KR101437739B1 - A method for introducing N-hydroxysuccinimide group on surface of material - Google Patents

Abstract

The present invention relates to a method for introducing an N-hydroxyimide group onto the surface of a material. More specifically, the present invention relates to a method for introducing an N-hydroxysuccinimide group onto the surface of a substance, characterized by mixing a compound having an N-hydroxysuccinimide group with lipid and introducing the compound into the surface of the substance.
The method according to the present invention is simple and economical and has the effect of increasing efficiency and reproducibility in receptor fixation. These effects are very advantageous for introducing NHS groups on the surfaces of biosensors, gas sensors, chromatographic media, nanomaterials for drug delivery, and diagnostic nanomaterials.

Description

[0001] The present invention relates to a method for introducing an N-hydroxyimide group onto a surface of a substance,

The present invention relates to a method for introducing an N-hydroxyimide group onto the surface of a material.

Introduction of an N-hydroxysuccinimide (NHS) group on the surface of a substance (e.g., solid or particle) can be used for biosensor, gas sensor, drug delivery, liposome-based diagnosis or drug delivery, chromatography, and the like. NHS groups are most frequently used to immobilize antibodies, enzymes, and DNA because they react with amino groups at high speed to form stable covalent bonds.

A conventional method of introducing an NHS group onto the surface of a substance is to add N-ethyl-N '- (3-dimethylaminopropyl) carbodiimide [N-ethyl-N' 3-dimethylaminopropyl) carbodiimide; EDC] and N-hydroxysuccinimide (NHS) in this order, which is cumbersome, difficult to obtain a constant result, and takes a long time.

Further, since the NHS group is hydrolyzed well, there is a disadvantage that a lipid or lipophilic compound containing an NHS group is decomposed in the course of introducing the liposome into the surface of the substance.

KR 10-1108578

Accordingly, the present invention provides a method for introducing an NHS group into a surface of a substance without introducing a chemical reaction at various stages.

Further, the present invention is to improve the efficiency by inhibiting the hydrolysis of the NHS group in the process of introducing the NHS group.

The present invention relates to a method for introducing a compound having an NHS group into a surface of a substance by mixing with a lipid.

More specifically, the present invention relates to a method for introducing an N-hydroxysuccinimide group into the surface of a substance, characterized by introducing a compound having an N-hydroxysuccinimide group into a surface of a substance by mixing with a lipid.

The method according to the present invention is simple and economical and has the effect of increasing efficiency and reproducibility in receptor fixation. These effects are very advantageous for introducing NHS groups on the surfaces of biosensors, gas sensors, chromatographic media, nanomaterials for drug delivery, and diagnostic nanomaterials.

FIG. 1 shows a method of fixing a protein by converting a carboxyl group into an NHS group by a continuous reaction between EDC and NHS according to a conventional technique and then reacting with an amino group of the protein.
Figure 2 is a schematic representation of the structure of the lipid.
Figure 3 is a schematic representation of the structure of a lipid bilayer and liposome.
Figure 4 schematically illustrates the formation of a lipid monolayer and a lipid bilayer on a hydrophobic surface and a hydrophilic surface, respectively.
Figure 5 is a schematic representation of the structure of (A) a lipid monolayer or (B) a lipid bilayer formed when liposomes are mixed with a compound having an NHS group in the background lipid and the liposome is added on a hydrophobic surface or a hydrophilic surface.
Fig. 6 shows the structure of hydrocarbons having (A) a lipid having an NHS group and (B) an NHS group, which can be used for each application.
Figure 7 shows the structure of palmitic acid NHS ester.
Figure 8 shows the structure of the lipid bilayer formed when the palmitic acid NHS ester is added to the QCM surface in the lipid mixture.
9 shows the results of evaluating the minimum time required for hydration by changing the hydration time of the lipid film to 10, 20 and 30 minutes.
FIG. 10 shows the evaluation of the minimum time required for coating by changing the liposome coating time to 10, 20, 30, and 40 minutes.
Figure 11 shows that a QCM sensor chip functionalized with liposomes containing a palmitic acid NHS ester was immobilized with streptavidin and a biotin anti-goat IgG antibody against biotinylated goat IgG and repeatedly injected with goat IgG And the frequency changes with time.
FIG. 12 shows the results of a QCM sensor chip functionalized with liposomes containing only DPPC, in which sequestrated avidin and biotin anti-goat IgG antibody against biotin anti-goat IgG were sequentially immobilized and repeatedly injected with goat IgG Fig.
FIG. 13 is a graph showing an average of the frequency change values when the chlorine IgG is repeatedly injected after repeating the experiment of FIGS. 11 and 12 three times or more.
FIG. 14 shows the results of immunoprecipitation of DPPC-coated liposomes (DPPC only) and DPPC-coated liposomes (DPPC + NHS) containing palmitic acid NHS ester, followed by addition of streptavidin and biotin groups And HRP enzyme activity of each well was measured. The result of repeating 6 experiments was shown.

The present invention provides a method for introducing an N-hydroxyimide group onto the surface of a substance. More specifically, the present invention relates to a method for introducing an N-hydroxysuccinimide group into the surface of a substance, characterized by introducing a compound having an N-hydroxysuccinimide group into a surface of a substance by mixing with a lipid.

The method comprises mixing a compound having an N-hydroxysuccinimide group with a lipid to form a liposome; And introducing the liposome to the surface of the material.

The method also includes the steps of: drying a mixture of a compound having an N-hydroxysuccinimide group with lipid; Forming a liposome in the buffer solution using the dried mixture; And introducing the liposome to the surface of the material.

Further, a mixture of the N-hydroxysuccinimide group-containing compound and the lipid may be dried in a film form to form a liposome from the film.

When the liposome is formed, the pH can be adjusted to a pH of 4 to 6 in a weakly acidic buffer solution. The buffer solution includes, but is not limited to, 2- (N-morpholino) ethanesulfonic acid (2- N-morpholino) ethanesulfonic acid, MES) buffer solution.

When liposomes are formed from the film, a buffer solution may be added and hydrated, followed by mixing using a vortex mixer.

The compound having the N-hydroxysuccinimide group may be a lipid having an N-hydroxysuccinimide group and / or a hydrocarbon having an N-hydroxysuccinimide group.

The " material " according to the present invention may be any one of metals, ceramics, liposomes, semiconductors and polymers. These materials may be substances which form the surface of a biosensor, a gas sensor, a nanoparticle, a nanorod, a chromatography medium, a semiconductor, a plastic or a glass, and more specifically, a modified microbalance biosensor, A surface plasmon resonance biosensor, an immunoassay plate, a cell culture dish, or a plate.

NHS groups are the most commonly used functional groups for receptor fixation. Herein, the term " receptor " according to the invention can be interpreted broadly as a substance which can be directly or indirectly connected to the surface of a substance and interact with the target substance.

Examples of such receptors are biopolymers or organic functional groups used in various fields such as biosensors, gas sensors, diagnosis or drug delivery using nanoparticles, diagnosis using liposomes, drug delivery, chromatography, and the like. Biomolecules can be proteins, such as antibodies, enzymes, and hormones, as well as nucleic acids or carbohydrates. Organic functional groups such as biotin, folic acid, vitamins, fluorescent substances, and dye substances can be used in some forms.

As shown in FIG. 2, membrane lipid, which is a main constituent of the cell membrane, can be represented by a structure having a polar head and a non-polar tail, and a polar head having two nonpolar tails. When the lipid is placed in the aqueous solution environment due to the structural characteristic of the lipid, a lipid bilayer is formed as shown in FIG. 3 in order to conceal the hydrophobic tail, and as a result, a liposome is formed do.

Adding liposomes to the solid surface results in a lipid monolayer on the hydrophobic surface and a lipid bilayer on the hydrophilic surface, respectively, as shown in Figure 4, depending on the state of the solid surface. Therefore, when a compound containing an NHS group is mixed with a base lipid to form a liposome and then added to a solid surface, the NHS group can be directly introduced onto the surface of the substance as shown in FIG.

Herein, the term "background lipid" refers to a liposome without a NHS group, such as phosphatidylcholine (PC) or phosphatidylethanolamine (PE), which can be mixed with a compound having an NHS group to form a liposome.

As a compound including an NHS group, as shown in FIG. 6, a hydrocarbon including an NHS group and a lipid including an NHS group is a representative example. In the case of lipids containing NHS groups, they can be used independently without mixing with the background lipids. To expose the NHS groups to the surface of the lipid layer, NHS groups should be present at the head of the lipid as shown in the left picture of FIG.

In hydrocarbons including NHS groups, the form of hydrocarbons can be either linear, cyclic or aromatic if they can be stably inserted into the lipid layer, but the number of carbon atoms is between 6 and 20 for effective interaction with the lipid tail, Linear saturated hydrocarbons. In the case of such linear hydrocarbons, as shown in FIG. 6 (B), the hydrocarbons can be used in combination with the background lipids, and the storage stability is higher than that of the lipids containing NHS groups, and the synthesis is easy and economical.

In one embodiment of the present invention, two methods were used to minimize the hydrolysis of NHS groups. First, since the NHS group is hydrolyzed under basic conditions, a weakly acidic buffer solution was used to make liposomes. Second, the time taken to fix the receptor using the NHS group introduced on the surface of the substance after the buffer solution was added to the compound containing the NHS group was minimized.

In the present invention, liposomes can be dried and stored in the form of a film in an amount that can coat one or a certain number of sensor chips in one vial during the production of liposomes, and liposomes can be prepared using them when necessary.

In addition, a method of mixing a compound having an NHS group with a lipid and introducing the same into a surface of a substance includes a step of mixing a substance having an already hydrophobic surface, all substances capable of converting the surface into a hydrophobic substance, lipid particles such as liposome, It can be applied to surface chemistry such as all materials that can be. For example, the present invention is applicable to a substance (usually metal) constituting the surface of a biosensor or quartz crystal microbalance (QCM) biosensor using a surface plasmon resonance (SPR) principle .

In addition to the sensor having a metal surface, such as the modified microbalance, the surface of various types of sensors such as carbon nanotube gas sensor, semiconductor type gas sensor, nanowire FET (nanowire FET) gas sensor, NHS group can be introduced by the method of the present invention.

In addition, since the liposome is a lipid bilayer, a compound having an NHS group can be directly introduced. When liposomes are prepared by incorporating a compound having an NHS group, targeting ligands including antibodies can easily be introduced into liposomes. Liposomes with such goal-directed ligands can be used for delivery or diagnosis of therapeutic agents.

Hydrophobic poly (dimethylsiloxane); The PDMS surface can be coated with a lipid monolayer, while the PDMS surface can be plasma-oxidized to hydrophilic to be coated with a lipid bilayer. Therefore, it is possible to introduce an NHS group on the surface of the PDMS by various methods as described above.

Since a multi-well plate or culture dish used for immunoassay or cell culture is made of plastic such as polystyrene or polypropylene, it is possible to introduce a single layer of lipid. Therefore, NHS group is formed on the surface of a multi-well plate and a culture dish by the method of the present invention Can be introduced.

Nanomaterials can be applied to ultra-sensitive biosensors, diagnostics, therapeutic substance delivery, life science research, etc. Biofunctionalization is a key technology for such applications. The method according to the present invention can also be used for imparting biofunctionality of nanomaterials.

For example, in the case of TOPO-stabilized quantum dots stabilized with trioctylphosphine oxide (TOPO), TOPO can form a lipid monolayer directly on top of it with the property of having a hydrophobic surface . In particular, this method has the advantage that it does not replace the TOPO layer and does not expose the sensitive surface of the quantum dot because it is a layered layer on top of it. Gold or silver nanoparticles can form hydrophobic SAMs by using alkanethiol in the same manner as QCM or SPR surfaces, and a lipid monolayer can be formed thereon. Recently, methods for synthesizing nanoparticles directly surrounded by lipids have also been reported . Carbon nanotubes have a hydrophobic surface and a lipid layer can be formed on the surface. Therefore, it is possible to introduce an NHS group on the surface of the materials according to the method of the present invention, thereby conferring biofunctionality to the nanomaterial.

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail with reference to the following examples. However, the following examples are intended to illustrate the contents of the present invention, but the scope of the present invention is not limited to the following examples. Embodiments of the present invention are provided to more fully describe the present invention to those skilled in the art.

< Example  1> Palmitic acid NHS  Ester, QCM  On the surface Streptavidin  fixing

In this example, a quartz crystal microbalance (QCM) biosensor using a dipalmitoylphosphatidylcholine as a background lipid and a palmitic acid NHS ester (see FIG. 7) as a compound having an NHS group After the introduction, streptavidin protein was immobilized using NHS group. Here, since the QCM surface is made of hydrophilic gold, a lipid bilayer containing hydrocarbon having an NHS group may be formed on the QCM surface as shown in FIG. The lipid used in this example was purchased from Avanti Polar Lipids (Alabaster, USA) and the remaining proteins and reagents were purchased from Sigma-Aldrich (St. Louis, USA).

Preparation of measuring instrument for biosensor

The quartz crystal microbalance (QCM) was purchased from Japan Crystal Sunlife. The quadrilateral modified microbalance size is 8x8 mm, the diameter of the circular gold electrode is 5 mm, and the natural frequency of the quartz microbalance is about 10 MHz. Since the frequency of the micromanipulator decreases in proportion to the mass increase, if the receptor capable of binding the analyte is immobilized on the surface of the micromanipulator, it can be used as a biosensor . The modified microbalance biosensor system was prepared by the present inventor.

In this experiment, a well cell with a well to which a solution can be added and a flow cell with a solution flowing through the tube and brought into contact with the crystal scale electrode are connected together Respectively. First, a receptor protein that specifically binds to a detection substance is immobilized in a well cell. Subsequently, a buffer solution is injected through a pump and a sample to be detected is injected through an injection valve. When the analyte passes through the surface of the QCM mounted on the flow cell and binds to the antibody, the mass changes and the oscillation frequency changes. The frequency is measured by the frequency meter and sent to the computer so that the data can be analyzed.

Production of liposomes

In this example, liposomes were prepared by mixing dipalmitoyl phosphatidylcholine (DPPC) with palmitic acid N-hydroxysuccinimide ester (PA-NHS) in a molar ratio of 3: 1, Method. Here, DPPC was used as a background lipid to form a lipid layer, and PA-NHS was used as a hydrocarbon compound with an NHS group to cause NHS groups to appear in this lipid layer.

When liposomes are made, it is difficult to weigh a very small amount of lipid or NHS-containing compound, so it is necessary to make a large amount of lipid mixture as follows: A liposome was prepared from the retrieved film. 0.733 mL of a 3 mg / mL DPPC solution dissolved in chloroform and 0.354 mL of a 1 mg / mL PA-NHS solution dissolved in chloroform were added and 0.2 mL was added to each glass vial and the mixture was dried with nitrogen gas to prepare a lipid film. As a result, each vial contains 0.4 mg of DPPC and 0.0652 mg of PA-NHS, which corresponds to the amount of coating the four QCM sensor chips. The vials were sealed and stored at -20 ° C or -80 ° C until use.

To make the liposomes, 0.48 mL of 0.1 M M [2- (N-morpholino) ethanesulfonic acid] buffer (pH 6.0) was added to the vial containing the lipid film, the vial was filled with nitrogen, sealed and left at 50-60 ° C To hydrate the lipid mixture.

In order to minimize the hydrolysis of the NHS group in the hydration process, it is necessary to shorten the hydration time as much as possible. Therefore, the minimum time required for hydration was evaluated by varying the hydration time to 10, 20, and 30 minutes. As a result, since a sufficiently stable signal was obtained at 20 minutes, the hydration time was determined to be 20 minutes (see FIG. 9). After hydration, the vial solution was vigorously mixed with a vortex mixer for 1 minute or ultrasonication was applied to the solution to form a lipid in the form of a small unilamella vesicle (SUV) from the lipid.

Liposome coating

In order to clean the electrode surface of the modified microbalance, the modified microbalance was immersed in a piranha solution (H ₂ SO ₄ : H ₂ O ₂ = 7: 3) for 1 minute, rinsed with distilled water and ethanol, dried with nitrogen gas .

The QCM sensor chip was coated with the prepared liposome by the following method.

The cleaned QCM was mounted on a well-shaped cell, 100 μL of a liposome solution was added, and the solution was allowed to stand at 50 to 60 ° C. to form a lipid bilayer spontaneously on the surface of the gold electrode. Since the NHS group was hydrolyzed during the liposome coating process, the liposome coating time was changed to 10, 20, 30, 40 minutes. As a result, since a sufficiently stable signal was obtained at 10 minutes, the coating time was determined to be 10 minutes (see FIG. 10). The liposome solution was removed from the well-shaped cells and washed three times with phosphate buffered saline (PBS).

Streptavidin  Protein fixation

After changing the well cell into a flow cell, PBS was flowed at a flow rate of 20 μL / min to stabilize the frequency. The flow rate was changed to 10 μL / min and 0.1 mL of 25 μg / mL streptavidin protein solution was injected to allow the streptavidin protein solution to contact the QCM surface for 10 minutes. As a result, FIG. 11 shows that the streptavidin is fixed on the surface of the sensor chip and the frequency is decreased. On the other hand, when streptavidin was flowed into QCM coated with liposomes made only of DPPC without PA-NHS, frequency change was almost not observed as shown in FIG. It is believed that some of the frequency changes observed here are caused by nonspecific adsorption. These results demonstrate that streptavidin is immobilized in the QCM coated with liposome containing PA-NHS while forming covalent bonds through the NHS group.

Immobilization of antibodies and detection of antigens

To confirm whether the streptavidin was stabilized, the flow rate was changed to 20 μL / min and the biotinylated anti-goat immunoglobulin G antibody (Bt-Ab) was injected and fixed. 11, in QCM functionalized with liposome containing PA-NHS, a frequency change of about 80 Hz was observed when Bt-Ab was injected. However, in QCM functionalized with liposomes containing only DPPC, a frequency change of about 40 Hz was observed and the signal was unstable (Fig. 12). Some of the Bt-Ab immobilized on the liposome-functionalized QCM containing DPPC alone would be immobilized by nonspecifically adsorbed streptavidin, while the rest would have adsorbed Bt-Ab itself nonspecifically.

Two 20 μg / mL solutions of the goat IgG protein were injected into the two sensor chips immobilized on Bt-Ab. As a result, in the QCM functionalized with liposomes containing PA-NHS, a frequency change of about 50 Hz was continuously observed repeatedly, but in the QCM functionalized with DPPC, the frequency change was small and unstable at less than 30 Hz (FIGS. 11 and 12) .

FIG. 13 shows only the frequency change value when the above experiment was repeated 3 times or more and a goat IgG 20 μg / mL solution was injected. In both QCMs, when the goat IgG was injected for the first time, the frequency change was particularly large, which seems to be due to non-specific adsorption. However, we can observe almost constant frequency change from then on. Especially, the frequency change in QCM functionalized with liposome containing PA-NHS is much larger than QCM functionalized with liposome containing only DPPC. This experiment confirmed that the NHS group introduced through PA-NHS was effectively used for antibody immobilization.

The above results show that it is possible to introduce an NHS group on the surface of a sensor chip by adding a compound containing NHS group to the surface of the sensor chip by mixing with lipid.

< Example  2> on the immune plate Streptavidin  fixing

In this example, dipalmitoylphosphatidylcholine was used as a background lipid and a palmitoic acid NHS ester (see FIG. 7) was used as a compound having an NHS group, NHS group was introduced into an immunoplate well, and streptavidin ) Protein was immobilized. Here, since the surface of the immune plate is made of hydrophobic polystyrene, a lipid monolayer containing a hydrocarbon having an NHS group may be formed on the surface of the immune plate as shown in FIG. 5A. The lipid used in this example was purchased from Avanti Polar Lipids (Alabaster, USA) and the remaining proteins and reagents were purchased from Sigma-Aldrich (St. Louis, USA). The immune plate was a clear polystyrene 8-well strip plate from Corning (Corning, USA).

Production of liposomes

The same method as in Example 1 was used.

With liposome coating Streptavidin  Protein fixation

100 [mu] L of the prepared liposome solution was placed in each well and allowed to stand at 50 to 60 DEG C for 10 minutes so that a single lipid layer was spontaneously formed on the surface of the well. For comparison, the same experiment was carried out using liposomes prepared only with dipalmitoylphosphatidylcholine without including palmitic acid NHS ester.

Liposomal solution was removed from the wells and washed three times with PBS. 0.1 mL of 10 mg / mL streptavidin protein solution was added thereto, followed by reaction at room temperature for 10 minutes. After washing three times with PBS, 0.1 mL of 0.1% bovine serum albumin (BSA) solution was added and reacted at room temperature for 10 minutes.

horseradish peroxidase  Enzyme fixation and enzymatic activity evaluation

To evaluate the amount of fixed streptavidin protein, 0.01 mg / mL of horseradish peroxidase (HRP) enzyme with biotin group attached to each well was added to each well. After washing three times with PBS, 0.1 mL of 3,3 ', 5,5'-Tetramethylbenzidine (TMB) solution, which is a substrate of HRP enzyme, was added to each well and reacted at room temperature for 5 minutes. The HRP enzyme activity bound to each well was estimated by measuring the absorbance at 600 nm. This experiment was repeated six times and the results of the experiment are shown in Fig. In the graph, activity was also measured in liposome-coated wells (DPPC only) prepared only with dipalmitoylphosphatidylcholine, which may be the result of nonspecific adsorption of streptavidin or HRP enzyme in wells. However, in all experiments, it was confirmed that a much higher amount of HRP enzyme was bound to the liposome-coated wells (DPPC + NHS) containing palmitic acid NHS ester, &Lt; / RTI &gt; NHS). &Lt; / RTI &gt;

Claims (9)

Mixing a compound having an N-hydroxysuccinimide group with a lipid to form a liposome; And
Introducing the liposome to the surface of the material to form a lipid bilayer or a monolayer on the surface of the material; Wherein the N-hydroxysuccinimide group is introduced into the surface of the substance. delete The method according to claim 1,
Wherein the compound having the N-hydroxysuccinimide group is a lipid having an N-hydroxysuccinimide group or a hydrocarbyl group having an N-hydroxysuccinimide group. How to introduce. The method according to claim 1,
Wherein the material is a metal, a ceramic, a semiconductor or a polymer. The method according to claim 1,
Wherein a mixture of the N-hydroxysuccinimide group-containing compound and the lipid is dried in the form of a film, and a liposome is formed from the film, wherein the N-hydroxysuccinimide group is introduced into the surface of the substance. The method according to claim 1,
A method for introducing an N-hydroxysuccinimide group onto a surface of a substance, characterized in that the liposome is formed in a buffer solution having a pH of 4 to 6. The method according to claim 6,
Wherein the buffer solution is 2- (N-morpholino) ethanesulfonic acid. 2. A method for introducing an N-hydroxysuccinimide group into a surface of a substance, characterized in that the buffer solution is 2- (N-morpholino) ethanesulfonic acid. 6. The method of claim 5,
A method for introducing an N-hydroxysuccinimide group into a surface of a substance, wherein a liposome is formed from the film by adding a buffer solution, hydrating and mixing using a vortex mixer. The method according to claim 1,
Characterized in that the substance constitutes the surface of any one of a modified microbalance biosensor, a surface plasmon resonance biosensor, an immunoassay plate, a cell culture dish, or a plate. The N-hydroxysuccinimide group .

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