Method for modifying nad(p), immobilization method, and continuous analysis sensor

Abstract

The application discloses a modification method of NAD(P), comprising the following steps: S1, using Br-(CH2) x -NH2·HBr to react with NAD(P) to obtain N 1 -(x-(CH2) x NH2)-NAD(P) + ; S2, N 1 -(x-(CH2) x NH2)-NAD(P) + is subjected to a rearrangement reaction to obtain N 6 -(x-(CH2) x NH2)-NAD(P) + ; wherein x=2-6. The application further provides a fixing method of the modified NAD(P) and a continuous analysis sensor. The method provided by the application is environment-friendly and harmless, amino groups are added to the NAD(P) through modification, the NAD(P) can be effectively fixed, and therefore 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, in particular to an NAD(P) modification method, a fixing method and a continuous analysis sensor. BACKGROUND

[0002] Diabetes is the most common endocrine disorder disease in clinical, and its etiology and pathogenesis are complex. The main performance is that glucose in blood cannot be effectively utilized and stored, so that blood glucose is in a high level for a long time. Long-term high blood glucose state can cause functional damage of multiple tissues and organs such as heart, kidney and eye, and even diabetic ketosis (DK) or diabetic ketoacidosis (DKA) due to stress, improper treatment and other inducements. By monitoring the concentration of hydroxybutyric acid in interstitial fluid to indirectly reflect the change of blood ketone concentration, the risk of diabetic ketosis and diabetic ketoacidosis can be effectively prompted, so as to rescue in time and accurately.

[0003] In the prior art, a sensor system is used to monitor physiological indexes related to diabetes, such as blood glucose, blood ketone (β hydroxybutyric acid). Taking the blood ketone index as an example, the sensor system for detecting blood ketone includes a working electrode, a β hydroxybutyric acid enzyme sensing layer arranged on the working electrode, and a polymer limiting membrane of β hydroxybutyric acid arranged on the enzyme sensing layer. Among them, the enzyme sensing layer mainly includes β hydroxybutyric acid dehydrogenase, coenzyme nicotinamide adenine dinucleotide (NAD) or coenzyme nicotinamide adenine dinucleotide phosphate (NADP) in operable contact with β hydroxybutyric acid dehydrogenase, myocardial yellow enzyme for regenerating NAD(P), and electron mediator.

[0004] NAD and NADP mainly act as coenzymes of dehydrogenase, play the role of hydrogen transfer body in enzymatic reaction, are single hydrogen transfer body, are organic small molecules, and are a kind of non-protein compound which is loosely combined with enzyme. It does not directly promote the catalytic ability of enzyme, but participates in catalytic reaction together with enzyme. However, in the process of continuous monitoring of β hydroxybutyric acid, NAD(P) will penetrate out of the polymer limiting membrane with the passage of time, which will have a certain influence 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 influence on the performance of the sensor is a technical problem to be solved by those skilled in the art. SUMMARY ​​

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

[0007] The technical solutions provided by the present application are as follows:

[0008] A method for modifying NAD(P) comprises the following steps: S1, using Br-(CH2) x -NH2·HBr to react with NAD(P) to obtain N 1 -(x-(CH2) x NH2)-NAD(P) + ;

[0009] S2, N 1 -(x-(CH2) x NH2)-NAD(P) + undergoes a rearrangement reaction to obtain N 6 -(x-(CH2) x NH2)-NAD(P) + ;

[0010] wherein x = 2-6.

[0011] Preferably, in step S1, Br-(CH2) x -NH2·HBr and NAD(P) are respectively dissolved in a first solvent, then the Br-(CH2) x -NH2·HBr solution is added dropwise into the NAD(P) solution, and the reaction is carried out under the protection of an inert gas at 20-30°C for 18-24 h, and N 1 -(x-(CH2) x NH2)-NAD(P) + is obtained by separation.

[0012] Preferably, the first solvent is any one or more of water or methanol.

[0013] The inert gas is any one of nitrogen or helium.

[0014] Preferably, after the reaction is completed, the solvent is removed by vacuum rotary evaporation, the product is washed with ethanol for 2-3 times, centrifuged and precipitated, dried by vacuum rotary evaporation, and then subjected to ion exchange chromatography to obtain N 1 -(x-(CH2) x NH2)-NAD(P) + .

[0015] Preferably, in step S2, N 1 -(x-(CH2)x NH2)-NAD(P) + Dissolved in water, 1-5 mM LiOH solution was added to adjust pH to 6.4-6.6, reacted at 45-55℃ for 4-6 h, and N 6 -(x-(CH2) x NH2)-NAD(P) + .

[0016] Preferably, after the reaction, N 6 -(x-(CH2) x NH2)-NAD(P) + .

[0017] Preferably, Br-(CH2) x -NH2·HBr is any one of bromoethylamine hydrobromide, bromopropylamine hydrobromide, bromobutylamine hydrobromide, bromopentylamine hydrobromide, and bromohexylamine hydrobromide.

[0018] A modified NAD(P) immobilization method, N 6 -(x-(CH2) x NH2)-NAD(P) + is coupled to a carrier material with amino or carboxyl functional groups;

[0019] wherein x=2-6.

[0020] Preferably, the carrier material is any one of polylysine, polyethyleneimine, polylactic acid copolymer, polyaspartic acid-polyethylene glycol-carboxyl, amino, or carboxyl compound modified nanomaterial.

[0021] A continuous analysis sensor, comprising a substrate, an electrode layer provided on the substrate, an enzyme sensing film provided on the electrode layer, and a biocompatible outer film provided on the enzyme sensing film; the enzyme sensing film is the NAD(P) immobilized enzyme sensing film prepared by the above immobilization method.

[0022] The present application first provides a modified method of NAD(P), which utilizes Br-(CH2) x -NH2·HBr to react with NAD(P) (i.e., nicotinamide adenine dinucleotide or nicotinamide adenine dinucleotide phosphate) to obtain N 1 -(x-(CH2) x NH2)-NAD(P) + After rearrangement reaction, N 6 -(x-(CH2) x NH2)-NAD(P)+ The prepared N 6 -(x-(CH2) x NH2)-NAD(P) + The modification of NAD(P) adds an amino group, which is easier to be fixed to the carrier material compared to unmodified NAD(P), thereby effectively fixing it; and N 6 -(x-(CH2) x NH2)-NAD(P) + After being fixed, it is difficult to overflow through the polymer limiting film, so as to ensure that the concentration of NAD(P) is maintained at a high level during continuous monitoring of the sensor system, effectively enhancing the stability of the sensor and prolonging the service life of the sensor. Moreover, the modification method provided by the present application uses Br-(CH2) x -NH2·HBr (x=2~6), compared to the route of introducing an amino group on NAD(P) by using ethylene imine for alkylation, the modification route used in the present application is more environmentally friendly and harmless, and is suitable for mass production.

[0023] Taking x=2, i.e. using Br-(CH2)2-NH2·HBr (bromoethylamine hydrobromide) as an example, the modification method provided by the present application is shown in the following figure:

[0024]

[0025]

[0026] Preferably, in the modification method provided by the present application, in step S1, Br-(CH2) x -NH2·HBr and NAD(P) are dissolved in a first solvent, and then the Br-(CH2) x -NH2·HBr solution is added dropwise into the NAD(P) solution, and the reaction is carried out at 20~30℃ for 18~24h under inert gas protection, to obtain N 1 -(x-(CH2) x NH2)-NAD(P) + In step S2, the N 1 -(x-(CH2) x NH2)-NAD(P) + is dissolved in water, and a 1~5mM LiOH solution is added to adjust the pH to 6.4~6.6, and the reaction is carried out at 45~55℃ for 4~6h, to obtain N 6 -(x-(CH2) x NH2)-NAD(P) + Preferably, the Br-(CH2) x -NH2·HBr is dissolved in methanol, and the NAD(P) is dissolved in water.

[0027] The Br-(CH2) used in this application x -NH₂·HBr, preferably any one of bromoethylamine hydrobromide, bromopropylamine hydrobromide, bromobutylamine hydrobromide, bromopentamine hydrobromide, and bromohexamine hydrobromide. The prepared N 6 -(x-(CH2) x NH2)-NAD(P) + N 6 -(2-(CH2)2NH2)-NAD(P) + N 6 -(3-(CH2)3NH2)-NAD(P) + N 6 -(4-(CH2)4NH2)-NAD(P) + N 6 -(5-(CH2)5NH2)-NAD(P) + N 6 -(6-(CH2)6NH2)-NAD(P) + More preferably, N is prepared by using bromoethylamine hydrobromide, bromopropylamine hydrobromide, and bromobutylamine hydrobromide, respectively. 6 -(2-(CH2)2NH2)-NAD(P) + N 6 -(3-(CH2)3NH2)-NAD(P) + N 6 -(4-(CH2)4NH2)-NAD(P) + .

[0028] The method provided in this application can also be used for the modification of NAD(P)-NH2. Although NAD(P)-NH2 itself contains amino groups, its activity is low and it is still difficult to fix. However, the modification by the method provided in this application can improve the activity of fixing and linking with the support material, especially the products modified by reacting NAD(P)-NH2 with bromoethylamine hydrobromide, bromopropylamine hydrobromide, or bromobutylamine hydrobromide.

[0029] In this application, the first solvent is preferably one or more of water or methanol, more preferably Br-(CH2). x -NH2·HBr dissolves in methanol, and NAD(P) dissolves in water.

[0030] This application also provides a method for fixing modified NAD(P), wherein N 6 -(x-(CH2) x NH2)-NAD(P) +The coupling is fixed on the carrier material with amino or carboxyl functional groups, an amide bond is formed between the modified NAD(P) and the carrier material, and the NAD(P) is firmly fixed on the carrier material by using a covalent bond, thereby reducing the overflow thereof.

[0031] Preferably, the carrier material is polylysine, polyethyleneimine, polylactic acid The amino compound modified nanomaterial can be polyethyleneimine modified nanosilica or other nanomaterials with amino groups, or a carboxyl compound modified nanosilica, etc.

[0032] The nanomaterial has a large specific surface area, can adsorb protease, and creates a relatively more moderate reaction microenvironment around the enzyme protein, so that the enzyme protein always reacts under suitable conditions, is free from interference from the external environment, and the stability thereof is enhanced; the nanomaterial can also introduce nanoscale effects into the biological catalyst, so that the reaction of the enzyme protein is more active, thereby improving the catalytic activity of the enzyme. The amino compound modified nanomaterial can covalently bond the modified NAD(P) by forming an amide bond, thereby improving the stability and catalytic activity of the protease, and maintaining the non-overflow of the coenzyme, which is conducive to the long-term stable operation of the sensor system.

[0033] The amino or carboxyl compound modified nanomaterial can be obtained by purchasing existing products, or can be prepared by referring to the methods of the prior art.

[0034] The modified N 6 -(x-(CH2) x NH2)-NAD(P) + When coupling and fixing on the carrier material with amino or carboxyl functional groups, methods known in the art can be used. When the carrier material has an amino functional group, a coupling agent (such as glutaraldehyde, PEGDGE-400) can be added for assistance.

[0035] The modification method and the fixing method of the NAD(P) provided in the application, and the prepared immobilized enzyme sensor film are suitable for all sensors that need to use NAD(P) as a coenzyme, and are sensors that can be continuously and stably analyzed. For example, glucose reaction enzymes for detecting glucose, lactic acid reaction enzymes for detecting lactic acid, or β ketoreaction enzymes for detecting β

[0036] In the application, the ketoreaction enzyme is taken as an example, and the enzyme sensing layer mainly includes β hydroxybutyric acid responsive β hydroxybutyric acid dehydrogenase, β The hydroxybutyric acid dehydrogenase is operatively linked to a coenzyme, a coenzyme regenerator, and an electron mediator, which are combined to form a continuous analysis sensor. The coenzyme can be NAD(P)H provided by the present application, and the coenzyme regenerator can be diaphorase. 6 -(x-(CH2) x NH2)-NAD(P) + , or NAD(P)H immobilized on a carrier material with an amino functional group 6 -(x-(CH2) x NH2)-NAD(P) + . BRIEF DESCRIPTION OF DRAWINGS

[0037] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments or prior art description will be briefly introduced. Obviously, the drawings in the following description are only some embodiments described in the present application, and other drawings can be obtained by those skilled in the art without any creative effort.

[0038] Figure 1 The response current-test concentration curve of each sensor system prepared in Example 4 of the present application;

[0039] Figure 2 The response current-test time curve of each sensor system prepared in Example 4 of the present application;

[0040] Figure 3 The response current-test concentration curve of the stability test of each sensor system prepared in Example 4 of the present application. DETAILED DESCRIPTION

[0041] In order to make those skilled in the art better understand the technical solutions in the present application, the technical solutions in the embodiments of the present application will be clearly and completely described below. Obviously, the described embodiments are only some of the embodiments of the present application, not all. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without any creative effort are within the scope of protection of the present application.

[0042] The difference between NAD and NADP is that NADP has an additional phosphate group than NAD, and their chemical structures are shown in the following figure. The first step S1 of the modification of NAD or NADP, the reaction uses an alkylating agent Br-(CH2) x-NH2·HBr alkylates the position on the adenine ring of NAD or NADP; in the second step S2, the reaction is a Dimroth rearrangement reaction, which also does not involve the phosphate group on NADP. 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, and the phosphate group on NADP will not affect the modification and immobilization effect in the present application. Therefore, whether it is NAD or NADP, it is suitable for the modification method and immobilization method of the present application, and the technical effect after being prepared into an enzyme sensing film is consistent.

[0043]

[0044] Based on the above, NAD is taken as an example in the present application for experimental effect verification, and the experimental effect of NADP is equivalent to that of NAD. For details, see the following example description.

[0045] Example 1 Synthesis of N 6 -(2-(CH2)2NH2)-NAD +

[0046] S1, 1.2 grams of bromoethylamine hydrobromide was dissolved in 2 mL of methanol, 4.8 g of NAD was dissolved in 5 mL of water, the bromoethylamine hydrobromide solution was added dropwise into the flask containing the NAD solution, and the reaction was carried out at 25°C for 20 hours under nitrogen protection. After the reaction was completed, vacuum rotary evaporation was performed, and 5 mL of ethanol was used for washing 3 times, centrifugal precipitation was performed, and vacuum rotary evaporation was performed after drying to obtain white powder product. After ion exchange chromatography, the collected components were concentrated by rotary evaporation to obtain N 1 -(2-(CH2)2NH2)-NAD + , yield 47.8% (2.3 g);

[0047] S2, the N 1 -(2-(CH2)2NH2)-NAD + 2.0 g of which was dissolved in 5 mL of deionized water and added to 1 mL of LiOH solution, the pH was adjusted to 6.5, and the reaction was carried out at 50°C for 5 h. After the reaction was completed, vacuum rotary evaporation was performed to obtain white powder product. After ion exchange chromatography, the collected components were concentrated by rotary evaporation to obtain N 6 -(2-(CH2)2NH2)-NAD + , yield 62% (1.24 g).

[0048] Example 2 N 6 -(2-(CH2)2NH2)-NAD + immobilization

[0049] The N 6-(2-(CH2)2NH2)-NAD + It is fixed to the polylysine polymer linked to the electron mediator by the coupling agent glutaraldehyde.

[0050] Example 3N 6 -(2-(CH2)2NH2)-NAD + Fixed

[0051] Take N prepared in Example 1 6 -(2-(CH2)2NH2)-NAD + The glutaraldehyde was used as a coupling agent to fix the glutaraldehyde onto a silica nanocarrier material with a size of 300-500 nm modified with polyethyleneimine.

[0052] The finished products prepared in Examples 1 and 3, as well as unmodified NAD, were used as Comparative Example 1 to prepare a blood ketone sensor system according to the materials in Table 1.

[0053] Table 1

[0054]

[0055] A β-hydroxybutyric acid (HHA) sensitive layer solution was prepared according to the materials in Table 1. The HHA sensitive layer solution was then deposited onto the electrode layer to obtain an enzyme sensing membrane. A diffusion-limiting membrane solution was then coated onto the enzyme sensing membrane to form a biocompatible outer membrane, thus obtaining the sensor system.

[0056] The above sensor system was used for testing, and the linear response current was observed. The concentration curve is shown below. Figure 1 As shown, the response current Test time curve as shown Figure 2 As shown, the response current-test concentration curve for the stability test is as follows: Figure 3 As shown.

[0057] like Figure 1 As shown, the response of each sensor system at 37℃ to β-hydroxybutyric acid at concentrations of 0 mM, 0.5 mM, 1.0 mM, 2.0 mM, 4.0 mM, 6.0 mM, and 8.0 mM is illustrated by the linear current curve R of the response to β-hydroxybutyric acid. 2 Both values ​​are greater than 0.98, indicating that both immobilized and non-immobilized NAD sensors have a good linear relationship. Therefore, it is shown that immobilized NAD does not reduce the linear relationship between current and concentration.

[0058] But from Figure 2It can be seen that with the increase of test time, the response current of Comparative Example 1 is significantly reduced from the 50th minute, indicating that the unmodified NAD is exuded from the sensor system during the long-time detection process, affecting the catalytic reaction ability of the sensor system and the detection results.

[0059] Similarly, as shown in Figure 3 the stability test, Comparative Example 1 cannot maintain the stability in 8mM β-hydroxybutyric acid at 37℃ for 15 days, and the performance begins to decline on the 2nd day, while the sensors of Example 1 and Example 3 respectively use modified NAD and immobilized modified NAD, which can maintain the stability for 15 days, realizing the longer stability of the blood ketone sensor, thereby increasing the service life of the blood ketone sensor.

[0060] The above description of the disclosed embodiments enables a person skilled in the art to implement or use the present application. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present application. Therefore, the present application will not be limited to these embodiments shown herein, but will conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for modifying NAD(P), characterized by, Comprising the following steps: S1, using Br-(CH2) x -NH2·HBr and NAD(P) in a first solvent under inert gas protection, and 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) + ; wherein x = 2-6.

2. The modification method according to claim 1, characterized by, In step S1, Br-(CH2) x -NH2·HBr, NAD(P) is dissolved in the first solvent, and then Br-(CH2) x -NH2·HBr solution is added dropwise into the NAD(P) solution, and the reaction is carried out at 20-30°C for 18-24h under inert gas protection to obtain N 1 -(x-(CH2) x NH2)-NAD(P) + .

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

4. The modification method according to claim 2, wherein, In step S1, after the reaction is completed, the solvent is removed by vacuum rotary evaporation, washed with ethanol for 2-3 times, centrifuged to precipitate, 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 by, In step S2, N 1 -(x-(CH2) x NH2)-NAD(P) + dissolved in water, 1-5 mM LiOH solution was added, the pH was adjusted to 6.4-6.6, and the reaction was carried out at 45-55°C for 4-6 h to obtain N 6 -(x-(CH2) x NH2)-NAD(P) + .

6. The modification method according to claim 5, wherein, In step S2, after the reaction is completed, N 6 -(x-(CH2) x NH2)-NAD(P) + .

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

8. A method for immobilization of modified NAD(P), characterized by, The modified N 6 -(x-(CH2) x NH2)-NAD(P) + coupled to a carrier material having an amino or carboxyl functional group wherein x = 2-6.

9. The method of claim 8, wherein, The carrier material is polylysine, polyethylenimine, polylactic acid Any of a hydroxyacetic acid copolymer, polyaspartic acid-polyethylene glycol-carboxyl, amino or carboxyl compound modified nanomaterial.

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; wherein, The enzyme sensing membrane is an NAD(P) immobilized enzyme sensing membrane prepared by the immobilization method of any one of claims 8-9.

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