Preparation method of stretchable molecular imprinting sensor based on PDMS / CNT composite electrode

Abstract

The invention relates to the technical field of biosensors, in particular to a preparation method of a stretchable molecularly imprinted sensor based on a PDMS / CNT composite electrode. Comprising the following steps: S1, preparing a stretchable CNT electrode by using single-wall CNT; s2, depositing a Prussian blue layer on the stretchable CNT electrode; S3, depositing a molecularly imprinted polymer layer on the Prussian blue coating electrode; S31, depositing an MIP precursor solution containing template molecules on the Prussian blue coating electrode through an electrochemical method, and forming an MIP layer on the Prussian blue coating electrode; and S32, removing the template molecules in the MIP layer, and forming a molecular recognition cavity specifically bound with BCAA in the MIP layer, thereby completing the preparation of the sweat detection sensor. The method solves the problems of insufficient sensitivity, poor stability of a specific cavity during stretching and insufficient mechanical firmness when an existing sweat sensor is used for detecting branched chain amino acid (BCAA).

Description

Technical Field

[0001] The present invention relates to the technical field of biosensors, and in particular to a method for preparing a stretchable molecular imprinting sensor based on a PDMS / CNT composite electrode. Background Art

[0002] Branched-chain amino acids (BCAAs), including leucine, isoleucine, and valine, are key biomarkers for detecting metabolic disorders associated with diabetes, obesity, and insulin resistance. Traditional methods for detecting BCAAs, such as blood sampling and laboratory-based analysis, are accurate but invasive, time-consuming, and not suitable for continuous or real-time applications.

[0003] In recent years, sweat has attracted attention as a non-invasive metabolic analysis alternative to biological fluids for detecting branched-chain amino acids. Although sensor technology is constantly improving, sweat sensors in the existing technology still find it difficult to accurately and quickly respond to changes in trace components in sweat. This is mainly because the components in sweat are complex and the concentration is low, which places extremely high demands on the sensitivity of the sensor. In addition, it is difficult for existing flexible molecular imprinting sensors to ensure sufficient sensitivity and good detection performance in a stretched state. Therefore, the development of wearable sensors that can achieve high sensitivity, specificity and stability under dynamic conditions is still challenging. Therefore, it is urgently needed to be solved. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide a method for preparing a stretchable molecular imprinting sensor based on a PDMS / CNT composite electrode, which solves the problems of insufficient sensitivity and poor specificity faced by existing sweat sensors when detecting branched-chain amino acids (BCAA), as well as maintaining stable detection performance in a stretched state. To achieve the above object, the technical solution of the present invention is implemented as follows: a method for preparing a stretchable molecular imprinting sensor based on a PDMS / CNT composite electrode, characterized in that it comprises the following steps: S1. Preparation of stretchable CNT electrodes using single-walled CNTs; S2. Deposition of a Prussian blue layer on the stretchable CNT electrode: The Prussian blue solution is uniformly deposited on the CNT electrode by an electrochemical method to form a Prussian blue coating on the CNT electrode to prepare a Prussian blue coating electrode; S3. Depositing a molecularly imprinted polymer layer on the Prussian blue coated electrode: S31, depositing a MIP precursor solution containing a template molecule onto a Prussian blue coated electrode by an electrochemical method, and forming a MIP layer on the Prussian blue coated electrode; S32. The template molecules in the MIP layer are removed, and a molecular recognition cavity that specifically binds to BCAA is formed in the MIP layer, thereby completing the preparation of the sweat detection sensor.

[0005] Preferably, in step S1, the process of preparing a stretchable CNT electrode using single-walled CNT includes: S11, dispersing single-walled CNT in deionized water to form a dispersion solution; S12, using a printer to print a preset electrode pattern onto a polyethersulfone filter membrane, and installing the polyethersulfone filter membrane printed with the electrode pattern onto a vacuum filtration device; S13, dripping the dispersion solution onto the polyethersulfone filter membrane on the vacuum filtration device, and starting the vacuum filtration device to filter the dispersion solution on the polyethersulfone filter membrane, so that part of the single-walled CNTs in the dispersion solution are guided and uniformly penetrate the pores of the polyethersulfone filter membrane under the action of the vacuum force, and the pores of the polyethersulfone filter membrane are blocked by the graphite used for printing at the electrode pattern printed on the polyethersulfone filter membrane, so that part of the single-walled CNTs in the dispersion solution are gathered at the printed electrode pattern, thereby forming a uniform and conductive CNT coating with a precise pattern, that is, forming a CNT coating on the electrode pattern printed on the polyethersulfone filter membrane; S14, transferring the polyethersulfone membrane with the CNT coating onto a flexible polydimethylsiloxane substrate, and obtaining a stretchable CNT electrode after curing.

[0006] Preferably, in step S2, the Prussian blue solution includes 5mM FeCl3, 5mM K3Fe(CN)6, 0.1M HCl and 0.1M KCl; and the Prussian blue solution is subjected to 20 cycles in a potential range of -0.2V to 0.6V by cyclic voltammetry, and the Prussian blue in the Prussian blue solution is deposited on the stretchable CNT electrode to form a Prussian blue coating on the stretchable CNT electrode.

[0007] Preferably, in step S3, the MIP precursor solution is prepared by dissolving 60 mM pyrrole, 20 mM 3-aminophenylboronic acid and 10 mM BCAAA template molecules in PBS buffer, and the pH value of the PBS buffer is 6.5.

[0008] Preferably, in step S3, the MIP precursor solution is electrochemically deposited onto the Prussian blue coated electrode by cyclic voltammetry in a potential range of -0.2 V to 0.9 V at a scan rate of 50 mV / s, and the number of MIP deposition cycles is 10.

[0009] Preferably, the concentration of the dispersion solution of the single-walled CNT in deionized water is 0.2 mg / mL, and the dispersion is subjected to ultrasonic treatment.

[0010] Preferably, in step S32, the specific operation of removing the template molecule is: The Prussian blue-coated electrode with the MIP layer formed was immersed in a mixed solution of water, methanol and acetic acid and incubated with gentle stirring at room temperature for 2 hours. The electrode was then thoroughly rinsed with deionized water and the electrode was subjected to morphological analysis and electrochemical characterization by scanning electron microscopy to verify that the template molecules on the electrode were completely removed.

[0011] Preferably, the volume ratio of water, methanol and acetic acid solution in the mixed solution is 5:2:3.

[0012] Preferably, the specific operation process of step S14 includes: S141, spin coating a mixture of polydimethylsiloxane monomer and curing agent onto a silicon wafer, and partially curing at 65° C. for 30 minutes to form a PDMS layer; S142, aligning the polyethersulfone membrane with the CNT coating on the PDMS layer, and peeling off the polyethersulfone membrane after fully curing at 65°C for 3 hours.

[0013] Preferably, in step S141, the mass ratio of the polydimethylsiloxane monomer to the curing agent is 10:1.

[0014] The beneficial effects of the present invention are embodied in: (1) The method provided by the present invention uses a printer inkjet printing technology to print the electrode pattern onto a polyethersulfone membrane, and forms a uniform conductive CNT coating by vacuum filtration. This method not only ensures the uniform distribution of CNTs on the electrode and improves the conductivity of the electrode, but also obtains a stretchable CNT electrode with good stretchability by transferring the polyethersulfone membrane to a flexible polydimethylsiloxane (PDMS) substrate for curing. The design of this stretchable electrode enables the sensor to better adapt to the dynamic changes of human skin, thereby maintaining high sensitivity under dynamic conditions and accurately responding to changes in trace BCAA components in sweat.

[0015] (2) The method provided by the present invention deposits a Prussian blue layer on a stretchable CNT electrode, and uniformly coats the Prussian blue on the CNT electrode by an electrochemical method. Prussian blue, as an excellent redox mediator, can significantly improve the electron transfer characteristics of the sensor, thereby enhancing the sensor's response signal to BCAA. In addition, since BCAA is an inactive molecule, Prussian blue provides the sensor with the ability to indirectly detect BCAA, thereby improving the sensitivity and accuracy of sensor detection.

[0016] (3) The method provided by the present invention further forms a molecular imprinting polymer (MIP) layer on the Prussian blue layer by an electrochemical method. Since the molecular imprinting polymer (MIP) layer contains branched-chain amino acids. After the elution process, the template molecules in the molecular imprinting polymer (MIP) layer are removed, that is, the amino acid molecules are eluted, and then a cavity matching the shape and size of the branched-chain amino acid (BCAA) is formed, and the cavity can be connected to the branched-chain amino acid through hydrogen bonds, so it can specifically recognize the branched-chain amino acid, thereby forming a molecular recognition cavity that specifically binds to the BCAA in the MIP layer. The design of this MIP layer enables the sensor to highly specifically recognize BCAA when in use, avoiding interference from other components, thereby improving the specificity of the sensor.

[0017] (4) The method provided by the present invention adopts a flexible PDMS substrate as the supporting material of the sensor. PDMS has good flexibility and elasticity and can withstand large mechanical strain without breaking or falling off. This not only ensures the structural integrity of the sensor under long-term wearing and dynamic conditions, but also enables the molecular recognition cavity to have a large deformation ability, ensuring that the working effect of the molecular recognition cavity is not affected by the deformation when the sensor is used, thereby improving the stability of the sensor.

[0018] (5) The method provided by the present invention combines process technologies such as precision inkjet printing and electrochemical deposition. During the preparation process of the sensor of the present invention, the bonding force and stability between the layers are fully considered, thereby further improving the mechanical robustness of the sensor.

[0019] (6) The preparation method of the stretchable carbon nanotube BCAA sweat detection sensor of the present invention effectively solves the problems of existing sweat sensors in terms of sensitivity, specificity and mechanical robustness through innovative designs such as optimizing electrode structure, improving electron transfer characteristics and enhancing specific recognition ability, and provides a new solution for non-invasive, real-time and highly sensitive BCAA sweat detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Schematic diagram of the structure of the wearable sensor of the present invention; Figure 2 A schematic diagram of the manufacturing process of the sensor of the present invention; Figure 3 Schematic diagram of evaluating the morphology, electrochemical and mechanical properties of the molecular imprinting sensor based on stretchable carbon nanotubes (CNTs) for the present invention; Figure 4 It is a schematic diagram of the electrochemical performance of the present invention; Figure 5 This is a schematic diagram of the stretchability and stability of the present invention; Figure 6 Schematic diagram of the present invention for evaluating BCAA absorption and sweat analysis using stretchable molecular imprinting sensors. DETAILED DESCRIPTION

[0021] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. In the absence of conflict, the embodiments in this application and the features in the embodiments can be combined with each other. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0022] Example like Figure 1-Figure 6 As shown, the present invention provides a method for preparing a stretchable molecular imprinting sensor based on a PDMS / CNT composite electrode.

[0023] Figure 1 The structural design of the wearable sensor shown has a flexible CNT electrode, a Prussian blue (PB) signal transduction layer, and a molecularly imprinted polymer (MIP) layer. Figure 2 Schematic diagram of the sensor fabrication process, including CNT filtration, PDMS transfer, and curing steps. Figure 3 (a) shows the EIS spectra of impedance changes during the fabrication steps. Figure 3 The (bc) CV curves in the figure indicate the electrochemical characteristics at different stages. Figure 3 The d in the figure is the long-term stability of Prussian blue (PB) for 6 days. Figure 3 (ef) Sensitivity analysis showing the relationship between deposition cycle and sensor response.

[0024] It should also be noted that in the prepared electrode structure, the Prussian blue layer not only promotes the effective transfer of electrons, but also plays a key role as an indirect signal transmission layer. Since branched-chain amino acids are non-electroactive substances, they cannot directly undergo oxidation reactions in the molecular imprinting cavities. When these cavities combine with the target molecules, branched-chain amino acids, the contact area between the Prussian blue layer and the electrolyte solution will be reduced accordingly, resulting in a significant change in the signal. This mechanism provides an effective signal response for the detection of branched-chain amino acids.

[0025] Molecular imprinting layers are deposited on carbon nanotubes (especially single-walled carbon nanotubes). These molecular imprinting specific cavities are tightly wrapped on the surface of carbon nanotubes. It is worth noting that there is a certain stacking phenomenon between carbon nanotubes. When the electrode is stretched, these carbon nanotubes will have relative slippage without breaking, thus ensuring that the conductivity of the electrode is maintained. At the same time, this structure also effectively protects the integrity of the molecular imprinting specific cavities. Figure 5 As shown, when the stretching amount is 50%, the performance of the electrode is basically the same as that when it is not stretched, which fully verifies the stability and reliability of the structure.

[0026] Finally, considering that branched-chain amino acids are usually negatively charged in sweat, the IT method was used to apply a voltage of -0.2V to the electrode for 50 seconds. This operation can effectively expel the target molecules, branched-chain amino acids, from the specific cavity, thereby achieving reusable electrodes. This design not only improves the utilization rate of the electrode, but also provides the possibility for continuous detection in practical applications.

[0027] The present invention can be further configured as follows in a preferred embodiment: in step S1, the dispersion concentration of single-walled CNT in deionized water is 0.2 mg / mL, and the uniformity of the dispersion is ensured by ultrasonic treatment. In step S1, after the precision inkjet printing prints the required electrode pattern onto the polyethersulfone membrane, the polyethersulfone membrane is installed on a vacuum filtration device, wherein the CNT dispersion is filtered to form a uniform conductive CNT layer. Too high a concentration will cause CNT to agglomerate in the dispersion, affecting the subsequent printing effect and the conductivity of the electrode; while too low a concentration will make the distribution of CNT on the electrode too sparse, which is also not conducive to the improvement of conductivity. The concentration of 0.2 mg / mL can ensure that the CNT is fully dispersed while ensuring that it forms a uniform and dense conductive layer on the electrode, thereby effectively improving the sensitivity of the sensor. In addition, the uniformity of the dispersion is further ensured by ultrasonic treatment, avoiding the agglomeration and uneven deposition of CNT, and laying a good foundation for the subsequent filtration steps.

[0028] In the precision inkjet printing step, the desired electrode pattern is accurately printed onto the polyethersulfone membrane. This process not only requires high printing precision, but also requires ensuring the stability and continuity of the printing ink during the printing process. Subsequently, the polyethersulfone membrane is mounted on a vacuum filtration device, the dispersion solution is dripped onto the polyethersulfone membrane, and the vacuum filtration device is started. Through the action of vacuum filtration, the solvent in the CNT dispersion is quickly drawn away, and the CNTs are evenly deposited on the electrode pattern of the polyethersulfone membrane to form a uniform conductive CNT layer. This process not only improves the uniformity of the distribution of CNTs on the electrode, but also further removes impurities and bubbles in the CNT dispersion through the action of vacuum filtration, thereby improving the conductivity and stability of the electrode. For example, in actual operation, an inkjet printing device with high precision and stability can be selected to ensure accurate printing of the electrode pattern.

[0029] In a preferred embodiment, the present invention can be further configured as follows: In step S1, the specific operation of transferring the polyethersulfone membrane of the CNT coating to the flexible polydimethylsiloxane substrate is: spin coating a 10:1 mixture of PDMS monomer and curing agent onto a silicon wafer, which ensures the uniformity and thickness consistency of the PDMS layer. Subsequently, partial curing is performed at 65°C for 30 minutes so that the PDMS layer is initially formed and has a certain viscosity, which is convenient for the subsequent transfer of the polyethersulfone membrane of the CNT coating. Next, the polyethersulfone membrane with the CNT coating is aligned and attached to the partially cured PDMS layer, and fully cured at 65°C for 3 hours. This long curing process ensures the close bonding between the PDMS layer and the CNT coating, avoiding delamination or falling off during use. Finally, the polyethersulfone membrane is peeled off to obtain a flexible PDMS substrate sensor with CNT electrodes.

[0030] The sensor made in this way can better adapt to the dynamic changes of human skin, such as bending and stretching, without causing the electrode to break or performance degradation. This greatly improves the stability and reliability of the sensor under dynamic conditions, enabling it to continuously and accurately detect BCAA in sweat in various complex environments. In addition, the flexibility of the PDMS substrate also allows the sensor to fit closely to the skin, reducing sweat evaporation and external interference, further improving the accuracy and sensitivity of detection.

[0031] In a preferred embodiment, the present invention can be further configured as follows: in step S2, the Prussian blue layer solution contains 5mM FeCl3, 5mM K3Fe(CN)6, 0.1M HCl and 0.1M KCl; 20 cycles are performed in the potential range of -0.2V to 0.6V by cyclic voltammetry to deposit a uniform and stable Prussian blue coating on the CNT electrode. Prussian blue, as an excellent electrochemically active material, has high specific capacitance and good ionic conductivity, and can efficiently capture and convert biomarkers such as BCAA (branched chain amino acids) in sweat. 20 cycles are performed in the potential range of -0.2V to 0.6V by cyclic voltammetry, which not only ensures the uniformity and compactness of the Prussian blue coating, but also promotes its close bonding with the CNT electrode and improves the efficiency of electron transfer. This improvement enables the sensor to respond more accurately to changes in BCAA concentration in sweat, improving the accuracy and reliability of detection.

[0032] The solution of the Prussian blue layer contains 5mM FeCl3 and 5mM K3Fe(CN)6 as precursors of Prussian blue, and 0.1M HCl and 0.1M KCl are added to adjust the pH value and ionic strength of the solution to optimize the electrochemical deposition process. Cyclic voltammetry was performed for 20 cycles in the potential range of -0.2V to 0.6V. The selection of this potential range not only ensures the effective deposition of Prussian blue, but also avoids damage to the electrode caused by too high or too low potential.

[0033] In a preferred embodiment, the present invention can be further configured as follows: after depositing the Prussian blue layer, the electron transfer efficiency enhancement factor (η) is obtained by electrochemical impedance spectroscopy measurement, wherein the electron transfer efficiency enhancement factor η is obtained by the following formula:

[0034] in represents the current response after the deposition of the Prussian blue layer, The current response before deposition, the electron transfer efficiency enhancement factor calculated by this formula, and the electron transfer efficiency enhancement factor measured by electrochemical impedance spectroscopy not only provide an objective basis for evaluating the deposition effect of the Prussian blue layer, but also provide guidance for further optimizing the sensor design. The introduction of this indicator makes the evaluation and comparison of sensor performance more scientific and accurate, which helps promote the development and application of sweat sensor technology.

[0035] In practical applications, electrochemical impedance spectroscopy can be measured by electrochemical workstations and other equipment, and the current response values ​​before and after the deposition of the Prussian blue layer can be recorded and substituted into the formula for calculation. For example, assuming that the current response before the deposition of the Prussian blue layer is I0 and the current response after deposition is I, the electron transfer efficiency enhancement factor η can be calculated by the formula η=(I / I0)-1. Through this calculation process, we can intuitively understand the degree of improvement in the electron transfer efficiency caused by the deposition of the Prussian blue layer, and then adjust the deposition conditions or optimize the sensor structure according to actual needs to achieve the best performance.

[0036] The present invention can be further configured as follows in a preferred embodiment: in step S3, MIP is a polymer with specific recognition ability, and the factors such as template molecule selection, precursor solution composition and electrochemical deposition conditions in its preparation process have an important influence on the performance of the final sensor. The MIP precursor solution contains pyrrole, 3-aminophenylboronic acid and BCAA template molecules, which are dissolved in PBS buffer and undergo polymerization reaction to form a MIP membrane with specific recognition sites. Electrochemical deposition is performed at a scanning rate of 50mV / s in a potential range of -0.2V to 0.9V by cyclic voltammetry, which not only ensures the uniformity and density of the MIP membrane, but also promotes its close binding with the Prussian blue coated electrode, thereby improving the electron transfer efficiency. In addition, through the template molecule removal step, that is, the electrode is immersed in a specific proportion of water, methanol and acetic acid solution for incubation, and then the electrode is thoroughly rinsed, which ensures the effective removal of the template molecule, thereby retaining the specific recognition ability of the MIP membrane for BCAA.

[0037] The concentrations of pyrrole, 3-aminophenylboronic acid and BCAA template molecules in the MIP precursor solution were selected to form a MIP membrane with excellent recognition performance and stability. The pH value of the PBS buffer was adjusted to 6.5, providing a suitable acid-base environment for the polymerization reaction. During the electrochemical deposition process, the selection of the potential range and scan rate ensured the effective deposition of the MIP membrane while avoiding damage to the electrode caused by excessively high or low potentials. In the template molecule removal step, the volume ratio of water, methanol and acetic acid solution was designed to be 5:2:3. This ratio was selected to ensure the effective dissolution and removal of the template molecules while avoiding damage to the structure of the MIP membrane.

[0038] In a preferred embodiment, the present invention can be further configured as follows: it also includes morphological analysis and electrochemical characterization by scanning electron microscopy to verify the removal of template molecules. Among them, the scanning electron microscope image shows the significant change of surface morphology before and after template removal, and the electrochemical impedance measurement reveals the obvious difference in interface characteristics; and the sensor can maintain its structural integrity and electrochemical function when subjected to a strain level of 0% to 50%, and exhibits stable performance in long-term stability tests. At the same time, the sensitivity analysis indicates that the optimal number of cycles for molecular imprinting polymer (MIP) deposition is 10 times, and the scanning electron microscope image shows the significant change of surface morphology before and after template removal, which intuitively verifies the effective removal of template molecules. The high-resolution imaging capability of the scanning electron microscope enables the microstructure and morphological changes of the MIP membrane surface to be clearly observed, thereby providing intuitive and reliable evidence for the evaluation of the effect of template molecule removal. At the same time, the introduction of electrochemical impedance measurement reveals the obvious difference in interface characteristics, further confirms the change of electrochemical performance of the MIP membrane after template molecule removal, and provides an important reference for the performance optimization of the sensor. This double verification method not only ensures the complete removal of the template molecules, but also retains the MIP membrane's specific recognition ability for the target molecules, thereby improving the selectivity and sensitivity of the sensor.

[0039] The sensor was fully characterized to evaluate its morphological, electrochemical, and mechanical properties. Figure 3 As shown in Figure 2, these analyses are critical to verifying the performance and structural integrity of the sensor throughout the fabrication process and operating conditions. Morphological analysis was performed using scanning electron microscopy (SEM) to observe the surface structure of the sensor during key fabrication stages, and electrochemical performance was evaluated using linear sweep voltammetry (LSV) and electrochemical impedance spectroscopy (EIS). Figure 3 As shown in (a), the EIS spectrum shows that the impedance is significantly reduced after the deposition of the PB layer and the MIP layer, indicating that the electron transfer efficiency is enhanced. The CV curve ( Figure 3 Figure 2 (b-c) further reveals the improvement of electrochemical properties throughout the fabrication stage, with distinct oxidation and reduction peaks reflecting the integration of the functional layer. Notably, after template removal, the MIP layer exhibits selective binding to BCAA, as evidenced by the change in current response.

[0040] Mechanical robustness was assessed by tensile testing, where the sensor was subjected to strain levels ranging from 0% to 50%. The LSV curves recorded under these conditions (e.g. Figure 5 The results show that the sensor can maintain its structural integrity and electrochemical functionality even under huge mechanical stress. These results highlight the suitability of the sensor for wearable applications where dynamic deformation is inevitable.

[0041] The long-term stability and sensitivity of the sensor were also examined. Over the 9-day test period, the sensor showed stable performance with negligible signal degradation ( Figure 5 Sensitivity analysis ( Figure 3 Figure ef) shows that the optimal number of MIP deposition cycles is 10, which provides the highest signal response. Beyond 10 cycles, the sensitivity decreases slightly due to excessive layer thickness hindering electron transfer, demonstrating the importance of accurate optimization.

[0042] The present invention uses the comprehensive results of SEM imaging, electrochemical analysis and mechanical testing ( Figure 3 ) verified the structural, functional, and mechanical reliability of the sensor fabricated in this invention. These findings highlight the potential of this sensor as a powerful platform for non-invasive, real-time monitoring of BCAAs in sweat, making it a promising tool for wearable healthcare applications and metabolic disease management.

[0043] like Figure 4 As shown, (a) Schematic diagram of the molecular imprinting process: the upper panel shows the specific binding of BCAA to the molecular imprinting polymer (MIP) cavity, while the lower panel shows the non-binding behavior of non-target molecules. (bd) Electrochemical detection of leucine, valine, and isoleucine at different concentrations (0 μM to 300 μM) using linear sweep voltammetry (LSV) demonstrated the high sensitivity and specificity of the sensor. For example, the selective analysis of BCAA (leucine, valine, and isoleucine) and interfering molecules (including ascorbic acid, tryptophan, dopamine, and glucose) illustrates the specific recognition ability of the sensor. (h) Non-imprinted polymer (NIP) control test showed negligible response to BCAA, highlighting the importance of the specific binding cavity generated by the molecular imprinting process. (i) Regeneration performance of the sensor over 10 cycles, showing consistent detection ability after repeated template removal and rebinding processes. (j) Stability testing over 9 days showed minimal degradation of the current response with a relative standard deviation (RSD) of 6.6%, confirming the long-term stability and reliability of the sensor.

[0044] The electrochemical performance of the sensor was comprehensively evaluated to assess its sensitivity and selectivity for the detection of branched-chain amino acids (BCAAs), including leucine, valine, and isoleucine. The sensor demonstrated excellent reliability, specificity, and suitability for noninvasive sweat analysis through linear sweep voltammetry (LSV) and interference studies.

[0045] The sensitivity of the sensor was studied over a range of BCAA concentrations (50 μM to 300 μM). Figure 4As shown in (bd), the current response (ΔI) showed a clear linear relationship with the analyte concentration of leucine, valine, and isoleucine. For leucine, the peak current increased from 6.3 μA at 50 μM to 15.6 μA at 300 μM, following the linear regression equation y=0.052x+1.12y=0.052x+1.12y=0.052x+1.12 with a correlation coefficient R2=0.998R^2=0.998R2=0.998. Similarly, valine and isoleucine showed linear responses with R2R^2R2 values ​​of 0.995 and 0.996, respectively, confirming the high sensitivity and consistent detection ability of the sensor. The observed sensitivity is attributed to the molecularly imprinted polymer (MIP) layer, which contains highly specific recognition cavities tailored for the BCAA structure. These cavities are formed during the imprinting process and provide strong selective binding to the target molecules, thereby minimizing interference from non-target molecules. Furthermore, the optimized polymer composition, combining pyrrole and 3-aminophenylboronic acid, enhanced the binding affinity and promoted efficient electron transfer, thereby amplifying the electrochemical signal.

[0046] The present invention evaluates the selectivity of the sensor by analyzing the response of the sensor to common interfering molecules in sweat, such as ascorbic acid, uric acid, dopamine, and glucose. Figure 4 As shown in (eg), the current responses of these non-target molecules were negligible (<1 μA) even at concentrations comparable to BCAA. In contrast, BCAA produced significantly higher current responses, reaching 15 μA at 300 μM for leucine. These results indicate that the sensor has excellent specificity for BCAA and is able to distinguish between target analytes and potential interferents. To further validate the role of the molecular imprinting process, a non-imprinted polymer (NIP)-based sensor was used as a control. Figure 4 As shown in Figure 3h, the current response of the NIP sensor to BCAA is significantly lower, with a maximum response of only 2.4 μA at 300 μM, compared to 15.6 μA for the MIP-based sensor. This confirms that the specific binding cavity within the MIP layer is critical for achieving high selectivity.

[0047] The sensor fabricated in this invention exhibits excellent electrochemical performance, characterized by high sensitivity and specificity for BCAA detection. These properties, coupled with its ability to minimize interference from non-target molecules, make it a promising tool for real-time, non-invasive monitoring of metabolic biomarkers in sweat. Figure 4 As shown, the results highlight the potential of the sensor in wearable healthcare applications and its ability to meet the needs of reliable, continuous metabolic monitoring.

[0048] In a preferred embodiment, the present invention can be further configured as follows: the sensitivity S of the sensor is calculated by the formula:

[0049] in represents the change in current response, Represents the change in BCAA concentration. Sensitivity analysis shows that when the number of MIP deposition cycles is 10, by introducing the quantitative formula of sensitivity S, that is, the ratio of the change in current response ΔI to the change in BCAA concentration ΔC, an objective and accurate quantitative indicator is provided for the performance evaluation of the sensor. The application of this formula enables the accurate measurement of the change in current response of the sensor at different BCAA concentrations, and then the sensitivity of the sensor is calculated, providing a scientific basis for the performance optimization and comparison of the sensor. Through sensitivity analysis, it was found that when the number of MIP deposition cycles was 10, the sensitivity of the sensor reached the optimal level. This result not only verifies the important influence of MIP deposition conditions on sensor performance, but also provides a clear direction for optimizing the sensitivity and response speed of the sensor.

[0050] In actual operation, the current response of the sensor under different BCAA concentrations can be measured, the corresponding current response change ΔI and the BCAA concentration change ΔC can be calculated, and then these two values ​​can be substituted into the sensitivity formula for calculation to obtain the sensitivity S of the sensor. For example, in the experiment, a series of known BCAA concentration gradients can be set, and then the current response of the sensor under these concentrations can be measured respectively, and the current value at each concentration can be recorded. By calculating the current response change between adjacent concentrations and the change in BCAA concentration, a series of sensitivity data can be obtained. By comparing the sensitivity data under different MIP deposition cycle numbers, it can be found that when the MIP deposition cycle number is 10 times, the sensitivity of the sensor is the highest. This result not only verifies the influence of MIP deposition conditions on sensor sensitivity, but also provides an important reference for subsequent experiments and sensor design.

[0051] For example, five BCAA concentration gradients were set in the experiment, namely 0.1mM, 0.2mM, 0.3mM, 0.4mM and 0.5mM, and then the current response of the sensor at these concentrations was measured. Four sensitivity data points were obtained by calculating the change in current response between adjacent concentrations and the change in BCAA concentration. By comparing the average values ​​of these four sensitivity data points under different MIP deposition cycles, it was found that when the MIP deposition cycle was 10 times, the average sensitivity was the highest, reaching XXμA / mM (the specific value is determined according to the experimental data). This result not only proves the important influence of MIP deposition conditions on sensor sensitivity, but also provides a clear optimization direction for subsequent experiments and sensor design, that is, the sensitivity of the sensor can be optimized by adjusting the number of MIP deposition cycles.

[0052] In actual applications, when BCAA in human sweat comes into contact with the MIP layer on the surface of the sensor, the BCAA molecules will specifically bind to the molecular recognition cavity in the MIP layer, resulting in a change in the current response of the sensor. By measuring the change in this current response and combining it with the known change in BCAA concentration, the sensitivity S of the sensor can be calculated. This working principle enables the sensor to achieve high-sensitivity and high-selectivity detection of BCAA in human sweat, providing strong technical support for health monitoring and disease diagnosis.

[0053] like Figure 5 Shown are (a, d, g) Linear sweep voltammetry (LSV) curves for leucine, valine, and isoleucine detection in the concentration range of 50 to 300 μM, showing that the peak current increases proportionally with the analyte concentration. (b, e, h) Calibration curves for BCAA detection, showing a strong linear relationship between the current response (ΔI) and analyte concentration with a determination coefficient (R²) exceeding 0.995. (c, f, i) Stability of ΔI under mechanical strain (0%–50%) demonstrates the robustness and consistent electrochemical performance of the sensor during stretching.

[0054] The stretchable carbon nanotube BCAA sweat detection sensor exhibits excellent mechanical strength and electrochemical stability, making it a promising candidate for wearable health monitoring applications. Its performance was systematically evaluated at different mechanical strain levels and during extended storage to confirm its reliability in sweat-based real-time biomarker detection.

[0055] The stretchability of the sensor was evaluated by applying mechanical strain ranging from 0% to 50% while monitoring its electrochemical response to branched-chain amino acids (BCAA). Figure 5 As shown in c, f, i, the current responses (ΔI) for leucine, valine, and isoleucine detection all show minimal changes compared to the unstretched state. After linear sweep voltammetry (LSV) measurements under these conditions, there was no significant attenuation of the signal, indicating that the sensor is able to maintain consistent electrochemical performance during dynamic mechanical deformation. This excellent mechanical stability is attributed to the synergistic integration of carbon nanotube (CNT) electrodes, Prussian blue (PB) layers, and molecularly imprinted polymer (MIP) layers, which together retain molecular recognition and signal transduction capabilities under strain. This strong stretchability ensures the adaptability of the sensor to real-world wearable scenarios, where mechanical deformation is inevitable.

[0056] The long-term stability of the sensor during a 9-day storage period was evaluated by regularly monitoring the electrochemical performance of the sensor. Figure 5As shown in Figure 1, the current response (ΔI) remained stable with a relative standard deviation (RSD) of 6.6%, indicating negligible degradation over time. This high stability reflects the durability of the sensor's functional layers, which retain their structural and functional properties throughout storage. The molecular recognition cavities within the MIP layer formed during the imprinting process are preserved, ensuring reliable and precise detection of BCAAs over extended periods of use.

[0057] From the above analysis, it can be seen that the sensor provided by the present invention exhibits excellent mechanical strength and long-term stability, making it a promising platform for wearable health monitoring applications. Its ability to maintain sensitive and accurate detection of BCAA during mechanical strain and extended storage emphasizes its suitability for continuous, non-invasive analysis of metabolic biomarkers in sweat. These findings highlight the potential of sensors for real-time, practical applications in wearable healthcare systems, meeting the need for reliable and durable diagnostic tools in dynamic environments.

[0058] like Figure 6 As shown, the sensor was evaluated for BCAA absorption and sweat analysis: (a) Schematic diagram of the relationship between BCAA intake, metabolism and subsequent detection in sweat. The process includes oral BCAA, absorption through the gastrointestinal tract, metabolic processing and excretion through sweat glands, thus achieving non-invasive monitoring. (bd) Electrochemical detection of leucine in sweat after BCAA intake. (b) LSV curves showing the current response at different time points (10-60 minutes). (c) Time-dependent changes in leucine concentration in sweat of three subjects. (d) Control experiment showing leucine levels in sweat of three subjects without BCAA intake. (e) Electrochemical detection of valine in sweat after BCAA intake. (e) LSV curves showing the current response at different time points (10-60 minutes). (f) Time-dependent changes in valine concentration in sweat of three subjects. (g) Control experiment showing valine levels in sweat of three subjects without BCAA intake. (hj) Electrochemical detection of isoleucine in sweat after BCAA intake. (h) LSV curves showing the current response at different time points (10-60 min). (i) Time-dependent changes in isoleucine concentration in sweat of three subjects. (j) Control experiment showing isoleucine levels in sweat of three subjects without BCAA intake.

[0059] Sweat provides an ideal medium for metabolic monitoring because it directly reflects the body's physiological state, including key biomarkers such as BCAA. Figure 6As shown in a in Figure 1: BCAAs such as leucine, valine, and isoleucine are absorbed through the gastrointestinal tract, metabolized by the body, and excreted through sweat glands, making them detectable using sweat-based sensors. This feature eliminates the need for invasive blood sampling, providing a more convenient and portable way to track metabolic activity. Figure 6 The (bd) in the figure demonstrates the sensor's ability to detect BCAA fluctuations in real time, where the electrochemical response to leucine increases significantly over time (10-60 min) after BCAA ingestion. This dynamic monitoring is further extended to valine and isoleucine, respectively. Figure 6 (eg) and Figure 6 These findings highlight the sensor's ability to provide real-time insights into metabolic changes, making it suitable for applications such as exercise monitoring or metabolic testing.

[0060] In the field of sports performance, real-time monitoring of BCAA levels can provide valuable insights into an athlete's metabolic state, energy expenditure, and recovery process. Elevated BCAA levels in sweat are often associated with muscle breakdown during intense physical activity, so monitoring them is essential for optimizing training and recovery regimens. The sensor was able to track BCAA concentrations over time in multiple subjects ( Figure 6 BD, EG, HJ in the study demonstrated its usefulness in providing real-time feedback during and after exercise. Unlike traditional laboratory methods that are invasive and time-consuming, the sensor enables continuous mobile monitoring, helping athletes adjust exercise intensity, avoid overtraining, optimize recovery, and ultimately improve performance and reduce injury risk.

[0061] The sensor also offers significant advantages for managing metabolic disorders such as obesity, type 2 diabetes, and insulin resistance, where BCAAs play a key role in insulin signaling and mitochondrial function. Elevated BCAA levels have been associated with the progression of these diseases, making their monitoring critical for disease management. Figure 6 As shown, the sensor can track fluctuations in sweat BCAA levels, providing real-time data to evaluate the effectiveness of dietary interventions, exercise regimens, and drug treatments. By continuously monitoring BCAA concentrations, patients can receive immediate feedback about their metabolic status, enabling timely adjustments to their lifestyle or treatment plans. In addition, the sensor can be integrated into personalized healthcare systems to provide non-invasive tools for managing chronic diseases. For example, individuals can track the impact of specific foods or medications on their metabolic health, thereby promoting more informed decisions and improving long-term outcomes.

[0062] Compared to existing SWEAT-based technologies, the proposed sensor solves challenges related to sensitivity, specificity, and mechanical robustness. Conventional sensors are often interfered by common sweat components such as ascorbic acid, glucose, and uric acid, resulting in inaccurate readings. The molecularly imprinted polymer (MIP) technology employed in this study significantly improves specificity by creating recognition sites tailored for BCAAs, such as Figure 6 As shown in (eg), the target molecule and the interfering species are effectively distinguished. In addition, the stretchability of the sensor (eg Figure 5 The combination of a flexible polydimethylsiloxane (PDMS) substrate and carbon nanotube (CNT) electrodes provides the necessary mechanical stability and reliable electrochemical performance, making it ideal for dynamic real-world applications.

[0063] From the above analysis, it can be seen that the high flexibility and low power consumption requirements of the sensor provided by the present invention make it an ideal choice for integration into wearable systems such as smart clothing, fitness trackers, and health monitoring devices. These devices can continuously monitor sweat BCAA levels and provide valuable data to users and healthcare providers. The sensor can be combined with wireless communication for remote monitoring, and metabolic health can be continuously assessed without regular visits or invasive surgery. This integration with wearable consumer technology has the potential to democratize health monitoring and enable individuals to take a proactive approach to managing their own health.

[0064] In summary, the sensor made by the method provided by the present invention, the PB layer (i.e., Prussian blue layer) uniformly deposited on the CNT electrode by cyclic voltammetry acts as a signal sensor, and its redox characteristics can achieve precise electron transfer and signal amplification. By promoting redox reactions, the PB layer can ensure stable and reliable detection of electrochemical changes caused by analyte binding. The uniformity and stability of this layer are crucial to achieving reproducible and accurate sensor responses.

[0065] On top of the sensor, the MIP layer introduces molecular recognition specificity. During the manufacturing process, BCAA molecules act as templates to form complementary recognition cavities within the polymer matrix. These cavities match the target molecules in shape, size and functional groups, selectively binding. When BCAA interact with these cavities, they hinder electron transfer in the PB layer, resulting in a measurable change in the electrochemical signal. This design enables the sensor to achieve high sensitivity and specificity. The integration of these three layers creates a sensor that can still function properly under significant mechanical deformation. Performance testing shows that it can still function properly even under 50% strain, and its signal detection results are basically consistent with when it is not stretched, highlighting its mechanical elasticity. The flexibility of CNTs, signal transduction of PBs and molecular recognition of MIPs combine to form a powerful platform that can detect BCAAs in real time and with high accuracy. This makes the sensor particularly suitable for wearable health monitoring applications, meeting the need for non-invasive tools in metabolic health management.

[0066] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for preparing a stretchable molecular imprinting sensor based on PDMS / CNT composite electrode, characterized in that: The following steps are involved: S1. Preparation of stretchable CNT electrodes using single-walled CNTs; S2. Deposition of a Prussian blue layer on the stretchable CNT electrode: Depositing a Prussian blue solution onto a CNT electrode by an electrochemical method to form a Prussian blue coating on the CNT electrode to prepare a Prussian blue coated electrode; S3. Depositing a molecularly imprinted polymer layer on the Prussian blue coated electrode: S31, depositing a MIP precursor solution containing a template molecule onto a Prussian blue coated electrode by an electrochemical method, and forming a MIP layer on the Prussian blue coated electrode; S32. Remove the template molecules in the MIP layer, form a molecular recognition cavity that specifically binds to BCAA in the MIP layer, and complete the preparation of the sweat detection sensor.

2. The method for preparing a stretchable molecular imprinting sensor based on a PDMS / CNT composite electrode according to claim 1, characterized in that: In step S1, the process of preparing a stretchable CNT electrode using single-walled CNT includes: S11, dispersing single-walled CNT in deionized water to form a dispersion solution; S12, using a printer to print a preset electrode pattern onto a polyethersulfone filter membrane, and installing the polyethersulfone filter membrane printed with the electrode pattern onto a vacuum filtration device; S13, dropping the dispersion solution onto the polyethersulfone filter membrane on the vacuum filtration device, and starting the vacuum filtration device to filter the dispersion solution on the polyethersulfone filter membrane, so as to form a CNT coating on the electrode pattern printed on the polyethersulfone filter membrane; S14, transferring the polyethersulfone membrane forming the CNT coating onto a flexible polydimethylsiloxane substrate, and obtaining a stretchable CNT electrode after curing.

3. The method for preparing a stretchable molecular imprinting sensor based on a PDMS / CNT composite electrode according to claim 1, characterized in that: In step S2, the Prussian blue solution includes 5mM FeCl3, 5mM K3Fe(CN)6, 0.1M HCl and 0.1M KCl; and the Prussian blue solution is subjected to 20 cycles in a potential range of -0.2V to 0.6V by cyclic voltammetry, and the Prussian blue in the Prussian blue solution is deposited on the stretchable CNT electrode to form a Prussian blue coating on the stretchable CNT electrode.

4. The method for preparing a stretchable molecular imprinting sensor based on a PDMS / CNT composite electrode according to claim 1, characterized in that: In step S3, the MIP precursor solution is prepared by dissolving 60 mM pyrrole, 20 mM 3-aminophenylboronic acid and 10 mM BCAAA template molecules in PBS buffer, and the pH value of the PBS buffer is 6.

5.

5. The method for preparing a stretchable molecular imprinting sensor based on a PDMS / CNT composite electrode according to claim 1, characterized in that: In step S3, the MIP precursor solution is electrochemically deposited onto the Prussian blue coating electrode by cyclic voltammetry in a potential range of -0.2 V to 0.9 V at a scan rate of 50 mV / s, and the number of MIP deposition cycles is 10.

6. The method for preparing a stretchable molecular imprinting sensor based on a PDMS / CNT composite electrode according to claim 1, characterized in that: The concentration of the dispersion solution of the single-walled CNT in deionized water is 0.2 mg / mL, and the dispersion is subjected to ultrasonic treatment.

7. The method for preparing a stretchable molecular imprinting sensor based on PDMS / CNT composite electrode according to claim 1, characterized in that: In step S32, the specific operation of removing the template molecules is as follows: immersing the Prussian blue coated electrode formed with the MIP layer in a mixed solution of water, methanol and acetic acid, gently stirring and incubating for 2 hours at room temperature, then thoroughly rinsing the electrode with deionized water, and performing morphological analysis and electrochemical characterization of the electrode by scanning electron microscopy to verify that the template molecules on the electrode are completely removed.

8. The method for preparing a stretchable molecular imprinting sensor based on PDMS / CNT composite electrode according to claim 7, characterized in that: The volume ratio of water, methanol and acetic acid solution in the mixed solution is 5:2:

3.

9. The method for preparing a stretchable molecular imprinting sensor based on PDMS / CNT composite electrode according to claim 2, characterized in that: The specific operation process of step S14 includes: S141, spin coating a mixture of polydimethylsiloxane monomer and curing agent onto a silicon wafer, and partially curing at 65° C. for 30 minutes to form a PDMS layer; S142, aligning the polyethersulfone membrane with the CNT coating on the PDMS layer, and peeling off the polyethersulfone membrane after fully curing at 65°C for 3 hours.

10. The method for preparing a stretchable molecular imprinting sensor based on PDMS / CNT composite electrode according to claim 9, characterized in that: In step S141, the mass ratio of the polydimethylsiloxane monomer to the curing agent is 10:1.

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