Preparation and application of modified carbon material electrode containing quaternary ammonium salt nanoclusters

Abstract

The invention discloses preparation and application of a modified carbon material electrode containing quaternary ammonium salt nanoclusters, the carbon material electrode is placed in a solution containing 1-100 mM of quaternary ammonium salt for electrochemical oxidation, tetravalent nitrogen of the quaternary ammonium salt contains one or more hydroxyl-containing aliphatic chains, and the quaternary ammonium salt contains one or more hydroxyl-containing aliphatic chains. The modified carbon material electrode containing the quaternary ammonium salt nanocluster is obtained, and synchronous high-precision detection of NMN and NR in a complex environment can be realized without various complex pre-treatments. Response is fast, stability is good, and excellent detection precision can be maintained in a normal temperature working condition for a long time. And the detection limits of NMN and NR in the range of 2-256 [mu] M are 1.36 [mu] M and 0.969 [mu] M respectively.

Description

Technical field:

[0001] The present invention relates to the field of electrode material modification and electrochemical sensors, and in particular to the preparation and application of a modified carbon material electrode containing quaternary ammonium salt nanoclusters. Background technology:

[0002] Nicotinamide mononucleotide (NMN) can be converted into coenzyme I in vivo. It is widely distributed in cells and participates in various enzymatic processes, making it an indispensable substance in the body. A decrease in coenzyme I levels in organisms is considered one of the main characteristics of aging and is also considered one of the main causes of hearing and vision loss, cognitive and motor dysfunction, and immune system impairment or imbalance (Belenky et al., Trends Biochem. Sci. 2007.32.12-19). Nicotinamide riboside (NR) is a precursor for NMN biosynthesis and has similar effects to NMN. Therefore, supplementing with NR and NMN can help increase coenzyme I levels in the body, thereby delaying, improving, or preventing various diseases and functional disorders associated with aging.

[0003] At present, NMN has been widely added to various health products as a functional substance (Ozaki et al., Anal. Biochem. 2022.655.114837). At the same time, given the anti-aging functions of NMN and NR, NMN and NR are often added to various cosmetics and facial masks. However, there is still no national standard for the determination of the amount of NMN and NR added to products. Therefore, there is an urgent need to develop an efficient analytical method for the quantitative analysis of NMN and NR.

[0004] Regarding the detection methods of NMN and NR, the main method reported in the literature is high performance liquid chromatography (HPLC), but this method is limited by the high cost and complex pretreatment of HPLC equipment, which greatly limits its application and reduces its practicality. Due to the high hydrophilicity of NMN and NR, their retention rate on the C18 column is low during HPLC detection, which reduces the separation effect (Ozaki et.al, Anal. Biochem. 2022.655.114837). In addition, due to the strong electron-withdrawing effect of quaternary nitrogen, the degree of ionization of hydroxyl hydrogen is large, and the molecule as a whole is easily electrically neutral, and the response degree in the mass spectrometry is low. For example, a method for simultaneously determining the content of nicotinamide mononucleotide α, β isomers and nicotinamide adenine dinucleotide in food mentioned in patent CN115876932A can achieve high-precision simultaneous detection of NMN and NR, but it requires a complex pretreatment process (ultrasonic extraction, solid phase extraction and elution filtration). Patent CN 118883781 A mentions a method for the simultaneous detection of NMN and PQQ in health foods or cosmetics, but it cannot be used for the simultaneous detection of NMN and NR. In addition, the capillary electrophoresis determination method for NMN in Enoki mushrooms mentioned in patent CN 113189185A, although it does not rely on HPLC, also requires a complex pretreatment process (activation with NaOH solution for 30 minutes, followed by 8 washes of 5 minutes each), and does not achieve the simultaneous detection of NMN and NR. In summary, there is currently no efficient and low-cost analytical method for the simultaneous detection of NMN and NR.

[0005] Electrochemical sensors have attracted widespread attention due to their fast response speed, low cost, good selectivity, and sensitivity. Carbon materials are the most common electrode materials due to their excellent conductivity, stability, and ease of modification. Electrochemical sensors rely on the reduction current generated by the reduction of quaternary ammonium salts to detect both NR and NMN. However, due to the similar chemical structures of NR and NMN, the current peaks generated by them merge into a single current peak. Therefore, existing electrochemical sensors are unable to distinguish between NR and NMN during detection, let alone achieve simultaneous detection. Summary of the invention:

[0006] The purpose of the present invention is to provide a preparation and application of a modified carbon material electrode containing quaternary ammonium salt nanoclusters, which solves the problem that the existing electrochemical sensors cannot distinguish NR and NMN during detection, and cannot achieve synchronous detection.

[0007] The present invention is achieved through the following technical solutions:

[0008] A method for preparing a modified carbon material electrode containing quaternary ammonium salt nanoclusters comprises the following steps: placing a carbon material electrode in a quaternary ammonium salt solution containing 1-100 mM for electrochemical oxidation, wherein the tetravalent nitrogen of the quaternary ammonium salt contains one or more hydroxyl-containing fatty chains, to obtain a modified carbon material electrode containing quaternary ammonium salt nanoclusters.

[0009] Carbon material electrodes include glassy carbon electrodes, carbon fiber felt electrodes, etc.

[0010] The size of quaternary ammonium salt nanoclusters is 10-100 nm.

[0011] The electrochemical oxidation is constant potential oxidation, pulse electrooxidation or cyclic voltammetric oxidation. The voltage during constant potential oxidation is +1.4 to +2.0V, the oxidation potential during pulse electrooxidation is +1.4 to +2.0V, and the cyclic voltammetric oxidation is carried out within a potential range of -1.7V to +2.0V, with a scan rate of 25mV / s and 6 cycles of scanning.

[0012] Preferably, the quaternary ammonium salt cationic portion is selected from one of choline, N,N,N,N-trimethylhydroxypropylammonium, N,N,N,N-diisopropylmethylhydroxyethylammonium, N,N,N,N-dimethylisopropylhydroxyethylammonium, N,N,N,N-triethylhydroxyethylammonium, N,N,N,N-diethyldihydroxyethylammonium, N,N,N,N-trihydroxyethylethylammonium and N,N,N,N-tetrahydroxyethylammonium.

[0013] Preferably, the quaternary ammonium salt solution further contains a phosphate buffer solution (PBS) of pH 7.

[0014] During electrochemical oxidation, due to the strong electron-withdrawing effect of quaternary nitrogen, hydroxyl hydrogen easily leaves to generate free radicals. The formed quaternary ammonium salt free radicals spontaneously form nanoclusters on the electrode surface to achieve surface modification. The prepared modified carbon material electrode does not require various complex pretreatments and can achieve simultaneous high-precision detection of NMN and NR in complex environments. It has fast response and good stability, and can maintain excellent detection accuracy even at room temperature for a long time. When performing simultaneous detection of NMN and NR in the range of 2-256μM, the detection limits are 1.36μM and 0.969μM, respectively.

[0015] Therefore, the present invention also protects the use of modified carbon material electrodes containing quaternary ammonium salt nanoclusters obtained by the above-mentioned preparation method for the electrochemical simultaneous detection of nicotinamide mononucleotide and nicotinamide riboside. During detection, the modified carbon material electrode does not require various complex pretreatments. It only needs to mix the test sample with a pH 9 phosphate buffer solution to achieve simultaneous high-precision detection of NMN and NR in the test sample. Some test samples, such as milk and freshly squeezed juice, contain suspended matter, which needs to be removed from the test sample. Otherwise, electrocoagulation is likely to occur on the electrode surface, which may significantly affect the detection effect.

[0016] Moreover, in complex environments such as juice and dairy products, the modified carbon material electrode prepared by this method can also maintain excellent detection effects.

[0017] The beneficial effects of the present invention are as follows:

[0018] 1. The modified carbon material electrode containing quaternary ammonium salt nanoclusters of the present invention is simple to prepare and can achieve simultaneous detection of NR and NMN in the sample to be tested without complex pretreatment.

[0019] 2. The modified carbon material electrode containing quaternary ammonium salt nanoclusters of the present invention has high detection accuracy for NR and NMN. When performing simultaneous detection, the detection limits (LODs) in the range of 2-256 μM are as low as 1.36 μM and 0.969 μM, respectively.

[0020] 3. The modified carbon material electrode containing quaternary ammonium salt nanoclusters of the present invention has good discrimination ability between NR and NMN, and is not interfered by similar structural compounds such as nicotinamide, nicotinic acid and adenine nucleotide.

[0021] 4. The modified carbon material electrode containing quaternary ammonium salt nanoclusters of the present invention has fast response and good stability, and can maintain excellent detection accuracy even under room temperature conditions for a long time. Description of the drawings:

[0022] Figure 1 This is a microscopic morphology of the modified carbon material electrode in Example 1 of the present invention;

[0023] Figure 2 This is a microscopic morphology of the carbon material electrode of Example 1 of the present invention before modification;

[0024] Figure 3 The DPV results of the carbon material electrode obtained in Example 1 of the present invention in test solutions containing different concentrations of NMN and NR are shown;

[0025] Figure 4 NR peak current fitting curve of the carbon material electrode obtained in Example 1 of the present invention in test solutions containing different concentrations of NMN and NR;

[0026] Figure 5 This is the NMN peak current fitting curve of the carbon material electrode obtained in Example 1 of the present invention in a test solution containing 200 μM NMN and NR;

[0027] Figure 6 This is a microscopic morphology of the modified carbon material electrode in Example 2 of the present invention;

[0028] Figure 7 This is the DPV result of the carbon material electrode obtained in Example 2 of the present invention in a test solution containing 200 μM NMN and NR;

[0029] Figure 8 This is the DPV result of the carbon material electrode obtained in Example 3 of the present invention in a spiked juice sample. Specific implementation method:

[0030] The following is a further description of the present invention, but not a limitation of the present invention.

[0031] Example 1:

[0032] The glassy carbon electrode was oxidized in pH 7 PBS containing 4 mM choline at +1.7 V for 1200 s. After oxidation, it was washed to obtain a glassy carbon electrode modified with quaternary ammonium salt. The surface morphology of the modified electrode was observed by scanning electron microscopy. Figure 1 As shown in Figure 2, a large number of nanoclusters can be observed to be formed on the electrode surface.

[0033] The sensor was placed in PBS at pH 9 to detect NMN and NR. The test results were as follows: Figure 3 As shown. It can be seen that NR and NMN present two different current peaks, and as the addition amount increases, the peak current also increases. This shows that the sensor can effectively detect NMN and NR simultaneously. The fitting curve of the test results is shown as follows Figure 4 、 Figure 5 As shown in the figure, the response current of the modified carbon material sensor obtained by this method has a significant linear relationship with the NR / NMN concentration, R2>0.995.

[0034] Example 2

[0035] The glassy carbon electrode was subjected to pulse electrooxidation at +2.0V for 10 minutes in PBS containing 100mM N,N,N,N-trimethylhydroxypropylammonium at pH 7, and then washed to obtain a glassy carbon electrode modified with quaternary ammonium salt. The surface morphology of the modified electrode was observed by scanning electron microscopy. Figure 6 As shown. A large number of nanoclusters can be observed on the electrode surface. The sensor can be placed in PBS with pH 9 to achieve simultaneous detection of NMN and NR. The test results are shown in Figure 7As shown, it can be seen that NR and NMN present two different current peaks.

[0036] Example 3

[0037] Cyclic voltammetry was performed on a glassy carbon electrode in pH 7 PBS containing 10 mM N,N,N,N-tetrahydroxyethylammonium over a potential range of -1.7 V to +1.8 V at a scan rate of 25 mV / s for six cycles. After the cycles were completed, the electrode was cleaned to obtain a quaternary ammonium salt-grafted glassy carbon electrode.

[0038] After centrifuging the sample juice to remove suspended matter, the supernatant was mixed with pH 9 PBS to form a 1:3 mixture. The mixture was used as the test solution and the modified electrode was used for detection to achieve the simultaneous detection of NMN and NR content in the sample. The test results are as follows Figure 8 As shown, it can be seen that NR and NMN present two different current peaks.

[0039] Example 4

[0040] The carbon fiber felt electrode was pulse electrooxidated in PBS containing 2 mM N,N,N,N-diisopropylmethylhydroxyethylammonium at pH 7 for 10 min at +1.4 V. After oxidation, the electrode was washed to obtain a quaternary ammonium salt-grafted glassy carbon electrode.

[0041] After centrifuging the dairy sample to remove suspended matter, the supernatant was mixed with pH 9 PBS to form a 1:3 mixture. This mixture was used as the test solution and the modified electrode was used for detection, achieving simultaneous detection of the NMN and NR content in the sample.

[0042] Example 5

[0043] The glassy carbon electrode was oxidized in pH 7 PBS containing 10 mM N,N,N,N-dimethylisopropylhydroxyethylammonium at a voltage of +2.0 V for 1200 s, and then washed to obtain a quaternary ammonium salt grafted glassy carbon electrode.

[0044] After centrifuging the sample juice to remove suspended matter, the supernatant was mixed with pH 9 PBS to form a 1:3 mixture. This mixture was used as the test solution and the modified electrode was used for detection, achieving simultaneous detection of the NMN and NR content in the sample.

[0045] Example 6

[0046] The glassy carbon electrode was oxidized in pH 7 PBS containing 10 mM N,N,N,N-triethylhydroxyethylammonium at a voltage of +1.4 V for 1200 s and then washed to obtain a glassy carbon electrode modified with quaternary ammonium salt.

[0047] After centrifuging the sample juice to remove suspended matter, the supernatant was mixed with pH 9 PBS to form a 1:3 mixture. This mixture was used as the test solution and the modified electrode was used for detection, achieving simultaneous detection of the NMN and NR content in the sample.

[0048] Example 7

[0049] Cyclic voltammetry was performed on a glassy carbon electrode in pH 7 PBS containing 10 mM N,N,N,N-diethyldihydroxyethylammonium over a potential range of -1.7 V to +1.8 V at a scan rate of 25 mV / s for six cycles. After the cycles were completed, the electrode was cleaned to obtain a quaternary ammonium salt-grafted glassy carbon electrode.

[0050] After centrifuging the sample juice to remove suspended matter, the supernatant was mixed with pH 9 PBS to form a 1:3 mixture. This mixture was used as the test solution and the modified electrode was used for detection, achieving simultaneous detection of the NMN and NR content in the sample.

[0051] Example 8

[0052] Cyclic voltammetry was performed on a glassy carbon electrode in pH 7 PBS containing 10 mM N,N,N,N-trishydroxyethylammonium over a potential range of -1.6 V to +1.6 V at a scan rate of 25 mV / s for six cycles. After the cycles were completed, the electrode was washed to obtain a quaternary ammonium salt-grafted glassy carbon electrode.

[0053] After centrifuging the sample juice to remove suspended matter, the supernatant was mixed with pH 9 PBS to form a 1:3 mixture. This mixture was used as the test solution and the modified electrode was used for detection, achieving simultaneous detection of the NMN and NR content in the sample.

[0054] Example 9

[0055] Cyclic voltammetry was performed on a glassy carbon electrode in pH 7 PBS containing 10 mM N,N,N,N-tetrahydroxyethylammonium over a potential range of -1.7 V to +2.0 V at a scan rate of 25 mV / s for six cycles. After the cycles were completed, the electrode was washed to obtain a quaternary ammonium salt-grafted glassy carbon electrode.

[0056] After centrifuging the sample juice to remove suspended matter, the supernatant was mixed with pH 9 PBS to form a 1:3 mixture. This mixture was used as the test solution and the modified electrode was used for detection, achieving simultaneous detection of the NMN and NR content in the sample.

[0057] The above exemplary embodiments describe the present invention in a relatively specific and detailed manner, but are not intended to limit the present invention to the detailed descriptions given. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. A method for preparing a modified carbon material electrode containing quaternary ammonium salt nanoclusters, characterized in that: The method comprises the following steps: The carbon material electrode is placed in a quaternary ammonium salt solution containing 1-100 mM for electrochemical oxidation, wherein the tetravalent nitrogen of the quaternary ammonium salt contains one or more hydroxyl-containing fatty chains to obtain a modified carbon material electrode containing quaternary ammonium salt nanoclusters.

2. The preparation method according to claim 1, characterized in that The size of quaternary ammonium salt nanoclusters is 10-100 nm.

3. The preparation method according to claim 1, characterized in that The electrochemical oxidation is constant potential oxidation, pulse electrooxidation or cyclic voltammetric oxidation. The voltage during constant potential oxidation is +1.4 to +2.0V, the oxidation potential during pulse electrooxidation is +1.4 to +2.0V, and the cyclic voltammetric oxidation is carried out within a potential range of -1.7V to +2.0V, with a scan rate of 25mV / s and 6 cycles of scanning.

4. The preparation method according to claim 1, characterized in that The quaternary ammonium salt cationic portion is selected from one of choline, N,N,N,N-trimethylhydroxypropylammonium, N,N,N,N-diisopropylmethylhydroxyethylammonium, N,N,N,N-dimethylisopropylhydroxyethylammonium, N,N,N,N-triethylhydroxyethylammonium, N,N,N,N-diethyldihydroxyethylammonium, N,N,N,N-trihydroxyethylethylammonium and N,N,N,N-tetrahydroxyethylammonium.

5. The preparation method according to claim 1, characterized in that The quaternary ammonium salt solution also contains a phosphate buffer solution of pH 7.

6. Application of the modified carbon material electrode containing quaternary ammonium salt nanoclusters obtained by the preparation method of claim 1, characterized in that: Used for the electrochemical simultaneous detection of nicotinamide mononucleotide and nicotinamide riboside.

7. The use according to claim 6, characterized in that During detection, the modified carbon material electrode does not require complex pretreatment, and only needs to mix the sample to be tested with a pH 9 phosphate buffer solution.

8. The use according to claim 7, characterized in that When the sample to be tested is milk or freshly squeezed juice, it is also necessary to remove suspended matter in the sample to be tested.

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