A ratiometric fluorescent sensing system and method for detecting Cu 2+ ​

Abstract

The application relates to the technical field of detection, and provides a ratio fluorescent sensing system and method for detecting Cu2+. The application relates to the technical field of detection, and provides a ratio fluorescent sensing system and method for detecting Cu2+. The application relates to the technical field of detection, and provides a ratio fluorescent sensing system and method for detecting Cu2+. The application relates to the technical field of detection, and provides a ratio fluorescent sensing system and method for detecting Cu2+. The application relates to the technical field of detection, and provides a ratio fluorescent sensing system and method for detecting Cu2+. The application relates to the technical field of detection, and provides a ratio fluorescent sensing system and method for detecting Cu2+. The application relates to the technical field of detection, and provides a ratio fluorescent sensing system and method for detecting Cu2+. The application relates to the technical field of detection, and provides a ratio fluorescent sensing system and method for detecting Cu2+. The application relates to the technical field of detection, and provides a ratio fluorescent sensing system and method for detecting Cu2+. The application relates to the technical field of detection, and provides a ratio fluorescent sensing system and method for detecting Cu2+.

Description

Technical Field

[0001] This invention relates to the field of detection technology, and more specifically, to a ratio fluorescence sensing system and method for detecting Cu²⁺. Background Technology

[0002] With the rapid pace of industrialization and urbanization, large quantities of wastewater containing heavy metals are discharged into water bodies without adequate treatment, leading to severe pollution of surface water, groundwater, and even drinking water sources. Copper ions, a typical toxic heavy metal pollutant, are widely found in industrial activities such as electroplating, mining, metallurgy, printing and dyeing, and the use of agricultural fungicides. Although copper is an essential trace element for the human body, long-term exposure to concentrations exceeding the limits stipulated in the national standard "Surface Water Environmental Quality Standard" (GB 3838-2002) (Class III water bodies Cu²⁺ ≤ 1.0 mg / L, approximately 15.7 μM) can cause health problems such as nausea, liver damage, and nervous system disorders, and also exhibits significant toxicity to aquatic organisms.

[0003] Therefore, establishing a rapid, accurate, and applicable on-site detection technology for Cu²⁺ in complex water bodies has become an urgent need in the field of environmental monitoring.

[0004] Traditional water quality testing methods, such as atomic absorption spectrometry (AAS), inductively coupled plasma mass spectrometry (ICP-MS), and anodic stripping voltammetry (ASV), while possessing high sensitivity and accuracy, generally suffer from limitations such as expensive equipment, complex operation, and the need for professional personnel and laboratory support. These limitations make it difficult to meet the real-time, in-situ testing needs of scenarios such as field inspections, emergency response, and grassroots water management. In contrast, portable detection technologies based on optical sensors have gained widespread attention in recent years due to their advantages of low cost, fast response, and ease of operation.

[0005] Among numerous optical methods, fluorescence sensing has become an important means of heavy metal detection due to its high sensitivity (reaching the nM or even pM level), good selectivity, and visual output. In particular, ratiometric fluorescence, by introducing an internal standard signal that does not respond to the target analyte (such as red light emission) and a response signal (such as blue light emission) and calculating the ratio (B / R), effectively eliminates common interferences such as light source intensity fluctuations, uneven probe loading, and imaging system noise, significantly improving the stability and reliability of detection. However, existing ratiometric fluorescence test strips or sensors still face multiple challenges in actual water quality detection, severely restricting their widespread application in real-world environments.

[0006] The complexity of real-world water bodies far exceeds the ideal conditions of a laboratory. Natural water bodies typically contain natural organic matter (NOM) such as humic acid and fulvic acid, resulting in a yellow or brownish color; suspended particulate matter causes increased turbidity; pH ​​fluctuations (such as acidic mine drainage or alkaline industrial wastewater) affect the protonation state and metal-binding ability of probe molecules; and temperature changes affect fluorescence quantum yield and reaction kinetics. All these factors interfere with the excitation, emission, and acquisition of fluorescence signals, leading to distorted B / R ratios and significant quantitative errors. For example, the strong absorption of humic acid in the blue light band (<500nm) significantly weakens the blue fluorescence channel signal, causing false positives. Furthermore, the inhomogeneity of the sensing interface limits signal repeatability and sensitivity. Traditional test strips rely on the physical adsorption or simple coating of probe molecules on the substrate, resulting in uneven probe distribution, easy detachment, and a lack of effective control over the mass transfer process, leading to long response times and large signal fluctuations. Although some studies have used molecular imprinting technology to improve selectivity, the control over the microstructure of the sensing layer remains insufficient.

[0007] Furthermore, while some studies have introduced internal standards or background subtraction to improve stability, most rely on additional optical components, multi-channel detection systems, or complex preprocessing procedures, increasing equipment costs and operational complexity, thus contradicting the design principles of "portability and low cost." In particular, there is still no effective and low-cost quantitative compensation mechanism for background color interference.

[0008] Therefore, it is necessary to design a ratio fluorescence sensing system and method for detecting Cu²⁺ to solve the technical problem that existing technologies struggle to detect Cu²⁺ quickly and effectively in complex environments like real water bodies. Summary of the Invention

[0009] In view of this, the present invention proposes a ratio fluorescence sensing system and method for detecting Cu²⁺, aiming to solve the technical problem of difficulty in rapid and effective detection in the complex environment of real water bodies in the prior art.

[0010] In one aspect, the present invention proposes a ratiometric fluorescence sensing system for detecting Cu²⁺, comprising:

[0011] A functional film layer, including a blue response probe B and a red rare earth internal standard R, is fixed on the surface of a magnetic molecular imprinted composite material. The wavelength difference between the emission peaks of B and R is ≥80nm, thus constructing a ratio response system composed of blue light emission signal and red light emission signal.

[0012] A magnetron splicing platform is used to enrich the magnetic molecularly imprinted composite material onto the surface of a sensing film using an electrically controlled gradient magnetic field, forming a uniform fluorescent response layer. The sensing film has a multi-point microarray structure, with each microarray unit used for Cu²⁺. + The test or as a quality control point;

[0013] The imaging module is used to acquire the signal intensity of the blue light emission and the red light emission;

[0014] The environmental compensation unit acquires environmental data detection results of the background color and corrects the blue / red ratio signal based on the environmental data detection results. The background color compensation is preset with a background interference index S. color The background interference index S color The interference index S represents the proportion of blue component in the background RGB brightness. color Used to compensate for and correct the initial mass of water bodies;

[0015] The calculation module calculates the blue / red intensity ratio and, in conjunction with a standard curve, obtains the concentration of the analyte.

[0016] Preferably, the computing module includes:

[0017] When the standard curve is fitted using a four-parameter Logistic equation or a ratio Stern-Volmer equation, MIIMs test strips are immersed in each standard solution, fluorescence images are captured after the reaction, and the B / R ratio at each concentration is calculated. The resulting concentration and corresponding B / R ratio represent a set of discrete observation points. in, Where i is the i-th data set, This is the Cu²⁺ standard solution for the i-th data set. It is the B / R ratio of the i-th data set;

[0018] Specifically, when the fluorescence of the blue response probe B is dynamically quenched by Cu²⁺, including electron transfer and energy transfer, the B / R ratio monotonically decreases with increasing Cu²⁺ concentration. This was fitted using the Stern-Volmer equation, which includes:

[0019] ;

[0020] in, For none The B / R ratio at that time; I is the addition The B / R ratio after that, The Stern-Volmer quenching constant is used to reflect the sensitivity of the sensor. The concentration of Cu²⁺.

[0021] Preferably, the computing module further includes:

[0022] The standard curve is fitted using a four-parameter Logistic equation or a ratio Stern-Volmer equation. When the response exhibits an S-shaped curve (i.e., a rapid response at low concentrations and a tendency to saturate at high concentrations), the Logistic equation is used for fitting, including:

[0023] ;

[0024] in, For none The B / R ratio at that time; For none The B / R ratio at time t, i.e., the upper asymptote; D is... Approaching infinity The ratio, also known as the lower asymptote, is the signal saturation value. half-saturation concentration ,Right now time , which reflects the midpoint of the detection; H is the slope of the curve, the hill coefficient, which reflects the response sensitivity.

[0025] Preferably, the emission peak of the blue response probe is located at 440–470 nm, and the emission peak of the red rare earth internal standard is located at 610–620 nm.

[0026] Preferably, the magnetic molecularly imprinted composite material has a core-shell structure, consisting of a Fe3O4 magnetic core, a SiO2 isolation layer, and an ion-imprinted polymer (IIPs) recognition layer, denoted as Fe3O4@SiO2@IIPs structure, with a particle size of 120-300 nm and an imprinted site density of 30–120 μmol·g. ﹣ ¹.

[0027] Preferably, the magnetron assembly platform includes:

[0028] Planar Helmholtz coils and miniature permanent magnet arrays;

[0029] Power supply and control unit, used for 1-15T·m ﹣ Under gradient magnetic field conditions, the composite material is enriched onto the membrane surface and forms a microarray.

[0030] Preferably, the functional film layer is disposed on an elastic substrate, which is selected from TPU, PDMS or SEBS, and the ultimate tensile strain of the elastic substrate is greater than 150% to adapt to different curved surface detection scenarios.

[0031] Preferably, the functional film layer undergoes a pre-stretch-release cycle treatment to form an island-bridge microstructure on an elastic substrate, enabling the functional material to maintain electrical and optical connectivity under tension and maintain a stable blue / red ratio signal.

[0032] Preferably, the imaging module includes:

[0033] An imaging unit is used to acquire fluorescence images of the sensing membrane;

[0034] Algorithm unit, used to perform the following steps:

[0035] Image preprocessing and region of interest extraction;

[0036] The RGB channel intensities were normalized, and the fluorescence intensities corresponding to blue light emission 440–470 nm and red light emission 610–620 nm were extracted using a pre-established RGB value-fluorescence intensity calibration model.

[0037] Calculate the blue / red ratio.

[0038] On the other hand, the present invention also provides a ratio fluorescence sensing method for detecting Cu²⁺, comprising:

[0039] Step S1: The blue response probe B and the red rare earth internal standard R are fixed together on the surface of the magnetic molecular imprinted composite material, wherein the wavelength difference between the emission peaks of B and R is ≥80nm, and a ratio response system composed of blue light emission signal and red light emission signal is constructed.

[0040] Step S2: The magnetron assembly platform enriches the magnetic molecularly imprinted composite material onto the surface of the sensing film using an electrically controlled gradient magnetic field, forming a uniform fluorescent response layer. The sensing film has a multi-point microarray structure, with each microarray unit used for Cu²⁺. + The test or as a quality control point;

[0041] Step S3: Used to collect the signal strength of the blue light emission and red light emission;

[0042] Step S4: Obtain the environmental data detection results of the background color, and correct the blue / red ratio signal according to the environmental data detection results. The background color compensation is preset with a background interference index S. color The background interference index S color The interference index S represents the proportion of blue component in the background RGB brightness. color Used to compensate for and correct the initial mass of water bodies;

[0043] Step S5: Calculate the blue / red intensity ratio and obtain the concentration of the analyte by combining it with the standard curve.

[0044] Compared with existing technologies, the advantages of this invention lie in the use of a blue response probe combined with a red rare-earth internal standard to construct a true B / R dual emission ratio system. Since the wavelength difference Δλ between the blue and red emission peaks is ≥80nm, spectral overlap and signal crosstalk problems can be significantly reduced, achieving high signal-to-noise ratio detection. This design overcomes the limitations of existing single-wavelength or narrow-interval ratio detection. Fe3O4@SiO2@IIPs nanocomposite materials are enriched onto the sensing film surface using an electrically controlled gradient magnetic field, forming a uniform microarray fluorescent response layer, ensuring controllable probe distribution and consistent response. Compared to traditional drop-coating, spin-coating, or self-assembled films, the magnetron array method produces films with higher uniformity and significantly improved repeatability of detection results. The standard curve of this invention supports both the Stern–Volmer equation for linear quenching processes and the four-parameter Logistic equation for S-shaped response curve fitting. Through the dual-model fitting mechanism, different concentration ranges and response modes can be covered, broadening the detection concentration range and improving fitting accuracy. Utilizing TPU, PDMS, or SEBS elastic substrates and employing a pre-stretch-release process to form an island-bridge microstructure, the sensing membrane maintains ratio signal stability even under tensile strain exceeding 150%, making it suitable for curved surface detection, such as wearable sensors and portable inspection sheets, thus expanding practical application scenarios. The imaging module, combined with an RGB camera and image algorithms, allows direct acquisition of ratio signals using mobile phones or portable imaging devices, avoiding expensive spectrometers. With environmental compensation and standard curve calculation, rapid detection can be performed in the field or on-site, enabling low-cost, intelligent, and portable applications.

[0045] Furthermore, compared to existing ratio sensing methods that do not consider environmental factors, the detection results of this invention remain stable and accurate even in complex environments. Traditional fluorescence sensors are susceptible to "dirty water" because in real water body detection (such as rivers, lakes, and industrial wastewater), samples often contain humic acid, fulvic acid, algae, and iron and manganese oxides. These background colors strongly interfere with the fluorescence signal, causing blue light to be absorbed and misinterpreted as high concentrations of Cu²⁺, resulting in false positives. Therefore, the detection results are unreliable and cannot be used for on-site law enforcement or emergency monitoring. This invention collects environmental data such as temperature, pH, turbidity, and background color, and combines them with a compensation model to correct the B / R ratio signal, especially through the background interference index S. color Achieve quantitative elimination of background optical interference.

[0046] It is understandable that the ratio fluorescence sensing system of the present invention not only has superior sensitivity and stability in laboratory testing, but also demonstrates multi-dimensional application value in commercial applications:

[0047] It can be used for online monitoring of Cu²⁺ in drinking water, surface water and industrial wastewater, providing a portable, low-cost and rapid-response detection method to meet the daily monitoring needs of environmental regulatory departments and enterprises.

[0048] It can be embedded in the food processing and drinking water supply chain to help companies establish a quality traceability system, meeting the urgent need for rapid screening under food safety regulations.

[0049] Based on a flexible substrate and an imaging module, the system of this invention can be combined with mobile phones, portable terminals, and even wearable devices to form a personal water quality testing tool, targeting the mass consumer market.

[0050] The Fe3O4@SiO2@IIPs material used has a mature preparation process and controllable cost. The magnetron assembly and microarray film formation process is easy to scale up and can be directly connected to the existing thin film and micro-nano processing industry chain, making it feasible for commercial production.

[0051] With the support of an app or cloud platform, the detection data can be stored, analyzed and shared, supporting smart water management, environmental big data platforms and home-level water quality monitoring systems, further expanding commercial application models. Attached Figure Description

[0052] Various other advantages and benefits will become apparent to those skilled in the art upon reading the following detailed description of preferred embodiments. The accompanying drawings are for illustrative purposes only and are not intended to limit the invention. Furthermore, the same reference numerals denote the same parts throughout the drawings. In the drawings:

[0053] Figure 1 This is a functional block diagram of a ratio fluorescence sensing system for detecting Cu²⁺ provided in an embodiment of the present invention;

[0054] Figure 2 The present invention provides a flowchart and a detection schematic diagram of the preparation of MIIMs for a ratiometric fluorescence sensing system for detecting Cu²⁺.

[0055] Figure 3 This is a flowchart of a ratio fluorescence sensing method for detecting Cu²⁺ provided in an embodiment of the present invention. specific Application method

[0056] Exemplary embodiments of the present disclosure will now be described in more detail with reference to the accompanying drawings. While exemplary embodiments of the present disclosure are shown in the drawings, it should be understood that the present disclosure may be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided to enable a more thorough understanding of the present disclosure and to fully convey the scope of the disclosure to those skilled in the art. It should be noted that, unless otherwise specified, the embodiments and features described herein can be combined with each other. The present invention will now be described in detail with reference to the accompanying drawings and embodiments.

[0057] See Figure 1 As shown, this embodiment proposes a ratiometric fluorescence sensing system for detecting Cu²⁺, comprising:

[0058] A functional film layer, including a blue response probe B and a red rare earth internal standard R, is fixed on the surface of a magnetic molecular imprinted composite material. The wavelength difference between the emission peaks of B and R is ≥80nm, thus constructing a ratio response system composed of blue light emission signal and red light emission signal.

[0059] A magnetron splicing platform is used to enrich the magnetic molecularly imprinted composite material onto the surface of a sensing film using an electrically controlled gradient magnetic field, forming a uniform fluorescent response layer. The sensing film has a multi-point microarray structure, with each microarray unit used for Cu²⁺. + The test or as a quality control point;

[0060] The imaging module is used to acquire the signal intensity of the blue light emission and the red light emission;

[0061] The environmental compensation unit acquires environmental data detection results of the background color and corrects the blue / red ratio signal based on the environmental data detection results. The background color compensation is preset with a background interference index S. color The background interference index S color The interference index S represents the proportion of blue component in the background RGB brightness. color Used to compensate for and correct the initial mass of water bodies;

[0062] The calculation module calculates the blue / red intensity ratio and, in conjunction with a standard curve, obtains the concentration of the analyte.

[0063] It is understood that this embodiment proposes an intelligent ratio fluorescence sensing system for detecting Cu²⁺, which not only achieves highly sensitive and selective quantitative analysis, but also overcomes the application bottleneck of traditional fluorescent test strips in complex real-world environments through multimodal integrated design. Using a magnetic molecularly imprinted composite material as a carrier, a blue response probe B with Cu²⁺-specific recognition capability and a stable red rare-earth internal standard R are co-immobilized on the material surface, constructing a dual-emission fluorescence system with Δλ≥80nm, effectively avoiding spectral crosstalk and ensuring accurate extraction of the B / R ratio signal. The magnetron splicing platform, by applying an electrically controlled gradient magnetic field, drives magnetic nanoparticles to rapidly accumulate on the surface of the sensing film and self-assemble into a highly uniform microarray structure, significantly improving fluorescence signal density and spatial consistency, while shortening the response time to less than 5 minutes. The imaging module uses a smartphone or CMOS camera to capture images in 365°. Fluorescence images are acquired under nm ultraviolet excitation, and image processing algorithms automatically identify each microarray unit and extract the average intensity values ​​of the blue and red channels, achieving non-contact, visual detection. Crucially, the system incorporates an environmental compensation unit, integrating temperature, pH, and turbidity sensors to monitor water physicochemical parameters in real time. It also innovatively utilizes images of the sensor membrane captured by a smartphone under white light illumination to calculate the background interference index Scolor. This index quantifies the absorption interference of water color on blue light excitation / emission without additional hardware, thereby establishing a correction model between Scolor and B / R signal attenuation. Under conditions where natural organic matter such as humic acid and fulvic acid causes water to turn yellow or become turbid, the system automatically and dynamically compensates for the original B / R value, significantly reducing the risk of false positives. The calculation module, based on the corrected B / R ratio and combined with a pre-calibrated Stern-Volmer or four-parameter Logistic standard curve, outputs Cu²⁺ concentration in real time and can generate a detection report via Bluetooth or an app. It features intelligent functions such as multi-point parallel detection, quality control self-verification, and environmental self-adaptation. It can work stably in complex scenarios such as rivers, lakes, and industrial wastewater. The detection limit can reach the sub-nM level, and the relative error is less than 5%. It achieves laboratory-level accuracy and convenient on-site rapid detection of heavy metals, providing a low-cost and highly robust technical solution for environmental monitoring, food safety, and emergency response.

[0064] In some embodiments of this application, the environmental compensation module has an interference index S. color This is used to compensate for and correct the initial mass of the water body, where the initial mass represents the color cast of the water body. This includes taking an image of the sensing membrane area using a smartphone under white light illumination. In this case, the fluorescent probe does not emit light and only records background color information.

[0065] Use image processing software (such as the Python OpenCV library) to extract the average RGB values ​​of the membrane region. These three values ​​represent the average brightness of the red, green, and blue channels, respectively. The background interference index S... color The definition includes: where, The range of values ​​is ;

[0066] Understandably, when the background is entirely yellow (without any blue component), .

[0067] When the background is white hour, .

[0068] When the background is pure blue .

[0069] A series of water samples containing different concentrations of humic acid or other colored substances were prepared to simulate the actual detection environment. During calibration, multiple types of interference (humic acid, Fe³⁺, algae, etc.) were included to improve the model's generalization ability.

[0070] For each water sample, in the same At different concentrations, the B / R ratio of the fluorescence image and the white light image were measured respectively. ;

[0071] by Plot a scatter plot with B / R as the x-axis and fit a curve, the relationship of which is denoted as: where a is the first fitting parameter and b is the second fitting parameter;

[0072] Based on the above relationships, the following compensation model is established: where, When it is pure water ; This is an empirical compensation coefficient, reflecting the degree of influence of background color changes on the B / R ratio;

[0073] B / R corrected This is the ratio of blue to red light intensity after background color interference compensation. This ratio is used to measure fluorescence signals. In the presence of background interference, the original measured B / R ratio may be distorted, so it needs to be corrected to more accurately reflect the concentration or other parameters under actual conditions.

[0074] B / R measured This is the ratio of blue to red light intensity directly obtained from the sensor without any compensation processing. It is based on directly observed data.

[0075] when When the background is yellowish / brown, more blue light is absorbed, thus affecting the B / R ratio. measuredThe value is too low and needs to be compensated. It is understandable that natural organic matter such as humic acid and fulvic acid in water samples mainly absorb ultraviolet and blue light <500nm, so they appear yellow or brown. This color change is directly reflected in the RGB imaging under white light.

[0076] Specifically, in this embodiment, an example compensation calculation process is given:

[0077] Measured

[0078] Extracted (i.e., the background is yellowish)

[0079] Known

[0080] Experience compensation coefficient

[0081] Substituting into the compensation formula, it can be seen that after background color compensation, the B / R ratio increases from 0.8 to 0.852.

[0082] In some embodiments of this application, the environmental compensation unit can also acquire real-time temperature, pH, and turbidity, and preset correction algorithms for each item, and provides examples of practical applications:

[0083] Example: Temperature Compensation

[0084] In this embodiment, the environmental compensation unit includes a temperature sensor DS18B20, which is used to collect and detect the temperature value of the environment in real time.

[0085] During the system calibration phase, the sensing membrane is placed in... Under these conditions, the blue / red ratio (B / R) was measured to obtain the temperature sensitivity coefficient. .

[0086] In actual testing, if the measured temperature is T, the ratio signal is corrected as follows:

[0087]

[0088] in, .

[0089] Example: pH compensation

[0090] In this embodiment, the environmental compensation unit further includes a miniature pH glass electrode for real-time monitoring of the acidity or alkalinity of the sample solution.

[0091] During the calibration phase, respectively The detection was performed in the buffer solution, and the pH compensation function was obtained by fitting:

[0092]

[0093] In actual testing, the corrected formula is as follows:

[0094]

[0095] Example: Turbidity Compensation

[0096] In this embodiment, the environmental compensation unit also includes a light scattering turbidity sensor for measuring the turbidity value of the sample solution.

[0097] Calibration experiments show that the attenuation of the turbidity ratio signal can be described by a linear relationship, with a compensation coefficient of [value missing]. .

[0098] In actual testing, the corrected formula is as follows:

[0099] ;

[0100] In some embodiments of this application, when the standard curve is fitted using a four-parameter Logistic equation or a ratio Stern-Volmer equation, MIIMs test strips are immersed in each standard solution, fluorescence images are captured after the reaction, and the B / R ratio at each concentration is calculated. The resulting concentration and corresponding B / R ratio are then used to obtain a set of discrete observation points. in, Where i is the i-th data set, This is the Cu²⁺ standard solution for the i-th data set. It is the B / R ratio of the i-th data set;

[0101] Specifically, when the fluorescence of the blue response probe B is dynamically quenched by Cu²⁺, including electron transfer and energy transfer, the B / R ratio monotonically decreases with increasing Cu²⁺ concentration. This was fitted using the Stern-Volmer equation, which includes:

[0102] ;

[0103] in, For none The B / R ratio at that time; I is the addition The B / R ratio after that, The Stern-Volmer quenching constant is used to reflect the sensitivity of the sensor. The concentration of Cu²⁺.

[0104] In some embodiments of this application, the standard curve is fitted using a four-parameter Logistic equation or a ratio Stern-Volmer equation. When the response exhibits an S-shaped curve (i.e., a rapid response at low concentrations and a tendency to saturate at high concentrations), the Logistic equation is used for fitting, including:

[0105] ;

[0106] in, For none The B / R ratio at that time; For none The B / R ratio at time t, i.e., the upper asymptote; D is... Approaching infinity The ratio, also known as the lower asymptote, is the signal saturation value. half-saturation concentration ,Right now time , which reflects the midpoint of the detection; H is the slope of the curve, the hill coefficient, which reflects the response sensitivity.

[0107] Specifically, this application significantly improves the accuracy and applicability of Cu²⁺ detection by adaptively fitting the standard curve using a four-parameter Logistic equation or a ratio Stern-Volmer equation. When the fluorescence of the blue probe B is quenched by Cu²⁺ and the B / R ratio decreases monotonically with concentration, a linear fit is performed using the physically meaningful Stern-Volmer equation, which is suitable for rapid detection at low concentrations, computationally efficient, and convenient for real-time on-site analysis. When the response exhibits an S-shaped pattern, sensitive at low concentrations and saturated at high concentrations, a nonlinear fit is performed using the four-parameter Logistic equation, accurately describing the response behavior over a wide dynamic range and improving the full-range fitting accuracy. By selecting the optimal model as needed based on the actual response characteristics, the system combines high sensitivity, a wide detection range, and strong robustness, achieving an upgrade from empirical table lookup to mathematical modeling. This provides a reliable data processing foundation for intelligent quantitative detection on-site, demonstrating outstanding technical effects and practical value.

[0108] In some embodiments of this application, the emission peak of the blue response probe is located at 440–470 nm, and the emission peak of the red rare earth internal standard is located at 610–620 nm.

[0109] In some embodiments of this application, the magnetic molecularly imprinted composite material has a core-shell structure, consisting of a Fe3O4 magnetic core, a SiO2 isolation layer, and an ion-imprinted polymer (IIPs) recognition layer, denoted as Fe3O4@SiO2@IIPs structure, with a particle size of 120-300 nm and an imprinted site density of 30–120 μmol·g. ﹣ ¹.

[0110] Specifically, the preparation of Fe3O4@SiO2@IIPs includes:

[0111] 0.724 g 7-HACA and 0.1345 g CuCl2 were ultrasonically dispersed in a three-necked flask containing 20 mL acetonitrile and allowed to stand for 2 h to construct a prepolymerization system. Then, 100 mg vinylated Fe3O4@SiO2 was added to 30 mL acetonitrile and ultrasonically dispersed for 30 min. The two solutions were mixed and 0.792 mL of crosslinking agent EGDMA was added. Nitrogen gas was purged for 30 min to remove oxygen from the reaction vessel. Subsequently, 10 mg of initiator AIBN was added, and the mixture was mechanically stirred at 65 °C under sealed conditions for 24 h. The reaction product was collected by centrifugation. The reaction product was then subjected to Soxhlet extraction with 0.2 mol / L EDTA as solvent to remove the template ion Cu2+. The product was washed three times each with deionized water and anhydrous ethanol, and then vacuum dried to obtain Fe3O4@SiO2@IIPs.

[0112] See Figure 2 As shown in some embodiments of this application, the preparation flowchart of MIIMs includes: first, preparing a functionalized substrate material Fe3O4@SiO2-KH570; then, using 7-HAC as the fluorescent functional monomer, Cu2+ as the template ion, and EGDMA as the crosslinking agent, preparing Fe3O4@SiO2@IIPs with different imprint layer thicknesses on the surface of Fe3O4@SiO2-KH570. Next, Fe3O4@SiO2@IIPs and Eu(MAA)3phen are added to the casting solution respectively, and MIIMs with uniformly loaded Fe3O4@SiO2@IIPs are prepared under the guidance of an external magnetic field. When Cu2+ is absent, the overall fluorescence color of the MIIMs is blue, the fluorescence color of 7-HAC. As the concentration of Cu2+ gradually increases, the fluorescence emission of 7-HAC is suppressed, and the overall fluorescence color of the MIIMs gradually changes to red, the fluorescence color of Eu(MAA)3phen.

[0113] MIIMs were prepared by a blending phase inversion method, where the Fe3O4@SiO2@IIPs were guided and loaded onto the rough surface of the imprinted film during the casting stage by applying a magnetic field. The preparation process was as follows: 4.0 g of PVDF and 0.1 g of PVP were accurately measured and added to a 50 mL three-necked flask containing 20 mL of DMAc. After ultrasonic dispersion for 30 min, the mixture was mechanically stirred until homogeneous. 100 mg of Eu(MAA)3phen and 200 mg of Fe3O4@SiO2@IIPs were added to the casting solution, and the mixture was mechanically stirred at 50°C for 1 day. After the reaction, the mixture was vacuum dried at 60°C for 8 h to remove air bubbles. The casting solution was slowly and uniformly poured onto a glass scraping plate, and the film was scraped at uniform speeds of 100 mm, 200 mm, 300 mm and 400 mm thicknesses, respectively. A strong magnet was placed 2.0 cm above the casting solution on the glass plate surface for 5.0 min to complete the magnetic guidance process. After that, the glass scraping plate was slowly immersed in deionized water for 2 h to complete the phase conversion process and obtain MIIMs of different thicknesses.

[0114] In some embodiments of this application, the magnetically controlled assembly platform includes:

[0115] Planar Helmholtz coils and miniature permanent magnet arrays;

[0116] Power supply and control unit, used for 1-15T·m ﹣ Under gradient magnetic field conditions, the composite material is enriched onto the membrane surface and forms a microarray.

[0117] Specifically, the planar Helmholtz coil consists of a pair of parallel circular or polygonal coils carrying current in the same direction, mounted below the detection cell. By adjusting the input current, a uniform and programmable gradient magnetic field can be generated in the sensing area, enabling remote, non-contact manipulation of magnetic nanoparticles. This coil has a simple structure and highly controllable magnetic field distribution, making it suitable for dynamic adjustment of magnetic field strength and direction in laboratory or portable devices.

[0118] The miniature permanent magnet array consists of multiple microscale neodymium iron boron (NdFeB) magnets arranged in a specific geometric pattern (such as a lattice or concentric circles) and fixed beneath the sensing film substrate. This array forms a static but highly localized strong gradient magnetic field in space, enabling the rapid adsorption and self-assembly of MIIMs (Mild Imaging Injection Molding Materials) into a multi-point microarray corresponding to the array structure without the need for power supply. Each micropoint corresponds to a detection unit or quality control point. This design features low power consumption, fast response, and a compact structure, making it particularly suitable for disposable, low-cost on-site testing equipment.

[0119] The power supply and control unit includes a programmable DC power supply, a current drive module, and a microcontroller (such as STM32 or Arduino) for precise control of the current magnitude and on / off timing of the Helmholtz coil, thereby enabling dynamic activation, intensity adjustment, and gradient optimization of the gradient magnetic field. During operation, the sensing membrane with added MIIMs dispersion is placed at the detection position, the power is turned on, and the temperature is within the range of 1-15 T·m. ﹣ ¹ Under the action of a gradient magnetic field (preferably 5-10 T·m) ﹣ ¹), under the action of magnetic force, the magnetic composite material rapidly migrates and accumulates on the film surface, forming a high-density, uniformly distributed fluorescent response layer, with an accumulation time of usually less than 2 minutes.

[0120] In some embodiments of this application, the functional film layer is disposed on an elastic substrate, which is selected from TPU, PDMS or SEBS, and the ultimate tensile strain of the elastic substrate is greater than 150% to adapt to different curved surface detection scenarios.

[0121] In some embodiments of this application, the functional film layer undergoes a pre-stretch-release cycle treatment to form an island-bridge microstructure on an elastic substrate, thereby maintaining the electrical and optical connection of the functional material under tension and stabilizing the blue / red ratio signal.

[0122] In some embodiments of this application, the imaging module includes:

[0123] An imaging unit is used to acquire fluorescence images of the sensing membrane;

[0124] Algorithm unit, used to perform the following steps:

[0125] Image preprocessing and region of interest extraction;

[0126] The RGB channel intensities were normalized, and the fluorescence intensities corresponding to blue light emission 440–470 nm and red light emission 610–620 nm were extracted using a pre-established RGB value-fluorescence intensity calibration model.

[0127] Calculate the blue / red ratio.

[0128] See Figure 3 As shown, this embodiment also provides a ratiometric fluorescence sensing method for detecting Cu²⁺, comprising:

[0129] Step S1: The blue response probe B and the red rare earth internal standard R are fixed together on the surface of the magnetic molecular imprinted composite material, wherein the wavelength difference between the emission peaks of B and R is ≥80nm, and a ratio response system composed of blue light emission signal and red light emission signal is constructed.

[0130] Step S2: The magnetron assembly platform enriches the magnetic molecularly imprinted composite material onto the surface of the sensing film using an electrically controlled gradient magnetic field, forming a uniform fluorescent response layer. The sensing film has a multi-point microarray structure, with each microarray unit used for Cu²⁺. + The test or as a quality control point;

[0131] Step S3: Used to collect the signal strength of the blue light emission and red light emission;

[0132] Step S4: Obtain the environmental data detection results of the background color, and correct the blue / red ratio signal according to the environmental data detection results. The background color compensation is preset with a background interference index S. color The background interference index S color The interference index S represents the proportion of blue component in the background RGB brightness. color Used to compensate for and correct the initial mass of water bodies;

[0133] Step S5: Calculate the blue / red intensity ratio and obtain the concentration of the analyte by combining it with the standard curve.

[0134] Compared with existing technologies, the beneficial effect of this embodiment lies in the use of a blue response probe combined with a red rare-earth internal standard to construct a true B / R dual emission ratio system. Since the wavelength difference Δλ between the blue and red emission peaks is ≥80nm, spectral overlap and signal crosstalk problems can be significantly reduced, achieving high signal-to-noise ratio detection. This design overcomes the limitations of existing single-wavelength or narrow-interval ratio detection. Fe3O4@SiO2@IIPs nanocomposite materials are enriched onto the sensing film surface using an electrically controlled gradient magnetic field, forming a uniform microarray fluorescent response layer, ensuring controllable probe distribution and response consistency. Compared with traditional drop-coating, spin-coating, or self-assembled film layers, the film layer prepared by the magnetron array method has higher uniformity, and the repeatability of detection results is significantly improved. The standard curve in this embodiment supports both the Stern–Volmer equation for linear quenching processes and the four-parameter Logistic equation for S-shaped response curve fitting. Through the dual-model fitting mechanism, different concentration ranges and response modes can be covered, broadening the detection concentration range and improving fitting accuracy. Utilizing TPU, PDMS, or SEBS elastic substrates and employing a pre-stretch-release process to form an island-bridge microstructure, the sensing membrane maintains ratio signal stability even under tensile strain exceeding 150%, making it suitable for curved surface detection, such as wearable sensors and portable inspection sheets, thus expanding practical application scenarios. The imaging module, combined with an RGB camera and image algorithms, allows direct acquisition of ratio signals using mobile phones or portable imaging devices, avoiding expensive spectrometers. With environmental compensation and standard curve calculation, rapid detection can be performed in the field or on-site, enabling low-cost, intelligent, and portable applications.

[0135] Furthermore, compared to existing ratio sensing methods that do not consider environmental factors, the detection results of this embodiment remain stable and accurate even in complex environments. Traditional fluorescence sensors are susceptible to "dirty water" because in real water body detection (such as rivers, lakes, and industrial wastewater), samples often contain humic acid, fulvic acid, algae, and iron and manganese oxides. These background colors strongly interfere with the fluorescence signal, causing blue light to be absorbed and misinterpreted as high concentrations of Cu²⁺, resulting in false positives. Therefore, the detection results are unreliable and cannot be used for on-site law enforcement or emergency monitoring. This embodiment collects environmental data such as temperature, pH, turbidity, and background color, and combines them with a compensation model to correct the B / R ratio signal, especially through the background interference index S. color Achieve quantitative elimination of background optical interference.

[0136] It is understandable that the ratio fluorescence sensing system of this embodiment not only has superior sensitivity and stability in laboratory testing, but also demonstrates multi-dimensional application value in commercial applications:

[0137] It can be used for online monitoring of Cu²⁺ in drinking water, surface water and industrial wastewater, providing a portable, low-cost and rapid-response detection method to meet the daily monitoring needs of environmental regulatory departments and enterprises.

[0138] It can be embedded in the food processing and drinking water supply chain to help companies establish a quality traceability system, meeting the urgent need for rapid screening under food safety regulations.

[0139] Based on the flexible substrate and imaging module, the system in this embodiment can be combined with mobile phones, portable terminals, and even wearable devices to form a personal water quality testing tool, targeting the mass consumer market.

[0140] The Fe3O4@SiO2@IIPs material used has a mature preparation process and controllable cost. The magnetron assembly and microarray film formation process is easy to scale up and can be directly connected to the existing thin film and micro-nano processing industry chain, making it feasible for commercial production.

[0141] With the support of an app or cloud platform, the detection data can be stored, analyzed and shared, supporting smart water management, environmental big data platforms and home-level water quality monitoring systems, further expanding commercial application models.

[0142] Those skilled in the art will understand that embodiments of this application can be provided as methods, systems, or computer program products. Therefore, this application can take the form of a completely hardware embodiment, a completely software embodiment, or an embodiment combining software and hardware aspects. Furthermore, this application can take the form of a computer program product embodied on one or more computer-usable storage media (including but not limited to disk storage, CD-ROM, optical storage, etc.) containing computer-usable program code.

[0143] This application is described with reference to flowchart illustrations and / or block diagrams of methods, apparatus (systems), and computer program products according to embodiments of this application. It will be understood that each block of the flowchart illustrations and / or block diagrams, and combinations of blocks in the flowchart illustrations and / or block diagrams, can be implemented by computer program instructions. These computer program instructions can be provided to a processor of a general-purpose computer, special-purpose computer, embedded processor, or other programmable data processing apparatus to produce a machine, such that the instructions, which execute via the processor of the computer or other programmable data processing apparatus, generate instructions for implementing the flowchart... Figure 1 One or more processes and / or boxes Figure 1 A device that provides the functions specified in one or more boxes.

[0144] These computer program instructions may also be stored in a computer-readable storage medium that can direct a computer or other programmable data processing device to function in a particular manner, such that the instructions stored in the computer-readable storage medium produce an article of manufacture including instruction means, which are implemented in a process Figure 1 One or more processes and / or boxes Figure 1 The function specified in one or more boxes.

[0145] These computer program instructions may also be loaded onto a computer or other programmable data processing equipment to cause a series of operational steps to be performed on the computer or other programmable equipment to produce a computer-implemented process, thereby providing instructions that execute on the computer or other programmable equipment for implementing the process. Figure 1 One or more processes and / or boxes Figure 1 The steps of the function specified in one or more boxes.

[0146] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the specific implementation of the present invention. Any modifications or equivalent substitutions that do not depart from the spirit and scope of the present invention should be covered within the scope of protection of the claims of the present invention.

Claims

1. A ratiometric fluorescence sensing system for detecting Cu²⁺, characterized in that, include: A functional film layer, including a blue response probe B and a red rare earth internal standard R, is fixed on the surface of a magnetic molecular imprinted composite material. The wavelength difference between the emission peaks of B and R is ≥80nm, thus constructing a ratio response system composed of blue light emission signal and red light emission signal. A magnetron assembly platform is used to enrich the magnetic molecular imprinted composite material onto the surface of the sensing membrane by an electrically controlled gradient magnetic field, forming a uniform fluorescent response layer. The sensing membrane is a multi-point microarray structure, and each microarray unit is used for the detection of Cu²⁺ or as a quality control point. The imaging module is used to acquire the signal strengths of the blue light emission signal and the red light emission signal; The environmental compensation unit acquires real-time temperature, pH, and turbidity, and pre-sets correction algorithms for each item to correct the blue / red ratio signal. The environmental compensation unit includes a DS18B20 temperature sensor for real-time acquisition of ambient temperature values. During the system calibration phase, the sensing membrane is placed in... Under these conditions, the blue / red ratio signal values ​​were measured to obtain the temperature sensitivity coefficient. ; In actual testing, if the measured temperature is T, the ratio signal is corrected as follows: Among them, T ref =25℃; The environmental compensation unit also includes a miniature pH glass electrode for real-time monitoring of the sample solution's acidity and alkalinity; during the calibration phase, respectively... The detection was performed in the buffer solution, and the pH compensation function was obtained by fitting: f(pH) = 0.01·(pH-7)²; The corrected formula is: (B / R) corr =(B / R) meas +f(pH); The environmental compensation unit also includes a light scattering turbidity sensor for measuring the turbidity value of the sample solution; Calibration experiments show that the attenuation of the turbidity-to-ratio signal can be described by a linear relationship. The compensation coefficient is β = 0.001 / NTU; The corrected formula is: ； The calculation module calculates the concentration of the analyte based on the corrected blue / red ratio signal and combines it with a pre-calibrated Stern-Volmer or four-parameter Logistic standard curve.

2. The ratio fluorescence sensing system for detecting Cu²⁺ according to claim 1, characterized in that, The computing module includes: When the standard curve is fitted using a four-parameter Logistic equation or a ratio Stern-Volmer equation, MIIMs test strips are immersed in each standard solution, fluorescence images are captured after the reaction, and the B / R ratio at each concentration is calculated. The obtained concentration and corresponding B / R ratio are a set of discrete observation points. in, Where i is the i-th data set, This is the Cu²⁺ standard solution for the i-th data set. It is the B / R ratio of the i-th data set; Specifically, when the fluorescence of the blue response probe B is dynamically quenched by Cu²⁺, including electron transfer and energy transfer, the B / R ratio monotonically decreases with increasing Cu²⁺ concentration. This was fitted using the Stern-Volmer equation, which includes: ； in, For none The B / R ratio at that time; I is the addition The B / R ratio after that, The Stern-Volmer quenching constant is used to reflect the sensitivity of the sensor. The concentration of Cu²⁺.

3. The ratio fluorescence sensing system for detecting Cu²⁺ according to claim 2, characterized in that, The computing module further includes: The standard curve is fitted using a four-parameter Logistic equation or a ratio Stern-Volmer equation. When the response exhibits an S-shaped curve (i.e., a rapid response at low concentrations and a tendency to saturate at high concentrations), the Logistic equation is used for fitting, including: ； in, For none The B / R ratio at that time; D is Approaching infinity The ratio, also known as the lower asymptote, is the signal saturation value. half-saturation concentration , which reflects the midpoint of the detection; H is the slope of the curve, the hill coefficient, which reflects the response sensitivity.

4. The ratio fluorescence sensing system for detecting Cu²⁺ according to claim 3, characterized in that, The functional film layer includes: The emission peak of the blue response probe is located at 440–470 nm, and the emission peak of the red rare earth internal standard is located at 610–620 nm.

5. The ratio fluorescence sensing system for detecting Cu²⁺ according to claim 4, characterized in that, The magnetic molecularly imprinted composite material has a core-shell structure, consisting of a Fe3O4 magnetic core, a SiO2 isolation layer, and an ion-imprinted polymer (IIPs) recognition layer, denoted as Fe3O4@SiO2@IIPs structure. The particle size is 120-300 nm, and the imprinted site density is 30–120 μmol·g⁻¹. ﹣ ¹.

6. The ratio fluorescence sensing system for detecting Cu²⁺ according to claim 5, characterized in that, The magnetically controlled assembly platform includes: Planar Helmholtz coils and miniature permanent magnet arrays; Power supply and control unit, used for 1-15T·m ﹣ Under gradient magnetic field conditions, the magnetic molecular imprinted composite material is enriched onto the surface of the sensing membrane and forms a microarray.

7. The ratio fluorescence sensing system for detecting Cu²⁺ according to claim 6, characterized in that, The functional film layer is disposed on an elastic substrate, which is selected from TPU, PDMS or SEBS, and the ultimate tensile strain of the elastic substrate is greater than 150% to adapt to different curved surface detection scenarios.

8. The ratio fluorescence sensing system for detecting Cu²⁺ according to claim 7, characterized in that, The functional film is subjected to a pre-stretch-release cycle to form an island-bridge microstructure on an elastic substrate, which enables the functional material to maintain electrical and optical connectivity under tension and maintain a stable blue / red ratio signal.

9. The ratio fluorescence sensing system for detecting Cu²⁺ according to claim 8, characterized in that, The imaging module includes: An imaging unit is used to acquire fluorescence images of the sensing membrane; Algorithm unit, used to perform the following steps: Image preprocessing and region of interest extraction; The RGB channel intensities were normalized, and the fluorescence intensities corresponding to blue light emission 440-470nm and red light emission 610–620nm were extracted respectively using a pre-established RGB value-fluorescence intensity calibration model. Calculate the blue / red ratio signal.

10. A ratiometric fluorescence sensing method for detecting Cu²⁺, applied to the ratiometric fluorescence sensing system for detecting Cu²⁺ as described in any one of claims 1-9, characterized in that, include: Step S1: The blue response probe B and the red rare earth internal standard R are fixed together on the surface of the magnetic molecular imprinted composite material, wherein the wavelength difference between the emission peaks of B and R is ≥80nm, and a ratio response system composed of blue light emission signal and red light emission signal is constructed. Step S2: The magnetron assembly platform enriches the magnetic molecular imprinted composite material onto the surface of the sensing film using an electrically controlled gradient magnetic field to form a uniform fluorescent response layer. The sensing film is a multi-point microarray structure, and each microarray unit is used for the detection of Cu²⁺ or as a quality control point. Step S3: Used to collect the signal strength of the blue light emission signal and the red light emission signal; Step S4: Obtain environmental data detection results for temperature, pH, and turbidity; correct the blue / red ratio signal based on the environmental data detection results. The environmental compensation unit acquires real-time temperature, pH, and turbidity, and pre-sets correction algorithms for each item to correct the blue / red ratio signal. The environmental compensation unit includes a DS18B20 temperature sensor for real-time acquisition of ambient temperature values. During the system calibration phase, the sensing membrane is placed in... Under these conditions, the blue / red ratio signal values ​​were measured to obtain the temperature sensitivity coefficient. ; In actual testing, if the measured temperature is T, the ratio signal is corrected as follows: Among them, T ref =25℃; The environmental compensation unit also includes a miniature pH glass electrode for real-time monitoring of the sample solution's acidity and alkalinity; during the calibration phase, respectively... The detection was performed in the buffer solution, and the pH compensation function was obtained by fitting: f(pH) = 0.01·(pH-7)²; The corrected formula is: (B / R) corr =(B / R) meas +f(pH); The environmental compensation unit also includes a light scattering turbidity sensor for measuring the turbidity value of the sample solution; Calibration experiments show that the attenuation of the turbidity-to-ratio signal can be described by a linear relationship. The compensation coefficient is β = 0.001 / NTU; The corrected formula is: ； The calculation module calculates the concentration of the analyte based on the corrected blue / red ratio signal and combines it with a pre-calibrated Stern-Volmer or four-parameter Logistic standard curve.

Images