sensor
Using the test apparatus, the optical fiber SPR microfluidic sensor was evaluated for refractive index sensing. Different types of glycerol solutions were set up, with refractive indices ranging from 1.335 to 1.385. The solutions with varied refractive indices were pumped into the channel using a microfluidic pump. The capillary fiber groove on the outside of the D-type sensing probe wasn't filled with PDMS to better test the sensing probe's performance in measuring refractive index. The test results and sensing spectra for various refractive index solutions are gathered in Fig. 4(a). The refractive index sensing sensitivity is 2640 nm/RIU, and the SPR resonance valley wavelength shift range is 620.2-752.2 nm.
Fig. 4. Sensitization modification of microfluidic sensor (a) Test results when the sensing probe was not sensitized, (b)Scanning electron microscopy of MOF material modified on the surface of gold film, (c) Test results after sensitization of the sensing probe, (d) Before and after modification, the connection between resonance wavelength and refractive index.
In order to further improve the refractive index sensing sensitivity of the optical fiber microfluidic sensor, the metal organic framework material (MOF) were used to sensitize the sensing probe [16,17]. The following is the change process: A microflow pump was used to inject 50 mg of the metal-organic framework material (MOF-74, Nanjing XFNANO Materials Tech) into the optical fiber microfluidic channel after it had been dissolved in 50 ml of anhydrous ethanol. After standing for 30 min, the MOF material adsorbed on the surface of the sensing film. To remove the MOF material that hadn't been modified from the surface of the sensing film, distilled water was pumped into the microchannel. Nitrogen dried the microchannel. Scanning electron microscopy was used to study the sensing region at this point. As shown in Fig. 4(b), it can be seen that the MOF material was modified on the surface of the sensing metal film. Then, we tested the refractive index sensing of the fiber optic microfluidic sensor modified with MOF material. In Fig. 4(c), the test results were displayed, the wavelength shift range of the SPR resonance valley was 650.0-845.3 nm, and the refractive index sensitivity was increased by 1.48 times, reaching 3906 nm/RIU. We repeated three sets of refractive index test experiments. The relationship between refractive index and resonance wavelength was shown in Fig. 4(d).
2.6 Surface functionalization of microfluidic channel for microfluidic sensor
The primary active component of the traditional Chinese medication Rhizoma Coptidis is berberine, which has anti-inflammatory and antibacterial effects [18]. Usually, the concentration of berberine in unprocessed Rhizoma Coptidis is 20 µg/ml-80 µg/ml. The content of berberine will affect the quality of Rhizoma Coptidis, therefore, determining the amount of berberine in Rhizoma Coptidis is extremely important. In order to specifically detect the active ingredients of berberine in the sensing area of the sensor, it is necessary to modify the recognition molecules in the sensing area. Studies have shown that human vascular endothelial growth factor receptor 2 (VEGFR2, Yaji biological) had a strong binding force with berberine [19,20]. At the same time, the previously modified MOF material also has the adsorption ability to adsorb VEGFR2 [21,22]. The precise technique for alteration was as follows: A microfluidic pump was used to inject 1 mg/ml VEGFR2 into the modified MOF material's microfluidic sensor channel, and the material was then left in a 4 °C environment for one hour. Phosphate buffer solution (PBS, Yaji biological) was injected to rinse, dry with nitrogen. Finally, the scanning electron microscope was used to photograph the sensing area of the microchannel, as shown in Fig. 5. As can be observed, the gold film was uniformly applied to the fiber's surface, and the metal surface's VEGFR2 protein had a similar uniform modification.
3. Experimental results and discussion
3.1 Concentration measurement of active molecule berberine in traditional Chinese medicine
The concentration of berberine was tested by the modified optical fiber microfluidic sensor. At 50 °C, berberine from Yaji biological was dissolved in a tiny amount of dimethyl sulfoxide and diluted with PBS solution to create berberine solutions with concentrations of 1 µg/ml, 20 µg/ml, 40 µg/ml, 60 µg/ml, 80 µg/ml, and 100 µg/ml. The temperature of the temperature platform was fixed at 20 °C, and then different concentrations of berberine solution were injected into the fiber optic microfluidic sensor using a microfluidic pump in the order from low to high. When measuring each concentration of berberine solution, the sensing spectrum was collected every 5 min until the reaction was stable for a total of 45 min of reaction. Figure 6(a) depicts the resonance wavelength change curve over time at various concentrations. The wavelength shift was large in the first 20 min, indicating that a large amount of berberine was combined with the target protein, The combination of berberine with the target protein on the surface of the gold film caused a slight increase in the refractive index of the surface environment of the sensing metal layer, which in turn caused a red shift of the resonance wavelength of the sensing valley 1. The final stable test results after the reaction were shown in Fig. 6(b). The SPR valley produced by the detection of berberine corresponded to the sensing valley 1 with the wavelength shift range as 602.5-628.7 nm, the sensitivity of the sensing berberine as 0.2646 nm /(µg/ml), the detection limit of the sensing probe was 0.38 µg/ml. The SPR valley generated by the detection temperature corresponded to the sensing valley 2. At this time, the temperature did not change, and the wavelength of the sensing valley 2 did not move. The sensor performed the same experiments three times, and Fig. 6(c) depicts the resonance wavelength and berberine concentration fitting curve.
Fig. 6. Test results of berberine concentration and temperature detected by microfluidic sensor (a) SPR resonance wavelength's time-dependent curve, (b) Data of berberine concentration detected by microfluidic sensor, (c) Plot of fit between berberine concentration and the amount of resonance wavelength shift, (d) Data of temperature measured by microfluidic sensor.
Next, we conducted an experiment to test how the detection spectrum would change when the ambient temperature changed. After injecting a berberine solution into the microfluidic channel at a concentration of 1 µg/ml, the temperature of the temperature stage was changed after 45 minutes of standing response. The temperature range was from 20 °C to 70 °C, and every 10 °C was a gradient. We collected the SPR spectrum at each temperature after the reaction was stable. The test results were shown in Fig. 6(d). Because polydimethylsiloxane (PDMS, Yaji biological) has a high thermo-optical coefficient, its refractive index decreased rapidly with the increase of temperature [23,24]. Therefore, the refractive index environment of the temperature sensing area on the microfluidic sensing probe decreases with the increase of temperature, and the resonance wavelength of the corresponding sensing valley 2 has a significant blue shift. and the SPR wavelength shift range was 860.5-751.2 nm. The temperature sensitivity was -2.18 nm/°C. Meanwhile, the biosensing region's sensitivity to temperature was -0.126 nm/°C, The refractive index of the biological sample solution decreased slightly with the increase of temperature, causing the resonance valley 1 to blue shift with the moving range as 604.5-598.2 nm.
3.2 Comparative experiment of berberine specificity detection
In addition to berberine, the main active ingredients in Coptidis Rhizoma also include jatrorrhizine and palmatine. In order to test the specific adsorption effect of VEGFR2 on berberine, the modified microfluidic sensor was used to test the concentration of jatrorrhizine hydrochloride (Yaji biological) and palmatine hydrochloride (Yaji biological). Dissolve jatrorrhizine in a small amount of distilled water, then dilute with PBS solution to prepare jatrorrhizine solutions of the same concentration as berberine samples. The aforesaid berberine detection method was used to identify various concentrations of jatrorrhizine solution in a 20 °C setting. In Fig. 7(a), the test results were displayed. The wavelength shift range of sensor valley 1 was 605.2-607.1 nm, which indicates that there was no obvious shift. Similar procedures were used to dissolve palmatine, which was then diluted with phosphate buffered saline (PBS) solution and set up with the same sample concentrations as previously mentioned. The test results were shown in Fig. 7(b). The wavelength shift range of sensor valley 1 was 608.5-610.4 nm and there was no obvious shift, which indicating that VEGFR2 had weak binding force to jatrorrhizine and palmatine.
Fig. 7. Experimental results of detection of jatrorrhizine and palmatine by microfluidic sensor. (a) SPR spectra of detection of jatrorrhizine, (b) SPR spectra of detection of palmatine.
In order to test the specific detection effect of optical fiber microflow sensing sensor modified with VEGFR2 protein on berberine in mixed solution containing various active ingredients of traditional Chinese medicine. 1 ml each of rhizophylline solution and palmatine solution was taken, the jatrorrhizine (100 µg/ml) and palmatine (100 µg/ml) solutions were mixed uniformly to form a mixed solution 1. 1 ml each of berberine solution, jatrorrhizine solution and palmatine solution was taken, berberine (150 µg/ml), jatrorrhizine (150 µg/ml) and palmatine (150 µg/ml) solutions were uniformly mixed to form a mixed solution 2. The temperature of the temperature stage was kept at 20 °C. A microflow pump was used to first inject the solution 1 into the fiber microflow channel, and the sensing spectra were acquired every 10 minutes after that. In Fig. 8(a), the test results were displayed. The biosensor region's resonant wavelength ranged from 604.2 to 607.9 nm, indicating that the sensing probe modified with VEGFR2 protein had no obvious binding to jatrorrhizine and palmatine. The external temperature was stable, and the resonance wavelength of the temperature sensing area did not move. Similar to above, mixed solution 2 was injected, and every 10 minutes, sensing spectra were gathered. In Fig. 8(b), the test results were displayed. The biosensing area's resonance wavelength ranged from 609.8 to 628.3 nm, indicating that the VEGFR2 protein-modified sensing probe was obviously combined with berberine. The external temperature did not change, and the resonance wavelength of the temperature sensing area did not move.
Fig. 8. Specific detection data of microfluidic sensor in mixed solution. (a) SPR spectrum of jatrorrhizine and palmatine mixed solution experiment, (b) SPR spectrum of berberine, jatrorrhizine and palmatine mixed solution experiment.
3.3 Temperature compensation
In light of the aforementioned experimental findings, the resonance wavelength shift ($\Delta {\lambda _{spr - 1}}$) in the biosensing region on the upper side of the D-type fiber is related to the change in the refractive index of the metal surface caused by the binding of biomolecules and target proteins ($\Delta n$) and the change in the external temperature ($\Delta T$). The resonance wavelength shift ($\Delta {\lambda _{spr - 2}}$) of the temperature sensing area was only related to the change of the external temperature ($\Delta T$). Therefore, the sensitivity, temperature sensitivity and wavelength shift of the sensing berberine can form a matrix relationship:
Then the temperature compensation matrix of the final sensing probe can be obtained:
3.4 Discussion
In order to implement temperature compensation in the fiber-optic microfluidic chip, we suggested the temperature-compensated fiber-optic SPR microfluidic sensor based on micro-nano 3D printing in this paper. Compared with the currently proposed open temperature-compensated fiber-optic SPR sensor, it has the advantage of more accurate compensation results. Because the biosensor for specific detection by modifying the target protein requires a temperature control device to ensure that the temperature of the target protein modified on the sensing probe and the solution to be tested is in an appropriate temperature range. The temperature of the sensing area where the probe is located is often different from the room temperature, and temperature compensation is required for the sensing area. The temperature control device is usually a small constant temperature platform, and the sample solution of the active ingredient of traditional Chinese medicine is usually contained in a small container, resulting in a small area of temperature stability detection area. Beyond the temperature stability detection area, the ambient temperature will be unevenly distributed. At present, the biomolecular sensing area of the temperature-compensated optical fiber biosensor has a certain lateral distance from the temperature sensing area. The temperature collected at the temperature sensing area is inconsistent with the temperature of the biosensing area, and there is a compensation error.
At the same time, the performance comparison results of the sensing probe were shown in Table 1. It can be seen that the temperature-compensated sensor proposed in this paper was modified with a metal-organic framework material in the sensing area, the metal-organic framework material belonged to the nanomaterial with a large specific surface area, high electron mobility and light absorption rate, and the nanomaterial was used to construct a sensitive layer to enhance the SPR signal. Therefore, compared with other temperature-compensated fiber-optic SPR biosensors, it has the advantage of high refractive index sensitivity and is sensitive to the refractive index change of the sensing surface caused by biomolecules and temperature changes.
Table 1. Compares how well temperature-compensated fiber-optic SPR biosensors perform with various sensing architectures.
4. Conclusion
In this study, we suggested a micro-nano 3D-printed temperature-compensated fiber-optic SPR microfluidic sensor. Two SPR sensing areas were constructed on both sides of the fiber D-type microfluidic channel, one for biological detection and the other one for temperature sensing. The temperature compensation sensing area and the biosensing area were located in the same spatial position, which ensured that the temperature measured by the temperature sensing area was the temperature of the biosensing area. The specific modification was carried out in the biosensor area to realize the specific detection of berberine, the main active ingredient in Rhizoma Coptidis, and the temperature compensation function. The microfluidic sensor's temperature sensitivity was 2.18 nm/°C, the sensitivity of berberine was 0.2646 nm /(µg/ml). The fiber optic microfluidic sensor proposed in this paper has a temperature compensation function, which further reduces the external influence on the measurement results, improves the detection accuracy while maintaining the advantage of low sample consumption, and provides a solution for the temperature compensation detection of active pharmaceutical ingredients in the fiber optic micro-flow sensing channel.
Funding
Chongqing Three Gorges Medical College Project (XJ2023000703); Chongqing Postgraduate Research and Innovation Project; The Foundation of Intelligent Ecotourism Subject Group of Chongqing Three Gorges University (zhlv20221008); Open Project Program of Key Laboratories of Sensing and Application of Intelligent Optoelectronic System in Sichuan Provincial Universities; Fundamental Research Funds for Chongqing Three Gorges University of China (19ZDPY08); Open Project Program of Chongqing Key Laboratory of Geological Environment Monitoring and Disaster Early-Warning in Three Gorges Reservoir Area (ZD2020A0103, ZD2020A0102); The Science and Technology Project Affiliated to the Education Department of Chongqing Municipality (KJZD-M202201201); Open Project Program of Chongqing Key Laboratory of Development and Utilization of Genuine Medicinal Materials in Three Gorges Reservoir Area (KFKT2022005); National Natural Science Foundation of China (61705025).
Disclosures
The authors declare no conflict of interest.
Data availability
Data underlying the results presented in this paper are not publicly available at this time but may be obtained from the authors upon reasonable request.
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