A fiber optic sensor and detection system for detecting protamine sulfate

Abstract

This invention discloses a fiber optic sensor and detection system for detecting protamine sulfate. The sensor comprises a fiber optic interference structure and a functionalized thin film of chitosan and heparin self-assembled layer by layer. The fiber optic interference structure uses a cascade of single-mode fiber-coreless fiber-single-mode fiber, generating mode interference and exciting the evanescent field of the cladding mode at the coreless fiber to form a fiber optic sensing structure. Chitosan and heparin are self-assembled into a molecular film through layer-by-layer stacking based on the principle of electrostatic adsorption. The target biological protein molecule is fixed to the surface of the molecular film by the specific binding of heparin to the target protein molecule in the molecular film. This invention utilizes the sensitivity of the evanescent field of the fiber optic interference structure to the refractive index of the environment. The specific binding of the target protein to the surface molecular film amplifies the specific binding signal, which not only greatly improves the detection sensitivity and detection limit, but also is simple and easy to implement, with excellent detection repeatability.

Description

Technical Field

[0001] This invention relates to a fiber optic biosensor, and more particularly to a surface-functionalized fiber optic interferometric structure biosensor and a method for detecting the concentration of biological protein molecules, belonging to the field of fiber optic biosensor design. Background Technology

[0002] In existing technologies, the detection of protamine sulfate often employs biochemical detection methods (such as enzyme-linked immunosorbent assay), which are cumbersome and costly. Summary of the Invention

[0003] In view of the defects or deficiencies of the prior art, the present invention provides an optical fiber sensor for the detection of protamine sulfate.

[0004] Therefore, the method for fabricating the optical fiber sensor provided by the present invention includes:

[0005] Step 1: Sequentially fuse the first single-mode fiber, the coreless fiber, and the second single-mode fiber to obtain the initial device.

[0006] Step 2: Immerse the initial device in a hydroxide solution to introduce hydroxyl groups onto the surface of the initial device.

[0007] Step 3: Immerse the device treated in Step 2 in acetic acid solution of chitosan and sodium heparin injection solution in turn to form a single-layer functional membrane.

[0008] Step 4: Repeat Step 3 multiple times to form a multilayer functional membrane and obtain a fiber optic sensor for protamine sulfate detection.

[0009] An alternative is that the length of the coreless optical fiber is L, and 0.5cm≤L≤2cm.

[0010] An alternative approach is to repeat Step 3 multiple times in Step 4 to form 3 to 15 functional membranes.

[0011] Alternatively, the hydroxide solution may be selected from sodium hydroxide solution.

[0012] Alternatively, the concentration of the hydroxide solution is less than or equal to 1.0 mol / L; the concentration of the chitosan acetic acid solution is less than or equal to 0.01 mg / mL; and the concentration of the heparin sodium injection is 2.5–10 mg / mL.

[0013] This invention also provides a protamine sulfate detection system, which includes a supercontinuum light source, the aforementioned fiber optic sensor, and a spectrometer. A first single-mode fiber at one end of the fiber optic sensor is connected to the output of the supercontinuum light source, and a second single-mode fiber at the other end is connected to the spectrometer. The supercontinuum light source generates incident light that is transmitted to the fiber optic sensor. The fiber optic sensor captures protamine sulfate in the sample solution and generates an interference spectrum that is transmitted to the spectrometer. The spectrometer converts the interference spectral signal into spectral data.

[0014] In a further embodiment, the detection system of the present invention also includes an optical fiber clamp that holds both ends of the optical fiber sensor and keeps the optical fiber sensor in an extended state.

[0015] In a further embodiment, the detection system of the present invention also includes a lifting platform with a sample slot, wherein the fiber optic sensor is inserted into the sample to be tested in the sample slot by adjusting the lifting platform.

[0016] This invention also provides a method for detecting protamine sulfate. The provided method uses the aforementioned sensor or detection system to detect the protamine sulfate content in a sample to be tested. The method includes: acquiring the spectral data of the sample to be tested, and calculating the protamine sulfate content in the sample to be tested based on the wavelength of the highest-order interference peak when testing the sample to be tested and the wavelength of the highest-order interference peak when testing a standard sample (i.e., a sample with a known gradient concentration of the analyte). Specifically, the sample to be tested is a pharmaceutical product or blood.

[0017] The present invention has the following advantages over the prior art:

[0018] (1) The sensor surface of this invention is modified with a chitosan / heparin layer-by-layer self-assembled biosensitive membrane as a carrier for specific recognition, thereby achieving selective capture of target analytes. Utilizing the evanescent field of the cladding mode of the interference structure, which is sensitive to refractive index, a sensitization effect is achieved. This method not only possesses excellent detection limits and sensitivity but is also simple to implement and exhibits highly stable detection results. Compared with the traditional ELISA method (enzyme-linked immunosorbent assay), this invention saves a significant amount of preparation time for processing test samples and avoids the cumbersome process of handling large amounts of test data using statistical methods, greatly reducing detection costs and simplifying the detection process.

[0019] (2) Compared with traditional electrochemical sensors, the optical wave sensing method of the present invention, which uses low-loss single-mode communication optical fiber as a carrier, has the characteristics of fast signal speed and low power consumption. Therefore, it can realize long-distance real-time online sensing of biological protein molecule concentration according to actual use needs.

[0020] (3) The present invention is able to eliminate the influence of temperature changes on the detection results because the transmission mode of the single-mode fiber itself is not sensitive to temperature and the interference peak of the transmission spectrum of the measuring device has good resistance to temperature changes.

[0021] (4) Compared with traditional optical and electrical detection methods, this invention utilizes the feature that the fiber optic sensor and the detection signal are integrated into a single fiber, which can greatly improve the accuracy of the detection results and the stability of the system.

[0022] (5) Compared with the traditional BCA method (diquinoline carboxylic acid assay), this invention can achieve specific detection of the target molecule. It eliminates the background by eliminating the inherent interference peak wavelength change caused by the sample refractive index, thus eliminating interference from other protein molecules.

[0023] (6) The present invention obtains the concentration information of protamine sulfate molecules in solution by observing the wavelength change of the interference peak in the transmission spectrum of the sensor. The detection sensitivity reaches 5.7 nm / (mg / mL) and the detection limit reaches 0.475 μg / mL (0.765 μM). Attached Figure Description

[0024] Figure 1 This is a schematic diagram of the detection system of the present invention; the cladding and core are components of a single-mode optical fiber, and the light yellow shading is a schematic diagram of a chitosan / heparin sodium self-assembled membrane in a coreless optical fiber segment.

[0025] Figure 2 This is the linear fitting result of the sensing spectrum and wavelength change of the refractive index of sucrose liquid obtained by the 10-layer functional film fiber optic sensor in the embodiment of the present invention.

[0026] Figure 3 The detection results are from an embodiment of the present invention, wherein, Figure 3 (a) shows the wavelength change of the highest-order interference peak in the transmission spectrum and the piecewise fitting curve when the 5-layer functional film fiber optic sensor obtained in the embodiment of the present invention detects a sulfuric acid protamine concentration gradient solution. Figure 3 (b) shows the wavelength change of the highest-order interference peak and the piecewise fitting curve in the transmission spectrum of the 10-layer functional membrane sensor obtained in the embodiment of the present invention for detecting sulfuric acid protamine concentration gradient solution.

[0027] Figure 4 The wavelength changes in the detection spectrum of the 10-layer functional membrane sensor obtained in this embodiment of the invention for 400 μg / mL solutions of urea, glucose, ascorbic acid, hemoglobin, and protamine sulfate are shown.

[0028] Figure 5This is a bar chart showing the center wavelength distribution of the transmission spectrum of a certain interference mode for a 400 μg / mL protamine sulfate solution obtained by the 10-layer functional membrane sensor in this embodiment of the invention, obtained in five repeated concentration tests. Detailed Implementation

[0029] Unless otherwise specified, the scientific and technical terms used herein are for the understanding of those skilled in the art. It should be noted that, based on the present invention, those skilled in the art can optimize and select aspects of the method involved in the present invention, including but not limited to solution concentration, immersion time, device size, and number of functional film layers, to achieve the effects of the present invention. Examples include, but are not limited to, the specific technical means provided by the present invention.

[0030] The present invention will be further described below with reference to the accompanying drawings and specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Operating methods not specifically specified in the following embodiments are generally performed under conventional conditions or as recommended by the manufacturer.

[0031] The supercontinuum light source used in the following embodiments is the SUPERK EXTREMEFIANIUM from NKT Photonics, with a wavelength range of 260nm-2400nm; the spectrometer is the Yokogawa AQ6375B; however, this invention is not limited to these.

[0032] Example 1:

[0033] The fiber optic sensor fabrication method in this embodiment is as follows:

[0034] S1. A coreless fiber (cladding diameter 125 micrometers) is fused together between two standard single-mode optical fibers for communication (model: Corning SMF28, core diameter 8.3 micrometers / cladding diameter 125 micrometers) to prepare the initial device.

[0035] S2, the initial device surface is thoroughly rinsed with ethanol and deionized water to remove residual contaminants; then the device is immersed in a 1.0 mol / L sodium hydroxide solution for 30 minutes to introduce hydroxyl groups;

[0036] S3. Immerse the device treated in S2 in a chitosan acetic acid solution (0.2g chitosan powder dissolved in 20mL of 4% acetic acid and stirred until the bubbles disappear) for 10 minutes. Chitosan molecules form a uniform self-assembled monolayer on the device surface. Then, use a cleaning solution to remove excess chitosan molecules remaining on the optical fiber surface.

[0037] Next, the device, after being soaked in chitosan acetic acid solution, is immersed in a 2.5 mg / m³ heparin sodium solution for 10 minutes. Based on the principle of positive and negative charge binding, the heparin sodium molecules will fully bind with chitosan to form a uniform chitosan / heparin sodium molecule monolayer functional film. Then, the unbound heparin sodium molecules are cleaned with a cleaning solution.

[0038] S4, repeat S3 to form a multilayer functional film on the device surface; in this embodiment, the chitosan / heparin sodium molecular film has 5 layers and 10 layers.

[0039] Example 2:

[0040] A detection system is built using the sensor from Example 1, such as... Figure 1 As shown, the supercontinuum light source is connected to the single-mode fiber through a jumper. The incident light generated by the supercontinuum light source enters the sensor from the single-mode fiber at one end of the fiber optic sensor prepared in Example 1, and the outgoing light is output from the single-mode fiber at the other end through the jumper to the spectrometer to record data.

[0041] During operation, the incident light generates mode coupling at the first fusion splice between the single-mode fiber and the coreless fiber. The functional film that satisfies the phase matching condition is coupled to the interface between the coreless fiber and the air and propagates as an evanescent traveling wave. It is coupled back to the single-mode fiber at the second fusion splice between the coreless fiber and the single-mode fiber and generates an interference spectrum with the fundamental mode. Then, the spectrum analyzer records multiple interference peaks at different wavelengths in the output spectrum. The peak with the largest wavelength is the highest-order interference peak. Based on the fact that the refractive index sensitivity of the higher-order cladding mode is the strongest, this invention selects the highest-order interference peak as the detection basis.

[0042] The wavelength change of the spectral interference peak (i.e., the wavelength change corresponding to the highest-order interference peak) is obtained by subtracting the wavelengths corresponding to the highest-order interference peak in two spectral acquisitions (e.g., a blank sample and a sample to be tested).

[0043] When using the fiber optic sensor, ensure it is in an extended state to avoid introducing cross-sensitivity due to bending or stress. In some solutions, both ends of the sensor can be fixed in a naturally extended state using fiber optic clamps.

[0044] In a further design, a lifting platform and a U-shaped trough are placed below the fiber optic sensor area. The top of the U-shaped trough is open, and the fiber optic sensor is positioned within the U-shaped area. During measurement, the sample to be tested is injected into the U-shaped trough using a pipette, and the sensor is kept submerged in the sample by adjusting the height of the lifting platform.

[0045] Example 3:

[0046] This embodiment uses the detection system of Example 2 to detect the concentration of protamine sulfate molecules in a series of gradient concentrations of protamine sulfate sample solutions:

[0047] In each of the following tests, the spectral acquisition time was calibrated to be recorded one minute after the sensor reacted with the sample. After each test, the sample solution was aspirated and the sensor surface and U-groove were rinsed with 10 mMoL phosphate buffer.

[0048] First, the refractive index sensitivity of the device was determined using sucrose solutions with gradient refractive indices. The spectrometer output detection spectra for solutions with different refractive indices. The change in the center wavelength of the highest-order interference peak at different refractive indices was fitted, and the results are as follows: Figure 2 As shown, the linear fitting results indicate that the refractive index sensitivity of the device is 136.1 nm / RIU. Figure 2 The highest-order interference peak in the spectrum is the one with the largest center wavelength among the multiple interference peaks in the transmission spectrum of the sensor described in Example 1. The sample concentration will be detected based on this highest interference peak in the following tests.

[0049] Detection of protamine sulfate molecular concentration in a series of gradient concentration sample solutions: The fiber optic sensor was used to detect the concentration of protamine sulfate in a series of gradient concentrations (specifically 6.25 μg / mL, 12.5 μg / mL, 25 μg / mL, 50 μg / mL, 100 μg / mL, 200 μg / mL, 400 μg / mL, 800 μg / mL, and 1000 μg / mL).

[0050] Due to the strong and specific binding between protamine sulfate and sodium heparin molecules, the effective refractive index of the surface functional film changes significantly, thereby amplifying the binding signal between molecules. At this time, the interference peak wavelength of the sensor's transmission spectrum changes significantly compared to a bare fiber optic sensor without a surface functional film. The concentration of protamine sulfate molecules in the sample solution can be detected based on the magnitude of the wavelength change after calibration for the same reaction time.

[0051] Five-layer and ten-layer functional membrane sensors were used to sense a series of protamine sulfate solutions with varying concentrations, obtaining interference spectra for samples of different concentrations. The wavelength changes of the interference peaks in the spectra at different concentrations were calculated, and the wavelength changes were fitted using data. The detection results of the device with the obtained five-layer self-assembled membrane are shown below. Figure 3 As shown in (a); the device testing results for the 10-layer self-assembled film are as follows. Figure 3As shown in (b), the results indicate that with increasing sample concentration, the interference peak of the sensor's output spectrum exhibits a red shift. Both the 5-layer and 10-layer functional membrane sensors satisfy the Langemore adsorption model within the range of 6.25-200 μg / mL. In the detection range of 200-1000 μg / mL protamine sulfate samples, the wavelength change of the interference peak satisfies a linear fit. The Langemore adsorption model is as follows:

[0052]

[0053] Where Δλ represents the change in wavelength of the spectral interference peak caused by the binding of the chitosan / heparin sodium functional membrane to protamine (i.e., the change in wavelength of the spectral interference peak during detection); Q m The maximum adsorption capacity of the chitosan / heparin sodium functional membrane is indicated; K and C are the steady-state equilibrium binding constant and analyte concentration, respectively (K has no unit, C is in μg / mL, and the analyte concentration in this example refers to the sample concentration of protamine sulfate).

[0054] By fitting Δλ, we obtain K = 0.0079, Q m =5.34.

[0055] The detection limit (LOD) of the sensor is calculated using the following formula:

[0056]

[0057] Where, Δλ min The main limitation comes from the minimum resolution of the spectrometer itself, which is Δλ in this embodiment. min The value is set to 0.02nm; Figure 3 (a) The LOD calculation result of the 5-layer membrane sensor is 0.475 μg / mL (0.765 μM). In the range of 200-1000 μg / mL, the linear sensing sensitivity of the sensor is 3 nm / (mg / mL). Figure 3 (b) The LOD calculation result of the 10-layer membrane sensor is 0.75 μg / mL (1.2 μM), and its linear sensing sensitivity is 5.7 nm / (mg / mL) in the range of 200-1000 μg / mL.

[0058] Example 4:

[0059] This embodiment uses the detection system of Example 2 to detect the concentration of other biomolecules to characterize the selectivity of the sensor:

[0060] Specifically, analytical grade urea, glucose, ascorbic acid, and hemoglobin were prepared into various sample solutions with a concentration of 400 μg / mL using deionized water. The concentration of protamine sulfate solution for comparison was also 400 μg / mL.

[0061] The spectral changes caused by these sample solutions were tested sequentially using the test method described in Example 3. The refractive index of each sample was measured in advance using a refractive index meter. After detecting the change in the wavelength of the interference peak in the spectrum, the wavelength change caused by the inherent refractive index difference of the mixed samples was subtracted from the wavelength change to detect the wavelength change caused by the functional film of the sensor.

[0062] The results are as follows Figure 4 As shown, due to the low concentration, the refractive index of each sample solution did not change significantly compared to deionized water. Compared to the interference peak wavelength detected in deionized water, the wavelength changes for each sample were as follows: urea: -0.03 nm, glucose: 0.26 nm, ascorbic acid: 0.4 nm, hemoglobin: 0.29 nm, and protamine sulfate: 4.51 nm. Therefore, the sensor of this invention exhibits good selectivity for the detection of protamine sulfate.

[0063] Example 5:

[0064] This embodiment uses the detection system of Example 2 to detect the sulfuric acid protamine molecule solution described in Example 4, and examines the spectral stability of repeated tests.

[0065] Specifically, a 400 μg / mL protamine sulfate solution was used for five tests. After each test, the device surface was rinsed with the phosphate buffer solution described in Example 3. The spectra and corresponding wavelength changes of the five tests are as follows: Figure 5 As shown in the figure, the detection wavelength has good stability.

Claims

1. A fiber-optic sensor for detecting protamine sulfate, characterized by, The preparation method of the sensor comprises: Step 1, sequentially fusing a first single-mode optical fiber, a coreless optical fiber and a second single-mode optical fiber to obtain an initial device; Step 2, soaking the initial device in a hydroxide solution to introduce hydroxyl groups on the surface of the initial device; Step 3, sequentially soaking the device treated in Step 2 in a chitosan acetic acid solution and a heparin sodium injection to form a single-layer functional film; Step 4, repeatedly performing Step 3 multiple times to form a multi-layer functional film, thereby obtaining a protamine sulfate detection optical fiber sensor.

2. The fiber-optic sensor for detecting protamine sulfate according to claim 1, characterized in that, The coreless optical fiber has a length L, and 0.5 cm≤L≤2 cm.

3. The fiber optic sensor for detecting protamine sulfate according to claim 1, wherein, In Step 4, Step 3 is repeatedly performed multiple times to form 3-15 layers of functional films.

4. The fiber optic sensor for detecting protamine sulfate according to claim 1, wherein, The hydroxide solution is selected from a sodium hydroxide solution.

5. The pepsin detection optical fiber sensor according to claim 1, wherein, The concentration of the hydroxide solution is less than or equal to 1.0 mol / L; the concentration of the chitosan acetic acid solution is less than or equal to 0.01 mg / mL; and the concentration of the heparin sodium injection is 2.5-10 mg / mL.

6. A protamine sulfate detection system characterized by, The system comprises an ultrabroadband light source, the optical fiber sensor of claim 1 and a spectrum analyzer; the first single-mode optical fiber at one end of the optical fiber sensor is connected to the output end of the ultrabroadband light source, and the second single-mode optical fiber at the other end is connected to the spectrum analyzer.

7. The detection system of claim 6, wherein, The system further comprises a fiber clamp that holds the two ends of the optical fiber sensor and makes the optical fiber sensor in an extended state.

8. The detection system of claim 6, wherein, The system further comprises a lifting platform, which is provided with a sample tank; the optical fiber sensor is adjusted to enter the sample to be measured in the sample tank.

9. A method for detecting protamine sulfate, characterized by, The sensor of claim 1 or the system of claim 6 is used to detect the content of protamine sulfate in a sample to be measured. The method comprises: obtaining the spectral data of the sample to be measured, and calculating the content of protamine sulfate in the sample to be measured according to the highest-order interference peak wavelength of the sample to be measured and the highest-order interference peak wavelength of a standard sample.

10. The protamine sulfate detection method according to claim 9, characterized in that, The sample to be measured is a drug or blood.

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