Optical fiber SPR-fluorescence calcium ion detection method and device

Abstract

The invention belongs to the technical field of ion detection, and relates to a calcium ion detection method and device based on optical fiber SPR and fluorescence double signals. The method comprises the following steps: detecting a calcium-calcein standard solution by using an optical fiber sensing probe, and establishing a double-signal quantitative model of SPR signals and fluorescence signals; synchronously collecting double signals of the liquid sample to be detected; and substituting a signal value into a model to calculate the calcium ion concentration. The device comprises a heterogeneous optical fiber probe module integrating SPR and a fluorescence sensing unit, a microfluidic module used for fixing a probe and transporting a sample, and a signal acquisition and processing module used for synchronously exciting and acquiring double signals. By utilizing the characteristic that the complex reaction of the same indicator synchronously initiates refractive index and fluorescence change, an SPR signal and a fluorescence signal are obtained at the same time, and the accuracy, the specificity and the anti-interference capability of detection are remarkably improved through cooperative verification of the SPR signal and the fluorescence signal. The device integrates a microfluidic technology, and has the advantages of small volume, low sample demand, quick response and simplicity and convenience in operation.

Description

Technical Field

[0001] This invention belongs to the field of ion detection technology and relates to a method and device for calcium ion detection using a combination of fiber surface plasmon resonance (SPR) and fluorescence dual signals. Background Technology

[0002] Calcium ions (Ca²) + Concentration is a key parameter for water environment monitoring, ecological research, and resource utilization. Achieving accurate, rapid, and on-site detection of this concentration is of great significance.

[0003] Currently, calcium ion detection mainly relies on methods such as atomic absorption spectrometry, inductively coupled plasma mass spectrometry, and colorimetry. While these methods are accurate, they involve bulky equipment, complex operation, and high costs, making them unsuitable for on-site, in-situ, and rapid monitoring. Therefore, fiber optic sensing technology has been introduced into this field due to its advantages such as small size, resistance to electromagnetic interference, and suitability for remote monitoring.

[0004] Existing fiber optic sensing solutions mostly employ a single detection principle. For example, sensors based on fiber optic surface plasmon resonance (SPR), such as the protein detection device based on fiber optic SPR disclosed in Chinese patent CN106526195A, have high sensitivity, but their SPR signals are easily interfered with by temperature, salinity changes, and other non-specific adsorptions in complex matrices in water, resulting in insufficient selectivity and quantitative accuracy for calcium ions. On the other hand, sensors based on fiber optic fluorescence have high specificity and fast response, but their fluorescence signal intensity is easily affected by light source fluctuations, sample turbidity, and scattering, posing challenges to detection stability and sensitivity, as exemplified by the fluorescence-based fiber optic biochemical sensor disclosed in CN113030035A.

[0005] In addition, some studies have attempted to combine different principles to improve performance. For example, CN105548102A discloses a method for monitoring calcium ion concentration in deep tissues using the photoacoustic-fluorescence complementary principle. However, this technology is geared towards biomedical tissue imaging, relies on photoacoustic effects and ultrasound detection, has complex devices, and is not suitable for complex matrices or environments requiring rapid on-site detection.

[0006] Therefore, there is an urgent need for a Ca² method that can simultaneously acquire spectral and fluorescence signals using fiber optic probes under label-free conditions, and fully exploit the complementary characteristics of multimodal information through an adaptive fusion mechanism. + The detection technology solution aims to improve detection sensitivity, stability, and anti-interference capabilities. Summary of the Invention

[0007] In view of this, the present invention aims to overcome the shortcomings of existing calcium ion detection technologies, such as the complexity and inconvenience of operating large-scale instruments, and the weak anti-interference ability, poor selectivity, and insufficient detection accuracy of fiber optic sensors based on a single principle, such as pure SPR or pure fluorescence, in complex liquid matrices, making it difficult to achieve rapid and micro-level detection on site. The present invention provides a fiber optic SPR-fluorescence calcium ion detection method and device.

[0008] The detection method includes the following steps: S1. Establish a dual-signal model: Use fiber optic sensing probes to detect a series of calcium-calcichlorophyll standard solutions of known concentrations, simultaneously acquire their surface plasmon resonance (SPR) signal and fluorescence signal, and establish a dual-signal linear fitting curve model that reflects the relationship between calcium ion concentration and the SPR signal and fluorescence signal. S2. Synchronous acquisition of the signal to be tested: Using the fiber optic sensing probe, the SPR signal and fluorescence signal in the liquid sample to be tested are acquired simultaneously. S3. Calculate calcium ion concentration: Substitute the SPR signal value and fluorescence signal value collected in step S2 into the dual-signal linear fitting curve model to calculate the calcium ion concentration in the liquid sample to be tested.

[0009] The SPR signal is used to characterize the change in refractive index of the medium caused by the calcium-calcein complexation reaction, and the fluorescence signal is used to characterize the change in fluorescence intensity caused by the calcium-calcein complexation reaction. The two are independent physical quantities that work together to participate in the quantitative calculation of calcium ion concentration.

[0010] Furthermore, the calcein, as a fluorescent indicator, undergoes a specific complexation reaction with calcium ions, causing a change in the refractive index of the detection interface medium to excite and generate the SPR signal. Simultaneously, under the action of excitation light, it generates a change in fluorescence intensity to form the fluorescence signal.

[0011] Preferably, the concentration range of the series of calcium-calciferol standard solutions with known concentrations covers 200 μmol / L to 1400 μmol / L.

[0012] Preferably, after obtaining the detection concentration, the method further includes a step of verifying the accuracy of the detection result using the spiked recovery rate method, specifically: adding a known concentration of calcium-calcichlorophyll standard solution to the liquid sample to be tested, obtaining the SPR signal and fluorescence signal of the sample before and after spiking, and calculating the spiked recovery rate.

[0013] The detection device is used to implement the above method, including: The fiber optic sensing probe module adopts a heterogeneous fiber structure and integrates an SPR sensing unit for sensing changes in refractive index and a fluorescence sensing unit for receiving fluorescence signals. A microfluidic module includes a microfluidic chip 1, wherein the microfluidic chip 1 is provided with an adapter socket A1 for fixing the fiber optic sensing probe module and a microfluidic channel for accommodating and transporting the liquid sample to be tested. The signal acquisition and processing module includes an excitation light source 7 for exciting the fluorescence sensing unit, a broadband light source 8 for exciting the SPR sensing unit, an optical fiber coupling assembly for coupling light from the two light sources to the optical fiber sensing probe module and coupling the light signal containing SPR and fluorescence information returned from the probe to the detection end, a spectrometer 14 for receiving the light signal, and a data processing unit 15 for processing spectral data and calculating concentration.

[0014] Furthermore, the SPR sensing unit is a gold film region located on the end face and side of the optical fiber, responsible for sensing changes in the refractive index of the medium; the fluorescence sensing unit is located in the solution region surrounding the gold film and the optical fiber interaction region, responsible for receiving fluorescence emission signals.

[0015] Furthermore, the signal acquisition and processing module also includes a long-pass filter 12 disposed in front of the optical path entrance of the spectrometer 14, used to filter out the wavelength of the excitation light source 7 and allow the fluorescence signal to pass through.

[0016] Furthermore, the microfluidic module also includes an injection pump, a waste liquid reservoir, and a silicone capillary; the silicone capillary includes a first capillary for connecting the injection pump 2 to the sample inlet of the microfluidic channel, and a second capillary for connecting the sample outlet of the microfluidic channel to the waste liquid reservoir 3.

[0017] Preferably, the excitation source is a 405nm laser 7; the broadband source is a halogen source 8 with an emission wavelength range covering 350-1000nm.

[0018] Preferably, the heterogeneous fiber structure is formed by fusion splicing multimode fiber and thin-core fiber, and a gold film with a thickness of 40-50nm is deposited in the end region of the thin-core fiber to form the SPR sensing unit.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) By utilizing the characteristic that calcein and calcium ions simultaneously induce changes in SPR and fluorescence signals, a dual-signal model was established and validated to achieve mutual complementarity and calibration of two independent physical quantities (wavelength and light intensity). This effectively overcomes the problem that a single signal is easily interfered with by temperature, salinity, turbidity or non-specific adsorption in complex liquid matrices, and significantly improves the accuracy, reliability and anti-interference ability of the detection results.

[0020] (2) The method has a wide linear range (200-1400 μmol / L), covering the detection needs of common samples such as environmental water bodies; it has high accuracy, with a spiked recovery rate of 95%-105%; it has good repeatability, with a relative standard deviation (RSD) of less than 3%, which fully meets the accuracy requirements of practical applications.

[0021] (3) The device is highly integrated, combining fiber optic probes with microfluidic chips to achieve system miniaturization. It requires only trace amounts of sample (μL level), has a fast response speed, and can directly mix detection solutions, making it easy to operate. These features make it very suitable for rapid on-site detection on resource-constrained platforms such as shipborne, buoy, and field laboratories.

[0022] Other advantages, objectives, and features of the invention will be set forth in part in the description which follows, and in part will be apparent to those skilled in the art from the following examination, or may be learned from practice of the invention. The objectives and other advantages of the invention can be realized and obtained through the following description. Attached Figure Description

[0023] To make the objectives, technical solutions, and advantages of the present invention clearer, the preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings, wherein: Figure 1 This is a flowchart of the detection method of the present invention; Figure 2 This is a dual-signal spectrum of the detection method of the present invention; Figure 3 This is a diagram of the dual-signal linear fitting curve model of the detection method of the present invention; wherein... Figure 3 (a) is the SPR resonance wavelength shift fitting curve. Figure 3 (b) is the fitted curve of fluorescence intensity change; Figure 4 This is a verification diagram showing the specificity of the detection method of the present invention; Figure 5 This is a schematic diagram of the microfluidic chip structure in the detection device of the present invention; wherein, Figure 5 (a) is a three-dimensional diagram of the overall chip structure. Figure 5 (b) is a cross-sectional view of the chip along its central axis; The markings and corresponding components in the diagram are: A1 - Fiber optic adapter socket, A2 - Chip reservoir, A3 - Sample inlet channel, A4 - Reaction chamber, A5 - Sample outlet channel; Figure 6 This is a schematic diagram of the connection of the detection device of the present invention; The markings and corresponding components in the diagram are as follows: 1-Microfluidic chip, 2-Injection pump, 3-Waste liquid tank, 4-Silicone capillary, 5-Reaction chamber (located inside chip 1), 6-Bare fiber adapter, 7-405nm excitation light source, 8-Halogen light source, 9-First fiber coupler, 10-Second fiber coupler, 11-FC adapter, 12-Filter, 13-Fiber jumper, 14-Spectrometer, 15-Computer. Detailed Implementation

[0024] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that the illustrations provided in the following embodiments are only schematic representations of the basic concept of the present invention. Unless otherwise specified, the following embodiments and features can be combined with each other.

[0025] The accompanying drawings are for illustrative purposes only and are schematic diagrams, not actual pictures. They should not be construed as limiting the invention. To better illustrate the embodiments of the invention, some parts in the drawings may be omitted, enlarged, or reduced, and do not represent the actual product dimensions. It is understandable to those skilled in the art that some well-known structures and their descriptions may be omitted in the drawings.

[0026] Example 1 provides a method for calcium ion detection using fiber optic SPR-fluorescence, such as... Figure 1 As shown, it includes the following steps: S1. Establish a dual-signal model: Use fiber optic sensing probes to detect a series of calcium-calcichlorophyll standard solutions of known concentrations, simultaneously acquire their surface plasmon resonance (SPR) signal and fluorescence signal, and establish a dual-signal linear fitting curve model that reflects the relationship between calcium ion concentration and the SPR signal and fluorescence signal. S2. Synchronous acquisition of the signal to be tested: Using the fiber optic sensing probe, the SPR signal and fluorescence signal in the liquid sample to be tested are acquired simultaneously. S3. Calculate calcium ion concentration: Substitute the SPR signal value and fluorescence signal value collected in step S2 into the dual-signal linear fitting curve model to calculate the calcium ion concentration in the liquid sample to be tested.

[0027] Specifically, in step S1, a series of calcium-calcein standard solutions of known concentrations are prepared using deionized water, with a concentration gradient covering 200 μmol / L–1400 μmol / L. The fiber optic sensing probe is then sequentially placed into each standard solution, and SPR and fluorescence signals are acquired simultaneously, with a response time <0.5 min, to obtain the following results: Figure 2 The dual-signal spectrum shown indicates that as the concentration of calcium-calcein increases from 200 μmol / L to 1400 μmol / L, the SPR resonance wavelength continuously shifts towards longer wavelengths, indicating that the formation of the calcium-calcein complex effectively alters the refractive index of the medium at the sensing interface. At 520 nm, the fluorescence intensity increases gradient with increasing calcium ion concentration, indicating that the specific complexation reaction between calcium ions and calcein produces a significant fluorescence enhancement effect.

[0028] The resonance wavelength and fluorescence intensity at 520 nm were recorded respectively. A linear regression fitting was performed with calcium ion concentration as the abscissa and SPR resonance wavelength and fluorescence intensity as the ordinates, respectively, to obtain the following results: Figure 3 (a) and Figure 3 (b) The SPR resonance wavelength shift fitting curve and the fluorescence intensity change fitting curve shown together constitute the dual-signal linear fitting curve model. The fitting curves show that the two independent physical quantities, SPR resonance wavelength and fluorescence intensity, both increase synchronously and linearly under different concentration gradients. This change verifies the effectiveness of the detection reaction.

[0029] When this model is used to detect actual liquid samples, the two signals can complement and verify each other. When one signal deviates due to specific interference (such as the effect of interfering ions on SPR, or the effect of turbidity on fluorescence), the other signal can provide a reliable correction benchmark, thereby improving the accuracy and anti-interference ability of quantitative results in complex water matrices.

[0030] In step S2, the sample to be tested is taken and appropriately diluted with deionized water according to its estimated calcium ion concentration range, so that its calcium ion concentration falls within the linear range of 200-1400 μmol / L established in step S1. After pretreatment, the sample is refrigerated and used immediately. Subsequently, using the same fiber optic sensing probe, it is placed into the pretreated sample, and the SPR signal and fluorescence signal of the sample are acquired simultaneously to obtain the SPR resonance wavelength value λ of the sample. s and characteristic fluorescence intensity value I s .

[0031] λ s and I s Substituting these values ​​into the two linear equations obtained in step S1, we can calculate two concentration values, C1 and C2. The final detectable concentration C of calcium ions in the sample can be determined by averaging, weighted averaging, or by selecting one method based on signal quality.

[0032] Preferably, after step S3, a spiked recovery method verification experiment is conducted to verify the suitability and detection accuracy of the method described in Example 1 for a calcium-containing liquid matrix. Specifically, a known calcium-calcein concentration (within the range of 200 μmol / L-1400 μmol / L) is selected to spike the diluted sample to be tested. The spiked recovery rate reflects the method's recovery efficiency for the target ion, and the calculation formula is as follows: P = ×100%, where P is the water spiking recovery rate. The actual concentration after spiking was measured. To detect the background concentration of the sample, The theoretical scalar values ​​are 200 μmol / L, 400 μmol / L, and 600 μmol / L, respectively.

[0033] Each concentration point was measured at least three times, and the spiked recoveries of the SPR and fluorescence signals were calculated separately. During the calculation, considering the background interference and response fluctuations of the SPR signal in complex water matrices, which limit the quantitative accuracy, this embodiment preferentially uses the fluorescence method for recovery evaluation. The spiked recoveries of the fluorescence signal remained stable within the range of 95%-105%, with a relative standard deviation (RSD) of less than 3%, indicating that this method has good accuracy and reliability for the detection of calcium ions in the water matrix.

[0034] Preferably, an anti-interference ion experiment is used to further verify the high specificity of the method of the present invention for calcium ions. A series of solutions are prepared, each containing the same concentration (1000 μmol / L) of Ca²⁺. + Na + K + Mg² + Plasma solutions were prepared, and equal concentrations of calcein were added as fluorescence indicators. The corresponding fluorescence intensities were measured. Given that the refractive index change caused by the interfering ions was minimal and the SPR signal showed no significant wavelength-specific shift, the fluorescence intensity under the interfering ions was the primary focus. The results are as follows: Figure 4 As shown, the fluorescence signal only exhibits a significant response in the presence of calcium ions, while the signal changes caused by other interfering ions are negligible and not significantly different from those in the background solution. This directly reflects the excellent selectivity of calcein for calcium ions, thus ensuring the reliability of the fluorescence signal as a specific reference.

[0035] In this embodiment, calcein is used as a fluorescent indicator, which can specifically complex with calcium ions, thereby achieving specific detection of calcium ions in water. Alternatively, fluorescent indicators with specific complexing ability for other target ions can be selected to achieve the detection of corresponding ion concentrations.

[0036] Example 2 provides a fiber optic SPR-fluorescence calcium ion detection device, comprising: a fiber optic sensing probe module, a microfluidic module, and a signal acquisition and processing module.

[0037] The fiber optic sensing probe module is a heterogeneous fiber structure, consisting of a 10mm long multimode fiber and a 6mm long thin-core fiber fused together. A 40-50nm thick gold film is deposited on the end face and side 1mm region of the thin-core fiber using magnetron sputtering to form the SPR sensing unit. The surrounding solution and fiber interaction area constitute the fluorescence sensing unit.

[0038] The microfluidic module includes a microfluidic chip, a syringe pump, a waste reservoir, and a silicone capillary. The microfluidic chip, such as... Figure 5 As shown, Figure 5 (a) is a three-dimensional diagram of the overall chip structure. Figure 5 (b) is a cross-sectional view of the chip along its central axis. The microfluidic chip has a fiber optic adapter socket A1 and a chip reservoir A2. The chip reservoir includes an injection channel A3, a reaction chamber A4, and an outlet channel A5. The microfluidic chip is made entirely of quartz material and is a cuboid of 20mm × 20mm × 60mm. The fiber optic adapter socket has an overall thickness of 4mm, a T-shaped structure, and a central channel for fixing the fiber optic sensing probe. The reaction chamber A4 has a volume of approximately 4.5mL. Both the injection channel A3 and the outlet channel A5 are cylindrical. There are two silicone capillaries: one connects the injection channel A3 to the syringe pump 2 for injecting the sample; the other connects the outlet channel A5 to the waste liquid tank 3 for collecting waste liquid after detection, enabling micro-volume detection of 200μL-800μL.

[0039] Signal acquisition and processing module, such as Figure 6 As shown, it is used to acquire SPR and fluorescence signals. It includes: a 405nm excitation source 7, a 350-1000nm halogen source 8, a first fiber coupler 9, a second fiber coupler 10, a bare fiber adapter 6, an FC adapter 11, a 550nm long-pass filter 12, fiber optic patch cords 13, a spectrometer 14, and a computer 15.

[0040] The specific optical path connection is as follows: the two input ports of the first fiber coupler 9 are connected to the excitation source 7 and the halogen source 8, and its output port is connected to the input port of the second fiber coupler 10 via the FC adapter 11. One output port of the second fiber coupler 10 is connected to the fiber probe via the bare fiber adapter 6, and the other output port is connected to the spectrometer 14 via the filter 12 and the fiber optic patch cord 13. The spectrometer communicates with the computer 15.

[0041] During the detection process, the injection pump 2 injects the sample to be tested into the reaction chamber A4 of the microfluidic chip 1 through the silicone capillary tube 4. The spectrometer 14 transmits the collected SPR characteristic signal and fluorescence characteristic signal to the computer 15 for processing. After the detection is completed, the waste liquid pool 3 collects the tested sample.

[0042] In this example, a microfluidic chip was used to achieve micro-volume detection of the solution, ensuring that the entire system is leak-free and well-sealed. Only μL-level samples are required for detection, making it suitable for marine scenarios with limited sampling volumes. This also reduces reagent costs and matrix contamination. By combining the enhancement effects of SPR and fluorescence, the detection sensitivity and linear response range are significantly improved, while suppressing the interference of the water matrix on the dual signals, further ensuring detection accuracy and improving overall detection performance.

[0043] The synchronous acquisition action described in Example 1 is implemented in the device described in Example 2 in the following manner: the halogen light source and the 405 nm excitation light source are turned on simultaneously, and their light is combined through an optical fiber coupler before being transmitted to the sensing probe. The returned light signal contains the SPR signal and the fluorescence signal, which are simultaneously received by the same spectrometer. The spectral data is analyzed in real time using Spectral Analysis software to obtain the resonance wavelength and fluorescence intensity value at the same time point.

[0044] Preferred embodiments of the present invention are shown below: In a preferred embodiment of the present invention, the fiber optic sensing probe employs a multimode fiber-core fiber heterostructure, fabricated via fiber fusion splicing. This structure allows for better transmission of optical signals while enhancing the response performance of SPR and fluorescence signals. The optical sensor is a probe-type design, compact in size, and can be directly embedded into the fiber optic adapter socket of a microfluidic chip, fixed with Blu-Tack adhesive, enabling micro-level detection within the microfluidic chip. The optical sensor can also employ heterostructures such as multimode fiber-single-mode fiber, multimode fiber-single-multimode fiber, or multimode fiber-coreless fiber-multimode fiber heterostructures. Furthermore, fiber optic sensors can be fabricated using methods such as fiber tapering, fiber polishing, and fiber etching.

[0045] In a preferred embodiment of the present invention, a gold film is deposited on the end face and side face of the optical sensing probe using magnetron sputtering deposition technology. The process parameters are: sputtering pressure 6 Pa, sputtering current 60 mA, deposition time 14 s, and gold film thickness controlled at 40-50 nm. This thickness of gold film can effectively excite the SPR effect. Simultaneously, when detecting the solution, calcium around the gold film specifically complexes with calcein, significantly enhancing the fluorescence intensity. The gold film simultaneously possesses the excitation function of the SPR effect and the amplification function of the fluorescence signal, realizing the dual-signal synergistic detection of fiber optic SPR and fluorescence, thus improving detection accuracy.

[0046] In a preferred embodiment of the present invention, the fluorescent indicator calcein specifically complexes with calcium ions, and the optimal complexation ratio, determined by Job's curve method, is 1:2. At this ratio, the complexation reaction is rapid and stable, maintaining high specificity with calcium ions in water and effectively avoiding the formation of other ions (K+). + Na + Fe³ + Mg² + Li + Interference.

[0047] In a preferred embodiment of the present invention, a direct calcium-calcein mixed detection technique is employed. This eliminates the need to modify and fix calcein onto the surface of the fiber optic sensing probe, allowing the target detection reaction to be completed directly in the solution. This eliminates the complex modification process of the fiber optic probe, simplifies the detection procedure, and improves the applicability of the detection.

[0048] In a preferred embodiment of the present invention, the microfluidic chip is designed using SolidWorks and fabricated using 3D printing technology. With a compact structure, the microfluidic chip consists of a reaction chamber for storing the test liquid and an adapter holder for fixing the fiber optic sensing probe, facilitating the detection of minute amounts of ions efficiently. It is understood that the microfluidic module is not limited to the structure described in the embodiment; any cavity structure capable of fixing the fiber optic probe and enabling the injection, reaction, and discharge of minute amounts of liquid is applicable.

[0049] It should be noted that the technical solution of this invention is not limited to a specific liquid matrix. The core of the fiber optic SPR-fluorescence dual-signal detection method and device provided by this invention lies in utilizing the principle that a specific fluorescent indicator (such as calcein) binds to the target ion, simultaneously causing a change in refractive index (generating an SPR signal) and a change in fluorescence signal. This is achieved through the establishment of a dual-signal model, enabling accurate and interference-resistant quantitative analysis. Therefore, this method and device are also applicable to the detection of specific ions in different liquid environments, such as seawater, freshwater, pore water from sediments, industrial circulating water, biological fluids, or laboratory buffer solutions. The key is to select a fluorescent indicator that can specifically complex with the target ion and generate a corresponding change in optical signal, and to establish a corresponding dual-signal calibration model. The microfluidic module and signal acquisition and processing module of the device have good versatility and adaptability, and can be applied to the injection and detection of various liquid samples mentioned above.

[0050] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A method for detecting calcium ions using fiber optic SPR-fluorescence, characterized in that, Includes the following steps: S1. Establish a dual-signal model: Use fiber optic sensing probes to detect a series of calcium-calcichlorophyll standard solutions of known concentrations, simultaneously acquire their surface plasmon resonance (SPR) signal and fluorescence signal, and establish a dual-signal linear fitting curve model that reflects the relationship between calcium ion concentration and the SPR signal and fluorescence signal. S2. Synchronous acquisition of the signal to be tested: Using the fiber optic sensing probe, the SPR signal and fluorescence signal in the liquid sample to be tested are acquired simultaneously. S3. Calculate calcium ion concentration: Substitute the SPR signal value and fluorescence signal value collected in step S2 into the dual-signal linear fitting curve model to calculate the calcium ion concentration in the liquid sample to be tested. The SPR signal is used to characterize the change in refractive index of the medium caused by the calcium-calcein complexation reaction, and the fluorescence signal is used to characterize the change in fluorescence intensity caused by the calcium-calcein complexation reaction. The two are independent physical quantities that work together to participate in the quantitative calculation of calcium ion concentration.

2. The calcium ion detection method according to claim 1, characterized in that, The concentration range of the series of known calcium-calciferol standard solutions covers 200 μmol / L to 1400 μmol / L.

3. The calcium ion detection method according to claim 1, characterized in that, The calcein, as a fluorescent indicator, undergoes a specific complexation reaction with calcium ions, causing a change in the refractive index of the detection interface medium to generate the SPR signal. Simultaneously, it generates a change in fluorescence intensity under the action of excitation light to form the fluorescence signal.

4. The calcium ion detection method according to claim 1, characterized in that, After obtaining the detection concentration, the method further includes a step of verifying the accuracy of the detection results using the spiked recovery rate method. Specifically, a known concentration of calcium-calcichlorophyll standard solution is added to the liquid sample to be tested, and the SPR signal and fluorescence signal of the samples before and after spiking are obtained respectively, and the spiked recovery rate is calculated.

5. An apparatus for implementing the calcium ion detection method according to any one of claims 1-4, characterized in that, include: The fiber optic sensing probe module adopts a heterogeneous fiber structure and integrates an SPR sensing unit for sensing changes in refractive index and a fluorescence sensing unit for receiving fluorescence signals. A microfluidic module includes a microfluidic chip (1), wherein the microfluidic chip (1) is provided with an adapter holder (A1) for fixing the fiber optic sensing probe module and a microfluidic channel for accommodating and transporting the liquid sample to be tested. The signal acquisition and processing module includes an excitation light source (7) for exciting the fluorescence sensing unit, a broadband light source (8) for exciting the SPR sensing unit, an optical fiber coupling assembly for coupling light from the two light sources to the optical fiber sensing probe module and coupling the light signal containing SPR and fluorescence information returned from the probe to the detection end, a spectrometer (14) for receiving the light signal, and a data processing unit (15) for processing spectral data and calculating concentration.

6. The apparatus according to claim 5, characterized in that, The SPR sensing unit is a gold film area located on the end face and side of the optical fiber, responsible for sensing changes in the refractive index of the medium; the fluorescence sensing unit is located in the solution area around the gold film and the optical fiber interaction area, responsible for receiving fluorescence emission signals.

7. The apparatus according to claim 5, characterized in that, The excitation source is a 405nm laser (7); the broadband source is a halogen source (8) with an emission wavelength range covering 350-1000nm.

8. The apparatus according to claim 7, characterized in that, The signal acquisition and processing module also includes a long-pass filter (12) placed in front of the optical path entrance of the spectrometer (14) to filter out the wavelength of the excitation light source (7) and allow the fluorescence signal to pass through.

9. The apparatus according to claim 5, characterized in that, The heterogeneous fiber structure is formed by fusing multimode fiber and thin-core fiber, and a gold film with a thickness of 40-50nm is deposited in the end region of the thin-core fiber to form the SPR sensing unit.

10. The apparatus according to claim 5, characterized in that, The microfluidic module further includes an injection pump, a waste liquid reservoir, and a silicone capillary; the silicone capillary includes a first capillary for connecting the injection pump to the inlet of the microfluidic channel, and a second capillary for connecting the outlet of the microfluidic channel to the waste liquid reservoir.

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