NAD (P) modification method, fixing method and continuous analysis sensor

Abstract

The invention discloses a modification method of NAD (P), which comprises the following steps: S1, reacting Br <-> (CH2) x-NH2. HBr with NAD (P) to obtain N1-(x-(CH2) xNH2)-NAD (P) < + >; s2, carrying out rearrangement reaction on the N1-(x-(CH2) xNH2)-NAD (P) < + > to obtain N6-(x-(CH2) xNH2)-NAD (P) < + >; wherein x is equal to 2-6. The invention further provides a method for fixing the modified NAD (P) and a continuous analysis sensor. The method provided by the invention is environment-friendly and harmless, amino groups are modified and added on the NAD (P), and the NAD (P) can be effectively fixed, so that the stability of the sensor is effectively enhanced, and the service life of the sensor is prolonged.

Description

Technical Field

[0001] The present application relates to the technical field of biosensors, and in particular to a NAD(P) modification method, a fixation method and a continuous analysis sensor. Background Art

[0002] Diabetes is the most common endocrine dysfunction disease in clinical practice. Its etiology and pathogenesis are complex, mainly manifested in that glucose in the blood cannot be effectively utilized and stored, so blood sugar remains high for a long time. Long-term high blood sugar levels can lead to damage to multiple tissues and organs such as the heart, kidneys, and eyes, and even cause diabetic ketosis (DK) or diabetic ketoacidosis (DKA) due to stress, improper treatment and other factors. By monitoring the concentration of β-hydroxybutyric acid in tissue fluid to indirectly reflect changes in blood ketone concentrations, the risk of diabetic ketoacidosis and diabetic ketoacidosis can be effectively indicated, so that timely and accurate rescue can be provided.

[0003] In the prior art, a sensor system is used to monitor physiological indicators related to diabetes, such as blood sugar and blood ketones (β-hydroxybutyrate). Taking the blood ketone index as an example, the sensor system for detecting blood ketones includes a working electrode, a β-hydroxybutyrate enzyme sensing layer disposed on the working electrode, and a polymer limiting membrane of β-hydroxybutyrate disposed on the enzyme sensing layer. Among them, the enzyme sensing layer mainly includes β-hydroxybutyrate dehydrogenase that responds to β-hydroxybutyrate, coenzyme nicotinamide adenine dinucleotide (NAD) or coenzyme nicotinamide adenine dinucleotide phosphate (NADP) that can be operably contacted with β-hydroxybutyrate dehydrogenase, diaphorase that makes NAD (P) regenerative, and electron mediators, etc.

[0004] NAD and NADP are mainly used as coenzymes of dehydrogenases, and they act as hydrogen donors in enzymatic reactions. They are single hydrogen donors, both are small organic molecules, and are non-protein compounds that are loosely bound to the enzyme. It does not directly promote the catalytic ability of the enzyme, but participates in the catalytic reaction together with the enzyme. However, in the process of continuous monitoring of β-hydroxybutyrate, NAD(P) will permeate out of the polymer restriction membrane over time, which will have a certain impact on the catalytic reaction of the sensor, thereby affecting the performance of the sensor.

[0005] How to reduce the penetration of NAD(P) and reduce the impact on sensor performance is a technical problem that needs to be urgently solved by technical personnel in this field. Summary of the invention

[0006] In order to solve the above technical problems, the first object of the present invention is to provide a method for modifying NAD(P); the second object of the present invention is to provide a method for fixing modified NAD(P); and the third object of the present invention is to provide a continuous analysis sensor.

[0007] The technical solution provided by the present invention is as follows: A method for modifying NAD(P), comprising the following steps: S1, using Br-(CH 2 ) x -NH 2 HBr reacts with NAD(P) to give N 1 -(x-(CH 2 ) x NH 2 )-NAD(P) + ; S2, N 1 -(x-(CH 2 ) x NH 2 )-NAD(P) + Rearrangement reaction to obtain N 6 -(x-(CH 2 ) x NH 2 )-NAD(P) + ; Among them, x=2~6.

[0008] Preferably, in step S1, Br-(CH 2 ) x -NH 2 HBr and NAD(P) were dissolved in the first solvent, and then Br-(CH 2 ) x -NH 2 ·HBr solution was added dropwise to NAD(P) solution, and reacted at 20~30℃ for 18~24h under inert gas protection to separate N 1 -(x-(CH 2 ) x NH 2 )-NAD(P) + .

[0009] Preferably, the first solvent is any one or more of water or methanol; The inert gas is either nitrogen or helium.

[0010] Preferably, after the reaction is completed, the solvent is removed by vacuum rotary evaporation, washed with ethanol 2 to 3 times, centrifuged, dried by vacuum rotary evaporation, and then subjected to ion exchange chromatography to obtain N 1 -(x-(CH 2 ) x NH 2 )-NAD(P) + .

[0011] Preferably, in step S2, N 1 -(x-(CH 2 ) x NH 2 )-NAD(P) + Dissolve in water, add 1~5mM LiOH solution, adjust pH to 6.4~6.6, react at 45~55℃ for 4~6h, and separate N 6 -(x-(CH 2 ) x NH 2 )-NAD(P) + .

[0012] Preferably, after the reaction is completed, ion exchange chromatography is performed and then vacuum rotary evaporation is performed to obtain N 6 -(x-(CH 2 ) x NH 2 )-NAD(P) + .

[0013] Preferably, Br-(CH 2 ) x -NH 2 HBr is specifically any one of ethyl bromide, propyl bromide, butyl bromide, amyl bromide, and hexyl bromide.

[0014] A method for fixing modified NAD(P) 6 -(x-(CH 2 ) x NH 2 )-NAD(P) + Coupled and fixed to a carrier material with an amino or carboxyl functional group; Among them, x=2~6.

[0015] Preferably, the carrier material is any one of polylysine, polyethyleneimine, polylactic acid-co-glycolic acid, polyaspartic acid-polyethylene glycol-carboxyl, amino or carboxyl compound modified nanomaterials.

[0016] A continuous analysis sensor comprises a substrate, an electrode layer arranged on the substrate, an enzyme sensing membrane arranged on the electrode layer, and a biocompatible outer membrane arranged on the enzyme sensing membrane; the enzyme sensing membrane is a NAD(P) immobilized enzyme sensing membrane obtained by the above-mentioned fixing method.

[0017] The present application first provides a method for modifying NAD(P) by using Br-(CH 2 ) x -NH 2HBr reacts with NAD(P) (nicotinamide adenine dinucleotide or nicotinamide adenine dinucleotide phosphate) to produce N 1 -(x-(CH 2 ) x NH 2 )-NAD(P) + After rearrangement reaction, N 6 -(x-(CH 2 ) x NH 2 )-NAD(P) + The obtained N 6 -(x-(CH 2 ) x NH 2 )-NAD(P) + The modification of NAD(P) with the addition of an amino group makes it easier to be fixed to the carrier material than the unmodified NAD(P), thereby effectively fixing it; and N 6 -(x-(CH 2 ) x NH 2 )-NAD(P) + After fixation, it is difficult to overflow through the polymer limiting membrane, so as to ensure that the concentration of NAD(P) in the sensor system is maintained at a high level during continuous monitoring, effectively enhancing the stability of the sensor and extending the service life of the sensor. In addition, the modification method provided in the present application uses a reagent of Br-(CH 2 ) x -NH 2 HBr (x = 2~6), compared with the route of introducing amino groups on NAD(P) by alkylation with ethyleneimine, the modification route used in this scheme is more environmentally friendly, harmless and suitable for mass production.

[0018] With x=2, that is, using Br-(CH 2 ) 2 -NH 2 Taking HBr (bromoethylamine hydrobromide) as an example, the modification method provided in this application is shown in the following figure:

[0019]

[0020] Preferably, in the modification method provided by the present application, in step S1, Br-(CH 2 ) x -NH 2 HBr and NAD(P) were dissolved in the first solvent, and then Br-(CH 2 ) x -NH2 ·HBr solution was added dropwise to NAD(P) solution, and reacted at 20~30℃ for 18~24h under inert gas protection to separate N 1 -(x-(CH 2 ) x NH 2 )-NAD(P) + In step S2, N 1 -(x-(CH 2 ) x NH 2 )-NAD(P) + Dissolve in water, add 1~5mM LiOH solution, adjust pH to 6.4~6.6, react at 45~55℃ for 4~6h, and separate N 6 -(x-(CH 2 ) x NH 2 )-NAD(P) + Preferably, Br-(CH 2 ) x -NH 2 HBr was dissolved in methanol and NAD(P) was dissolved in water.

[0021] The Br-(CH 2 ) x -NH 2 HBr, preferably any one of bromoethylamine hydrobromide, bromopropylamine hydrobromide, bromobutylamine hydrobromide, bromopentylamine hydrobromide and bromohexylamine hydrobromide. 6 -(x-(CH 2 ) x NH 2 )-NAD(P) + N 6 -(2-(CH 2 ) 2 NH 2 )-NAD(P) + 、N 6 -(3-(CH 2 ) 3 NH 2 )-NAD(P) + 、N 6 -(4-(CH 2 ) 4 NH 2 )-NAD(P) + 、N 6 -(5-(CH 2 ) 5 NH 2 )-NAD(P)+ 、N 6 -(6-(CH 2 ) 6 NH 2 )-NAD(P) + More preferably, ethyl bromide, propyl bromide, and butyl bromide are used to prepare N 6 -(2-(CH 2 ) 2 NH 2 )-NAD(P) + 、N 6 -(3-(CH 2 ) 3 NH 2 )-NAD(P) + 、N 6 -(4-(CH 2 ) 4 NH 2 )-NAD(P) + .

[0022] The method provided in this application can also be used for NAD(P)-NH 2 modification, because NAD(P)-NH 2 Although it has an amino group, its activity is low and it is still difficult to fix. However, after modification by the method provided in the present application, the activity of fixation with the carrier material can be improved, especially by using bromoethylamine hydrobromide, bromopropylamine hydrobromide, bromobutylamine hydrobromide and NAD(P)-NH 2 The product of the modification reaction.

[0023] In the present application, the first solvent is preferably any one or more of water or methanol, more preferably Br-(CH 2 ) x -NH 2 HBr is soluble in methanol and NAD(P) is soluble in water.

[0024] The present application also provides a method for fixing modified NAD(P), wherein N 6 -(x-(CH 2 ) x NH 2 )-NAD(P) + The NAD(P) is coupled and fixed to a carrier material with an amino or carboxyl functional group, an amide bond is formed between the modified NAD(P) and the carrier material, and the NAD(P) is firmly fixed to the carrier material by a covalent bond to reduce its overflow.

[0025] The preferred carrier material is any one of polylysine, polyethyleneimine, polylactic acid-co-glycolic acid, polyaspartic acid-polyethylene glycol-carboxyl, amino or carboxyl compound modified nanomaterials. Among them, the amino compound modified nanomaterial can be polyethyleneimine modified nanosilica, or other nanomaterials with amino groups, and carboxyl compound modified nanosilica can also be used.

[0026] Nanomaterials have a large specific surface area and can adsorb proteases and create a relatively milder reaction microenvironment around the enzyme protein, so that the enzyme protein always reacts under suitable conditions, free from interference from the external environment, and enhances its stability; nanomaterials can also introduce nanoscale effects into biocatalysts, making the enzyme protein reaction more active, thereby improving the catalytic activity of the enzyme. Amino compound-modified nanomaterials can fix the modified NAD(P) by forming amide bonds covalently, improving the stability and catalytic activity of the protease while keeping the coenzyme from spilling out, which is conducive to the long-term stable operation of the sensor system.

[0027] The nanomaterials modified with amino or carboxyl compounds can be obtained by purchasing existing products, or can be prepared by referring to the methods in the prior art.

[0028] The modified N 6 -(x-(CH 2 ) x NH 2 )-NAD(P) + When coupling and fixing to a carrier material with an amino or carboxyl functional group, methods known in the art can be used. When coupling to a carrier material with an amino functional group, a coupling agent (such as glutaraldehyde, PEGDGE-400) can be added to assist.

[0029] The modification method and fixation method of NAD(P) provided in the present application and the obtained immobilized enzyme sensor membrane are applicable to all sensors that need to use NAD(P) as a coenzyme and are sensors that can perform continuous and stable analysis, such as glucose-reactive enzymes for detecting glucose, lactate-reactive enzymes for detecting lactate, or ketone-reactive enzymes for detecting β-hydroxybutyrate, etc.

[0030] In the present application, taking the ketone-reactive enzyme as an example, the enzyme sensing layer mainly includes β-hydroxybutyrate dehydrogenase that responds to β-hydroxybutyrate, a coenzyme that can be in operative contact with the β-hydroxybutyrate dehydrogenase, diaphorase that regenerates the coenzyme, and an electron mediator, which are combined to form a continuous analysis sensor. Among them, the coenzyme can use the N provided in the present application. 6 -(x-(CH 2 ) x NH 2 )-NAD(P) +, or N immobilized on a carrier material with amino functional groups 6 -(x-(CH 2 ) x NH 2 )-NAD(P) + . BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0032] Figure 1 The response current-test concentration curve of each sensor system prepared in Example 4 of the present invention; Figure 2 The response current-test time curve diagram of each sensor system prepared in Example 4 of the present invention; Figure 3 This is a response current-test concentration curve of the stability test of each sensor system prepared in Example 4 of the present invention. DETAILED DESCRIPTION

[0033] In order to enable those skilled in the art to better understand the technical solutions in this application, the technical solutions in the embodiments of this application will be clearly and completely described below. Obviously, the described embodiments are only part of the embodiments of this application, not all of them. Based on the embodiments in this application, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of this application.

[0034] The only difference between NAD and NADP is that NADP has one more phosphate group than NAD. The chemical structures of the two are shown in the figure below. In the first step S1 of the modification of NAD or NADP, the reaction is carried out using an alkylating agent Br-(CH 2 ) x -NH 2 HBr alkylates the adenine ring of NAD or NADP; in the second step S2, the reaction is a Dimroth rearrangement reaction, and the phosphate group on NADP is not involved. In summary, the phosphate group on NADP does not participate in the reaction process of step S1 and step S2, nor does it participate in the subsequent coupling reaction. The phosphate group on NADP will not affect the modification and immobilization effects in this application. Therefore, whether it is NAD or NADP, it is suitable for the modification method and immobilization method of this application, and the technical effect after being prepared into an enzyme sensor membrane is consistent.

[0035]

[0036] Based on the above, in this application, NAD is used as an example to verify the experimental effect, and the experimental effect of NADP is also equivalent to the experimental effect of NAD. Please see the following embodiment for details.

[0037] Example 1 Synthesis of N 6 -(2-(CH 2 ) 2 NH 2 )-NAD + S1. Dissolve 1.2 g of bromoethylamine hydrobromide in 2 mL of methanol, and dissolve 4.8 g of NAD in 5 mL of water. Add the bromoethylamine hydrobromide solution dropwise into the flask containing the NAD solution. React at 25 °C for 20 hours under nitrogen protection. After the reaction is completed, vacuum rotary evaporation is performed to remove the solvent, and the mixture is washed with 5 mL of ethanol three times. The precipitate is centrifuged and vacuum rotary evaporation is performed to obtain a white powder product. After ion exchange chromatography, the collected components are concentrated by rotary evaporation to obtain N 1 -(2-(CH 2 ) 2 NH 2 )-NAD + , yield 47.8% (2.3 g); S2, the N obtained in step S1 1 -(2-(CH 2 ) 2 NH 2 )-NAD + Take out 2.0g and dissolve it in 5mL deionized water, add it into 1mM LiOH solution, adjust the pH to 6.5, react at 50℃ for 5h, after the reaction, vacuum rotary evaporation and drying are carried out to obtain a white powder product, after ion exchange chromatography, the collected components are rotary evaporated and concentrated to obtain N 6 -(2-(CH 2 ) 2 NH 2 )-NAD + , yield 62% (1.24g).

[0038] Example 2 N 6 -(2-(CH 2 ) 2 NH 2 )-NAD + Fixed Take N prepared in Example 1 6 -(2-(CH 2 ) 2 NH 2 )-NAD+ , fixed to the polylysine polymer linked to the electron mediator via the coupling agent glutaraldehyde.

[0039] Example 3N 6 -(2-(CH 2 ) 2 NH 2 )-NAD + Fixed Take N prepared in Example 1 6 -(2-(CH 2 ) 2 NH 2 )-NAD + , fixed to the polyethyleneimine-modified silica nanocarrier material with a size of 300~500 nm through the coupling agent glutaraldehyde.

[0040] The finished products prepared in Example 1 and Example 3, and unmodified NAD were taken as Comparative Example 1, and a blood ketone sensor system was prepared according to the materials in Table 1.

[0041] Table 1

[0042] The β-hydroxybutyrate sensitive layer solution was prepared according to the materials in Table 1. The β-hydroxybutyrate sensitive layer solution was deposited on the electrode layer to obtain an enzyme sensing membrane. Then, a diffusion limiting membrane solution was coated on the enzyme sensing membrane to form a biocompatible outer membrane to obtain a sensor system.

[0043] The above sensor systems were used to perform tests respectively, and the linear response current-test concentration curves were shown in Figure 1 As shown, the response current-test time curve is as follows Figure 2 As shown, the response current-test concentration curve of the stability test is as follows Figure 3 shown.

[0044] like Figure 1 As shown, the response of each sensor system to β-hydroxybutyrate at 0 mM, 0.5 mM, 1.0 mM, 2.0 mM, 4.0 mM, 6.0 mM, and 8.0 mM concentrations at 37°C. The linear response current curve R 2 Both are greater than 0.98, indicating that both the immobilized NAD and non-immobilized NAD sensors have a good linear relationship, thus indicating that the immobilized NAD does not reduce the linear relationship between current and concentration.

[0045] But from Figure 2It can be seen that as the test time increases, starting from the 50th minute, the response current of Comparative Example 1 decreases significantly, indicating that unmodified NAD overflows from the sensor system during the long-term detection process, affecting the catalytic reaction ability of the sensor system and the detection results.

[0046] Similarly, if Figure 3 As shown, a stability test was conducted. Comparative Example 1 could not maintain stability for 15 days in 8 mM β-hydroxybutyrate at 37°C, and performance began to decline on the second day. However, the sensors of Example 1 and Example 3 using unmodified NAD, modified NAD, and immobilized modified NAD, respectively, could maintain stability for 15 days, thereby achieving longer stability of the blood ketone sensor and increasing the service life of the blood ketone sensor.

[0047] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for modifying NAD(P), characterized in that: The following steps are involved: S1. Use Br-(CH2) x -NH2·HBr reacts with NAD(P) to obtain N 1 -(x-(CH2) x NH2)-NAD(P) + ; S2, N 1 -(x-(CH2) x NH2)-NAD(P) + Rearrangement reaction to obtain N 6 -(x-(CH2) x NH2)-NAD(P) + ; Among them, x=2~6.

2. The modification method according to claim 1, characterized in that In step S1, Br-(CH2) x -NH2·HBr and NAD(P) are dissolved in the first solvent respectively, and then Br-(CH2) x -NH2·HBr solution was added dropwise to NAD(P) solution, and reacted at 20~30℃ for 18~24h under the protection of inert gas to separate N 1 -(x-(CH2) x NH2)-NAD(P) + .

3. The modification method according to claim 2, characterized in that The first solvent is any one or more of water or methanol; The inert gas is either nitrogen or helium.

4. The modification method according to claim 2, characterized in that In step S1, after the reaction is completed, the solvent is removed by vacuum rotary evaporation, washed with ethanol 2 to 3 times, centrifuged, dried by vacuum rotary evaporation, and then subjected to ion exchange chromatography to obtain N 1 -(x-(CH2) x NH2)-NAD(P) + .

5. The modification method according to any one of claims 1 to 4, characterized in that In step S2, N 1 -(x-(CH2) x NH2)-NAD(P) + Dissolve in water, add 1~5mM LiOH solution, adjust pH to 6.4~6.6, react at 45~55℃ for 4~6h, and separate N 6 -(x-(CH2) x NH2)-NAD(P) + .

6. The modification method according to claim 5, characterized in that In step S2, after the reaction is completed, ion exchange chromatography is performed and then vacuum rotary evaporation is performed to obtain N 6 -(x-(CH2) x NH2)-NAD(P) + .

7. The modification method according to claim 1, characterized in that Br-(CH2) x -NH2·HBr is specifically any one of bromoethylamine hydrobromide, bromopropylamine hydrobromide, bromobutylamine hydrobromide, bromopentylamine hydrobromide, and bromohexylamine hydrobromide.

8. A method for fixing modified NAD(P), characterized in that: The modified N according to any one of claims 1 to 7 6 -(x-(CH2) x NH2)-NAD(P) + Coupled and fixed to a carrier material with an amino or carboxyl functional group; Among them, x=2~6.

9. The fixing method according to claim 8, characterized in that: The carrier material is any one of polylysine, polyethyleneimine, polylactic acid-glycolic acid copolymer, polyaspartic acid-polyethylene glycol-carboxyl, amino or carboxyl compound modified nanomaterials.

10. A continuous analysis sensor, comprising a substrate, an electrode layer disposed on the substrate, an enzyme sensing membrane disposed on the electrode layer, and a biocompatible outer membrane disposed on the enzyme sensing membrane; characterized in that: The enzyme sensor membrane is a NAD(P) immobilized enzyme sensor membrane prepared by the immobilization method described in any one of claims 8-9.

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