Molecularly imprinted electrochemical sensor of inositol tripyrophosphate as well as preparation method and application of molecularly imprinted electrochemical sensor
By forming a polypyrrole molecularly imprinted film on the surface of a glassy carbon electrode and combining it with differential pulse testing, the problems of invasive sampling and time lag in ITPP detection are solved, realizing highly selective and rapid response inositol tripyrophosphate sweat detection, which is suitable for on-site real-time anti-doping detection.
Patent Information
- Application Number
- CN202511287750.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-09-10
- Publication Date
- 2026-02-06
AI Technical Summary
Existing ITPP detection technologies suffer from problems such as invasive sampling, time lag, and interference from complex matrices, especially lacking highly selective and rapid response solutions in sweat detection.
A molecularly imprinted electrochemical sensor was fabricated by forming a polypyrrole molecularly imprinted film on the surface of a glassy carbon electrode and using differential pulse testing to achieve highly selective detection of inositol tripyrophosphate. Combined with a stepwise elution process using methanol and NaOH, a specific recognition cavity was formed, enabling rapid and accurate sweat detection.
It achieves a detection limit of 50 ng/mL in real sweat matrix, with a linear range covering 50 ng/mL to 1500 ng/mL, a rapid response time of <25 minutes, good stability, low cost, and is suitable for on-site real-time anti-doping detection. It also has high specificity and anti-interference ability.
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Figure CN121476336A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of molecularly imprinted electrochemical detection technology, specifically to a molecularly imprinted electrochemical sensor of inositol tripyrophosphate, its preparation method, and its application. Background Technology
[0002] Inositol tripyrophosphate (ITPP) is a novel hemoglobin allosteric effector, which, especially under hypoxic conditions, can shift the oxygen dissociation curve to the right and increase oxygen release in target tissues.ITPP has been successfully used in cancer research, demonstrating its anti-cancer properties by preventing tumor hypoxia. ITPP also has broad prospects for treating hypoxic cardiovascular diseases. [[1] Görgens C, Guddat S, Schänzer W, et al. Screening and confirmation of myo-inositol trispyrophosphate (ITPP) in human urine by hydrophilic interaction liquid chromatography high resolution / high accuracy mass spectrometry for doping control purposes [J]. Drug Test Anal, 2014, 6(11-12):1102-7. [2] Lam G, Zhao S, Sandhu J, et al. Detection of myo-inositol trispyrophosphate (ITPP) in equine following an administration of ITPP [J]. Drug Test Anal, 2014, 6(3): 268-76. [3] Görgens C, Walker K, Boeser C, et al. Paperspray mass spectrometry - A potential complementary technique for the detection of polar compounds in sports drug testing [J]. Drug Test Anal, 2020, 12(11-12): 1658-65.[4] Wong ASY, Yuen BP, Wong COL, et al. Dopingcontrol analysis of myo-inositol trispyrophosphate and 10 bisphosphonates inequine plasma by ion chromatography-mass spectrometry and its application toclodronic acid horse administration [J]. Drug Test Anal.].
[0003] ITPP, as a potent oxygen carrier stimulant, presents significant challenges to existing detection technologies due to its rapid metabolism (half-life ≈ 5.7 hours) and ease of compounding and avoidance. While laboratory mass spectrometry (such as LC-MS / MS) offers high sensitivity, it relies on invasive blood / urine samples and has an analysis cycle exceeding 2 hours. Furthermore, ensuring the samples do not decompose during transport to a qualified laboratory indirectly impacts the accuracy and timeliness of doping detection. Electrochemical (EC) sensing technology, with its rapid response (minute-level), portable equipment, and low cost, has become an ideal medium for on-site screening; however, its insufficient selectivity for trace targets in complex biological matrices limits its application expansion.
[0004] Currently, molecular imprinting (MIP) technology, by constructing recognition cavities complementary to the spatial / functional groups of target molecules, can endow EC sensors with antibody-level specificity, especially suitable for molecules with characteristic phosphate group structures such as ITPP. Combining the high selectivity of MIP with the rapid detection advantages of EC (MIP-EC) can simultaneously overcome the dual bottlenecks of "specific recognition - rapid output," providing a technological foundation for non-invasive sweat detection. However, there are currently no reports of MIP-EC sensors specifically for the detection of ITPP in sweat, highlighting the urgent need to develop solutions that combine on-site speed with laboratory accuracy. Summary of the Invention
[0005] The technical problem to be solved by this invention is to provide a molecularly imprinted electrochemical sensor for inositol tripyrophosphate (ITPP), its preparation method, and its application. The aim is to address the three major shortcomings of existing ITPP detection technologies: invasive sampling, time lag (>2 hours), and interference from complex matrices.
[0006] The technical solution of the present invention to solve the above-mentioned technical problems is as follows: The first aspect is the preparation method of the molecularly imprinted electrochemical sensor of inositol tripyrophosphate, which includes the following steps: A glassy carbon electrode is immersed in an electrolyte containing pyrrole, and electropolymerization is carried out using cyclic voltammetry to form a polypyrrole molecularly imprinted film on the surface of the glassy carbon electrode. Then, the inositol tripyridine template in the glassy carbon electrode with the polypyrrole molecularly imprinted film on the surface is eluted to obtain an inositol tripyridine molecularly imprinted electrochemical sensor. The pyrrole-containing electrolyte includes pyrrole, inositol tripyrophosphate template, and salts.
[0007] Based on the above technical solution, the present invention can be further improved as follows.
[0008] Furthermore, the salts include at least one of LiClO4, KNO3, LiBF4, and LiPF6; The molar ratio of pyrrole, inositol tripyrophosphate template, and salt is 0.03~0.1:0.00136:0.1; The concentration of pyrrole in the electrolyte containing pyrrole is 0.03 M to 0.1 M; The concentration of inositol tripyrophosphate template in the pyrrole-containing electrolyte is 1 mM to 6.85 mM, preferably 1.36 mM; The concentration of salts in the pyrrole-containing electrolyte is 0.05M to 0.5M, with 0.1M being optimal.
[0009] Furthermore, the conditions for electropolymerization using cyclic voltammetry are as follows: scan range 0 V~1.2 V (vs. Ag / AgCl), scan rate 0.05 V / s~0.2 V / s, preferably 0.1 V / s, and 3~6 cycles, preferably 4 cycles.
[0010] Furthermore, the specific method for eluting the inositol tripyrophosphate template from the glassy carbon electrode with the polypyrrole molecularly imprinted film on its surface is as follows: the glassy carbon electrode with the polypyrrole molecularly imprinted film on its surface is sequentially immersed in a mixture of methanol and acetic acid, and then in a 0.08M~0.12M NaOH solution, and then rinsed with ultrapure water. The immersion time in the mixture or the 0.08M~0.12M NaOH solution is determined as needed, and can be 10 min, 15 min, 20 min, 25 min, etc.; the mixture includes methanol and acetic acid in a volume ratio of 7~9:1~3, preferably 7:3, 8:2, or 9:1.
[0011] Furthermore, the pretreatment of the glassy carbon electrode before immersing it in an electrolyte containing pyrrole includes the following steps: the glassy carbon electrode is polished sequentially with alumina paste (e.g., 0.3 μm, 0.05 μm) and ultrasonically cleaned (5 min each with ultrapure water and ethanol), then cyclically voltammetrically scanned in 0.1 M to 0.5 M H2SO4 solution at a scan rate of 0.1 V / s within a potential range of -1 V to 1 V until the electrochemical curves highly overlap, and finally dried (e.g., dried with nitrogen) for later use.
[0012] Secondly, a molecularly imprinted electrochemical sensor of inositol tripyrophosphate, wherein the molecularly imprinted electrochemical sensor of inositol tripyrophosphate is prepared by the aforementioned preparation method.
[0013] Thirdly, the application of a molecularly imprinted electrochemical sensor for inositol tripyrophosphate involves using the aforementioned molecularly imprinted electrochemical sensor for the detection of inositol tripyrophosphate.
[0014] Fourthly, a method for detecting inositol tripyrophosphate includes the following steps: The molecularly imprinted electrochemical sensor of inositol tripyrophosphate was immersed in a sweat sample and incubated at room temperature. Using the molecularly imprinted electrochemical sensor of inositol tripyrophosphate as the working electrode, differential pulse testing was performed in an electrolyte containing potassium hexacyanoferrate to achieve the detection of inositol tripyrophosphate in the liquid sample to be tested.
[0015] Furthermore, the detection linear range of the inositol tripyrophosphate is 50 ng / mL to 1500 ng / mL, and the linear regression equation is: I = 0.00253C + 0.26763, with a correlation coefficient R. 2 >0.998, where I is in μA and C is in ng / mL.
[0016] Furthermore, the volume of the sweat sample is 5 μL to 20 μL, preferably 10 μL; The incubation time at room temperature is 15 min to 20 min; The electrolyte containing potassium hexacyanoferrate includes K3[Fe(CN)6] and KCl; the concentration of K3[Fe(CN)6] in the electrolyte containing potassium hexacyanoferrate is 5 mM to 10 mM; the concentration of KCl in the electrolyte containing potassium hexacyanoferrate is 0.1 M to 1.0 M, preferably 0.5 M; The conditions for the differential pulse test are: potential range -0.2 V to 0.6 V, amplitude 0.01 V to 0.5 V, preferably 0.05 V, and pulse width 0.005 s to 0.5 s, preferably 0.05 s.
[0017] The fabrication process of the inositol tripyrophosphate (ITPP) molecularly imprinted (MIP) sensor of the present invention involves first immersing a pretreated glassy carbon electrode in a solution containing ITPP template molecules, pyrrole monomers, and LiClO4 electrolyte, and performing a cyclic scanning at 0 V to 1.2 V for 4 cycles to form a MIP film. Then, an elution step is performed to remove the ITPP template molecules, forming an ITPP-specific recognition cavity, thus completing the sensor fabrication. During the detection process, the sensor is immersed in sweat. ITPP molecules selectively embed into the imprinted cavity. Due to electron transfer inhibition, the bound ITPP molecules hinder the probe ions. (Yellow) diffuses to the electrode surface, causing the DPV peak current to decay, and finally the signal is quantified: the current decay value ΔI is positively correlated with the ITPP concentration, thus achieving quantitative detection.
[0018] The beneficial effects of this invention are: (1) Ultra-high sensitivity and wide dynamic range: A detection limit of 50 ng / mL (68.1 nM) is achieved in real sweat matrix, and the linear detection range covers 50 ng / mL to 1500 ng / mL, which is fully matched to the black market abuse dose (<0.5 mg / kg). However, it is more suitable for the actual concentration requirements of sweat than existing mass spectrometry methods (detection limit of 0.02 ng / mL, but requires >2 hours of analysis). (2) Excellent accuracy and anti-interference ability: The recovery rate of spiked real sweat reached 103.4%±1.76% (100 ng / mL) and 103.7%±3.24% (500 ng / mL), with RSD <5%, which meets the ISO 17025 certification standard (80%~120%), proving that it can overcome the interference of complex sweat matrix; (3) Rapid response: The total analysis time is less than 25 minutes (including 20 minutes of incubation), which is up to 83% faster than laboratory mass spectrometry (>2 hours), meeting the timeliness requirements of point-of-care testing (POCT) for the first time; (4) Long-term stability and low cost: The methanol / acetic acid → NaOH stepwise elution process enables the sensor to retain signal rate >94% after 30 days of storage at room temperature and can be reused ≥6 times (RSD=3.1%), reducing the cost of a single detection by more than 80%.
[0019] (5) High specificity: The cavity formed by the molecular imprint of ITPP template is used to realize "lock-key" recognition, effectively distinguishing ITPP from its structural analogs (such as Efaproxiral®) and interfering substances in the complex matrix of sweat.
[0020] (6) Low cost and easy operation: low equipment requirements (electrochemical workstation), easy to miniaturize and port, laying the foundation for the development of wearable real-time monitoring devices.
[0021] (7) Non-invasive sampling: Directly detect sweat, avoiding the invasiveness of blood / urine sampling, reducing subject resistance, and making it easier to accept.
[0022] (8) Its application areas are relatively wide. In addition to its application in the field of anti-doping, it can also be used for real-time medical drug monitoring based on the real-time test results of ITPP. Attached Figure Description
[0023] Figure 1 This is a schematic diagram illustrating the sensor fabrication and detection principle of the present invention; Figure 2Characterization diagrams of the MIP membrane of the present invention; including SEM images of electropolymerized MIP at pyrrole concentrations of (a to e) 0.5M, 0.1M, 0.05M, 0.03M and 0.01M respectively; (f) MIP; (g) EDAX elemental distribution diagram of NIP; (h) SEM diagram of electropolymerized NIP; (i) FT-IR spectra of ITPP, MIP and NIP; Figure 3 The following are electrochemical performance graphs of the sensor of the present invention: (a) DPV response curves of bare GCE, MIP before / after elution, and NIP; (b) schematic diagram of signal response intensity at different ITPP concentrations under pH 5-7 conditions; (c) linear graph of DPV response versus ITPP concentration under pH 5-7 conditions; (d) adsorption kinetic curves and pseudo-second-order linear fitting of the ITPP sensor (inset); (e) reusability of the MIP-EC sensor (n = 3) and (f) stability study of the developed ITPP sensor. Detailed Implementation
[0024] The principles and features of this invention are described below. The examples given are for illustrative purposes only and are not intended to limit the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they should be performed according to the techniques or conditions described in the literature in this field, or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.
[0025] Example like Figure 1 The molecularly imprinted (MIP) sensor of inositol tripyrophosphate (ITPP) of the present invention is prepared by first immersing a pretreated glassy carbon electrode in a solution containing ITPP template molecules (red hexagons), pyrrole monomers (blue units) and LiClO4 electrolyte, and cyclically scanning at 0 V to 1.2 V for 4 cycles to form a MIP film; then removing the template molecules through an elution step to form an ITPP-specific recognition cavity, thus completing the preparation of the sensor.
[0026] The detection process involves immersing the sensor in a sweat sample. ITPP molecules (red) selectively embed into the imprint cavity. Due to electron transfer inhibition, the bound ITPP molecules hinder the probe ions from binding. (Yellow) diffuses to the electrode surface, causing the DPV peak current to decay, and finally the signal is quantified: the current decay value ΔI is positively correlated with the ITPP concentration, thus achieving quantitative detection.
[0027] This embodiment relates to a method for preparing a molecularly imprinted electrochemical sensor of inositol tripyrophosphate, including the following steps: A glassy carbon electrode is immersed in an electrolyte containing pyrrole, and electropolymerization is carried out using cyclic voltammetry to form a polypyrrole molecularly imprinted film on the surface of the glassy carbon electrode. Then, the inositol tripyridine template in the glassy carbon electrode with the polypyrrole molecularly imprinted film on the surface is eluted to obtain an inositol tripyridine molecularly imprinted electrochemical sensor. The pyrrole-containing electrolyte includes pyrrole, inositol tripyrophosphate template, and salts.
[0028] Preferably, the salts in this embodiment include at least one of LiClO4, KNO3, LiBF4, and LiPF6; The molar ratio of pyrrole, inositol tripyrophosphate template, and salt is 0.03~0.1:0.00136:0.1; The concentration of pyrrole in the electrolyte containing pyrrole is 0.03 M to 0.1 M; for example, 0.5 M, 0.1 M, 0.05 M, 0.03 M, and 0.01 M, etc. The concentration of the inositol tripyrophosphate template in the pyrrole-containing electrolyte is 1 mM to 6.85 mM, for example, 1 mM, 1.36 mM, 6.85 mM, etc. The concentration of salts in the pyrrole-containing electrolyte is 0.05M to 0.5M, for example, 0.05M, 0.1M, 0.5M, etc.
[0029] Preferably, the conditions for electropolymerization using cyclic voltammetry in this embodiment are: scan range 0 V~1.2 V (vs. Ag / AgCl), scan rate 0.05 V / s~0.2 V / s, for example 0.1 V / s, etc., and 3~6 cycles, for example 4 cycles, etc.
[0030] Preferably, the specific method for eluting the inositol tripyrophosphate template from the glassy carbon electrode with a polypyrrole molecularly imprinted film on its surface in this embodiment is as follows: the glassy carbon electrode with the polypyrrole molecularly imprinted film on its surface is sequentially immersed in a mixture of methanol and acetic acid for 20 min, then in a 0.08M~0.12M NaOH solution for 20 min, and then rinsed with ultrapure water; the volume ratio of the methanol and acetic acid mixture is 7~9:1~3, for example 7:3, 8:2, 9:1, etc.
[0031] Preferably, the glassy carbon electrode pretreatment before immersing the glassy carbon electrode in the electrolyte containing pyrrole in this embodiment includes the following steps: polishing the glassy carbon electrode sequentially with alumina paste (e.g., 0.3 μm, 0.05 μm) and ultrasonic cleaning (5 min each with ultrapure water and ethanol), then performing cyclic voltammetry scans in 0.1 M~0.5 M H2SO4 solution at a scan rate of 0.1 V / s within a potential range of -1 V~1 V until the electrochemical curves highly overlap, and finally drying it (e.g., drying with nitrogen) for later use.
[0032] Specifically, the preparation method of the molecularly imprinted electrochemical sensor of inositol tripyrophosphate in this embodiment includes the following steps: Electrode pretreatment: The glassy carbon electrode (GCE, 3 mm in diameter) was polished sequentially with 0.3 μm and 0.05 μm alumina slurries, ultrasonically cleaned in ultrapure water and anhydrous ethanol for 5 minutes each, and then cyclically scanned in 0.5 M H2SO4 solution at a scan rate of 0.1 V / s in the potential range of -1 to 1 V until the electrochemical curves highly overlapped. Finally, it was dried with nitrogen gas for later use. Preparation of electropolymerization solution: Take 1 mL of ultrapure water, add 3.4 μL of 99% pyrrole (purchased from Shanghai Aladdin) (final concentration 0.05 M), 400 μL of 5 mg / mL ITPP sodium salt solution (purchased from Shanghai Glycosci) (final concentration 1.36 mM), and 600 μL of 0.167 M LiClO4 solution (final concentration 0.1 M). Electropolymerization: The pretreated GCE was immersed in the above solution, with Ag / AgCl as the reference electrode and platinum wire as the counter electrode, and cyclic voltammetry was performed for 4 cycles at a scan rate of 0.1 V / s in the potential range of 0 V to +1.2 V. Template elution: After polymerization, the electrode was successively immersed in methanol / acetic acid (7:3 v / v) for 20 minutes and 0.1 M NaOH for 20 minutes, and finally rinsed with ultrapure water and dried with nitrogen to obtain the MIP sensor.
[0033] This embodiment also relates to a molecularly imprinted electrochemical sensor of inositol tripyrophosphate, which is prepared by the aforementioned preparation method.
[0034] This embodiment also relates to the application of a molecularly imprinted electrochemical sensor for inositol tripyrophosphate, wherein the molecularly imprinted electrochemical sensor for inositol tripyrophosphate is used to detect inositol tripyrophosphate.
[0035] This embodiment also relates to a method for detecting inositol tripyrophosphate, comprising the following steps: The molecularly imprinted electrochemical sensor of inositol tripyrophosphate was immersed in a sweat sample and incubated at room temperature. Using the molecularly imprinted electrochemical sensor of inositol tripyrophosphate as the working electrode, differential pulse testing was performed in an electrolyte containing potassium hexacyanoferrate to achieve the detection of inositol tripyrophosphate in the liquid sample to be tested.
[0036] Furthermore, the detection linear range of the inositol tripyrophosphate is 50 ng / mL to 1500 ng / mL, and the linear regression equation is: I = 0.00253C + 0.26763, with a correlation coefficient R. 2>0.998, where I is in μA and C is in ng / mL.
[0037] Furthermore, the volume of the sweat sample is 5 μL to 20 μL, for example, 10 μL; The room temperature incubation conditions are as follows: the time is 15 min to 20 min; for example, 15 min, 20 min, etc. The electrolyte containing potassium hexacyanoferrate includes K3[Fe(CN)6] and KCl; the concentration of K3[Fe(CN)6] in the electrolyte containing potassium hexacyanoferrate is 5 mM to 10 mM; the concentration of KCl in the electrolyte containing potassium hexacyanoferrate is 0.1 M to 1.0 M, for example, 0.5 M; The conditions for the differential pulse test are: potential range -0.2~0.6 V, amplitude 0.01V~0.5V, for example 0.05V, pulse width 0.005s~0.5s, for example 0.05s.
[0038] Examples 2 and 3: MIP sensors prepared with different monomer concentrations The only difference between the preparation steps in this embodiment and those in Embodiment 1 of the present invention lies in the concentration of pyrrole monomer in the electropolymerization solution: Example 2: Using 0.03 M pyrrole; Example 3: Using 0.10 M pyrrole; *Note: The ITPP concentration of the template molecule (1.36 mM), the supporting electrolyte (0.1 M LiClO4), the electropolymerization parameters (0 V~1.2 V, 0.1 V / s, 4 cycles), and the subsequent elution steps (stepwise elution) are exactly the same as in this invention.
[0039] The sensors of Embodiments 1 to 3 of this invention were tested in parallel, and the key performance data are shown in Table 1 below: Table 1 As shown in Table 1, the optimal pyrrole concentration of this invention is 0.05 M. The sensor prepared at this concentration exhibits excellent sensitivity, detection limit, and detection accuracy in actual samples.
[0040] Comparative Example 1: Preparation of MIP Sensors by Single Solvent Elution Method The electrode pretreatment and electropolymerization steps were exactly the same as in Example 1 of this invention (i.e., using 50 mmol / L pyrrole, 1.36 mmol / L ITPP, and 0.1 M LiClO4, scanning 4 times at 0.1 V / s from 0 V to +1.2 V). Template elution step: The polymerized MIP / GCE electrode was immersed only in a methanol / acetic acid mixture (7:3 v / v) with shaking for 40 minutes (the total time was consistent with the 40 minutes of stepwise elution in this invention). It was then rinsed 3 times with ultrapure water and dried with nitrogen for later use.
[0041] The NIP sensors were fabricated using the same single elution method.
[0042] The results and data from the efficacy verification experiment are shown in Table 2. Table 2
[0043] As shown in Table 2, the experimental data demonstrate that the sensor prepared using only a single solvent elution method is significantly inferior to the "methanol / acetic acid, NaOH" stepwise elution process provided in this invention in several key performance indicators, including template elution efficiency, stability, reusability, and selectivity. This fully proves that stepwise elution is not a conventional technique in this field, and the resulting technical effects (high stability and high reproducibility) are unexpected, thus highlighting the inventiveness and progress of the technical solution of this invention.
[0044] Test case 1. Characterization of MIP membranes.
[0045] To elucidate the successful construction and specific recognition mechanism of ITPP-imprinted polypyrrole (PPy) films, this invention first used high-resolution scanning electron microscopy (SEM) to characterize the surface morphology of the molecularly imprinted polymer (MIP) of Example 1 before and after capturing the target molecule (ITPP). Figure 2 (a~f in the original text). Molecularly imprinted polymers (MIPs) form highly ordered "cauliflower-like" micro / nano structures at the optimal pyrrole concentration (0.05 M). Figure 2 c). Molecularly imprinted polymers (NIPs) Figure 2 h) exhibits morphological characteristics different from MIP, suggesting that it contains different components (excluding ITPP).
[0046] Fourier transform infrared spectroscopy (FT-IR) characterization Figure 2 i) reveals the molecular vibrational signature of inositol tripyrophosphate (ITPP): 1642 cm⁻¹ -1 The strong peak at 1250 cm⁻¹ is attributed to the CO bending vibration (δ(CO)) of the inositol ring. -1 The characteristic peak originates from the P=O asymmetric stretching vibration of the pyrophosphate group. ), 1060–1035 cm -1 The bimodal pattern corresponds to the antisymmetric stretching vibration of the PO3⁻ group. ), 870 cm -1 The peak represents the symmetrical stretching vibration of the POP (Pop). Together, these findings confirmed the pyrophosphate structure of ITPP. In molecularly imprinted polymers (MIPs), the PO3 group of ITPP... - Vibration (original 1060–1035 cm) -1 The double peak evolved to 1100 cm. -1 Single peak. The peak position variation and peak shape simplification indicate that the functional monomer (polypyrrole) within the imprinted cavity... ) and PO3 - The groups formed a strong hydrogen bond network ( ), leading to PO3 - The vibration modes tend to become homogenized under spatial constraints. It is worth noting that the ITPP's... Characteristic peak (1250 cm⁻¹) -1 The δ(CO) vibration of ITPP is clearly retained in MIPs but completely disappears in non-imprinted polymers (NIPs). Furthermore, the δ(CO) vibration (1642 cm⁻¹) of ITPPs is also clearly retained. -1 A blue shift occurs in MIP (up to 1620 cm). -1 This stems from the rigid constraint of the inositol ring by the cavity and Hydrogen bonds are formed; however, this shift phenomenon is not observed in NIP. In summary, FT-IR analysis confirms that ITPP molecules form hydrogen bonds through their pyrophosphate chains (…). ) forms a specific hydrogen bond network with the polypyrrole backbone ( Meanwhile, its inositol ring (δ(CO)) conformation is effectively locked by the imprinted cavity. NIP, lacking template molecules for guidance, cannot form this type of specific interaction, and therefore failed to reproduce the above-mentioned spectral shift and characteristic peak retention phenomena.
[0047] Energy-dispersive X-ray spectroscopy (EDAX) revealed the elemental distribution of the molecularly imprinted polymer (MIP) before and after the trapping of the target molecule (ITPP). Specifically, the characteristic elements O and P of ITPP were uniformly distributed on the layered surface of the MIP. Figure 2 The phosphorus (P) content in MIP is significantly higher than that in NIP. Figure 2The difference in h (weight percentage: 4.08 wt% vs 0.26 wt%, a difference of 15.7 times; atomic percentage: 1.67 at% vs 0.10 at%) directly confirms that the template molecule inositol tripyrophosphate (containing polyphosphate groups) was successfully embedded into the MIP backbone, and its phosphate group (PO) contributed additional oxygen atoms (O) (O: 5.60 wt% vs 3.72 wt%; 4.45 at% vs 2.86 at%), which also confirms the specific introduction mechanism of molecular imprinting.
[0048] 2. Electrochemical performance of the sensor.
[0049] The electrochemical performance of the sensor prepared in Example 1 was studied, and the results are as follows: Figure 3 (a) DPV response curves of naked GCE, MIP before / after elution, and NIP; (b) Schematic diagram of signal response intensity at different ITPP concentrations under pH 5–7 conditions; (c) Linear graph of DPV response versus ITPP concentration under pH 5–7 conditions; (d) Adsorption kinetic curves of the ITPP sensor and pseudo-second-order linear fitting (inset); (e) Reusability of the MIP–EC sensor (n = 3) and (f) Stability study of the developed ITPP sensor.
[0050] in, Figure 3 In the diagram, 'a' represents the bare glassy carbon electrode (black line), the non-imprinted polymer (NIP) sensor (green line), the MIP sensor after elution (red line), and the MIP before elution (i.e., the loaded template state, blue line) in the presence of... Differential pulse voltammetry (DPV) curves in the electrolyte. The similar current responses of MIP (red) and NIP (green) after elution demonstrate that template elution restores electron transfer pathways; the significantly reduced current of MIP (blue) before elution (peak current 54 μA → 10.2 μA, a decrease of 81.1%) confirms that template molecules hinder electron transfer; this comparison provides direct evidence for the label-free detection mechanism of "ITPP binding leading to current decay".
[0051] Figure 3 Figure b: Three-dimensional plot of the DPV response of the MIP sensor to different concentrations of ITPP (50–1500 ng / mL) under pH conditions of 5.0, 5.5, 6.0, 6.5, and 7.0. The sensor maintains a linear response within the physiological pH range of sweat (5.0–7.0), with a current change rate... Positively correlated with concentration; verifying the sensor's robustness against pH fluctuations in sweat.
[0052] Figure 3c: Calibration curve and linear equation of DPV peak current (vertical axis) versus ITPP concentration (horizontal axis, 0–1500 ng / mL). Quantitative sensitivity parameters are given: slope = 0.00253 μA / (ng / mL) (current change per ng / mL concentration change); the core detection formula is established as: I = 0.00253C + 0.26763 (R²>0.998).
[0053] Figure 3 The d-axis shows the ITPP adsorption kinetics curve (main plot) and the pseudo-second-order kinetic fitting line (inner plot). The binding rate reaches 90% in 15 minutes and equilibrium is reached in 20 minutes (the required incubation time is "15–20 minutes"). The pseudo-second-order kinetic fitting R²=0.9937 > the pseudo-first-order fitting R²=0.974, proving that the recognition process is dominated by chemisorption.
[0054] Figure 3 The bar chart (n=3) shows the current response of the MIP sensor to 500 ng / mL ITPP after 6 "detection → regeneration" cycles. The signal attenuation after 6 regenerations was ≤2.2% (RSD=3.1%), supporting the claim of "reusable ≥6 times." The small error bar (SD) demonstrates the stability of the stepwise elution process for regeneration. The regeneration process involved immersing the completed ITPP detection MIP sensor sequentially in the following solutions: a methanol / acetic acid mixture (7:3 v / v), with shaking and soaking at room temperature for 20 minutes; and a 0.1 M NaOH aqueous solution, with shaking and soaking at room temperature for 20 minutes. After removal, the sensor was gently rinsed 3 times with ultrapure water and dried under nitrogen. Storage conditions: Freshly prepared or regenerated MIP sensors were placed in a sealed desiccator and stored at 4°C, protected from light.
[0055] Figure 3 f in the figure: Line graph of the current response of the newly prepared sensor to detect 500 ng / mL ITPP after being stored at room temperature in the dark for different times (0 days, 5 days, 10 days, 20 days, 30 days); the signal retention rate was 94% after 30 days (initial current 100% → 94% on day 30), supporting the long-term storage effect; providing experimental basis for sensor pre-packaging and commercial storage.
[0056] 3. Accuracy and precision of actual sweat sample testing.
[0057] Sample collection: Volunteers disinfected their chest skin with alcohol, applied 3M™ Tegaderm™ dressings, and collected sweat into centrifuge tubes 40 minutes after exercise.
[0058] Spike treatment: Take 1 mL of sweat and add ITPP standard to a final concentration of 100 ng / mL.
[0059] Detection procedure: Immerse the sensor prepared in Example 1 in spiked sweat and incubate at room temperature for 20 minutes; rinse gently with ultrapure water; perform DPV test (parameters as before) and record the peak current; calculate the concentration based on the calibration curve; the results are shown in Table 3.
[0060] Results: The detected value was 103.4 ng / mL, and the recovery rate was 103.4% (RSD=1.76%).
[0061] Table 3. Accuracy and Precision of Detection Based on Actual Sweat Samples As shown in Table 3, in simulated sweat, the recoveries at all spiking levels ranged from 94.3% to 102.4%, and the RSD values for all parallel determinations were below 5%. In real human sweat, which has a more complex composition and greater variability, the recoveries of ITPP were 103.4% and 103.7% (RSDs 1.76% and 3.24%, respectively). These recovery results all fall within the acceptable range of the analytical method, fully demonstrating the accuracy, reliability, and repeatability of the MIP-EC sensor in the quantitative detection of ITPP in real sweat matrices, and meeting the requirements of actual sample analysis. Within the simulated sweat pH range of 5.0–7.0 (covering typical pH variations in human sweat), the sensor exhibited good recovery performance (94.3%–102.4%), further confirming its stability and applicability in real-world applications. This strongly validates the sensor's ability to process real biological samples. The slightly higher recovery rate in real sweat may be due to minor matrix effects or a slight overestimation of background contribution, but it remains at an excellent level. ITPP was not detected in any of the unspecified samples (ND), further confirming that the background sweat does not significantly interfere with ITPP detection.
[0062] In summary, this invention integrates the triple advantages of MIP's high specificity identification, EC's rapid response, and non-invasive dynamic monitoring of sweat to construct a closed-loop "sampling-detection-regeneration" system, providing a real-time, non-invasive, and field-deployable POCT solution for the anti-doping field. The established MIP-EC sweat detection paradigm can also be extended to other rapidly metabolized prohibited substances (such as Efaproxiral® structural analogs), promoting the transformation of doping monitoring from "post-event tracking" to "real-time early warning."
[0063] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.
Claims
1. A method for preparing a molecularly imprinted electrochemical sensor of inositol tripyrophosphate, characterized in that, Includes the following steps: A glassy carbon electrode is immersed in an electrolyte containing pyrrole, which includes pyrrole, an inositol tripyrophosphate template, and salts. Electropolymerization is performed using cyclic voltammetry to form a polypyrrole molecularly imprinted film on the surface of the glassy carbon electrode. The inositol tripyrophosphate template on the glassy carbon electrode with the polypyrrole molecularly imprinted film on its surface is then eluted to obtain an inositol tripyrophosphate molecularly imprinted electrochemical sensor.
2. The method for preparing the molecularly imprinted electrochemical sensor of inositol tripyrophosphate according to claim 1, characterized in that, The salts include at least one of LiClO4, KNO3, LiBF4, and LiPF6; The molar ratio of pyrrole, inositol tripyrophosphate template, and salt is 0.03~0.1:0.00136:0.1; The concentration of pyrrole in the electrolyte containing pyrrole is 0.03 M to 0.1 M; The concentration of the inositol tripyrophosphate template in the pyrrole-containing electrolyte is 1 mM to 6.85 mM; The concentration of salts in the pyrrole-containing electrolyte is 0.05M to 0.5M.
3. The method for preparing the molecularly imprinted electrochemical sensor of inositol tripyrophosphate according to claim 1, characterized in that, The conditions for electropolymerization using cyclic voltammetry are: scan range 0 V to 1.2 V, scan rate 0.05 V / s to 0.2 V / s, and 3 to 6 cycles.
4. The method for preparing the molecularly imprinted electrochemical sensor of inositol tripyrophosphate according to claim 1, characterized in that, The specific method for eluting the inositol tripyrophosphate template from the glassy carbon electrode with a polypyrrole molecularly imprinted film on its surface is as follows: the glassy carbon electrode with the polypyrrole molecularly imprinted film on its surface is immersed in a mixed solution and a 0.08M~0.12M NaOH solution in sequence, and then rinsed with ultrapure water; the mixed solution includes methanol and acetic acid in a volume ratio of 7~9:1~3.
5. The method for preparing the molecularly imprinted electrochemical sensor of inositol tripyrophosphate according to any one of claims 1 to 4, characterized in that, The pretreatment of the glassy carbon electrode before immersing it in an electrolyte containing pyrrole includes the following steps: the glassy carbon electrode is polished and ultrasonically cleaned sequentially with alumina slurry, and then cyclically scanned in a 0.1 M to 0.5 M H2SO4 solution at a scan rate of 0.1 V / s within a potential range of -1 V to 1 V until the electrochemical curves highly overlap, and finally dried for later use.
6. A molecularly imprinted electrochemical sensor for inositol tripyrophosphate, characterized in that, The molecularly imprinted electrochemical sensor of inositol tripyrophosphate is prepared by the preparation method according to any one of claims 1 to 5.
7. The application of a molecularly imprinted electrochemical sensor of inositol tripyrophosphate, characterized in that, The molecularly imprinted electrochemical sensor of inositol tripyrophosphate as described in claim 6 is used to detect inositol tripyrophosphate.
8. A method for detecting inositol tripyrophosphate, characterized in that, Includes the following steps: The molecularly imprinted electrochemical sensor of inositol tripyrophosphate as described in claim 6 was immersed in a sweat sample and incubated at room temperature. Using the molecularly imprinted electrochemical sensor of inositol tripyrophosphate as the working electrode, differential pulse testing was performed in an electrolyte containing potassium hexacyanoferrate to achieve the detection of inositol tripyrophosphate in the liquid sample to be tested.
9. The method for detecting inositol tripyrophosphate according to claim 8, characterized in that, The linear detection range of inositol tripyrophosphate was 50 ng / mL to 1500 ng / mL, and the linear regression equation was: I = 0.00253C + 0.26763, with a correlation coefficient R. 2 >0.998, where I is in μA and C is in ng / mL.
10. The method for detecting inositol tripyrophosphate according to claim 8, characterized in that, The volume of the sweat sample was 5 μL to 20 μL; The incubation time at room temperature is 15 min to 20 min; The electrolyte containing potassium hexacyanoferrate includes K3[Fe(CN)6] and KCl; the concentration of K3[Fe(CN)6] in the electrolyte containing potassium hexacyanoferrate is 5 mM to 10 mM; the concentration of KCl in the electrolyte containing potassium hexacyanoferrate is 0.1 M to 1.0 M. The conditions for the differential pulse test are: potential range -0.2 V to 0.6 V, amplitude 0.01 V to 0.5 V, and pulse width 0.005 s to 0.5 s.