Differential signal and MOFs molecular sieve-based anti-interference electrochemical sensor and preparation method thereof

Abstract

The invention relates to an anti-interference electrochemical sensor based on a differential signal and an MOFs molecular sieve and a preparation method. The anti-interference electrochemical sensor comprises a first working electrode, a second working electrode, a reference electrode and a counter electrode, wherein the surface of the first working electrode is modified with a first functional material, and the first functional material is used for performing non-selective electro-catalysis on a target object and an interferent to generate a total response signal; the surface of the second working electrode is modified with a second functional material, and the second functional material is used for physically screening the target object according to the molecular size, allowing the interferent to pass through and performing electro-catalysis on the interferent to generate an interferent response signal; wherein the second functional material comprises a metal organic framework material; and performing differential operation on the total response signal and the interferent response signal to obtain a target object response signal. According to the sensor, accurate stripping of interference signals is achieved, intrinsic response only related to the concentration of a target object is obtained, and accurate interference resistance from the source is achieved.

Description

Technical Field

[0001] This invention belongs to the field of biosensing technology and electrochemical analysis, specifically relating to an anti-interference electrochemical sensor based on differential signals and MOF molecular sieves and its preparation method. Background Technology

[0002] Lactic acid is a key indicator of anaerobic metabolism in the human body. In exercise physiology, it is used to assess training intensity and physical limits. In clinical medicine, it is used to monitor the tissue perfusion and oxygenation status of critically ill patients (such as those with sepsis or heart failure). It has extremely important monitoring value. The rapid and accurate detection of lactic acid in biological matrices such as sweat and blood is an urgent need for the development of wearable health devices and point-of-care diagnostic technologies.

[0003] Current methods for lactic acid detection mainly involve two approaches: modifying the electrode surface with selective membranes and developing highly selective catalysts. Modifying the electrode surface with selectively permeable membranes, such as Nafion membranes, utilizes the charge repulsion effect of the membrane itself to block negatively charged interfering substances from approaching the electrode surface. However, its selectivity depends on charge interaction, exhibiting poor resolution for neutral molecules or different molecules with the same charge, and its membrane stability is limited, easily degrading due to environmental factors. Developing novel nanocatalytic materials (such as metal oxides and alloy nanoparticles with specific morphologies) can enhance their catalytic activity towards lactic acid molecules, relatively reducing the response to interfering substances. However, the selectivity of nanomaterial catalysis is inherently relative; in complex biological samples, when the concentration of interfering substances is much higher than that of lactic acid, significant interference still occurs. Furthermore, the preparation processes of high-performance catalytic materials are often complex and lack reproducibility.

[0004] It is evident that existing sensors, due to the use of organic functional membranes or nanocatalytic materials, suffer from insufficient stability and are significantly affected by the concentration of interfering substances. Furthermore, current detection technologies also suffer from limitations in anti-interference capabilities due to their reliance on the inherent principle of chemoselectivity, and traditional multi-electrode systems suffer from inaccurate interference signal subtraction due to the lack of specific recognition units.

[0005] Therefore, providing a solution that can achieve high-precision and high-stability lactate detection in real complex biological samples has become an urgent problem to be solved. Summary of the Invention

[0006] To address the aforementioned problems in the prior art, this invention provides an anti-interference electrochemical sensor based on differential signaling and MOF molecular sieves, and its preparation method. The technical problem to be solved by this invention is achieved through the following technical solution: This invention provides an anti-interference electrochemical sensor based on differential signals and MOF molecular sieves, comprising, from bottom to top, a binder layer, a microfluidic layer, and an insulating layer. A dual working electrode system is disposed on the insulating layer, comprising a first working electrode, a second working electrode, a reference electrode, and a counter electrode. The surface of the first working electrode is modified with a first functional material, which is used to perform non-selective electrocatalysis on the target and interfering substances to generate a total response signal. The surface of the second working electrode is modified with a second functional material, which is used to physically sieve the target analyte according to its molecular size, while allowing the interfering analyte to pass through and electrocatalyzing the interfering analyte to generate an interfering analyte response signal; wherein, the second functional material includes a metal-organic framework material, and the molecular size of the interfering analyte is smaller than that of the target analyte; The total response signal and the interference response signal are used to obtain the target response signal through differential operation.

[0007] In one embodiment of the present invention, the target substance includes lactic acid.

[0008] In one embodiment of the present invention, the first functional material includes at least one of copper oxide and ruthenium oxide.

[0009] In one embodiment of the present invention, the first functional material further includes Prussian blue.

[0010] In one embodiment of the present invention, the metal-organic framework material includes at least one of ZIF-8 molecular sieve and UIO-66-NH2 molecular sieve, with a pore size of 3 Å to 6 Å.

[0011] In one embodiment of the present invention, the second functional material further includes NiCo2O4.

[0012] In one embodiment of the present invention, the second functional material is fixed to the surface of the second working electrode by Nafion adhesive.

[0013] In one embodiment of the present invention, the formula for calculating the target object response signal is as follows: I 差分 = I WE1 k × I WE2 in, I 差分 The response signal of the target object. I WE1For the total response signal, I WE2 For the response signal of the interfering object, k This is the calibration factor.

[0014] In one embodiment of the present invention, the first working electrode, the second working electrode, the reference electrode, and the counter electrode are all disposed on a flexible substrate; The electrochemical sensor is configured as a wearable device.

[0015] Another embodiment of the present invention provides a method for preparing an anti-interference electrochemical sensor based on differential signal and MOF molecular sieve, for realizing the preparation of the electrochemical sensor as described in the above embodiments, comprising the following steps: The first working electrode, the second working electrode, the reference electrode, and the counter electrode were prepared using screen printing technology. The first working electrode is modified with a first functional material, wherein the first functional material is used to perform non-selective electrocatalysis on the target and interfering substances to generate a total response signal; The second working electrode is modified with a second functional material, wherein the second functional material is used to selectively electrocatalyze the interfering substance to generate an interfering substance response signal, and the second functional material includes MOFs materials; the total response signal and the interfering substance response signal are differentially processed to obtain the target substance response signal.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. The electrochemical sensor of the present invention combines physical sieving with targeted differential to construct a dual working electrode system including a first working electrode and a second working electrode. The first working electrode is used to respond to the total signal of the target and the interfering substances, while the second working electrode uses metal-organic framework materials as molecular sieves to physically block the target molecules from passing through, while allowing specific interfering molecules to enter and be catalyzed, thereby realizing the detection of the interfering signal and establishing a pure reference channel. Finally, the interference signal is accurately stripped through the difference between the two signals, and the intrinsic response that is only related to the concentration of the target is obtained. This achieves accurate anti-interference from the source and enables high-precision and high-stability target detection in real complex biological samples. 2. The second functional material of the present invention includes metal-organic framework materials, which construct a sensing interface based on a physical sieving mechanism. By using the pore size of MOFs to exclude molecules by size, the specific recognition of target molecules can be achieved in the coexistence of interfering substances, thereby achieving a near-absolute anti-interference effect. Attached Figure Description

[0017] Figure 1A schematic diagram of the anti-interference electrochemical sensor based on differential signal and MOF molecular sieve provided in an embodiment of the present invention; Figure 2 This is a schematic diagram of the electrode structure of the anti-interference electrochemical sensor provided in an embodiment of the present invention; Figure 3 This is a schematic diagram of the operation of the anti-interference electrochemical sensor provided in an embodiment of the present invention; Figures 4a-4c The graph shows the response of a flexible lactic acid sensor based on ZIF-8 molecular sieve and copper oxide nanowires under ideal conditions. Figures 5a-5c The graph shows the response of the sensor based on UIO-66-NH2 molecular sieve and Prussian blue / ruthenium oxide under ideal conditions. Detailed Implementation

[0018] The present invention will be further described in detail below with reference to specific embodiments, but the implementation of the present invention is not limited thereto.

[0019] Please see Figure 1 , Figure 2 and Figure 3 , Figure 1 This is a schematic diagram of the anti-interference electrochemical sensor based on differential signals and MOF molecular sieves provided in an embodiment of the present invention. Figure 2 This is a schematic diagram of the electrode structure of the anti-interference electrochemical sensor provided in an embodiment of the present invention. Figure 3 This is a schematic diagram of the anti-interference electrochemical sensor provided in an embodiment of the present invention. The sensor combines differential signal processing with the molecular sieving effect of metal-organic framework materials to physically eliminate small molecule interferences, thereby achieving highly selective detection of biomarkers (such as lactic acid).

[0020] This invention relates to an anti-interference electrochemical sensor based on differential signals and MOF molecular sieves, comprising, from bottom to top, an adhesive layer, a microfluidic layer, and an insulating layer. The adhesive layer, which contacts the skin, has multiple pores to provide channels for sweat entry. The microfluidic layer and insulating layer control sweat flow, allowing sweat passing through the adhesive layer to flow through the channels to the electrode in the central circular hole. After the electrode completes its detection, the sweat is absorbed by cotton in another circular hole. The insulating layer can be made of PI film or other insulating materials.

[0021] Furthermore, a dual working electrode system is disposed on the insulating layer. The dual working electrode system includes: a first working electrode WE1, a second working electrode WE2, a reference electrode RE, and a counter electrode CE forming the dual working electrode system. The first working electrode WE1 and the second working electrode WE2 form a differential working electrode pair. The surface of the first working electrode WE1 is modified with a first functional material, which is used for non-selective electrocatalysis of the target and interfering substances to generate a total response signal. The surface of the second working electrode WE2 is modified with a second functional material, which is used for physical sieving of the target based on molecular size, while allowing interfering substances to pass through and electrocatalyzing the interfering substances to generate an interfering substance response signal. The second functional material includes metal-organic framework materials (MOFs), and the molecular size of the interfering substances is smaller than that of the target. The total response signal and the interfering substance response signal are differentially processed to obtain the target response signal.

[0022] Specifically, the target analyte is lactic acid, and interfering substances include uric acid, ascorbic acid, and other substances with molecular sizes smaller than lactic acid. It should be noted that this embodiment can achieve the detection of different target analytes by changing the pore size of the MOFs; the type of target analyte is not limited to one.

[0023] Specifically, the first functional material includes at least one of copper oxide and ruthenium oxide. Furthermore, the first functional material may also include Prussian blue, i.e., copper oxide and ruthenium oxide respectively form composite catalytic materials with Prussian blue.

[0024] Specifically, the metal-organic framework material includes at least one of ZIF-8 molecular sieve and UIO-66-NH2 molecular sieve, with a pore size of 3 Å to 6 Å. Further, the metal-organic framework material may also include NiCo2O4, i.e., ZIF-8 molecular sieve and UIO-66-NH2 molecular sieve are respectively combined with NiCo2O4 to form composite materials. Even further, the second functional material is fixed to the surface of the second working electrode WE2 using Nafion binder.

[0025] Specifically, the first working electrode WE1, the second working electrode WE2, the reference electrode RE, and the counter electrode CE are all disposed on a flexible substrate; the electrochemical sensor is configured as a wearable device for wearing on the skin surface to monitor the concentration of lactic acid in sweat. For example, the flexible substrate is selected from polyethylene terephthalate (PET).

[0026] Specifically, a first working electrode WE1, a second working electrode WE2, a reference electrode RE, and a counter electrode CE are simultaneously fabricated on a flexible substrate.

[0027] In other implementations, a ceramic substrate may also be used.

[0028] Specifically, the method for signal detection using the aforementioned sensor is as follows: the modified sensor is placed in the test liquid (such as sweat) and connected to an electrochemical workstation. Detection is performed using a chronoamperometric method at a constant operating potential of +0.5V to +0.7V (vs. Ag / AgCl). The electrochemical workstation simultaneously records the current response of WE1. I WE1 Current response of WE2 I WE2 Obtain I WE1 and I WE2 Then, differential calculations are performed in real time using a built-in algorithm or an external processor. Through differential operations, the interfering substance response signal is subtracted from the total response signal to obtain the pure target substance concentration response signal, i.e.: I 差分 = I WE1 k × I WE2 in, I 差分 The response signal of the target object. I WE1 For the total response signal, I WE2 For the response signal of the interfering object, k This is the calibration factor, typically close to 1.

[0029] This invention also provides a method for preparing an anti-interference electrochemical sensor based on differential signaling and MOF molecular sieves, used to prepare the aforementioned electrochemical sensor. The preparation method includes the following steps: S1. The first working electrode WE1, the second working electrode WE2, the reference electrode RE, and the counter electrode CE are prepared using screen printing technology; S2. Modify the first working electrode WE1 with a first functional material, wherein the first functional material is used to perform non-selective electrocatalysis on the target and interfering substances to generate a total response signal; S3. Modify the second working electrode WE2 with a second functional material, wherein the second functional material is used to selectively electrocatalyze the interfering substance to generate an interfering substance response signal, and the second functional material includes MOFs material; the total response signal and the interfering substance response signal are used to obtain the target substance response signal through differential operation.

[0030] This invention constructs a sensing interface based on a physical sieving mechanism, using MOF pore size to perform size exclusion of molecules, thereby achieving specific recognition of target molecules in the presence of interfering substances, achieving near-absolute anti-interference effect. It provides a differential sensing architecture with targeted recognition function, establishing a pure reference channel by dedicating the second working electrode to capturing interfering signals, and finally achieving precise removal of interfering signals through real-time differential sensing, obtaining an intrinsic response that is only related to the target analyte concentration. Furthermore, it develops an enzyme-free sensing system based on inorganic nanomaterials and a stable MOF framework, significantly improving the sensor's lifespan and environmental adaptability by eliminating failure mechanisms such as biological component inactivation and membrane structure aging, meeting the needs of long-term continuous monitoring in clinical applications. Therefore, this invention can achieve high-precision and high-stability target analyte detection in real, complex biological samples.

[0031] To facilitate understanding of the present invention, the following embodiments are provided. Those skilled in the art should understand that the embodiments are merely illustrative and should not be construed as limiting the scope of the invention.

[0032] Example 1 This embodiment provides a flexible lactic acid sensor based on ZIF-8 molecular sieve and copper oxide nanowires. This sensor is suitable for monitoring lactic acid in sweat, and its preparation method includes the following steps: S1. Fabricate the first working electrode WE1, the second working electrode WE2, the reference electrode RE, and the counter electrode CE using screen printing technology. This includes the following steps: 1) Substrate pretreatment: A flexible PET substrate with a size of 30 mm × 10 mm and a thickness of 0.25 mm was selected as the insulating substrate. The substrate was ultrasonically cleaned for 15 minutes with anhydrous ethanol and deionized water in sequence to remove organic matter and dust from the surface. Then it was placed in a 60°C oven to dry thoroughly.

[0033] 2) Electrode pattern printing: Using a 200-mesh polyester screen printing plate and a semi-automatic screen printing machine, the pattern of the three-electrode system is printed on the PET substrate. The pattern includes the prototype of two circular working electrodes (WE1 and WE2) with a diameter of 3 mm, a counter electrode CE, and a reference electrode RE. At the same time, the leads for connecting to the external circuit are printed.

[0034] 3) Heat treatment curing: Place the printed electrodes in a horizontal conveyor belt sintering furnace and perform a stepped heating program: first preheat at 80°C for 5 minutes, then raise to 120°C and hold for 15 minutes to completely remove the organic solvents in the slurry and form a stable conductive film.

[0035] 4) Functionalization of the reference electrode: The sintered electrode is immersed in a 0.1 M FeCl3 solution and polarized at a constant potential of +0.5 V for 30 seconds to partially chlorinate the surface of the carbon reference electrode, forming a stable Ag / AgCl quasi-reference electrode system.

[0036] 5) Insulation Layer Definition: Using a separate screen printing stencil, UV-curable insulating ink is precisely printed onto areas outside the electrode regions, specifically covering all leads, exposing only the 3 mm circular areas of the two working electrodes, most of the counter electrode area, and the tip of the reference electrode. The electrodes are then cured using a UV curing machine (intensity: 80 mW / cm²). 2 Irradiate for 30 seconds to fully cure the insulation layer.

[0037] S2. The first working electrode is modified with copper oxide nanowires.

[0038] 1) Hydrothermal Synthesis of Copper Oxide Nanowires: Weigh 0.726 g Cu(NO3)2·3H2O (3 mmol) and 1.200 g NaOH (30 mmol) into a polytetrafluoroethylene (PTFE) beaker. Add 80 mL of deionized water to the beaker and stir magnetically for 30 minutes until a uniform blue suspension is formed. Transfer the mixture to a 100 mL PTFE-lined stainless steel autoclave, seal it, and place it in a forced-air drying oven. Set the oven temperature to 150°C and react for 8 hours. After the reaction, allow it to cool naturally to room temperature. Open the autoclave; a black precipitate is visible at the bottom. Discard the supernatant, centrifuge at 8000 rpm to collect the precipitate, and wash it three times each with deionized water and anhydrous ethanol. Transfer the washed precipitate to a vacuum drying oven and dry it at 60°C for 6 hours to obtain fluffy black copper oxide nanowire powder.

[0039] 2) Preparation of the modification solution: Weigh 6.0 mg of the above copper oxide nanowire powder and dissolve it in 2.0 mL of anhydrous ethanol. Place the mixed solution in an ultrasonic cell disruptor and sonicate it at 400 W for 1 hour under ice-water bath conditions until a uniform, non-agglomerated black suspension with a concentration of approximately 3 mg / mL is formed, thus obtaining the copper oxide nanowire suspension.

[0040] 3) Electrode Drop Coating and Curing: The prepared screen-printed electrode was fixed on the platform. Using a calibrated micropipette, 3.0 μL of copper oxide nanowire suspension was precisely pipetted and slowly dropped vertically onto the center of the exposed circular area of ​​WE1, ensuring that the droplet completely covered the reaction window. Subsequently, the electrode was placed in a clean petri dish and allowed to stand at room temperature for 2 hours to allow the ethanol to completely evaporate, forming a firmly adhered and uniformly distributed copper oxide nanowire catalytic layer on the WE1 surface, thereby achieving efficient and non-selective electrocatalytic oxidation of lactic acid molecules and interfering substances (such as urea, ascorbic acid, etc.).

[0041] S3. The second working electrode is modified using ZIF-8 molecular sieve.

[0042] 1) Synthesis of ZIF-8 nanocrystals: 2.93 g of 2-methylimidazole was dissolved in 40 mL of methanol and magnetically stirred until completely clear, yielding solution A. 1.47 g of Zn(NO3)2·6H2O was dissolved in 40 mL of methanol, yielding solution B. Under room temperature and vigorous magnetic stirring (approximately 600 rpm), solution B was rapidly poured into solution A. The mixture immediately became white and turbid. The reaction was continued for 1 hour. After the reaction was complete, the mixture was transferred to a centrifuge tube and centrifuged at 10,000 rpm for 5 minutes to collect the white precipitate. The precipitate was washed three times with fresh methanol by repeated centrifugation. The washed product was placed in a vacuum drying oven and dried overnight at 60°C to obtain pure white ZIF-8 powder.

[0043] 2) Preparation of the modification solution: Accurately weigh 10.0 mg ZIF-8 powder and 0.5 mg Nafion powder (corresponding to a concentration of 0.5 wt%), and dissolve them together in a mixed solvent of 1.0 mL anhydrous ethanol and deionized water (V... 乙醇 :V 水 The mixture was then sonicated for 30 minutes to form a stable white suspension (ratio 9:1).

[0044] 3) Electrode drop coating and curing: Using a micropipette, accurately pipette 3.0 μL of ZIF-8 / Nafion suspension and drop it onto the exposed circular area of ​​WE2. Allow it to dry at room temperature for 4 hours to form a dense, uniform ZIF-8 modified layer with molecular sieving function.

[0045] Furthermore, to verify the performance of the sensor in this embodiment, the following tests were conducted: Using an electrochemical workstation, a constant working potential of +0.6V (vs. Ag / AgCl) was applied to 0.1M PBS buffer (pH=7.4), and the test was performed using a chronoamperometry method. The solution was kept gently stirred magnetically. Lactic acid, ascorbic acid (AA), and uric acid (UA) standard solutions (100 μM each time) were added sequentially to the continuously stirred buffer, and the current response values ​​of WE1 and WE2 were recorded simultaneously.

[0046] Please see Figures 4a-4c , Figures 4a-4c The graph shows the response of a flexible lactic acid sensor based on ZIF-8 molecular sieve and copper oxide nanowires under ideal conditions. Figure 4a This is the response diagram of the first working electrode. Figure 4b The response diagram of the second working electrode is shown. Figure 4c This is a differential signal diagram. Figure 4a In the study, the addition of WE1 to all three substances resulted in a significant increase in current, indicating that it has significant catalytic oxidation activity for both lactic acid and interfering substances (AA, UA). Figure 4b In the study, the addition of WE2 to lactic acid produced almost no change in current, but the addition of AA and UA produced a current step similar to that of WE1. This demonstrates that the ZIF-8 layer effectively blocks lactic acid molecules while allowing small molecule interfering substances to enter and be catalyzed. Figure 4c In the experiment, subtracting the current value of WE2 from the current value of WE1 revealed a differential signal that only responded to the addition of lactic acid, producing a clear current step, while maintaining a stable baseline for the addition of AA and UA. This result intuitively demonstrates that the flexible lactic acid sensor based on ZIF-8 molecular sieve and copper oxide nanowires in this embodiment successfully eliminated the interference of AA and UA.

[0047] Example 2 This embodiment provides a sensor based on UIO-66-NH2 molecular sieve and Prussian blue / ruthenium oxide. The preparation method of this sensor includes the following steps: S1. The first working electrode WE1, the second working electrode WE2, the reference electrode RE, and the counter electrode CE are prepared using screen printing technology.

[0048] The first working electrode WE1, the second working electrode WE2, the reference electrode RE, and the counter electrode CE were prepared using the same method as in Example 1.

[0049] S2. The first working electrode is modified with a Prussian blue / ruthenium oxide composite catalyst layer.

[0050] 1) Electrochemical deposition of Prussian blue thin film: The prepared screen-printed electrode was immersed in an acidic aqueous solution (pH ~2.0) containing 2.5 mM K3Fe(CN)6, 2.5 mM FeCl3, and 0.1 M KCl. Cyclic voltammetry was used, with continuous scanning at a scan rate of 50 mV / s for 10 cycles within a potential range of -0.2 V to +0.6 V (vs. Ag / AgCl). During the scanning process, a blue thin film, Prussian blue, was gradually formed on the WE1 surface. After completion, the electrode was gently rinsed with deionized water to remove physically adsorbed ions.

[0051] 2) Ruthenium oxide nanoparticle drop-coating: Ruthenium oxide nanoparticles were dispersed in anhydrous ethanol solution containing 0.1% Nafion to prepare a homogeneous suspension with a concentration of 1 mg / mL. 3 μL of this suspension was precisely pipetted onto the Prussian blue-modified WE1 surface using a micropipette. The mixture was dried at room temperature in the dark for 4 hours to form a Prussian blue-ruthenium oxide composite catalytic layer. Prussian blue, as a highly efficient electron mediator, reduces the overpotential of lactic acid oxidation; ruthenium oxide provides abundant catalytic sites, ensuring high reactivity.

[0052] S3. The second working electrode is modified using UIO-66-NH2 molecular sieve.

[0053] 1) Synthesis of UIO-66-NH2 using a solvothermal method: 0.233 g ZrCl4 and 0.271 g 2-aminoterephthalic acid were dissolved in 30 mL N,N-dimethylformamide (DMF) and sonicated until completely dissolved. 1 mL concentrated hydrochloric acid was added to the mixture to promote crystal nucleation, and then the solution was transferred to a 50 mL polytetrafluoroethylene-lined high-pressure reactor. The reactor was placed in a 120°C drying oven and reacted for 24 hours. After natural cooling, the yellow precipitate was collected by centrifugation and washed three times each with DMF and anhydrous methanol to remove unreacted ligands and solvent. The product was activated under vacuum at 150°C for 6 hours to obtain a highly crystalline yellow powder of UIO-66-NH2 with a pore size of approximately 6.0 Å, between the size of lactic acid molecules (~5.0 Å) and larger interfering molecules.

[0054] 2) Electrode modification: The synthesized UIO-66-NH2 powder was dispersed in an ethanol solution containing 0.5 wt% Nafion to prepare a homogeneous suspension of 5 mg / mL. 3 μL of this suspension was precisely pipetted onto the exposed reaction area of ​​WE2 using a micropipette. After drying at room temperature, a dense UIO-66-NH2 molecular sieve layer was formed.

[0055] Furthermore, to verify the performance of the sensor in this embodiment, the following tests were conducted: Using an electrochemical workstation, a constant working potential of +0.35V (vs. Ag / AgCl) was applied in 0.1M PBS buffer (pH=7.4), and the chronoamperometry method was used for testing. Lactic acid, ascorbic acid, uric acid, and dopamine (all 100 μM) were added sequentially to the basal solution, and the current response values ​​of WE1 and WE2 were recorded simultaneously.

[0056] Please see Figures 5a-5c , Figures 5a-5c The graph shows the response of the sensor based on UIO-66-NH2 molecular sieve and Prussian blue / ruthenium oxide under ideal conditions. Figure 5a This is the response diagram of the first working electrode. Figure 5b The response diagram of the second working electrode is shown. Figure 5c This is a differential signal diagram. Figure 5a Even at a relatively low operating potential of +0.35V, WE1 modified with the Prussian blue / ruthenium oxide composite catalyst layer exhibited significant current steps for lactic acid, AA, UA, and dopamine, demonstrating that the composite catalyst layer maintains broad-spectrum catalytic activity at low potentials while effectively reducing background interference in detection. Figure 5b In the study, WE2 showed no or extremely weak response to lactate and dopamine, but exhibited a significant current step for AA and UA. This result indicates that the pore size (~6.0 Å) of UIO-66-NH2 effectively blocks lactate and dopamine molecules with larger kinetic diameters, while allowing smaller AA and UA molecules to pass through. This contrasts sharply with the ZIF-8 system, demonstrating that the UIO-66-NH2 molecular sieve layer possesses different pore size selectivity characteristics, which can expand the range of interference exclusion and further enhance the selectivity of the sensor in complex biological samples. Figure 5c In this study, the differential signal responded to the addition of lactic acid and dopamine, but not to the addition of AA and UA. This result, compared to Example 1, demonstrates that the sensor's anti-interference range can be customized and extended by selecting MOF materials with different pore sizes (such as UIO-66-NH2). This fully illustrates the high flexibility and universality of the physical sieving-target differential strategy, providing a solid technical foundation for solving the problem of specific detection of targets in complex biological samples.

[0057] This embodiment utilizes metal-organic framework materials with different pore sizes: UIO-66-NH2 molecular sieves and composite catalytic materials that can reduce the working potential: Prussian blue / ruthenium oxide. By changing the pore size of MOFs, the sieving range of interfering substances can be adjusted, enabling targeted elimination of different combinations of interfering substances and addressing more complex biological sample environments. By introducing Prussian blue as an electron mediator, the working potential is reduced from +0.6V to +0.35V, significantly lowering the working potential for target analyte detection. This greatly reduces the non-specific oxidation of electroactive substances on the electrode, thereby reducing background current and interference from other substances with higher oxidation potentials, and improving the signal-to-noise ratio, stability, and reliability of the detection.

[0058] Example 3 This embodiment provides a flexible lactic acid sensor based on a composite material of NiCo2O4 and ZIF-8 molecular sieve and copper oxide nanowires. The preparation method of this sensor includes the following steps: S1. The first working electrode WE1, the second working electrode WE2, the reference electrode RE, and the counter electrode CE are prepared using screen printing technology.

[0059] Specifically, PET or ceramic is selected as the insulating substrate. Then, using carbon paste, the reference electrode (RE) and counter electrode (CE) are screen-printed onto the substrate; using platinum paste, the working electrode (WE) is screen-printed onto the substrate. Next, the printed carbon reference electrode is electrochemically chlorinated in a chloride-containing solution (such as FeCl3 or KCl solution) to form a stable Ag / AgCl quasi-reference electrode. Finally, insulating paste is printed on the electrode leads to define exposed and uniformly sized reaction regions WE1 and WE2.

[0060] S2. The first working electrode is modified with copper oxide nanowires.

[0061] 1) Hydrothermal Synthesis of Copper Oxide Nanowires: Copper oxide nanowires were synthesized using a hydrothermal method. The specific steps were as follows: Copper salts (such as Cu(NO3)2 or CuCl2) and an alkaline substance (such as NaOH) were dissolved in deionized water, transferred to a reaction vessel, and reacted at 120-180℃ for 6-12 hours. After the reaction was completed, the nanowires were centrifuged, washed, and dried to obtain copper oxide nanowire powder.

[0062] 2) Preparation of modification solution: Disperse the synthesized copper oxide nanowires in anhydrous ethanol to prepare a uniform suspension with a concentration of 1-5 mg / mL.

[0063] 3) Electrode drop-coating and curing: Using a micropipette, 2-5 μL of copper oxide suspension is precisely drop-coated onto the exposed reaction area of ​​WE1. Then, it is gently dried at room temperature or under an infrared lamp to allow the ethanol to evaporate, forming a stable copper oxide nanowire catalytic layer on the WE1 surface.

[0064] S3. The second working electrode is modified with NiCo2O4 and ZIF-8 molecular sieve.

[0065] 1) Synthesis of NiCo2O4 material: A co-precipitation method was used. Nickel salt Ni(NO3)2 and cobalt salt Co(NO3)2 were dissolved in deionized water at a molar ratio of 1:2. A precipitant (such as Na2CO3 or urea) was added under stirring to generate a precursor. After washing and drying the precursor, it was calcined at 300-400℃ for 2-4 hours to obtain spinel-structured NiCo2O4 nanomaterials.

[0066] 2) ZIF-8 with a pore size of ~3.4 Å was synthesized by room temperature stirring method: a methanol solution of 2-methylimidazole was quickly poured into a methanol solution of zinc salt (such as Zn(NO3)2), stirred vigorously for 5-30 minutes, the white precipitate was collected by centrifugation, washed with methanol and dried to obtain ZIF-8 nanocrystals.

[0067] 3) Preparation of composite material modification solution: The synthesized NiCo2O4 nanomaterials and ZIF-8 nanocrystals are mixed at a mass ratio of 1:1 to 1:3 and dispersed together in an ethanol solution containing 0.5-1.0wt% Nafion. The mixture is then ultrasonically treated to form a uniform composite modification slurry.

[0068] 4) Electrode Drop Coating and Film Formation: Using a micropipette, a volume (2-5 μL) of the composite modification slurry equal to that of WE1 was precisely drop-coated onto the exposed reaction area of ​​WE2. After drying at room temperature, Nafion was used as a binder to firmly fix NiCo2O4 and ZIF-8 onto the electrode surface, forming a dense composite catalyst-sieve layer.

[0069] In this embodiment, NiCo2O4 and ZIF-8 are modified on the surface of the second working electrode. While ZIF-8 blocks larger lactic acid molecules and allows small molecule interfering substances to enter and catalyze, NiCo2O4 can further catalyze small molecule interfering substances (such as urea), thereby constructing a molecular sieve interface that allows small molecule interfering substances such as urea to enter and be catalyzed, while physically blocking larger lactic acid molecules, thus achieving a better specific response to interfering substances.

[0070] Therefore, this invention revolutionizes the anti-interference strategy of lactic acid sensors from relying on chemically selective catalysis to physical size sieving, designing a differential sensing structure: the first working electrode responds to the total signal, while the second working electrode utilizes the precise pore size of MOFs specifically for capturing interfering signals, acting as a "molecular gatekeeper." Through the synergistic work of MOF sieving and real-time differential sensing, targeted identification and precise subtraction of interfering signals are achieved, ultimately outputting a pure signal that is only related to the concentration of the target analyte. Furthermore, by replacing MOFs with different pore sizes, the interfering sieving range can be customized, making the technology a platform with universality and adaptability to solve various detection scenarios.

[0071] The above description, in conjunction with specific preferred embodiments, provides a further detailed explanation of the present invention. It should not be construed that the specific implementation of the present invention is limited to these descriptions. For those skilled in the art, various simple deductions or substitutions can be made without departing from the concept of the present invention, and all such modifications and substitutions should be considered within the scope of protection of the present invention.

Claims

1. An anti-interference electrochemical sensor based on differential signaling and MOF molecular sieves, characterized in that, The system comprises, from bottom to top, an adhesive layer, a microfluidic layer, and an insulating layer. A dual-working-electrode system is disposed on the insulating layer. The dual-working-electrode system includes a first working electrode, a second working electrode, a reference electrode, and a counter electrode. The surface of the first working electrode is modified with a first functional material, which is used to perform non-selective electrocatalysis on the target and interfering substances to generate a total response signal. The surface of the second working electrode is modified with a second functional material, which is used to physically sieve the target analyte according to its molecular size, while allowing the interfering analyte to pass through and electrocatalyzing the interfering analyte to generate an interfering analyte response signal; wherein, the second functional material includes a metal-organic framework material, and the molecular size of the interfering analyte is smaller than that of the target analyte; The total response signal and the interference response signal are used to obtain the target response signal through differential operation.

2. The anti-interference electrochemical sensor based on differential signal and MOF molecular sieve according to claim 1, characterized in that, The target substance includes lactic acid.

3. The anti-interference electrochemical sensor based on differential signal and MOF molecular sieve according to claim 1, characterized in that, The first functional material includes at least one of copper oxide and ruthenium oxide.

4. The anti-interference electrochemical sensor based on differential signal and MOF molecular sieve according to claim 3, characterized in that, The first functional material also includes Prussian blue.

5. The anti-interference electrochemical sensor based on differential signal and MOF molecular sieve according to claim 1, characterized in that, The metal-organic framework material includes at least one of ZIF-8 molecular sieve and UIO-66-NH2 molecular sieve, with a pore size of 3 Å to 6 Å.

6. The anti-interference electrochemical sensor based on differential signal and MOF molecular sieve according to claim 4, characterized in that, The second functional material also includes NiCo2O4.

7. The anti-interference electrochemical sensor based on differential signal and MOF molecular sieve according to claim 5, characterized in that, The second functional material is fixed to the surface of the second working electrode using Nafion adhesive.

8. The anti-interference electrochemical sensor based on differential signal and MOF molecular sieve according to claim 1, characterized in that, The formula for calculating the target object's response signal is: I 差分 = I WE1 k × I WE2 in, I 差分 The response signal of the target object. I WE1 For the total response signal, I WE2 For the response signal of the interfering object, k This is the calibration factor.

9. The anti-interference electrochemical sensor based on differential signal and MOF molecular sieve according to claim 1, characterized in that, The first working electrode, the second working electrode, the reference electrode, and the counter electrode are all disposed on a flexible substrate; The electrochemical sensor is configured as a wearable device.

10. A method for preparing an anti-interference electrochemical sensor based on differential signaling and MOF molecular sieves, characterized in that, The method for fabricating an electrochemical sensor as described in any one of claims 1 to 9 includes the following steps: The first working electrode, the second working electrode, the reference electrode, and the counter electrode were prepared using screen printing technology. The first working electrode is modified with a first functional material, wherein the first functional material is used to perform non-selective electrocatalysis on the target and interfering substances to generate a total response signal; The second working electrode is modified with a second functional material, wherein the second functional material is used to selectively electrocatalyze the interfering substance to generate an interfering substance response signal, and the second functional material includes MOFs materials; the total response signal and the interfering substance response signal are differentially processed to obtain the target substance response signal.

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