WO2018000339A1

WO2018000339A1 - Modified cardiolipin-coated magnetic nanobead and preparation method therefor

Info

Publication number: WO2018000339A1
Application number: PCT/CN2016/087910
Authority: WO
Inventors: 胡鹍辉; 祝亮; 邹定标; 陈永棠; 夏福臻; 钱纯亘
Original assignee: 深圳市亚辉龙生物科技股份有限公司
Priority date: 2016-06-30
Filing date: 2016-06-30
Publication date: 2018-01-04
Also published as: KR102208218B1; KR20190022762A

Abstract

A modified cardiolipin-coated magnetic nanobead and a preparation method therefor. The modified cardiolipin-coated magnetic nanobead comprises: modified cardiolipin and a magnetic nanobead. The modified cardiolipin is obtained by oxidizing and aminating a hydrophobic fatty acid side chain of cardiolipin, and contains an amino group; the modified cardiolipin is connected with the magnetic nanobead by means of the amino group.

Description

Modified cardiolipin coated nano magnetic beads and preparation method thereof

Technical field:

The invention relates to the field of in vitro detection, in particular to a modified cardiolipin coated nano magnetic bead and a preparation method thereof.

Background technique:

An anticardiolipin antibody is an antibody that reacts with a variety of antigenic materials containing a phospholipid structure. The antigen is a negatively charged phospholipid component that is involved in a variety of cell membranes. It is mainly found in patients with antiphospholipid syndrome, various autoimmune diseases and syphilis infections.

Antiphospholipid syndrome (APS) includes a variety of autoimmune diseases, more common in young people, the ratio of male to female incidence is about 2:8. Patients may have one or more manifestations that may affect multiple systems and organs, including: venous and arterial thrombosis, thrombocytopenia, habitual abortion, cardiomyopathy, heart disease, brain and kidney infarction, and pulmonary hypertension. Malignant APS can be characterized by progressive extensive thrombosis in the short term, resulting in multiple organ failure and even death. It can be secondary to systemic lupus erythematosus or other autoimmune diseases, but it can also occur alone (primary antiphospholipid syndrome).

Anti-phospholipid antibodies can also be produced in patients with syphilis. Syphilis is a sexually transmitted disease caused by the spirochete syphilis. More than 5 million people are reported to be infected with syphilis each year, including 30,000 babies born through congenital infections. Syphilis can be lurking and hidden in patients for many years and can lead to a variety of clinical manifestations. Patients in the concealed period of syphilis have no clinical symptoms, and the secondary stage lasts for a lifetime in about two-thirds of untreated patients. Infected persons are not contagious during the concealment period, but children born to mothers in concealed periods may be infected with congenital syphilis.

Antiphospholipid antibody assays are widely used in the diagnosis of phospholipid syndrome and in the non-dense spiral test of syphilis. This detection has the advantage of being inexpensive, fast and convenient to perform on a large number of samples. In addition, since the concentration of antiphospholipid antibodies will gradually decrease with the successful treatment of syphilis, the specificity of syphilis infection Antibody Treponema antibodies can persist for years or even lifetimes. Therefore, antiphospholipid antibody test is considered to be a better choice for monitoring syphilis treatment.

The traditional method for detecting antiphospholipid antibodies is to apply cardiolipin to a specific solid such as a microplate by physical adsorption, and then combine the antiphospholipid antibodies in the sample to be tested with the cardiolipin attached to the plate. To achieve capture of antiphospholipid antibodies in the sample. The concentration of the antiphospholipid antibody in the sample can be indirectly read by coloring the captured antiphospholipid antibody and measuring the luminescent concentration.

Since the cardiolipin immobilized by physical adsorption is easily dissociated and detached from the solid plate under the influence of external conditions such as solvent and heating, the stability of the test product prepared by the method is poor.

Summary of the invention:

Based on this, it is necessary to provide a modified cardiolipin coated nano magnetic bead for detecting the stability of an anticardiolipin antibody and a preparation method thereof.

A modified cardiolipin coated nano magnetic bead, comprising: modified cardiolipin and nano magnetic beads;

The modified cardiolipin is obtained by oxidizing and aminating a hydrophobic fatty acid side chain of a cardiolipin, the modified cardiolipin containing an amino group, and the modified cardiolipin is linked to the nanomagnetic beads through the amino group.

In one embodiment, the surface of the nanomagnetic beads contains a carboxyl group that forms an amide ester structure with the carboxyl group to link the modified cardiolipin and the nanomagnetic beads together.

A method for preparing the modified cardiolipin coated nano magnetic beads, comprising the following steps:

The cardiolipin and the peroxide are sufficiently reacted in the presence of the first solvent to obtain an intermediate product;

Adding excess ammonia water to the intermediate product and reacting sufficiently to obtain a modified cardiolipid, wherein the hydrophobic fatty acid side chain of the modified cardiolipin is oxidized and aminated, and the modified cardiolipin contains an amino group;

The modified cardiolipin and the nano magnetic beads are mixed and fully reacted to obtain a modified cardiolipin coated nano magnetic bead.

The modified cardiolipin coated nano magnetic beads directly bond the modified cardiolipin to the nano magnetic beads through chemical bonds, and have the characteristics of strong stability and controllable connection amount. Modified cardiolipin coating The nano magnetic beads can be directly used for the detection of antiphospholipid antibodies, and have higher stability than the detection products prepared by the conventional physical adsorption of cardiolipin.

DRAWINGS

1 is a flow chart showing a method of preparing a modified cardiolipin coated nanomagnetic bead according to an embodiment.

detailed description

The embodiments of the present invention will be described in detail with reference to the drawings and specific embodiments. Numerous specific details are set forth in the description below in order to provide a thorough understanding of the invention. However, the present invention can be implemented in many other ways than those described herein, and those skilled in the art can make similar modifications without departing from the spirit of the invention, and thus the invention is not limited by the specific embodiments disclosed below.

The modified cardiolipin coated nanomagnetic beads of one embodiment include: modified cardiolipin and nano magnetic beads.

Cardiolipin is an ester composed of three glycerols, two phosphoric acids, and four long-chain unsaturated alkyl groups. The structure contains two hydrophilic centers and four hydrophobic side chains.

The structural formula of cardiolipin is as follows:

The modified cardiolipin is obtained by oxidizing and aminating the hydrophobic fatty acid side chain of the cardiolipin, the modified cardiolipin contains an amino group, and the modified cardiolipin is linked to the nanomagnetic beads through the amino group.

Since the cardiolipin has four side chains, the four side chains can be simultaneously oxidized when the cardiolipin is modified, so the modified cardiolipin may contain a plurality of amino groups. Specifically, the modified cardiolipin may contain one to eight amino groups (-NH ₂ ).

Preferably, the modified cardiolipin contains one -NH ₂ .

The surface of the nanomagnetic beads may contain at least one of a carboxyl group (-COOH) and a terminal epoxy group (-CH(O)CH ₂ ).

In one embodiment, the surface of the nanomagnetic beads contains a carboxyl group, and the amino group and the carboxyl group form an amide ester structure (-CO-NH-) to link the modified cardiolipin and the nanomagnetic beads together.

In a particular embodiment, the modified cardiolipin coated nanomagnetic beads have the following structural formula:

Preferably, the surface of the nanomagnetic beads contains a terminal epoxy group, and the amino group and the terminal epoxy group form a secondary amine structure (-NH-) to link the modified cardiolipin and the nanomagnetic beads together.

Since the size of the cardiolipin molecule is very small, the space that can be modified is seriously insufficient, and modification of the hydrophilic phosphate ester center reduces the affinity of the cardiolipin and the antiphospholipid antibody, and even the antigen activity disappears.

The modified cardiolipin-coated nanomagnetic beads modify the hydrophilic side chain of the cardiolipin, retain the modified cardiolipin of the cardiolipin hydrophilic phosphate center, and utilize the amino and nano magnetic of the modified cardiolipin. The beads combine to allow the modified cardiolipin to be firmly attached to the surface of the nanomagnetic beads.

The modified cardiolipin coated nano magnetic beads directly bond the modified cardiolipin to the nano magnetic beads through chemical bonds, and have the characteristics of stable and controllable connection amount. The modified cardiolipin coated nano magnetic beads can be directly used for the detection of antiphospholipid antibodies, and has higher stability than the detection products prepared by the conventional physical adsorption of cardiolipin.

The preparation method of the above modified cardiolipin coated nano magnetic beads as shown in FIG. 1 comprises the following steps:

S10. The cardiolipin and the peroxide are sufficiently reacted in the presence of the first solvent to obtain an intermediate product.

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

The peroxide is peroxybenzoic acid, m-chloroperoxybenzoic acid, peroxyacetic acid or peroxypropionic acid.

The molar ratio of cardiolipin to peroxide is from 1:1 to 8.

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

S10 also includes the operation of purifying the intermediate product, which may be purified by liquid phase preparative chromatography. After purification, a modified cardiolipin having a purity of about 80% can be obtained.

In S10, the reaction temperature is from 60 ° C to 100 ° C.

S20, adding excess ammonia water to the intermediate product obtained in S10 and sufficiently reacting to obtain a modified cardiolipin.

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

Since the cardiolipin has four side chains, the four side chains can be simultaneously oxidized when the cardiolipin is modified, so the modified cardiolipin may contain a plurality of amino groups. Specifically, the modified cardiolipin may contain from 1 to 8 amino groups.

Preferably, the modified cardiolipin contains one -NH ₂ .

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

In S20, the reaction temperature is from 60 ° C to 100 ° C.

S30, mixing the modified cardiolipin obtained by S20 and the nano magnetic beads and reacting sufficiently to obtain a modified magnetic cardiophore coated nano magnetic bead.

In the prepared modified cardiolipin-coated nanomagnetic beads, the modified cardiolipin is linked to the nanomagnetic beads through the amino group.

Nanomagnetic beads can be purchased directly, for example, the carboxyl magnetic beads of MagnaBind Company's Cat. No. 21353, MagnaMedics's product number MD01010, one of which may be selected, or a few may be mixed in a certain ratio.

When the surface of the nanomagnetic beads contains a carboxyl group, S30 is: nanomagnetic beads, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCi) and 3-sulfonic acid-N-hydroxyl group The succinimide (Sulfo-NHS) is reacted in the presence of a second solvent to form an active intermediate, followed by the addition of the modified cardiolipin and sufficient reaction to obtain a modified cardiolipin coated nanomagnetic bead. The second solvent is DMSO, DMF, tetrahydrofuran or a phosphate buffer having a pH of 6.5 to 8.5, and the concentration of the nano magnetic beads is 5 mg/mL to 10 mg/mL, and 1-ethyl-3-(3-dimethylamino) The concentration of propyl)carbodiimide is from 0.5 mg/mL to 1.5 mg/mL, and the concentration of 3-sulfonic acid-N-hydroxysuccinimide is from 0.5 mg/mL to 1.5 mg/mL.

Taking the active cardiolipin containing one amino group as an example, when the surface of the nanomagnetic beads contains a carboxyl group, the modified cardiolipin coated nanomagnetic beads obtained by the reaction of the modified cardiolipin and the nanomagnetic beads have the following structural formula:

When the surface of the nanomagnetic beads contains a terminal epoxy group, S30 is: the nanometer magnetic beads and the modified cardiolipin are sufficiently reacted in the presence of a phosphate buffer solution having a pH of 6.5 to 8.5 to obtain a modified cardiolipin coated nanometer. Magnetic beads. The concentration of the nano magnetic beads is 5 mg/mL to 10 mg/mL.

Taking the active cardiolipin containing one amino group as an example, when the surface of the nanomagnetic beads contains a terminal epoxy group, the modified cardiolipin coated nanomagnetic beads obtained by the reaction of the modified cardiolipin and the nano magnetic beads have the following structural formula:

The preparation method of the modified cardiolipin coated nano magnetic beads, by modifying the hydrophobic side chain of the cardiolipin, obtaining the modified cardiolipin which retains the hydrophilic phosphate ester center of the cardiolipin, and using the modified cardiolipin The amino group is combined with the nanomagnetic beads to firmly bond the modified cardiolipin to the surface of the nanomagnetic beads.

The preparation method of the modified cardiolipin coated nano magnetic beads directly bonds the modified cardiolipin to the nano magnetic beads through chemical bonds, and the prepared modified cardiolipin coated nano magnetic beads are stable and connected. The controllable characteristics can be directly used for the detection of antiphospholipid antibodies, and has higher stability than the detection products prepared by the conventional physical adsorption of cardiolipin.

The following are specific examples.

Example 1: Preparation of modified cardiolipin

Under argon protection, 1 mmol of cardiolipin was added to a 20 mL glass flask containing 5 mL of dichloromethane, and magnetically stirred at room temperature until completely dissolved. 3.5 mmol of m-chloroperoxybenzoic acid was added and the reaction was maintained at room temperature for 36 hours. The reaction solution was poured into an ice water bath, and the organic compound was extracted with petroleum ether. After evaporating the petroleum ether under reduced pressure and drying, 5 mL of 15% by mass aqueous ammonia was added and the mixture was warmed to reflux for 8 hours. After cooling, it was poured into ethyl acetate and washed with water three times. After drying, a colorless oil was obtained, which was separated and purified by liquid chromatography to obtain a modified cardiolipin having an amino group in a side chain. MS (ESI ⁺ , m/z): 1481.99142.

Example 2: Preparation of modified cardiolipin coated nanomagnetic beads

Take 50 μL of a magnetic bead suspension containing 8 mg of terminal epoxy groups on the surface, add 850 μL of 30 mmol/L phosphate buffer, add 100 μL of 1 mg/mL amino-modified modified cardiolipin, and react at 37 ° C with shaking. 4 hours. The unbound modified cardiolipin was magnetically separated, and the remaining magnetic beads were dispersed in 2 mL of 50 mmol/L Tris buffer.

Example 3: Preparation of modified cardiolipin coated nanomagnetic beads

200 μL of a magnetic bead suspension containing 10 mg of a terminal carboxyl group on the surface was added, and 400 μL of 20 mg/ml EDCi phosphate buffer and 400 uL of 20 mg/mL NHS phosphate buffer were sequentially added thereto, and the reaction was shaken at 37 ° C for 30 minutes. After magnetic separation, the magnetic beads were reconstituted to 900 uL with 20 mmol/L phosphate buffer, and 100 μL of 1 mg/mL amino-modified modified cardiolipin was added to the mixture, and reacted at 37 ° C for 2 hours. The unbound modified cardiolipin was magnetically separated, and the remaining magnetic beads were dispersed in 2 mL of 50 mmol/L Tris buffer.

Example 4: Modification of modified cardiolipin coated nanomagnetic beads with antiphospholipid samples

200 μL of the modified cardiolipin coated nanomagnetic bead solution prepared in Examples 1 to 3 was separately taken, and 5 μL of the antiphospholipid antibody serum sample was added and incubated at 37 ° C for 30 minutes. After magnetic separation, the modified cardiolipin coated nanomagnetic beads were reconstituted to 200 μL, and the horseradish peroxidase-labeled secondary antibody was added. After incubation at 37 ° C for 30 minutes, the cells were washed successively, added with TMB substrate solution and incubated for 10 minutes. Add 100 μL of stop solution, The OD value was read on the microplate reader within 10 minutes to obtain the luminescence signal value of the sample.

Three cardiolipin positive serum samples and three cardiolipin negative serum samples were taken separately, and the conventional physical adsorption method was used as a control. The measured OD values were compared to obtain the following Table 1.

Table 1: Examples 1 to 3 and control group (physical adsorption method) measure OD values of different samples

It can be seen from Table 1 that the modified cardiolipin coated nano-magnetic beads prepared in Examples 1 to 3 are compared with the magnetic beads (control group) adsorbed by the physical adsorption method in the test of the anti-phospholipid antibody sample, Example 1 The modified erythrocyte-coated nano-magnetic beads prepared by ~3 had significantly lower luminescence signal values than the control group when the negative samples were measured (4 to 8 times lower), and the luminescence signal was significantly higher than that of the control group when measuring positive samples. Improvement (up to 5 to 10 times).

Therefore, the modified cardiolipin coated nano magnetic beads prepared in Examples 1 to 3 have a significant improvement in detection sensitivity compared to the magnetic beads of the conventional physical adsorption method when measuring the antiphospholipid sample.

The above-mentioned embodiments are merely illustrative of one or more embodiments of the present invention, and the description thereof is not to be construed as limiting the scope of the invention. It should be noted that a number of variations and modifications may be made by those skilled in the art without departing from the spirit and scope of the invention. Therefore, the scope of the invention should be determined by the appended claims.

Claims

A modified cardiolipin coated nano magnetic bead, comprising: modified cardiolipin and nano magnetic beads;

The modified cardiolipin is obtained by oxidizing and aminating a hydrophobic fatty acid side chain of a cardiolipin, the modified cardiolipin containing an amino group, and the modified cardiolipin is linked to the nanomagnetic beads through the amino group.
The modified cardiolipin coated nanomagnetic bead according to claim 1, wherein the surface of the nanomagnetic bead contains a carboxyl group, and the amino group forms an amide ester structure with the carboxyl group to thereby modify the modified cardiolipin And the nano magnetic beads are connected together.
The modified cardiolipin coated nanomagnetic bead according to claim 2, wherein the modified cardiolipin coated nanomagnetic bead has the following structural formula:
The modified cardiolipin coated nanomagnetic bead according to claim 1, wherein the surface of the nanomagnetic bead contains a terminal epoxy group, and the amino group and the terminal epoxy group form a secondary amine structure, thereby The modified cardiolipin and the nanomagnetic beads are joined together.
The modified cardiolipin coated nanomagnetic bead according to claim 4, wherein the modified cardiolipin coated nanomagnetic bead has the following structural formula:
A method for preparing a modified cardiolipin coated nanomagnetic bead according to any one of claims 1 to 5, comprising the steps of:

The cardiolipin and the peroxide are sufficiently reacted in the presence of the first solvent to obtain an intermediate product;

Adding excess ammonia water to the intermediate product and reacting sufficiently to obtain a modified cardiolipid, wherein the hydrophobic fatty acid side chain of the modified cardiolipin is oxidized and aminated, and the modified cardiolipin contains an amino group;

The modified cardiolipin and the nano magnetic beads are mixed and fully reacted to obtain a modified cardiolipin coated nano magnetic bead.
The method for preparing a modified cardiolipin coated nano magnetic bead according to claim 6, wherein the operation of fully reacting cardiolipin and peroxide in the presence of the first solvent The peroxide is peroxybenzoic acid, m-chloroperoxybenzoic acid, peroxyacetic acid or peroxypropionic acid, and the molar ratio of the cardiolipin to the peroxide is 1:1 to 8, The first solvent is dichloromethane, chloroform, chloroform, benzene or toluene.
The method for preparing a modified cardiolipin coated nano magnetic bead according to claim 6, wherein in the operation of adding an excess amount of ammonia water to the intermediate product and sufficiently reacting, the mass concentration of the ammonia water is 10% to 30%.
The method for preparing a modified cardiolipin coated nano magnetic bead according to claim 6, wherein the surface of the nanomagnetic bead contains a carboxyl group;

The operation of mixing the modified cardiolipin and the nano magnetic beads and fully reacting to obtain the modified cardiolipin coated nano magnetic beads is: the nano magnetic beads, 1-ethyl-3-(3- The dimethylaminopropyl)carbodiimide and the 3-sulfonic acid-N-hydroxysuccinimide are reacted in the presence of a second solvent to form an active intermediate, followed by the addition of the modified cardiolipin and sufficient reaction The modified cardiolipin coated nano magnetic beads are obtained, wherein the second solvent is DMSO, DMF, tetrahydrofuran or a phosphate buffer having a pH of 6.5-8.5, and the concentration of the nano magnetic beads is 5 mg/mL. ～10 mg/mL, the concentration of the 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide is from 0.5 mg/mL to 1.5 mg/mL, and the 3-sulfonyl-N- The concentration of hydroxysuccinimide is from 0.5 mg/mL to 1.5 mg/mL.
The method for preparing a modified cardiolipin coated nano magnetic bead according to claim 6, wherein the surface of the nanomagnetic bead contains a terminal epoxy group;

The operation of mixing the modified cardiolipin and the nano magnetic beads and fully reacting to obtain the modified cardiolipin coated nano magnetic beads is: the nano magnetic beads and the modified cardiolipin are at a pH of 6.5 The modified cardiolipin-coated nanomagnetic beads are obtained by sufficiently reacting in the presence of a phosphate buffer of ～8.5, wherein the concentration of the nanomagnetic beads is 5 mg/mL to 10 mg/mL.