KR102208218B1 - Magnetic nanobead coated with modified cardiolipin and method for manufacturing the same - Google Patents

Abstract

The present invention discloses a magnetic nanobead coated with a modified cardiolipin and a method for producing the same, wherein the magnetic nanobead coated with a modified cardiolipin includes a modified cardiolipin and a magnetic nanobead; The modified cardiolipin is obtained by oxidation and amination of the hydrophobic fatty acid side chain of cardiolipin, the modified cardiolipin contains an amino group, and the modified cardiolipin is bonded to the magnetic nanobead through an amino group.

Description

Magnetic nanobead coated with modified cardiolipin and method for manufacturing the same

The present invention relates to the field of in vitro diagnostics, and in particular, to a modified cardiolipin coated magnetic nanobead and a method of manufacturing the same.

Anti-cardiolipin antibodies are antibodies capable of reacting with antigenic substances containing various phospholipid structures, and antigens are negatively charged phospholipid components involved in the construction of various cell membranes.

Antiphospholipid Antibody Syndrome (APS) includes a variety of autoimmune diseases and is more common in young people, with an incidence rate of about 2:8 in men and women. Patients may exhibit one or more symptoms that can affect several systems and organs, including venous and arterial thrombosis, thrombocytopenia, habitual abortion, cardiomyopathy, heart disease, brain and kidney infarction, and pulmonary hypertension. Malignant APS can manifest as a progressive and extensive thrombus formation in a short period of time, leading to multiple organ failure and even death. Systemic lupus erythematosus and other autoimmune diseases may be secondary or may occur alone (primary antiphospholipid antibody syndrome).

Antiphospholipid antibodies can also be generated in the body of a patient with syphilis, and syphilis is a transgressive disease caused by the spiral bacterium Treponema pallidum. Reportedly, more than 5 million people are infected with syphilis each year, including 30,000 infants from congenital infections. Syphilis can hide or hide in the patient's body for many years, and can cause a variety of clinical symptoms. Here, patients in the incubation phase of syphilis have no clinical symptoms, and the next stage lasts for life among about two-thirds of untreated patients. Although the infected are not contagious during the incubation period, newborns born by the mother in the incubation period are likely to become congenital syphilis.

Antiphospholipid antibody detection is widely used in the diagnosis of antiphospholipid antibody syndrome and treponemapalidum test of syphilis. This detection has advantages, such as inexpensive, fast, and convenient processing of multiple samples. Alternatively, the more successful the syphilis treatment is, the gradually the concentration of the antiphospholipid antibody decreases, but the concentration of the treponemapalidum antibody, a specific antibody infected with syphilis, not only lasts for many years, but can even be high during transmigration. Therefore, testing for detection of antiphospholipid antibodies is considered a better option to monitor syphilis treatment.

A typical method for detecting an antiphospholipid antibody is to apply cardiolipin on a specific solid such as an ELISA plate by physical adsorption, and then in a sample to detect cardiolipin attached to the ELISA plate. Combining with an antiphospholipid antibody results in capture of the antiphospholipid antibody in the sample. By detecting the luminescence concentration after coloring the captured antiphospholipid antibody, the concentration of the antiphospholipid antibody in the sample can be indirectly read.

Since the cardiolipin immobilized by the physical adsorption method is easily dissociated and separated from the solid plate by the influence of external conditions such as solvent and heating, the stability of the detection product manufactured according to this method is greatly reduced.

Accordingly, there is a need to provide a magnetic nanobead coated with a modified cardiolipin having excellent stability for detecting an anticardiopine antibody, and a method of manufacturing the same.

Magnetic nanobeads coated with modified cardiolipin include modified cardiolipin and magnetic nanobeads,

The modified cardiolipin is obtained through oxidation and amination of the side chain of the hydrophobic fatty acid of the cardiolipin, the modified cardiolipin contains an amino group, and the modified cardiolipin is bonded to the magnetic nanobead through the amino group. .

In one embodiment, the surface of the magnetic nanobead includes a carboxyl group, and the amino group forms an amide ester structure with the carboxyl group, thereby binding the cardiolipin to the nanobead.

The method for producing magnetic nanobeads coated with the modified cardiolipin,

Obtaining an intermediate product by sufficiently reacting cardiolipin and peroxide under conditions in the presence of a first solvent;

After adding an excess of ammonia water to the intermediate product, it is sufficiently reacted to obtain a modified cardiolipin, wherein the hydrophobic fatty acid side chain of the modified cardiolipin undergoes oxidation and amination, and the modified cardiolipin contains an amino group. step; And

And sufficiently reacting the modified cardiolipin and magnetic nanobeads to obtain a magnetic nanobead coated with the modified cardiolipin.

The magnetic nanobeads coated with such modified cardiolipin have excellent stability and the ability to control the amount of linkage by directly bonding the magnetic nanobeads and the modified cardiolipin through chemical bonding. Magnetic nanobeads coated with such modified cardiolipin can be used directly for detection of antiphospholipid antibodies, and have higher stability when compared to detection products prepared through conventional physical adsorption method for cardiolipin hops. .

1 is a flow chart illustrating a method of manufacturing magnetic nanobeads coated with modified cardiolipin according to an embodiment.

The above objects, characteristics and advantages of the present invention will become more apparent by describing embodiments of the detailed description with reference to the accompanying drawings. For a thorough understanding of the invention, many specific details have been set forth in the description. However, the present invention can be implemented in many other forms other than those described in the present specification, and those skilled in the art can make similar modifications without departing from the scope of the present invention, and accordingly, the present invention is provided by the specific embodiments disclosed below. Not limited.

Magnetic nanobeads coated with modified cardiolipin according to an embodiment include: modified cardiolipin and magnetic nanobeads.

Cardiolipin is an ester material composed of 3 glycerol, 2 phosphoric acid and 4 long-chain unsaturated alkyl groups, and its structure includes 2 hydrophilic centers and 4 hydrophobic side chains.

The structural formula of cardiolipin is as follows.

The modified cardiolipin is obtained by oxidation and amination of the hydrophobic fatty acid side chain of the cardiolipin, and the modified cardiolipin contains an amino group, and the modified cardiolipin is bonded to the magnetic nanobead through an amino group.

Since cardiolipin has four side chains, since cardiolipin can oxidize at the same time four side chains when modified, the modified cardiolipin may contain a plurality of amino groups. Specifically, the modified cardiolipin may include 1 to 8 amino groups (-NH ₂ ).

Preferably, the modified cardiolipin comprises one -NH ₂ .

The surface of the magnetic nanobead may include at least one of a carboxyl group (-COOH) and a terminal epoxy group (-CH (O) CH ₂ ).

In one embodiment, the surface of the magnetic nanobead includes a carboxyl group, and the amino group and carboxyl group form an amide ester structure (-CO-NH-) to connect the modified cardiolipin and the magnetic nanobead.

In one specific embodiment, the structural formula of the magnetic nanobead coated with modified cardiolipin is as follows.

Preferably, the surface of the magnetic nanobead includes a terminal epoxy group, and the amino group and the terminal epoxy group form a secondary amine structure (-NH-) to connect the modified cardiolipin and the magnetic nanobead.

In one specific embodiment, the structural formula of the magnetic nanobead coated with modified cardiolipin is as follows.

Due to the very small size of the cardiolipin molecule, the space for modification is extremely insufficient, and the modification of the hydrophilic phosphate center lowers the affinity of the cardiolipin and antiphospholipid antibody, and even causes the antigenic activity to disappear.

The magnetic nanobead coated with the modified cardiolipin maintains the modification of the cardiolipin hydrophilic phosphate center through the modification of the hydrophobic side chain of the cardiolipin, while the amino group of the modified cardiolipin is the magnetic nanobead and By binding, the modified cardiolipin is firmly connected to the surface of the magnetic nanobead.

The magnetic nanobeads coated with the modified cardiolipin have the characteristics of excellent stability and controllable amount of connection by directly and firmly connecting the modified cardiolipin to the magnetic nanobeads through chemical bonding. Magnetic nanobeads coated with such modified cardiolipin can be used directly for detection of antiphospholipid antibodies, and have higher stability when compared to detection products prepared by conventional physical adsorption method of hoping cardiolipin. .

As shown in Figure 1, the method of manufacturing the magnetic nanobead coated with the modified cardiolipin includes the following steps.

Step S10: The intermediate product is obtained by sufficiently reacting cardiolipin and peroxide under conditions in the presence of the first solvent.

The fatty acid side chain of cardiolipin is oxidized by the reaction of cardiolipin and peroxide.

The peroxide is benzoic acid, meta-chloroperbenzoic acid, peracetic acid, or peroxypropionic acid.

The molecular ratio of cardiolipin and peroxide is 1:1 to 8.

The first solvent is dichloromethane, trichloromethane, chloroform, benzene, or toluene.

Step S10 further includes a process of purifying an intermediate product that can be purified by liquid chromatography. After purification, a modified cardiolipin having a purity of about 80% can be obtained.

In step S10, the reaction temperature is 60 ℃ ~ 100 ℃

Step S20: The intermediate product obtained in step S10 is reacted sufficiently by adding an excess of aqueous ammonia to obtain a modified cardiolipin.

The hydrophobic fatty acid side chain of cardiolipin is oxidized and aminated by peroxide and aqueous ammonia, and the modified cardiolipin thus obtained contains an amino group.

Since cardiolipin has four side chains, when cardiolipin is modified, four side chains can be oxidized at the same time, so the modified cardiolipin may contain a plurality of amino groups. Specifically, the modified cardiolipin may include 1 to 8 amino groups (-NH ₂ ).

Preferably, the modified cardiolipin comprises one -NH ₂ .

The mass concentration of aqueous ammonia is 10% to 30%.

In step S20, the reaction temperature is 60 ℃ ~ 100 ℃.

Step S30: The modified cardiolipin and magnetic nanobeads obtained in step S20 are mixed and sufficiently reacted to obtain magnetic nanobeads coated with the modified cardiolipin.

In the obtained modified cardiolipin coated magnetic nanobeads, the modified cardiolipin is linked to the magnetic nanobeads through an amino group.

The surface of the magnetic nanobead may include at least one of a carboxyl group (-COOH) and a terminal epoxy group (-CH (O) CH ₂ ).

Magnetic nanobeads can be purchased directly from the market. For example, MagnaBind's Cat. No. 21353 carboxyl group magnetic beads and Cat. No. One of MD01010 can be selected, or multiple can be selected and mixed in a predetermined ratio.

When the surface of the magnetic nanobead contains a carboxyl group, in step S30, the magnetic nanobead, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) (EDCi) and 3-sulfonate-N-hydroxysuccinimide (Sulfo-NHS) were reacted in the presence of a second solvent to produce an active intermediate, and then modified cardio Lipin is added and reacted sufficiently to obtain magnetic nanobeads coated with modified cardiolipin. Here, the second solvent is DMSO, DMF, tetrahydrofuran, or a phosphate buffer with a pH of 6.5 to 8.5, and the concentration of the magnetic nanobeads is 5 mg/mL to 10 mg/mL, and 1-ethyl-3-(3- The concentration of dimethylpropyl)carbodiimide is 0.5 mg/mL to 1.5 mg/mL, and the concentration of 3-sulfonic acid-N-enehydroxysuccinimide is 0.5 mg/mL to 1.5 mg/mL.

For example, when the activated cardiolipin containing one amino group is included on the surface of the nanobead, the structural formula of the modified cardiolipin-coated magnetic nanobead obtained by the reaction of the modified cardiolipin and the magnetic nanobead is As in the following.

In the case of including a terminal epoxy group on the surface of the magnetic nanobeads, in step S30, the magnetic nanobeads and the modified cardiolipin are sufficiently reacted in a phosphate buffer of pH 6.5 to 8.5 to obtain a magnetic nanobead coated with the modified cardiolipin. Here, the concentration of the magnetic nanobead is 5mg/mL~10mg/mL.

For example, if an active cardiolipin containing one amino group is included on the surface of a magnetic nanobead, the structural formula of a nanobead coated with a modified cardiolipin obtained by reaction of the modified cardiolipin and magnetic nanobeads Is as in the following.

The method of manufacturing such modified cardiolipin-coated magnetic nanobeads maintains the modified cardiolipin in the center of the hydrophilic phosphate of cardiolipin through modification of the hydrophobic side chain of the cardiolipin, and the amino group of the modified cardiolipin is magnetic. Combined with the nanobeads, the modified cardiolipin is tightly bound to the surface of the magnetic nanobeads.

The method of manufacturing such modified cardiolipin coated magnetic nanobeads directly and tightly binds the modified cardiolipin to the magnetic nanobeads through chemical bonding, and has a property of being stable and capable of controlling the amount of connection. Magnetic nanobeads coated with such modified cardiolipin can be used directly for the detection of antiphospholipid antibodies, and have higher stability compared to detection products prepared by conventional physical adsorption method for cardiolipin hopping. .

A specific example is as follows.

Example 1: Preparation of modified cardiolipin

Under the protection of argon gas, 1 mmol of cardiolipin was added to a 20 mL glass flask containing 5 mL of dichloromethane, and sufficiently dissolved by magnetic stirring at room temperature. 3.5 mmol of m-chloroperoxybenzoic acid was added and reacted at room temperature for 36 hours. The reaction solution was poured into an ice-water bath, and organic compounds were extracted using petroleum ether. After drying by evaporation under reduced pressure on petroleum ether, ammonia water having a mass fraction of 5 mL of 15% was added, followed by heating and reacting for 8 hours to reflux. After cooling, poured into ethyl acetate and washed three times with water. After drying, a colorless oily substance is obtained, separated and purified by liquid chromatography to obtain a modified cardiolipin having an amino group in the side chain. MS (ESI ⁺ , m/z): 1481.99142.

Example 2: Preparation of magnetic nanobeads coated with modified cardiolipin

After taking 50 μ of a suspension containing magnetic beads having a terminal epoxy group on the surface of 8 mg, 850 μl of a 30 mmol/L phosphate buffer was added, and 100 μL of cardiolipin modified by an amino group of 1 mg/mL was added during mixing by shaking. Then, it was reacted at 37° C. for 4 hours. The unbound modified cardiolipin is magnetically separated, and the remaining magnetic beads are dispersed in 2 mL of 50 mmol/L Tris buffer.

Example 3: Preparation of magnetic nanobeads coated with modified cardiolipin

After taking 200 μL of a suspension containing magnetic beads having a terminal epoxy group on the surface of 10 mg, 400 μL of 20 mg/mL EDCi phosphate buffer and 400 μL of 20 mg/mL NHS phosphate buffer were sequentially added, followed by shaking reaction at 37° C. for 30 minutes. Perform. After magnetic separation, the magnetic beads were re-dissolved in 20 mmol/phosphate buffer until 900 uL, and in the process of shaking and uniformly mixing, 100 μL of cardiolipin modified by 1 mg/mL of aminomi group was added to 37°C. Reacted at for 2 hours. The unbound modified cardiolipin is magnetically separated, and the remaining magnetic beads are dispersed in 2 mL of 50 mmol/L Tris buffer.

Example 4: Reaction of magnetic nanobeads coated with modified cardiolipin and antiphospholipid samples

200 µl of the magnetic nanobead solution coated with the modified cardiolipin prepared in Examples 1 to 3 were each taken, 5 µl of an antiphospholipid antibody serum sample was added, and then incubated at 37°C for 30 minutes. After magnetic separation, the magnetic nanobeads coated with modified cardiolipin were redissolved until 200 μL, and then a secondary antibody labeled with Horseradish peroxidase was added, and incubated at 37°C for 30 minutes. , Wash sequentially, add TMB substrate solution, and incubate for 10 minutes, add 100 µl of stop solution, and the luminescence signal value of the sample through the OD value in a microplate reader within 10 minutes. To obtain.

Three cardiolipin positive serum samples and three cardiolipin negative serum samples were taken, respectively, and OD values detected as compared with the conventional physical adsorption method are shown in Table 1.

Table 1: OD values detected in different samples of Examples 1-3 and the control (physical adsorption method)

Referring to Table 1, the luminescence signal when detecting the antiphospholipid antibody sample from the magnetic nanobeads coated with modified cardiolipin prepared in Examples 1 to 3 and the magnetic beads (control) adsorbed by the physical adsorption method In contrast, the negative samples detected by the magnetic nanobeads coated with modified cardiolipin prepared according to Examples 1 to 3 had a significantly lower luminescence signal value than the control group (reduced 4 to 8 times), and detected positive In the sample, the emission signal value was significantly improved (4 to 8 times higher) than the control.

Therefore, the antiphospholipid samples detected by the nanomagnetic beads coated with modified cardiolipin prepared in Examples 1 to 3 have a significantly improved detection sensitivity compared to the magnetic beads hopped by the conventional physical adsorption method.

The above-described embodiments merely represent one or several embodiments of the present invention, and the description thereof is described in more detail and detail, but should not be understood as limiting the scope of the present invention. Those skilled in the art should know that modifications and improvements can be made without departing from the spirit and scope of the present invention, and all of them fall within the protection scope of the present invention. Accordingly, the scope of protection of the present invention will depend on the appended claims.

Claims (10)

Including modified cardiolipin and magnetic nanobead,
The modified cardiolipin is obtained by oxidation and amination of the hydrophobic fatty acid side chain of cardiolipin, the modified cardiolipin contains an amino group, and the modified cardiolipin is bonded to the magnetic nanobead through the amino group. ;
The surface of the magnetic nanobead includes a terminal epoxy group, and the amino group and the terminal epoxy group form a secondary amine structure to connect the modified cardiolipin and the magnetic nanobead, and a magnetic coated with the modified cardiolipin The structural formula of the nanobead is a magnetic nanobead coated with a modified cardiolipin, characterized in that the following.

In the method for producing magnetic nanobeads coated with the modified cardiolipin of claim 1,
Cardiolipin and peroxide are sufficiently reacted in the presence of a first solvent to obtain an intermediate product;
After sufficiently reacting by adding an excess of aqueous ammonia to the intermediate product, a modified cardiolipin is obtained, wherein the hydrophobic fatty acid side chain of the modified cardiolipin undergoes oxidation and amination, and the modified cardiolipin contains an amino group. ; And
Mixing and sufficiently reacting the modified cardiolipin and magnetic nanobeads to obtain a magnetic nanobead coated with the modified cardiolipin
A method of manufacturing magnetic nanobeads coated with modified cardiolipin, characterized in that it comprises a. The method of claim 2,
In the step of sufficiently reacting the cardiolipin and peroxide in the presence of a first solvent, the peroxide is benzoic acid peroxide, meta-chloroperbenzoic acid, peracetic acid or propionic peroxide, and the molecular ratio of cardiolipin and the peroxide is 1: 1 to 8, wherein the first solvent is dichloromethane, trichloromethane, chloroform, benzene, or toluene. Method for producing magnetic nanobeads coated with modified cardiolipin. The method of claim 2,
In the step of sufficiently reacting by adding an excess of aqueous ammonia to the intermediate product, the mass concentration of the aqueous ammonia is 10% to 30%, wherein the method of producing magnetic nanobeads coated with modified cardiolipin. The method of claim 2,
The surface of the magnetic nanobead contains a carboxyl group;
The step of obtaining a magnetic nanobead coated with the modified cardiolipin by mixing and sufficiently reacting the modified cardiolipin and the magnetic nanobead is, the magnetic nanobead, 1-ethyl-3-(3-dimethylaminopropyl)car After reacting bodyimide and 3-sulfonate-N-hydroxysuccinimide in the presence of a second solvent to produce an active intermediate, the modified cardiolipin is added and reacted sufficiently to coat the modified cardiolipin Obtained magnetic nanobeads;
Here, the second solvent is DMSO, DMF, tetrahydrofuran, or a phosphate buffer having a pH of 6.5 to 8.5, the concentration of the magnetic nanobeads is 5 mg/mL to 10 mg/mL, and the 1-ethyl-3-(3- The concentration of dimethylaminopropyl) carbodiimide is 0.5 mg/mL to 1.5 mg/mL, and the concentration of 3-sulfonate-N-hydroxysuccinimide is 0.5 mg/mL to 1.5 mg/mL. To, a method of manufacturing magnetic nanobeads coated with modified cardiolipin. The method of claim 2,
The surface of the magnetic nanobead includes a terminal epoxy group;
The step of obtaining a magnetic nanobead coated with the modified cardiolipin by mixing and sufficiently reacting the modified cardiolipin and the magnetic nanobead is, the magnetic nanobead and the modified cardiolipin are sufficiently mixed in a phosphate buffer solution having a pH of 6.5 to 8.5. Reacting to obtain the modified cardiolipin coated magnetic nanobeads;
Here, the concentration of the magnetic nanobeads is 5mg / mL ~ 10mg / mL, characterized in that, the method for producing a magnetic nanobead coated with modified cardiolipin. delete delete delete delete

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