A method for detecting monovalent copper based on EPR technology

Abstract

The present invention discloses a method for detecting monovalent copper based on EPR technology, belonging to the technical field of trace element detection, and comprising the following steps: connecting different chelating ligands of free radicals and Cu+ through a one-step coupling reaction to obtain an EPR probe; when Cu+ is added to the solution containing the EPR probe, new four-peak EPR spectral lines appear on both sides of the single-peak spectral line of the EPR probe, and the four-peak spectral line can be attributed to the specific EPR signal of Cu+, and the content of Cu+ in the system can be accurately quantified by the peak height or peak area of the specific EPR spectral line of Cu+. The present invention can realize specific quantitative detection and imaging of Cu+ through EPR technology, simultaneously distinguish the detection of Cu+ and Cu2+, and has higher specificity and more accurate quantification for detecting Cu+ by EPR.

Description

Technical Field

[0001] The present invention belongs to the technical field of trace element detection, and particularly relates to a method for detecting monovalent copper based on EPR technology. Background Art

[0002] Copper is one of the essential trace metal elements of biological organisms. It can cycle between Cu 2+ and Cu + , and Cu + is the main existing form of copper under physiological conditions.

[0003] Copper homeostasis imbalance is related to a variety of neurodegenerative diseases, such as Wilson's disease, Parkinson's disease, Alzheimer's disease, Menkes' disease, etc. Therefore, developing a method that can accurately detect the content of Cu + in biological systems is particularly important for the research of related diseases.

[0004] Free Cu + has poor stability and is very easy to oxidize to Cu 2+ in the air. Therefore, specific quantitative detection of Cu + faces great challenges. Most of the currently commonly used metal ion detection methods cannot distinguish different metal ions, and it is even more difficult to distinguish different valence states of metal ions.

[0005] Currently, the common methods for detecting Cu + mainly include fluorescence spectrophotometry, atomic absorption spectrometry, high-performance liquid chromatography (HPLC), electrochemistry, and inductively coupled plasma mass spectrometry (ICP-MS) methods, etc. Among them, atomic absorption spectrometry, HPLC method, electrochemistry, and ICP-MS method usually need to destroy biological samples and have a long analysis time when detecting Cu + , and it is difficult to accurately detect and image unstable Cu + . Therefore, many problems such as the high instrument cost, long sample analysis time cost, and difficulty in biological imaging of the above methods have made the research on the specific detection of Cu+ stagnant.

[0006] Fluorescence spectrophotometry is a technical method that is currently widely used for detecting Cu + . Its principle is to connect a fluorescent group with a Cu + chelating ligand to obtain a fluorescent probe. After the probe coordinates and binds with Cu + , it changes the electronic structure of the fluorescent probe molecule, thereby causing an increase or decrease in the fluorescence intensity of the probe, and thus realizing the detection of Cu +Quantitative detection. Its disadvantages are as follows: the background interference of fluorescence is relatively strong, and the detection accuracy remains to be investigated; photobleaching is prone to occur, quenching fluorescence, making it difficult to continuously and repeatedly detect samples; in addition, the tissue penetration ability of fluorescence is weak, posing challenges for in vivo applications. Summary of the Invention

[0007] The problem to be solved by the present invention is to provide a method for detecting cuprous ion based on EPR technology with higher specificity and more accurate quantification.

[0008] To solve the above technical problems, the technical solution adopted by the present invention is: a method for detecting cuprous ion based on EPR technology, comprising the following steps:

[0009] S1. Preparation of the EPR probe: Connect different chelating ligands of free radicals and Cu + through a one-step coupling reaction to obtain the EPR probe;

[0010] S2. Quantitative detection of Cu + by the EPR probe: When adding Cu + to the solution containing the EPR probe, new four-peak EPR spectral lines appear on both sides of the single-peak spectral line of the EPR probe. Accurately quantify the content of Cu + in the system through the peak height or peak area of the specific EPR spectral line of Cu + .

[0011] Before S2, monitor the reactivity of the EPR probe with Cu + by EPR: When adding Cu + to the solution containing the EPR probe, new four-peak EPR spectral lines appear on both sides of the single-peak spectral line of the EPR probe. Through EPR spectral simulation of the newly emerged EPR signal, attribute it to the specific EPR spectral line of Cu + . This result confirms that the EPR probe can react with Cu + and generate a new exclusive EPR signal of Cu + .

[0012] Preferably, the free radical is trityl (such as CT-03 or OX063) radical.

[0013] Preferably, the free radical is a nitroxide radical.

[0014] Preferably, when the chelating ligand is macrocyclic (such as Cyclen, DO3S, etc.), chain-like ether (such as ETA, etc.), heterocyclic (such as EDPA, etc.) (the molecular structures of the three types of ligands are as Figure 6 shown), connect them through a one-step coupling reaction to obtain corresponding EPR probes such as TAC, TAETA or TAEDPA.

[0015] Due to the above technical solution, the present invention can achieve specific quantitative detection and imaging of Cu + through the EPR technology, and simultaneously distinguish the detection of Cu + from Cu 2+ , and the specificity of detecting Cu + by EPR is higher and the quantification is more accurate. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The present invention will be specifically described below with reference to the accompanying drawings and examples. The advantages and implementation manners of the present invention will become more obvious. The content shown in the accompanying drawings is only used for the explanation of the present invention and does not constitute any limitation to the present invention. In the drawings:

[0017] Figure 1 is a schematic diagram of the synthesis method of the EPR-Cu + probe TAC of the present invention;

[0018] Figure 2 is the EPR spectrum of TAC and TAC-Cu + of the present invention;

[0019] Figure 3 is a reaction diagram of monitoring the reaction of TAC and Cu + by the EPR spectroscopy of the present invention;

[0020] Figure 4 is a quantitative detection ability diagram of monitoring the reaction of TAC and Cu + by the EPR spectroscopy of the present invention;

[0021] Figure 5 is a detection limit schematic diagram of detecting Cu + by TAC of the present invention;

[0022] Figure 6 is the molecular structure of the Cu + chelating ligand of the present invention.

[0023] Figure 7 is the EPR spectrum after chelation of two other such trityl probes and Cu + of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0024] The present invention will be further described below in conjunction with the embodiments and their accompanying drawings:

[0025] A method for detecting monovalent copper based on the EPR technology, comprising the following steps:

[0026] Preparation of the EPR probe:

[0027] As Figures 1 to 7 shown, the trityl radical CT-03 and Cu+ The chelating ligand Cyclen is linked by BOP, HOBT, and DIPEA through a one-step coupling reaction to obtain the EPR probe TAC;

[0028] The target molecule was purified by a C18 reversed-phase silica gel column, and the correctness of its structure was confirmed by high-performance liquid chromatography and high-resolution mass spectrometry-assisted analysis;

[0029] Currently, the probe molecule TAC has been synthesized. EPR spectroscopic studies show that (as Figure 2 shown), TAC has an EPR single-line signal. Due to the indistinguishable hyperfine splitting caused by amide-N, its linewidth is significantly greater than that of CT-03, which is consistent with the linewidth and coupling constant of amide-N of previously amide-derivatized trityl radicals.

[0030] Feasibility of TAC for detecting Cu + :

[0031] Using cuprous chloride obtained by reducing copper chloride with ascorbic acid as the Cu + source, the reactivity of TAC with Cu + was monitored by EPR (as Figure 3 shown). When 0.4 equivalents of Cu + was added to the TAC solution, new symmetric four-peak EPR spectral lines appeared on both sides of the single-peak spectrum of TAC, and the reaction was completed rapidly. The addition of excess Cu + completely transformed the single-line signal of TAC into four equally high EPR spectral lines. By simulating the newly emerged EPR signal, it could be attributed to the TAC-Cu + complex. Using the commercially available classic Cu + donor [(CH 3 CN) 4 Cu]PF 6 as the Cu + source gave the same result, which confirmed that TAC could react with Cu + and generate a new exclusive EPR signal for Cu + .

[0032] Quantitative detection and detection limit of TAC for Cu + :

[0033] Since the spectral lines on both sides of the complex TAC-Cu + are far from the single-line signal of the probe TAC in the EPR spectrum, the peak height or peak area of these two spectral lines can be used to accurately quantify the content of Cu + in the system. The results are as Figure 4 shown. TAC-Cu +The signal intensity is positively correlated with the Cu + concentration (0 - 1 eq). When the concentration of Cu(I) is equal to the concentration of TAC, the TAC signal almost completely disappears, indicating that the stoichiometric ratio of the complexation of TAC and Cu + is 1:1. However, when the Cu + concentration is greater than the TAC concentration, its signal intensity no longer changes.

[0034] Therefore, the detection limit of the probe TAC for Cu + is as Figure 5 shown, approximately 20 nM (S / N = 3.5).

[0035] The technical principle of the present invention:

[0036] Electron paramagnetic resonance (EPR) spectroscopy is a magnetic resonance technique specifically used for detecting or imaging paramagnetic substances (such as free radicals and transition metal complexes). It has characteristics such as appropriate magnetic field penetration depth, no background interference, non-invasiveness, and continuous repeatability of detection. Usually, paramagnetic Cu 2+ has an extremely broad EPR spectral line, so it is difficult to directly detect. When chelated with other stable free radicals, Cu 2+ even at very low concentrations will cause a significant broadening of the EPR spectral line of the stable free radical, resulting in the weakening or even disappearance of its EPR signal. However, Cu + is a diamagnetic and magnetic metal ion with a spin quantum number of I = 3 / 2, and it does not quench the EPR signal of the stable free radical. On the contrary, when it is chelated with a stable free radical (such as a tetrathio-substituted triphenylmethyl radical, trityl radical) through a metal ligand and the spatial distance is relatively close, Cu + will distribute a certain electron spin density and thus exhibit its specific EPR four-peak spectral line. Based on this, we can achieve specific quantitative detection and imaging of Cu+ through the EPR technique, and simultaneously distinguish between the detection of Cu + and Cu 2+ .

[0037] The above has described the embodiments of the present invention in detail, but the content described is only the preferred embodiments of the present invention and cannot be considered as limiting the scope of implementation of the present invention. All equivalent changes and improvements made within the scope of the present invention should still fall within the scope covered by this patent.

Claims

1. A method for detecting monovalent copper based on EPR technology, characterized in that: comprising the following steps: S1. Preparation of EPR probe: Connect free radicals and different chelating ligands of Cu + through a one-step coupling reaction to obtain the EPR probe; S2. Quantitative detection of Cu by EPR probe + When Cu was added to the solution containing the EPR probe, new four-peak EPR spectral lines appeared on both sides of the single-peak spectral line of the EPR probe. The content of Cu in the system was accurately quantified by the peak height or peak area of the specific EPR spectral line of Cu; + When Cu was added to the solution containing the EPR probe, new four-peak EPR spectral lines appeared on both sides of the single-peak spectral line of the EPR probe. The content of Cu in the system was accurately quantified by the peak height or peak area of the specific EPR spectral line of Cu; + When Cu was added to the solution containing the EPR probe, new four-peak EPR spectral lines appeared on both sides of the single-peak spectral line of the EPR probe. The content of Cu in the system was accurately quantified by the peak height or peak area of the specific EPR spectral line of Cu; + When Cu was added to the solution containing the EPR probe, new four-peak EPR spectral lines appeared on both sides of the single-peak spectral line of the EPR probe. The content of Cu in the system was accurately quantified by the peak height or peak area of the specific EPR spectral line of Cu; The free radical is a trityl radical or a nitroxide radical; The chelating ligand is a macrocyclic, chain-like ether or heterocyclic type, and is connected through a one-step coupling reaction to obtain corresponding EPR probes: TAC, TAETA or TAEDPA.

2. The method for detecting monovalent copper based on EPR technology according to claim 1, characterized in that: Before S2, EPR was used to monitor the reactivity of the EPR probe with Cu + : When Cu + was added to the solution containing the EPR probe, new four-peak EPR spectral lines appeared on both sides of the single-peak spectral line of the EPR probe. By simulating the EPR spectra of the newly emerged EPR signals, they were attributed to the specific EPR spectral lines of Cu + . The results confirmed that the EPR probe reacted with Cu + and generated new specific EPR signals of Cu + .

3. The method for detecting monovalent copper based on EPR technology according to claim 1, characterized in that: The trityl radical is CT-03 or OX063.

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