A type of Mg 2+ Ca 2+ Identification ligands and selective electrodes, their preparation methods and applications

Abstract

The application discloses high-selectivity and reversible Mg 2+ and Ca 2+ recognition ligands MPETH / PPETH, a preparation method thereof, and construction of a PANI / RGO / MPETH (PPETH) integrated ion selective electrode and application of the integrated ion selective electrode in detection of Mg 2+ and Ca 2+ . The method comprises the following steps: first, synthesizing high-selectivity and reversible Mg 2+ and Ca 2+ recognition ligands MPETH / PPETH; then, preparing PANI / RGO by utilizing the pi-pi interaction, hydrogen bond interaction and electrostatic interaction between polyaniline and graphene oxide; and finally, modifying the MPETH (PPETH) to the PANI / RGO by the pi-pi interaction to prepare the PANI / RGO / MPETH (PPETH) integrated ion selective electrode. The application further discloses that the high-selectivity and reversible Mg 2+ and Ca 2+ recognition ligands MPETH / PPETH have the characteristics of high selectivity and high reversibility for Mg 2+ and Ca 2+ respectively, the prepared integrated ion selective electrode PANI / RGO / MPETH (PPETH)-E has the characteristics of excellent anti-biological protein pollution, can realize long-term, reversible and stable real-time dynamic detection of the concentration of Mg 2+ and Ca 2+ , and has a wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of ion recognition sensing technology, and relates to highly selective and reversible Mg... 2+ and Ca 2+ The invention relates to the recognition of ligands MPETH and PPETH, an integrated ion-selective microelectrode with excellent resistance to biological protein contamination, its preparation method, and its application. Background Technology

[0002] Because of the calcium in the brain of living animals 2+ and Mg 2+ Ca is widely involved in many physiological and pathological events, therefore, simple and effective measurement of them is of significant physiological and pathological importance. 2+ It is an important signal transduction substance, intracellular Ca2+ 2+ As a second messenger, Mg is essential for many functions in the central nervous system, including gene expression, neurotransmitter release, regulation of neurite growth, synapsis, and synaptic transmission. Meanwhile, Mg... 2+ It is Ca 2+ Important mediators and regulators of signaling, under physiological conditions and Ca2+ 2+ It is an antagonistic effect. In addition, Mg 2+ It also plays a very important role in the utilization of adenosine triphosphate (ATP) by cells. Studies have shown that excess Ca in the extracellular fluid... 2+ Influx plays a major role in some brain pathologies, including those under ischemic / hypoxic conditions. Mg... 2+ It often plays the role of a "neuronal protectant." Therefore, developing a system that can track extracellular Ca2+ in real time is crucial. 2+ and Mg 2+ Tools that study concentration changes have significant physiological and pathological implications.

[0003] Many methods have been developed for in vitro measurement of Mg. 2+ Ca 2+ Methods, although atomic absorption spectrometry (AAS) and inductively coupled plasma mass spectrometry (ICPMS) have been used to detect Mg in mouse brains. 2+ Ca 2+ Discontinuous and continuous measurements are possible, but these methods are limited by expensive instrumentation, poor time resolution due to sample collection in AAS measurements, or complex pretreatment procedures in ICPMS measurements. In contrast, electrochemical methods have the advantage of relatively inexpensive instrumentation and simple procedures. More importantly, the ingenious design of the electrode sensing interface greatly improves the selectivity and stability of this method. Organic small molecule recognition ligands are currently widely used to recognize specific targets due to their good biocompatibility and designability. However, because Mg... 2+ Ca2+ The similarity in physicochemical properties makes it a significant challenge to measure these two species simply and efficiently in the brain system. Therefore, developing novel Mg compounds with high selectivity and reversibility remains crucial. 2+ Ca 2+ Ligand recognition provides a powerful tool for understanding the dynamic pathological or physiological processes within the brain.

[0004] For Mg 2+ Ca 2+ The most common method for electrochemical monitoring of non-electroactive ions is using potentiometric sensing with ion-selective electrodes. Traditional all-solid-state ion-selective electrodes consist of a conductive substrate, a solid transition layer, and an ion-selective membrane. The concentration of the target analyte in the sample solution is determined by the linear relationship between the potential of the ion-selective electrode and the concentration of the analyte. However, this non-integrated electrode fabrication method, requiring layer-by-layer modification, makes it difficult to achieve miniaturized sizes (~1 μm) to further improve spatial resolution. Furthermore, as the sensor is implanted in vivo for extended periods, tissue responses and non-specific adsorption of proteins can mask the sensing interface, leading to a loss of output signal sensitivity. Recently, graphene oxide, based on its high hydrophilicity and negative charge, has been introduced to modify electrode surfaces for the construction of biocontamination-resistant sensors. However, graphene oxide applied by coating or electrodeposition is prone to detachment, resulting in poor biocontamination resistance. Therefore, developing electrode materials with excellent resistance to bioprotein contamination is beneficial for constructing highly stable electrochemical sensors. Summary of the Invention

[0005] To overcome the shortcomings of existing technologies, the purpose of this invention is to provide a highly selective and reversible Mg 2+ Ca 2+ This invention relates to the identification ligands MPETH and PPETH, their preparation methods and applications, and the preparation method and application of an integrated ion-selective electrode with excellent resistance to biological protein contamination. The invention first synthesizes two highly selective and reversible Ca2+ ligands. 2+ and Mg 2+ The ligands MPETH and PPETH were identified, and then PANI / RGO electrode materials were prepared using non-covalent interactions. Furthermore, the MPETH and PPETH ion ligands were integrally modified into PANI / RGO through π-π interactions to obtain a reversible Mg2+ electrode with excellent resistance to biological protein contamination. 2+ Ca 2+ The selective electrode MPETH(PPETH)-E ultimately enabled the in vivo deposition of Mg. 2+ Ca 2+ Real-time dynamic monitoring.

[0006] The Mg of the present invention2+ Ca 2+ Recognizes ligands MPETH and PPETH for Mg 2+ Ca 2+ Exhibiting good selectivity and reversibility, the PANI / RGO / MPETH (PPETH) electrode material demonstrates excellent resistance to biological protein contamination. Mg... 2+ Ca 2+ The selective electrode MPETH (PPETH)-E can be used for in vivo Mg 2+ Ca 2+ Long-term stable real-time dynamic monitoring.

[0007] This invention provides a recognition ligand, including highly selective and reversible Mg... 2+ Recognizing the ligand MPETH, highly selective and reversible Ca 2+ Identify the ligand PPETH.

[0008] The highly selective, reversible Mg 2+ MPETH, a recognition ligand, is used for the specific recognition of Mg. 2+ The butyramide group and the phenyl group used to provide π-π interaction to construct the electrode constitute the electrode, which can be used to treat Mg 2+ It forms bonds and shows almost no response to other interfering substances in selectivity tests, but can react to Mg. 2+ Achieving a 95% response rate, it can not only indicate Mg 2+ An increase in concentration can also indicate Mg 2+ Reduced reversible response.

[0009] The highly selective and reversible Ca 2+ The recognition ligand PPETH is used for the specific recognition of Ca. 2+ The oxidized diacetyl group and the phenyl group used to provide π-π interaction to construct the electrode constitute the structure, which can be used to treat Ca 2+ It forms bonds and shows almost no response to other interfering substances in selectivity tests, but can respond to Ca. 2+ Achieving a 95% response rate, it can not only indicate Ca 2+ An increase in concentration can also indicate the presence of Ca. 2+ Reduced reversible response.

[0010] This invention also provides a method for preparing the aforementioned recognition ligand. Specifically, this invention provides a highly selective, reversible Mg... 2+ A method for preparing the identification ligand MPETH, the method comprising the following steps:

[0011] Step (1): Dimethylamine and succinic anhydride are mixed in a first organic solvent and stirred under nitrogen atmosphere to obtain the product 4-(dimethylamino)-4-oxobutyric acid; the reaction process is shown in reaction formula (1):

[0012]

[0013] Step (2): The 4-(dimethylamino)-4-oxobutyric acid, aniline, EDCI, and HOBT prepared in step (1) are mixed and stirred in a second organic solvent. After the reaction, the product N,N-dimethyl-N'-phenylbutyramide, i.e., Mg, is purified by column chromatography. 2+ Recognize the ligand MPETH; the reaction process is shown in reaction formula (2):

[0014]

[0015] The preparation of highly selective, reversible Mg 2+ The overall reaction formula for recognizing the ligand MPETH is shown below:

[0016]

[0017] In step (1), the molar ratio of dimethylamine to succinic anhydride is (0.5-2 mmol):(0.5-2 mmol); preferably, it is 1 mmol:1 mmol.

[0018] In step (1), the first organic solvent is one or more of tetrahydrofuran, dichloromethane, benzene, etc.; preferably, it is tetrahydrofuran.

[0019] In step (1), the amount of the first organic solvent added is 50-100 mL; preferably, it is 100 mL.

[0020] In step (1), the reaction temperature is 15-30℃; preferably, it is 25℃.

[0021] In step (1), the reaction time is 2-4 hours; preferably, it is 2 hours.

[0022] In step (2), the molar ratio of 4-(dimethylamino)-4-oxobutyric acid, aniline, EDCI and HOBT is (0.5-2 mmol):(0.5-2 mmol):(1-2 mmol):(1-2 mmol); preferably, it is 1:1:1.2:1.2.

[0023] In step (2), the second organic solvent is one or more of dichloromethane, tetrahydrofuran, benzene, etc.; preferably, it is dichloromethane.

[0024] In step (2), the amount of the second organic solvent added is 50-100 mL; preferably, it is 100 mL.

[0025] In step (2), the eluent for the column chromatography purification method is a mixed solvent of methanol and dichloromethane; the volume ratio of methanol to dichloromethane in the mixed solvent is 1:(20-50); preferably, it is 1:30.

[0026] In step (2), the reaction temperature is 15-30℃; preferably, it is 25℃.

[0027] In step (2), the reaction time is 6-12 hours; preferably, it is 12 hours.

[0028] In step (2), after stirring the reaction, a post-treatment process is also included: after the reaction is completed, the solution is washed with water several times and then washed with saturated sodium bicarbonate solution several times to adjust the pH of the solution to weak alkalinity. After separation, the organic phase is retained and evaporated to remove excess dichloromethane.

[0029] The water used for cleaning is ultrapure water.

[0030] This invention also provides highly selective, reversible Mg prepared by the above method. 2+ Identify the ligand MPETH.

[0031] This invention also provides a highly selective, reversible Ca 2+ A method for preparing the recognition ligand PPETH, wherein 2,2'-diacetyl chloride oxide, diphenylamine and pyridine are mixed in a third organic solvent and stirred to react, yielding 2,2'-di(N,N-diphenylacetamide).

[0032] The preparation of highly selective, reversible Ca 2+ The overall reaction formula for recognizing the ligand PPETH is shown below:

[0033]

[0034] Wherein, the molar ratio of 2,2'-diacetyl chloride, diphenylamine and pyridine is (0.5-2):(0.5-2):(1-3); preferably, it is 1:1:2.

[0035] The third organic solvent may be one or more of benzene, dichloromethane, tetrahydrofuran, etc.; preferably, it is benzene.

[0036] The amount of the third organic solvent added is 50-100 mL; preferably, it is 100 mL.

[0037] The reaction temperature is 15-30℃; preferably, it is 25℃.

[0038] The reaction time is 6-12 hours; preferably, it is 12 hours.

[0039] This invention also provides highly selective, reversible Ca prepared by the above method. 2+ Identify the ligand PPETH.

[0040] This invention also provides an integrated Mg with excellent resistance to biological protein contamination. 2+ Ca 2+ The selective electrode MPETH (PPETH)-E possesses superior resistance to non-specific adsorption of proteins into the electrode surface in vivo. It integrates a conductive substrate, a solid-state transition layer, and a recognition layer into a single structure, along with a specific ligand for Mg. 2+ Ca 2+ Highly selective recognition capability.

[0041] This invention also provides a reversible Mg with excellent resistance to biological protein contamination. 2+ Ca 2+ A method for preparing the selective electrode MPETH (PPETH)-E, the method comprising the following steps:

[0042] Step (X-1): Dissolve ammonium persulfate in deionized water to prepare solution a. Dissolve phytic acid and aniline in deionized water to prepare solution b. Mix the two solutions after cooling to obtain crude polyaniline hydrogel. Immerse the polyaniline hydrogel in deionized water to remove oligomers and excess ions. Vacuum dry to obtain PANI hydrogel.

[0043] Step (X-2): The PANI hydrogel prepared in step (X-1) is dispersed in a graphene oxide solution and ultrasonically treated to obtain PANI / GO. Then, Mg is added. 2+ (Ca 2+ The ligand MPETH(PPETH) was recognized to obtain PANI / GO / MPETH(PPETH). The addition of ascorbic acid yielded a PANI / RGO / MPETH(PPETH) hydrogel.

[0044] Step (X-3): Inject the PANI / RGO / MPETH (PPETH) hydrogel prepared in step (X-2) into a capillary, then seal both ends of the capillary. Place the capillary in an oven to allow the PANI / GO / MPETH (PPETH) hydrogel to revert to the PANI / RGO / MPETH (PPETH) hydrogel. After the reaction is complete, both ends of the capillary are opened. Then, gently flush the PANI / RGO / MPETH (PPETH) mixed hydrogel out with water using a syringe, and dry it in a refrigerator to obtain highly selective, reversible Mg. 2+ (Ca 2+ Selective electrode MPETH(PPETH)-E.

[0045] In step (X-1), the concentration of ammonium persulfate is 1-2M; preferably, it is 1.25M.

[0046] In step (X-1), the concentration of phytic acid is 0.2-0.8M; preferably, it is 0.5M.

[0047] In step (X-1), the concentration of aniline is 2-4M; preferably, it is 2.5M.

[0048] In step (X-1), the cooling temperature is 2-8°C; preferably, it is 4°C.

[0049] In step (X-1), the soaking time is 8-24 hours; preferably, it is 12 hours.

[0050] In step (X-1), the vacuum drying time is 40-60℃; preferably, it is 50℃.

[0051] In step (X-2), the concentration of the PANI hydrogel dispersion is 2-8 mg / mL; preferably, it is 4 mg / mL.

[0052] In step (X-2), the concentration of the graphene oxide solution is 2-8 mg / mL; preferably, it is 8 mg / mL.

[0053] In step (X-2), the concentration of the MPETH (PPETH) ion ligand is 1-4 mg / mL; preferably, it is 2 mg / mL.

[0054] In step (X-2), the concentration of ascorbic acid is 2-8 mg / mL; preferably, it is 8 mg / mL.

[0055] In step (X-3), the capillary diameter is 0.3–0.9 μm; preferably, it is 0.3 μm.

[0056] In step (X-3), the oven temperature is 100-180℃; preferably, it is 120℃.

[0057] In step (X-3), the baking time is 10-18 hours; preferably, it is 12 hours.

[0058] In step (X-3), the drying time is 4-10 hours; preferably, it is 6 hours.

[0059] This invention also provides a reversible Mg2+ with excellent resistance to biological protein contamination prepared by the above method. 2+ Ca 2+ Selective electrode MPETH(PPETH)-E.

[0060] In this invention, the highly selective, reversible Mg 2+ Ca 2+ The ligands MPETH and PPETH were identified by DFT calculations, and METH was associated with Mg. 2+ The binding free energy is -1.22 kcal / mol, and it is related to Ca. 2+ The binding free energy of PPETH and Mg is +2.27 kcal / mol. 2+ The binding free energy is -40.67 kcal / mol, and it is related to Ca. 2+ The binding free energy is -19.61 kcal / mol. A more negative binding free energy is more favorable for the binding of ligands and ions, thus achieving high selectivity. Therefore, MPETH can achieve the binding of Mg... 2+ PPETH can achieve highly selective identification of Ca. 2+ High selective recognition. Furthermore, MPETH and PPETH also demonstrated high selectivity for Mg. 2+ Ca 2 + Reversible recognition.

[0061] This invention also provides a highly selective, reversible Mg prepared by the above-described method. 2+ Ca 2+ Identify the ligands MPETH and / or PPETH.

[0062] The present invention also provides the aforementioned highly selective, reversible Mg 2+ Ca 2+ Recognizing ligands MPETH and PPETH in the preparation of Mg 2+ Ca 2+ Sensors and / or highly selective, reversible Mg 2+ Ca 2+ Application of selective electrode MPETH (PPETH)-E.

[0063] This invention also provides a reversible Mg2+ with excellent resistance to biological protein contamination prepared by the above-described method. 2+ Ca 2+ Selective electrode MPETH(PPETH)-E.

[0064] This invention also provides the aforementioned highly selective, reversible, and highly resistant Mg to biological protein contamination. 2+ Ca 2+ Selective electrode MPETH(PPETH)-E in the detection of Mg 2+ Ca 2+ And / or applications in monitoring and brain neural circuit studies, as well as in drug mechanism studies.

[0065] The Mg 2+ Ca 2+ The specific detection steps are as follows: The highly selective, reversible Mg obtained in the above steps... 2+ (Ca 2 + The selective electrode MPETH (PPETH)-E was connected as the working electrode to the three-electrode system. The electrode was inserted into a 10 mL detection cell. The bottom solution of the detection cell was prepared by removing Mg. 2+ (Ca 2+ Artificial cerebrospinal fluid was used, and 10 μL of Mg at different concentration gradients was added to the detection pool each time. 2+ (Ca 2+ The solution was used to detect the open-circuit potential of the electrode. With the addition of Mg... 2+ (Ca 2+ As the solution concentration increases, the open-circuit potential of the electrode also gradually increases. In the reversibility test, the detection cell was sequentially replaced with Mg solutions whose concentration gradient continuously decreased. 2+ (Ca 2 + In the artificial cerebrospinal fluid solution, the open-circuit potential of the electrodes gradually decreases. Finally, MPETH-E on Mg 2+ The concentration range of 10.0-10 mM showed a good linear relationship with Ca. 2+ The concentration exhibits a good linear relationship in the range of 10.0-31.6 mM, and can be used for the treatment of Mg. 2+ (Ca 2+ Real-time dynamic monitoring of ).

[0066] The present invention also provides the aforementioned highly selective, reversible Mg 2+ Ca 2+ Mg, which has excellent resistance to bioprotein contamination, is constructed by recognizing ligands MPETH and PPETH. 2+ Ca 2+Application of selective electrode MPETH (PPETH)-E in continuous detection of neural circuits in the rat brain.

[0067] The beneficial effects of this invention include: This invention discloses two highly selective and reversible Mg... 2+ and Ca 2+ The study identified the ligands MPETH and PPETH and their preparation methods, and constructed an integrated PANI / RGO / MPETH (PPETH) ion-selective electrode for in vivo detection of Mg. 2+ Ca 2+ Applications in [the field]. This invention first synthesizes two highly selective, reversible Mg [synthetic agents]. 2+ and Ca 2+ The ligands MPETH and PPETH were identified, and then the non-covalent interactions between these ligands and polyaniline and graphene oxide were utilized to construct an integrated ion-selective electrode, namely MPETH(PPETH)-E, exhibiting excellent resistance to biological protein contamination. In this invention, two highly selective and reversible Mg... 2+ and Ca 2+ The ligands MPETH and PPETH were identified, and then PANI / RGO was prepared using the π-π interactions, hydrogen bonding interactions, and electrostatic interactions between polyaniline (PANI) and graphene oxide (GO). Finally, MPETH (PPETH) was modified into PANI / RGO through π-π interactions to prepare an integrated PANI / RGO / MPETH (PPETH) ion-selective electrode. MPETH and PPETH are effective against Mg2+ and Mg2+, respectively. 2+ and Ca 2+ The recognition process exhibits high selectivity and reversibility, and the prepared MPETH(PPETH)-E demonstrates excellent resistance to biological protein contamination. Mg2+ constructed using this process... 2+ Ca 2+ The integrated ion-selective electrode MPETH (PPETH)-E can achieve the selective ionization of Mg 2+ Ca 2+ Long-term, stable, real-time dynamic monitoring of concentration. Attached Figure Description

[0068] Figure 1 This invention relates to an integrated Mg alloy with excellent resistance to biological protein contamination. 2+ Ca 2+UV-Vis spectral characterization of the selective electrode MPETH(PPETH)-E during its construction. The redshift after adding MPETH(PPETH) to PANI / rGO demonstrates that MPETH(PPETH) can self-assemble into PANI / rGO through π-π interactions to form an integrated MPETH(PPETH)-E electrode structure.

[0069] Figure 2 This invention relates to Mg, which exhibits excellent resistance to biological protein contamination. 2+ Ca 2+ Cyclic voltammetry (CV) of the selective electrode MPETH(PPETH)-E before and after immersion in 20 mg / mL BSA solution for 24 h. Test solution: 0.1 M H2SO4, scan rate: 10 mV / s. The essentially unchanged current response indicates that MPETH(PPETH)-E did not affect the sensor performance even after immersion in BSA solution; the current signal changed by only 0.8%, demonstrating that MPETH(PPETH)-E has good resistance to biological protein contamination.

[0070] Figure 3 This invention provides highly selective and reversible Mg 2+ (Ca 2+ Mg, which recognizes the ligand MPETH (PPETH), exhibits excellent resistance to biological protein contamination. 2+ (Ca 2+ Selective electrode MPETH (PPETH)-E for different concentrations of Mg 2+ (Ca 2+ Curve A (C) shows the increase in concentration, and curve B (D) shows the decrease in concentration. The smaller plots are linear fits to the concentration response curves. MPETH (PPETH)-E for Mg 2+ and Ca 2+ The linear partial responses are 28.5 mV / lg Mg. 2+ and 20.5mV / lg Ca 2+ The response is close to that of divalent ions, similar to the Nernst response of Mg. 2+ and Ca 2+ The reversible responses are 28.0 mV / lg Mg. 2+ and 20.1mV / lg Ca 2+ The values ​​represent 98.2% and 98.0% of the corresponding linear responses, respectively, demonstrating the effect of MPETH(PPETH)-E on Mg. 2+ and Ca 2+ The identification exhibits good linearity and reversibility.

[0071] Figure 4 This invention provides highly selective and reversible Mg2+ (Ca 2+ Mg, which recognizes the ligand MPETH (PPETH), exhibits excellent resistance to biological protein contamination. 2+ (Ca 2+ Selective electrode MPETH (PPETH)-E for Mg 2+ (Ca 2+ The selection and interference experiments were conducted. The bar chart on the left represents Mg. 2+ (Ca 2+ The diagram shows the situation when the Mg ion coexists with potential interfering metal ions (A), neurotransmitters (B), reactive oxygen species and reactive nitrogen species (C), and amino acids (D). The bar chart on the right shows the situation when the interfering ion exists alone. 2+ (Ca 2+ The concentration was 10 μM, K + and Na + The concentration is 100 μM, Cu 2+ and Fe 3+ The concentrations were 1 μM for Mg, 40 μM for AA, and 10 μM for the rest. Results showed that the responses of all interfering substances were less than 5.1%, with MPETH (PPETH) showing the best response to Mg. 2+ (Ca 2+ It achieves a specific response.

[0072] Figure 5 This invention provides highly selective and reversible Mg 2+ (Ca 2+ Mg, which recognizes the ligand MPETH (PPETH), exhibits excellent resistance to biological protein contamination. 2+ (Ca 2+ Selective electrode MPETH (PPETH)-E was used to monitor Mg levels in seven brain regions of epileptic mice. 2+ Ca 2+ The continuous changes were monitored using the open-circuit potential (OCP) method, which reduces stimulation of the mouse brain during in vivo monitoring by eliminating the need for external voltage application. Detailed Implementation

[0073] The present invention will be further described in detail below with reference to the specific embodiments and accompanying drawings. Except for the contents specifically mentioned below, the processes, conditions, and experimental methods for implementing the present invention are all common knowledge and general knowledge in the art, and the present invention does not have any particular limitations.

[0074] Example 1

[0075] Mg 2+Preparation of the recognition ligand MPETH: Dimethylamine (0.9012 g, 20 mmol) and succinic anhydride (2.00 g, 20 mmol) were mixed in 100 mL of tetrahydrofuran and stirred at 25 °C for 2 h. The mixture was then washed at least three times with ultrapure water, separated, and the organic phase was retained. Excess tetrahydrofuran was evaporated to obtain 4-(dimethylamino)-4-oxobutyric acid (2.47 g). Yield: 85%. Next, 4-(dimethylamino)-4-oxobutyric acid (0.725 g, 5 mmol), aniline (0.465 g, 5 mmol), EDCI (1.1982 g, 6 mmol), and HOBT (0.8445 g, 6 mmol) were stirred in 100 mL of dichloromethane at 25 °C for 12 h. The product was then purified by column chromatography with a mixture of methanol and dichloromethane in a volume ratio of 1:30 as the eluent. The final product was MPETH (0.99 g), yield: 90%.

[0076] Ca 2+ Preparation of the recognition ligand PPETH: 2,2'-diacetyl chloride oxide (3.42 g, 20 mmol), diphenylamine (0.9012 g, 20 mmol), and pyridine (3.16 g, 40 mmol) were mixed in 100 mL of benzene and reacted at 25 °C for 12 h to give 2,2'-bis(N,N-diphenylacetamide) oxide (3.23 g). Yield: 37%.

[0077] Example 2

[0078] Mg has excellent resistance to biological protein contamination 2+ Ca 2+ Preparation of the ion-selective electrode MPETH (PPETH)-E: First, PANI hydrogel was prepared. Ammonium persulfate (0.286 g, 1.25 mmol) was dissolved in deionized water (1 mL), labeled as solution a. A mixture of phytic acid (0.921 mL, 1 mmol), aniline (0.458 mL, 5 mmol), and deionized water (2 mL) was labeled as solution b. The two solutions were then cooled to 4°C and rapidly mixed. The hydrogel was then soaked in deionized water for 12 hours to remove oligomers and excess ions. Finally, the PANI hydrogel was dried overnight under vacuum at 50°C. Then, 4 mg of the prepared PANI hydrogel was dispersed in 1 mL of graphene oxide solution (8 mg / mL), and sonicated for a few seconds to obtain PANI / GO. Then, 2 mg / mL of MPETH (PPETH) prepared in Example 1 of this invention was added to obtain PANI / GO / MPETH (PPETH). Finally, adding 8 mg / mL of ascorbic acid can form PANI / RGO / MPETH (PPETH), which is a Mg group with excellent resistance to biological protein contamination. 2+ Ca2+ Ion-selective electrode MPETH(PPETH)-E.

[0079] Figure 1 The Mg of this invention has excellent resistance to biological protein contamination. 2+ Ca 2+ The selective electrode MPETH(PPETH)-E was prepared by stepwise UV-Vis spectroscopy characterization, and the UV-Vis spectra of PANI, GO, PANI / GO, PANI / rGO and MPETH(PPETH)-E were obtained respectively. Figure 1 It can be seen that the maximum absorption peak of MPETH(PPETH)-E has redshifted relative to PANI / rGO, proving that MPETH(PPETH) can self-assemble into PANI / rGO through π-π interaction to form an integrated MPETH(PPETH)-E electrode structure.

[0080] Example 3

[0081] Mg has excellent resistance to biological protein contamination 2+ Ca 2+ Characterization of the anti-bioprotein contamination properties of the ion-selective electrode MPETH (PPETH)-E: Figure 2 The cyclic voltammograms (CVs) of MPETH(PPETH)-E prepared in Example 2 of this invention before and after immersion in a 20 mg / mL bovine serum albumin (BSA) solution for 24 h are shown. Test solution: 0.1 M H₂SO₄, scan rate: 10 mV / s. The essentially unchanged current response indicates that MPETH(PPETH)-E did not affect the sensor performance even after immersion in BSA solution; the current signal changed by only 0.8%, demonstrating that MPETH(PPETH)-E has good resistance to biological protein contamination.

[0082] Example 4

[0083] Mg has excellent resistance to biological protein contamination 2+ MPETH-E ion-selective electrode for Mg 2+ Quantitative detection: The MPETH-E prepared in Example 2 of this invention was used as the working electrode and connected to an electrochemical three-electrode system. The three electrodes were inserted into a 10 mL detection cell containing Mg-removed... 2+ Artificial cerebrospinal fluid was prepared by adding 10 μL of Mg at different concentrations each time. 2+ The solution was then analyzed, and the open-circuit potential (OCP) of the electrode was measured. This was followed by the addition of Mg. 2+ As the solution volume increases, the open-circuit potential of the electrode gradually increases. In the reversibility test, the detection cell is sequentially replaced with Mg solutions of decreasing concentration. 2+In the solution of artificial cerebrospinal fluid, the measured open-circuit potential of the electrode gradually decreases.

[0084] Ca has excellent resistance to biological protein contamination. 2+ PPETH-E ion-selective electrode for Ca 2+ Quantitative detection: The PPETH-E prepared in Example 2 of this invention was used as the working electrode and connected to an electrochemical three-electrode system. The three electrodes were inserted into a 10 mL detection cell containing Ca2+. 2+ Artificial cerebrospinal fluid, each time 10 μL of Ca at different gradient concentrations was added. 2+ The solution was then analyzed, and the open-circuit potential (OCP) of the electrode was measured. With the addition of Ca... 2+ As the solution volume increases, the open-circuit potential of the electrode gradually increases. In the reversibility test, the detection cell is sequentially replaced with Ca solutions of decreasing concentration. 2+ In the solution of artificial cerebrospinal fluid, the measured open-circuit potential of the electrode gradually decreases.

[0085] Figure 3 It is a highly selective and reversible Mg 2+ (Ca 2+ Mg, which recognizes the ligand MPETH (PPETH), exhibits excellent resistance to biological protein contamination. 2+ (Ca 2+ Selective electrode MPETH (PPETH)-E for different concentrations of Mg 2+ (Ca 2+ Curve A (C) shows the increase in concentration, and curve B (D) shows the decrease in concentration. The smaller plots are linear fits to the concentration response curves. Finally... Figure 3 Mg in curves A and B 2+ The concentration and open-circuit potential exhibit good linearity in the ranges of 10.0 μM–10 mM and 10.0 μM–31.6 mM, respectively, with the linear equations being ΔE. ISE =E 0 +0.0285*lg a(Mg 2+ )-E RE -E IRE and ΔE ISE =E 0 +0.0205*lg a(Ca 2+ )-E RE -E IRE MPETH(PPETH)-E against Mg 2+ and Ca 2+ The linear partial responses are 28.5 mV / lg Mg. 2+ and 20.5mV / lg Ca 2+ The response is close to that of divalent ions, similar to the Nernst response of Mg.2+ and Ca 2+ The reversible responses are 28.0 mV / lgMg. 2+ and 20.1mV / lg Ca 2+ The values ​​represent 98.2% and 98.0% of the corresponding linear responses, respectively, demonstrating the effect of MPETH(PPETH)-E on Mg. 2+ and Ca 2+ The identification exhibits good linearity and reversibility. This demonstrates that MPETH(PPETH)-E in this invention can be used with Mg. 2+ (Ca 2+ Real-time reversible quantitative monitoring of ).

[0086] Example 5

[0087] Mg has excellent resistance to biological protein contamination 2+ Selectivity study of the ion-selective electrode MPETH-E: To investigate the effect of MPETH-E on common interfering substances in Example 2 of this invention, the effects of common metal ions (Ca) on common interfering substances were examined. 2+ Na + K + Zn 2+ Al 3+ NH4 + Cr 3+ Fe 2+ Ag + Hg 2+ Cu 2+ Fe 3+ Neurotransmitters (AA, UA, 5-HT, DA, DOPAC, GABA, ATP, EPI, NE), amino acids (Phe, Met, Gly, Glu, Cys, Arg, Lys, Leu, Ser, Thr, Val, His), and common reactive oxygen species / reactive nitrogen species (ClO) - ONOO - ·OH, NO2 - NO3 - H2O2, HNO, O2 ·- , 1 O2) exists alone with Mg 2+ When they coexist, the change in open-circuit potential is recorded. Among them, Mg... 2+ The concentration is 10 μM, K + and Na + The concentration is 100 μM, Cu 2+ and Fe 3+ The concentration was 1 μM, the AA concentration was 40 μM, and the rest were 10 μM.

[0088] Ca has excellent resistance to biological protein contamination.2+ Selectivity study of the ion-selective electrode PPETH-E: To investigate the effect of PPETH-E on common interfering substances in Example 2 of this invention, the effects of common metal ions (Mg) on ​​common interfering substances were examined. 2+ Na + K + Zn 2+ Al 3+ NH4 + Cr 3+ Fe 2+ Ag + Hg 2+ Cu 2+ Fe 3+ Neurotransmitters (AA, UA, 5-HT, DA, DOPAC, GABA, ATP, EPI, NE), amino acids (Phe, Met, Gly, Glu, Cys, Arg, Lys, Leu, Ser, Thr, Val, His), and common reactive oxygen species / reactive nitrogen species (ClO) - ONOO - ·OH, NO2 - NO3 - H2O2, HNO, O2 ·- , 1 O2) exists alone and with Ca 2+ When they coexist, the change in open-circuit potential is recorded. Among them, Ca... 2+ The concentration is 10 μM, K + and Na + The concentration is 100 μM, Cu 2+ and Fe 3+ The concentration was 1 μM, the AA concentration was 40 μM, and the rest were 10 μM.

[0089] like Figure 4 As shown, the bar chart on the left represents Mg. 2+ (Ca 2+ The study investigated the effects of MPETH (PPETH) on the coexistence of potential interfering metal ions (A), neurotransmitters (B), reactive oxygen species and reactive nitrogen species (C), and amino acids (D). The results showed that the response to all interfering substances was less than 5.1%, with MPETH (PPETH) showing the best response to Mg. 2+ (Ca 2+ It achieves a highly selective response.

[0090] Example 6

[0091] Example 2 of this invention prepared Mg with excellent resistance to biological protein contamination. 2+ Ca 2+ Ion-selective electrodes MPETH-E and PPETH-E were used to treat Mg in seven brain regions of mice.2+ Ca 2+ Continuous monitoring was conducted. The animal model was prepared with the approval of the Public Animal Care and Use Committee of East China Normal University. C57 rats used in the experiment were purchased from Shanghai Slack Laboratory Animal Co., Ltd. Adult male C57 mice (15-20g) were anesthetized with isoflurane gas (ABS small animal gas anesthesia machine). The isoflurane induction concentration was 5 mg / mL, and the sustained anesthesia concentration was 2 mg / mL. During the operation, the mice were wrapped in a heating pad to maintain their body temperature at 37°C. For brain measurements, the mice were fixed to a stereotaxic apparatus. Microelectrodes were implanted in the primary motor cortex (M1, AP = -1.2 mm, L = 1.0 mm, V = 0.52 mm), primary somatosensory barrel cortex (S1BF, AP = -1.2 mm, L = 2.8 mm, V = 1.2 mm), cornu ammonis 1 (CA1, AP = -1.2 mm, L = 0.6 mm, V = 1.3 mm), dentate gyrus (DG, AP = -1.2 mm, L = 0.3 mm, V = 1.8 mm), lateral dorsal (LD, AP = -1.2 mm, L = 1.2 mm, V = 2.5 mm), caudate-putamen (CPu, AP = -1.2 mm, L = 2.5 mm, V = 2.4 mm), and retinicular nuclei (RT, AP = -1.2 mm, L = 1.7 mm, V = 3.2 mm) of the mouse brain. Epilepsy modeling: Acute epileptic seizures were induced in mice by intraperitoneal injection of 2.5 mg / kg of 4-aminopyridine. During the seizures, Mg levels in the mouse brain were recorded. 2+ Ca 2+ Changes in chemical signals.

[0092] Figure 5 This is a highly selective and reversible Mg prepared in Example 1 of the present invention. 2+ (Ca 2+ Mg, which recognizes the ligand MPETH (PPETH), exhibits excellent resistance to biological protein contamination. 2+ (Ca 2+ Mg2+ levels were monitored in the hippocampal DG region of epileptic mice using the selective electrode MPETH (PPETH)-E. 2+ Ca 2+ Continuously varying potential-time curves. The monitoring method employed was the open-circuit potential (OCP) test, which reduces stimulation of the mouse brain during in vivo monitoring by eliminating the application of external voltage. During epileptic seizures, Mg... 2+ Ca 2+ The concentrations of [specific substances] will decrease to 40%-48% and 20%-45% of their initial values, respectively; Mg [specific substances] in the same brain region [will decrease]. 2+Change always precedes Ca. 2+ In Mg 2+ When the concentration of Ca begins to rise 2+ It has only just begun to decline; and in different brain regions, Mg... 2+ Ca 2+ The sequence of changes follows a loop: DG-CA1-M1-S1BF-CPu-RT-LD. These in vivo data indicate that, with the onset of epilepsy, extracellular Mg... 2+ Ca 2+ The concentration of Mg will decrease; as the epileptic seizures improve, the concentration of Mg... 2+ Ca 2+ The concentration will also rise back to baseline levels accordingly; and Mg 2+ The change is located in Ca 2+ The upstream of the brain; the time it takes for the same ions to change also varies in different brain regions.

[0093] The scope of protection of this invention is not limited to the above embodiments. Any variations and advantages that can be conceived by those skilled in the art without departing from the spirit and scope of this invention are included in this invention and are protected by the appended claims.

Claims

1. Integrated Mg 2+ Ca 2+ A method for preparing the selective electrode MPETH / PPETH-E, characterized in that, The preparation method includes the following steps: Step (X-1): Dissolve ammonium persulfate in deionized water to prepare solution a; dissolve phytic acid and aniline in deionized water to prepare solution b; cool and mix the two solutions a and b to obtain crude polyaniline hydrogel; immerse the polyaniline hydrogel in deionized water to remove oligomers and excess ions; vacuum dry to obtain PANI hydrogel. Step (X-2): The PANI hydrogel prepared in step (X-1) is dispersed in a graphene oxide solution and ultrasonically treated to obtain PANI / GO; then Mg is added. 2+ / Ca 2+ Recognize the ligand MPETH / PPETH to obtain PANI / GO / MPETH or PANI / GO / PPETH; add ascorbic acid to obtain PANI / RGO / MPETH or PANI / RGO / PPETH hydrogels; Step (X-3): Inject the PANI / RGO / MPETH or PANI / RGO / PPETH hydrogel prepared in step (X-2) into a capillary tube, and then seal both ends of the capillary tube. Place the capillary tube in an oven to reduce the PANI / GO / MPETH or PANI / GO / PPETH hydrogel back to PANI / RGO / MPETH or PANI / RGO / PPETH hydrogel. After the reaction is complete, open both ends of the capillary tube. Then, gently flush the PANI / RGO / MPETH or PANI / RGO / PPETH mixed hydrogel out with water using a syringe, and dry it in a refrigerator to obtain highly selective, reversible Mg. 2+ / Ca 2+ Selective electrode MPETH / PPETH-E; The structural formulas of MPETH and PPETH are shown below: ， 。 2. The preparation method according to claim 1, characterized in that, In step (X-1), the concentration of ammonium persulfate is 1-2 M; the concentration of phytic acid is 0.2-0.8 M; the concentration of aniline is 2-4 M; the cooling temperature is 2-8 ℃; the soaking time is 8-24 h; and the vacuum drying temperature is 40-60 ℃. In step (X-2), the concentration of the PANI hydrogel dispersion is 2-8 mg / mL; the concentration of the graphene oxide solution is 2-8 mg / mL; the concentration of the MPETH / PPETH ion ligand is 1-4 mg / mL; and the concentration of the ascorbic acid is 2-8 mg / mL. In step (X-3), the capillary diameter is 0.3-0.9 μm; the oven temperature is 100-180 ℃; the baking time is 10-18 h; and the drying time is 4-10 h.

3. The reversible Mg2+ with excellent resistance to biological protein contamination prepared by the method described in claim 1 or 2. 2+ Ca 2+ Selective electrode MPETH / PPETH-E.

4. The reversible Mg with excellent resistance to biological protein contamination as described in claim 3 2+ Ca 2+ The selective electrode MPETH / PPETH-E is characterized by, To enhance the ability to resist non-specific adsorption of proteins from living organisms onto the electrode surface, an integrated structure comprising a conductive substrate, a solid-state transition layer, and a recognition layer is employed, along with a specific ligand for Mg. 2+ Ca 2+ Highly selective recognition capability.

5. The reversible Mg with excellent resistance to biological protein contamination as described in claim 3 or 4 2+ Ca 2+ Selective electrode MPETH / PPETH-E in the determination of Mg 2+ Ca 2+ Applications in [the field].

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