TWI665447B - Detection microparticles and manufacturing method thereof - Google Patents

Abstract

A detection particle is used to capture pathogens contained in a test specimen. The detection particle includes a particle body, an affinity coating, a plurality of target antibodies, and a plurality of chain branches. The affinity coating is coated on the peripheral surface of the microparticle body, and the affinity coating is formed of a biocompatible material. The biocompatible material has at least one or more functional groups selected from the group consisting of an amine group, a carboxyl group, an alcohol group, and a thiol group. Several target antibodies are scattered on the peripheral surface of the particle body to bind to the pathogen. One end of the chain branch is fixed on the surface of the affinity membrane, and the other end is used to graft the target antibody. There are electron acceptors at each end of the chain branch to form a covalent bond with the electron supplier of the affinity coating and the target antibody, respectively.

Description

Detection particle and manufacturing method thereof

The present invention relates to a detection particle and a manufacturing method thereof, and in particular to a detection particle and a manufacturing method thereof that can be used to capture pathogens contained in a test specimen.

In the field of biosensors, many different types of biosensors have been developed that can be used in medical diagnosis, new drug development, cancer screening, food testing, and environmental assessment. The advantage of the biosensor is that the analysis speed is fast, only a small amount of samples and reagents are needed, and a large amount of analysis data can be obtained at the same time. In particular, for highly infectious diseases / viruses, such as dengue virus, if a biosensor can be used as an in vitro detection method, it will definitely accelerate the diagnosis time of the disease, quickly isolate the patient, and quickly establish Medical protection network to effectively disinfect infected areas.

However, the use of existing biosensors as a method for detecting epidemic diseases is not yet mature, and there are still many shortcomings to be overcome. For example, most of the existing biosensors use specific antibodies on grafts and carriers to capture pathogens in the test specimen, and then use reagents or fluorescent coloration, change in the frequency of vibration and other methods to interpret The final test result, however, the traditional grafting process between the specific antibody and the carrier still has the problem of poor binding between the two, which makes it impossible to effectively capture the pathogen and affect the actual detection effect. In view of this, it is indeed necessary to improve it.

The main object of the present invention is to provide a detection particle, which has a target antibody firmly bonded to its surface, and is used to effectively capture the target pathogen.

A second object of the present invention is to provide a method for manufacturing a detection particle, which can efficiently and stably bind a target antibody to the surface of a detection particle so that the surface of the detection particle has a better modification effect.

The detection particles of the present invention can be used to capture pathogens contained in a test specimen. The detection particles include a particle body, an affinity coating, a plurality of target antibodies, and a plurality of chain branches. The affinity coating is coated on the peripheral surface of the microparticle body, and the affinity coating is formed of a biocompatible material. The biocompatible material has at least one or more functional groups selected from the group consisting of an amine group, a carboxyl group, an alcohol group, and a thiol group. Several target antibodies are scattered on the peripheral surface of the particle body to bind to the pathogen. One end of the chain branch is fixed on the surface of the affinity membrane, and the other end is used to graft the target antibody. There are electron acceptors at each end of the chain branch to form a covalent bond with the electron supplier of the affinity coating and the target antibody, respectively.

In one embodiment of the present invention, the affinity film is formed of collagen and includes amine, carboxyl, alcohol, and thiol functional groups.

In an embodiment of the present invention, the diameter of the microparticle body is in a range of 30 nm to 500 nm.

In an embodiment of the present invention, the material of the particle body includes a material having magnetoelectricity under an electric field.

In an embodiment of the present invention, one end of the above-mentioned branched branch is formed by removing a diazonium group, and an electron acceptor is formed, and the electron acceptor is connected with a functional group on the affinity film. The carried electron supplier reacts to form a covalent bond.

In an embodiment of the present invention, the other end of the aforementioned branched branch is formed by another ring-opening reaction of an epoxy group, and another electron acceptor is formed with the electron acceptor on the target antibody. React to form covalent bonds.

The invention further provides a method for manufacturing detection particles. The detection particles produced can stably bind the target antibody to the surface thereof, so as to improve the effectiveness of capturing pathogens, and then obtain accurate detection results.

The method for producing a detection particle of the present invention includes the following steps. Provide particle body. The first surface modification step includes reacting the microparticle body with a bio-affinity material to form an affinity coating on the microparticle body, wherein the bio-affinity material includes at least one selected from the group consisting of amine, carboxyl, alcohol, and thiol groups At least one or more of the above functional groups. The second surface modification step includes reacting the particle body that has undergone the first surface modification step with a first compound, and an electron acceptor at one end of the first compound and an electron supplier on the affinity film form a covalent co-formation. Bonded so that the first compound is grafted onto the surface of the affinity film, and the other end of the first compound is exposed to another electron acceptor. Performing a third surface modification step includes reacting the particle body subjected to the second surface modification step with a plurality of target antibodies, so that the target antibody binds to the other end of the compound and passes through the other electron acceptor of the other end. Forms a covalent bond with the electron supplier on the target antibody.

In an embodiment of the present invention, the bio-affinity material is collagen, and the functional group of the collagen includes an amine group, a carboxyl group, an alcohol group, and a thiol group to self-assembly through the functional group. An affinity coating is formed on the microparticle body.

In an embodiment of the present invention, the first compound is a long-chain compound having a diazonium group at one end, and the diazo group of the first compound is removed during the second surface modification step. An electron acceptor, and the electron acceptor reacts with an electron supplier of a functional group on the affinity film to form a covalent bond.

In an embodiment of the present invention, the other end of the first compound is exposed to an epoxy group, and when the third surface modification step is performed, the epoxy group will open a ring to form another electron acceptor and contact the target antibody. Of electronic suppliers form covalent bonds.

In an embodiment of the present invention, after the second surface modification step and before the third surface modification step, the first compound bonded to the affinity film is reacted with oxygen and a metal catalyst to make the first The other end of a compound is converted into a structure having an epoxy group.

In an embodiment of the present invention, a diameter of the microparticle body ranges from 30 nm to 500 nm.

In an embodiment of the present invention, a material of the particle body includes a material having magnetoelectricity under an electric field.

Based on the above, the detection particle produced by the present invention has an affinity coating formed by a bio-affinity material and several target antibodies scattered on its peripheral surface and passing through the two ends of the branched electron acceptors, respectively. Forms covalent bonds with affinity membranes and target antibodies. Therefore, the detection particle of the present invention can improve the effectiveness of capturing pathogens through the target antibody firmly bonded to its surface, and then obtain accurate detection results.

In order to make the above features and advantages of the present invention more comprehensible, embodiments are hereinafter described in detail with reference to the accompanying drawings.

FIG. 1 is a flowchart of a method for manufacturing microparticles according to an embodiment of the present invention. Referring to FIG. 1, a manufacturing method of detecting particles in this embodiment includes providing a particle body 100. The diameter of the particle body 100 ranges from 30 nm to 500 nm, and the concentration of the particle body 100 ranges from 10 ^-1 M to 10 ^-5 M. In this embodiment, the material of the particle body 100 includes a material having magnetoelectricity under an electric field, but is not limited thereto. For example, when the particle body 100 is a material selected to have magnetoelectricity under an electric field, the particle body 100 can be endowed with a property that it can be grasped under an electric field. In other words, after the particle body 100 is used to capture pathogens, the target pathogens can be successfully purified using an electric field, and the in vitro detection can be effectively performed quickly. In the embodiment of the present invention, the microparticle body having magnetism is, for example, iron oxide nano particles, but it is not limited thereto. In addition, the particle body 100 of the present invention may include other types of materials. For example, when the particle body 100 is another kind of material, after the particle body 100 has successfully captured the pathogen, the pathogen can be purified, for example, by centrifugation.

Please continue to refer to FIG. 1, after the particle body 100 is provided, a first surface modification step S100 may be performed. The first surface modification step S100 includes reacting the microparticle body 100 with the bio-affinity material 105 to form an affinity coating 110 on the microparticle body 100. In this embodiment, the bio-affinity material 105 has at least one or more functional groups selected from the group consisting of an amine group, a carboxyl group, an alcohol group, and a thiol group, and the affinity coating 110 is made of the bio-affinity material 105. Covered. Since the affinity membrane 110 is composed of a bio-affinity material 105, and has high compatibility with organisms and has an affinity group, the affinity membrane 110 can be better with the target antibody and the pathogen to be tested subsequently. Affinity.

In one embodiment of the present invention, the bio-affinity material 105 is, for example, collagen. Among them, collagen has many types of functional groups, including functional groups such as amine, carboxyl, alcohol, and thiol groups. Therefore, Collagen has better plasticity. In addition, collagen is rich in functional groups such as amine, carboxyl, and alcohol groups, which can generate intra- and intermolecular hydrogen bonds. In other words, through the interactions such as hydrogen bonding, the collagen can form an affinity coating 110 on the microparticle body 100 in a self-assembly manner. However, the bio-affinity material 105 of the present invention is not limited to collagen, and other materials having bio-affinity / affinity groups can be selected to form a stable and coating affinity coating 110.

FIG. 2A is a flowchart of a method for synthesizing a first branched and branched compound according to an embodiment of the present invention. FIG. 2B is a flowchart of a second surface modification step according to an embodiment of the present invention. Next, please refer to FIG. 1, FIG. 2A and FIG. 2B at the same time. As shown in FIG. 1, after the first surface modification step S100 is performed, a second surface modification step S200 may be performed. The second surface modification step S200 includes reacting the microparticle body 100 having undergone the first surface modification step S100 with the first compound C1. In the present embodiment, the first compound C1 can be considered as a part of the chain branch 120. That is, the link branch 120 in the embodiment of the present invention includes the first compound C1. The type of the first compound C1 is not particularly limited, but it is a long-chain compound, and one end of the first compound C1 may have a leaving group. When the leaving group is removed, one end of the first compound C1 may have Electronic receiver. In this embodiment, as shown in FIG. 2A, the first compound C1 may be, for example, a compound having a diazonium group, and the first compound C1 is represented by the following formula (1). Formula 1)

2A, the first compound C1 is formed so that the 5-chloro-pentyl amine (5-chloro-1-pentanamine ) adding an alkaline solution, wherein the alkaline solution of hydroxy (OH ^-) will be a functional group with 5- The chlorine on chloropentylamine undergoes an elimination reaction and forms a double bond structure. Next, sodium nitrate (NaNO ₃ ) and hydrochloric acid (HCl) are added to prepare a first compound C1 having a diazonium group. Referring to FIG. 2B, in the second surface modification step S200, the microparticle body 100 having undergone the first surface modification step S100 is reacted with the first compound C1 so that the branch 120 including the first compound C1 is bonded to The biocompatible material 105 of the affinity film 110. More specifically, the branched branch 120 includes a first compound C1 having a diazonium group, and when the second surface modification step S200 is performed, the diazonium group on the first compound C1 is removed to form a nitrogen overflow. So that one end of the chain branch 120 has an electron acceptor, and the electron acceptor reacts with an electron supplier of a functional group on the affinity film 110 to form a covalent bond. In particular, in this embodiment, the carbon atom connected to the diazo group on the first compound C1 itself is an electron acceptor, and can accept lone pair electrons from the biocompatible material 105 to form a covalent bond therewith. .

In addition, referring to FIG. 1 and FIG. 2B, after the second surface modification step S200 and before the third surface modification step S300, the first compound C1 further including a link branch 120 bonded to the affinity film 110 is further included. It reacts with oxygen and a metal catalyst to convert the other end of the first compound C1 into a first compound C1 ′ including an epoxy group. That is, after the second surface modification step S200 is performed, the chain branch 120 may include a first compound C1 'that exposes an epoxy group. Accordingly, the epoxy group can be successfully bonded to the surface of the affinity coating 110 (ie, the bio-affinity material 105). In this embodiment, since the ring tension of the epoxy group structure is large, it can react with the functional group of the target antibody A1 used in the subsequent steps to bond and open the ring, and form a stable covalent bond. In this embodiment, the first compound C1 'having an epoxy group is coated on the surface of the affinity film 110 and is used as a part of the link branch 120. Although the embodiment of the present invention forms a covalent bond after reacting a compound including a diazo group with the affinity film 110 (removing the diazo group), and reacting with a target antibody A1 by a compound including an epoxy group Bonding to form a linking branch 120 connecting the affinity coating 110 and the target antibody A1, but the present invention is not limited thereto. For example, the chain branch 120 of the present invention can also form covalent bonds through other chemical reactions to connect the affinity coating 110 to the target antibody A1. It can be understood that the two ends of the chain branch 120 may have electron acceptors at different steps to form a covalent bond with the electron supplier of the affinity coating 110 and the target antibody A1, respectively.

3 is a flowchart of a third surface modification step according to an embodiment of the present invention. Next, please refer to FIG. 1 and FIG. 3 simultaneously to perform a third surface modification step S300. The third surface modification step S300 includes mixing / reacting the microparticle body 100 (including the epoxy group) after the second surface modification step S200 with the target antibody A1 so that the target antibody A1 is bonded to the affinity film 110. In more detail, when the third surface modification step S300 is performed, the chain branch 120 passes through the ring-opening reaction of the epoxy group of the first compound C1 ′ so that the other end thereof forms an electron acceptor, and the electron acceptor Reacts with an electron supplier on the target antibody A1 to form a covalent bond. In the example of FIG. 3, the target antibody A1 includes an amine group (NH ₂ ), and the epoxy group of the first compound C1 ′ reacts with the amine group of the target antibody A1, and then the ring is opened to form a stable co-polymer. Price bond. However, the present invention is not limited to the above embodiments. The target antibody A1 and the first compound C1 can also form covalent bonds through other chemical reactions. In addition, the type of the target antibody A1 can be changed according to the type of the pathogen to be detected, and is applicable to the detection of various diseases. In addition, in the embodiments of FIG. 2B and FIG. 3, only one chain branch 120 (the first compound C1 / C1 ′) is connected to the particle body 110 as an example, but it should be noted that the present invention The detection particle actually has a plurality of chain branches 120 and a plurality of target antibodies A1 connected to the peripheral surface of the particle body 100.

Accordingly, by performing the first surface modification step S100, the second surface modification step S200, and the third surface modification step S300, affinity modification, link modification, and target antibody modification can be performed on the surface of the microparticle body 100. Therefore, the detection particle can enhance the effectiveness of capturing pathogens through the target antibody A1 firmly bonded to its surface, thereby obtaining accurate detection results and achieving the purpose of rapid detection of the target disease.

Hereinafter, the method for forming the detection particles and the method for using the detection particles according to the embodiments of the present invention will be described by experimental examples. Experimental example

In this experimental example, detection particles produced with the goal of detecting dengue virus. In detail, there are seven segments of dengue virus non-structural proteins. Among them, the non-structural protein NS1 (Nonstructural protein) is a hydrophilic protein. It is a highly conserved glycoprotein and will be present in high concentrations in patients infected with dengue virus. Early clinical stage serum. For patients with primary and secondary infections of dengue virus, the NS1 antigen can be found in the specimens on the 1st to 9th days of fever symptoms. Therefore, in this experimental example, superparamagnetic nanometer magnetic beads were selected to have the characteristics of high surface area, and affinity modification, chain modification and target antibody modification were performed on the surface of the magnetic beads, and a large amount of NS1 antibodies were attached to the surface of the magnetic beads. . In addition, based on the magnetic and electrical properties of the magnetic beads, the NS1 antigen in the specimen can be purified by using an electric field to achieve the purpose of detecting the NS1 antigen in the specimen. Specifically, the detection particles of this experimental example are made and used by the following steps and methods. Microparticle synthesis

The microparticles selected in this experimental example are superparamagnetic nano-magnetic beads (iron oxide nano-particles). Superparamagnetic nano-magnetic beads are synthesized using a conventional hydrothermal synthesis method. A mixture of 50 mg of ferric chloride (Fe ₂ O ₃ ), 0.2M ethylene glycol (C ₂ H ₆ O ₂ ), 2M sodium acetate (CH ₃ COONa), and 0.1M polyethylene glycol (polyoxyethylene) was passed through Liquid-solid-solution reaction (liquid-solid-solution reaction) three-phase reaction, from time to time stirring and heating to 200 ℃ for 8-72 hours, iron oxide nano particles can be made. The diameter range of the produced iron oxide nano particles can be controlled in the range of 30nm to 500nm. First surface modification step

First, the iron oxide nano-particles formed by the above method were placed in 1.2M hydrochloric acid (HCl) and 1.2M sodium hydroxide (NaOH) aqueous solutions for 5 minutes, respectively. Deionized water was required during the process. rinse. Next, the iron oxide nano-particles with a concentration of 10 ^-5 M were immersed in a 1% to 2% collagen phosphate buffer solution, and immersed for 2 hours. By using functional groups such as amine, carboxyl, and alcohol groups contained in collagen, intramolecular and intermolecular hydrogen bonds can be generated in collagen, and stable coating can be formed on the surface of nanoparticle by self-assembly Film (affinity film). According to this, the first layer surface modification can be completed by the above method. Second surface modification step

The purified iron oxide nano particles that have completed the first surface modification step are added to a methanol / hexane organic solvent including the first compound C1 including a diazo group as shown in FIG. 2A for a mixing reaction. At this time, the first compound C1 will remove the diazo group so that the first compound C1 has an electron acceptor, and the electron supplier of the functional group on the collagen surface will react with the first compound C1 to bond the first compound C1. It binds to collagen and simultaneously removes a nitrogen molecule (N ₂ ). Next, oxygen (O2) and a metal catalyst are further added to convert the first compound C1 into an epoxide (first compound C1 ′) having an epoxy group. According to this, the epoxy group can be successfully bonded to the collagen and the second surface modification can be completed. Third surface modification step

Add the iron oxide nano particles that have completed the second surface modification step to the PBS buffer solution of NS1 protein antibody (target antibody), stir and react uniformly with a magnet for about 2-4 hours to make the functional groups on the surface of the NS1 antibody contact the iron oxide nano. Epoxy groups on the particle surface. During the reaction, the functional groups on the surface of the NS1 antibody will react with the epoxy group to bond and open the ring, forming a stable covalent bond. According to this, the NS1 protein antibody can be successfully bonded to collagen through the epoxy group, and the third surface modification can be completed. Detection method

In order to confirm the sensitivity of the detection particles of the embodiment of the present invention to dengue virus, the detection will be performed in the following manner. First, the iron oxide nano particles (that is, detection particles) that have completed the third surface modification step and the specimen (the blood of a patient with a pathogen) are dropped on the optical test strip and mixed, and left for about 1-2 minutes. To perform the reaction. After the reaction is completed, an electric field is placed under the optical test strip to adsorb iron oxide nano particles, and the test strip is rinsed with physiological saline at the same time. Accordingly, a purified specimen can be obtained. After the specimens purified through the above steps are detected by a Raman spectrometer, it can be confirmed that the detection particles of the present invention can effectively capture the dengue virus, thereby obtaining accurate detection results.

In summary, the detection particle produced by the present invention has an affinity coating formed by a bio-affinity material and several target antibodies scattered on its peripheral surface, and electrons are received through the two ends of the branched branches. They form covalent bonds with affinity membranes and target antibodies, respectively. Therefore, the binding stability of the microparticle body and the target antibody of the present invention can be improved, thereby improving the effectiveness of capturing pathogens, and obtaining accurate detection results. In addition, since the particle body of the present invention has high binding stability to a target antibody and can directly capture pathogens, the detection particle of the present invention is not easily affected by the environment. In other words, when the detection particle of the present invention is used as a biosensor, it can have extremely high specificity with the target antigen (pathogen), so as to achieve the purpose of rapid detection of the target disease with good efficiency and accuracy.

Although the present invention has been disclosed as above with the examples, it is not intended to limit the present invention. Any person with ordinary knowledge in the technical field can make some modifications and retouching without departing from the spirit and scope of the present invention. The protection scope of the present invention shall be determined by the scope of the attached patent application.

100‧‧‧ particle body

105‧‧‧Bio-affinity materials

110‧‧‧ affinity film

115‧‧‧metal catalyst

120‧‧‧ Linked Branches

A1‧‧‧Target antibody

C1, C1’‧‧‧ first compound

S100‧‧‧First surface modification step

S200‧‧‧Second surface modification step

S300‧‧‧The third surface modification step

FIG. 1 is a flowchart of a method for manufacturing microparticles according to an embodiment of the present invention. FIG. 2A is a flowchart of a method for synthesizing a first branched and branched compound according to an embodiment of the present invention. FIG. 2B is a flowchart of a second surface modification step according to an embodiment of the present invention. 3 is a flowchart of a third surface modification step according to an embodiment of the present invention.

Claims (11)

A detection particle is used to capture pathogens contained in a test object, which comprises: a particle body; an affinity film covering the peripheral surface of the particle body; and the affinity film is biocompatible The bio-affinity material has at least one or more functional groups selected from the group consisting of an amine group, a carboxyl group, an alcohol group, and a thiol group; and several target antibodies are dispersed on the peripheral surface of the microparticle body. To combine with the pathogen; several chain branches, one end of which is formed by removing a diazonium (diazonium) to form an electron acceptor, and the electron acceptor is compatible with the affinity An electron supplier carried by the functional group on the membrane reacts to form a covalent bond, the other end of the chain branch is used to graft the target antibody, and the other end of the chain branch Having another electron acceptor to form a covalent bond with an electron supplier of the target antibody. The detection particle according to item 1 of the scope of the patent application, wherein the affinity coating is formed of collagen and includes amine, carboxyl, alcohol, and thiol functional groups. The detection particle according to item 1 of the scope of patent application, wherein the diameter of the particle body is between 30 nm and 500 nm. The detection particle according to item 1 of the patent application scope, wherein a material of the particle body includes a material having magnetoelectricity under an electric field. The detection particle according to item 1 of the patent application scope, wherein the other end of the branched branch is formed by the ring-opening reaction of the epoxy group with the other electron acceptor, and the other electron acceptor and the target antibody The electron supplier on the substrate reacts to form the covalent bond. A manufacturing method for detecting particles includes: providing a particle body; and performing a first surface modification step including reacting the particle body with a bio-affinity material to form an affinity film on the particle body, wherein the organism The affinity material includes at least one or more functional groups selected from the group consisting of an amine group, a carboxyl group, an alcohol group, and a thiol group; performing a second surface modification step, including the microparticle body after the first surface modification step; Reacts with a first compound, the first compound is a long-chain compound having a diazonium group at one end, and the diazo group of the first compound is removed during the second surface modification step; An electron acceptor, and the electron acceptor reacts with an electron supplier of the functional group on the affinity film to form a covalent bond, so that the first compound is grafted on the surface of the affinity film, and The other end of the first compound is exposed to another electron acceptor; and a third surface modification step is performed, including the particles subjected to the second surface modification step. The reaction body with a plurality of the antibody to make the antibody bound to the other end of the first compound, and together form a covalent bond with an electron provider on the antibody via another end of the electron acceptor. The manufacturing method according to item 6 of the scope of the patent application, wherein the bio-affinity material is collagen, and the collagen has functional groups including amine, carboxyl, alcohol, and thiol groups to self-assemble through the functional group ( self-assembly) to form the affinity coating on the particle body. The manufacturing method according to item 6 of the application, wherein the other end of the first compound is exposed to an epoxy group, and when the third surface modification step is performed, the epoxy group will open a ring to form the other electron acceptor Or form a covalent bond with the electron supplier on the target antibody. The manufacturing method according to item 8 of the scope of patent application, wherein after the second surface modification step is performed and before the third surface modification step is performed, the first compound and oxygen bonded to the affinity film are bonded. And a metal catalyst is reacted to convert the other end of the first compound into a structure having the epoxy group. The manufacturing method according to item 6 of the patent application range, wherein the diameter of the particle body is between 30 nm and 500 nm. The manufacturing method according to item 6 of the patent application scope, wherein the material of the particle body includes a material having magnetoelectricity under an electric field.