This application claims the priority benefit of Taiwan application serial no. 110132354, filed on Aug. 31, 2021. The entirety of the above-mentioned patent application is hereby incorporated by reference herein and made a part of this specification.
The disclosure relates to a method for immobilizing a glycoprotein and an antibody chip, and particularly relates to an oriented and covalent method for immobilizing a glycoprotein and an antibody chip.
The antibody has tight and specific binding power with the epitope, so the antibody has been widely used in immunoaffinity separation, targeted therapy delivery, enzyme-linked immunosorbent assay (ELISA), inspection arrays, and other biomedical technology. The applications require the antibody to be bound onto a solid support as a carrier (for example, a microtiter plate, a nanoparticle, or a glass surface) while retaining the analyte binding activity.
Based on the chemical complexity of the antibody, the immobilization mechanism of the antibody is quite complex. The conventional antibody cross-linking method, such as the immobilized antibody prepared by physical adsorption, has relatively weak antibody binding ability. The substance contained in a complex specimen sample (for example, a blood sample) causes the antibody bound to boronic acid to dissociate, which affects the sensitivity of subsequent analysis of the antibody. If residues such as lysine, arginine, aspartate, and glutamate of the antibody are randomly selected for Schiff base or amide bond modification, there will be the disadvantage of random orientation, which can easily cause the antibody to lose the antigen binding activity.
Based on the above, developing a method for immobilizing a glycoprotein that can form a covalent bond with specific orientation, have high activity, have strong binding affinity, and increase the detection sensitivity is an important subject for current development of biomedical detection.
The disclosure provides an oriented and covalent method for immobilizing a glycoprotein and an antibody chip, which have high activity, have strong binding affinity, and increase the detection sensitivity.
The oriented and covalent method for immobilizing the glycoprotein of the disclosure includes the following steps. A silver-coated solid surface is provided. Multiple cuprous oxide nanoparticles are disposed on the silver-coated solid surface. Then, a solid surface (including the silver-coated solid surface and the cuprous oxide nanoparticles) is alkynylated. Next, an azido boronic acid tosyl probe is provided to the silver-coated solid surface. The boronic acid tosyl probe is bound to the solid surface by the cuprous oxide nanoparticles through self-catalyzed azide-alkyne cycloaddition (SAAC) reaction. Then, the glycoprotein is provided to the boronic acid tosyl probe. The glycoprotein may include an Fc fragment (crystallizable). An alcohol group of a glycan of the glycoprotein or a glycan chain of the Fc fragment and a boronic acid group of the boronic acid tosyl probe form organic boronate ester to immobilize the glycoprotein. Next, a nucleophilic residue on the glycoprotein is replaced with a tosyl group in the boronic acid tosyl probe by an SN2 reaction, and the tosyl group is released from a terminal azide group to immobilize the glycoprotein through a covalent bond between the nucleophilic residue and the terminal azide group. Finally, the organic boronate ester is released.
In an embodiment of the disclosure, a structure of the azido boronic acid tosyl probe is represented by Formula (1) or Formula (1A):
where in Formula (1) and Formula (1A), R1 is a boron-containing group, and a structure of an aromatic group with R1 group is represented by Formula (2), Formula (3), or Formula (4):
where X1 is NH,
m is a positive integer from 1 to 8, a is a positive integer from 2 to 10, b is a positive integer from 2 to 10, c is a positive integer from 1 to 15, and d is a positive integer from 1 to 15.
In an embodiment of the disclosure, the structure of the boronic acid tosyl probe is represented by Formula (1-1), Formula (1-2), or Formula (1-3):
In an embodiment of the disclosure, a material of the silver-coated solid surface includes glass.
In an embodiment of the disclosure, a thickness of a silver coating layer of the silver-coated solid surface is 5 nm to 200 nm.
In an embodiment of the disclosure, alkynylation is to react the silver-coated solid surface disposed with the cuprous oxide nanoparticles with alkyne thiol whose structure is represented by Formula (A) or Formula (B) and thiol whose structure is represented by Formula
where in Formula (A), n is a positive integer from 5 to 15, and in Formula (B), x is a positive integer from 1 to 15, and y is a positive integer from 1 to 15.
In an embodiment of the disclosure, when providing the boronic acid tosyl probe to the silver-coated solid surface, azido-linked tri(ethylene glycol) is provided to the silver-coated solid surface at a same time, and azido-linked tri(ethylene glycol) is bonded to the cuprous oxide nanoparticles by self-catalyzed azide-alkyne cycloaddition reaction.
In an embodiment of the disclosure, the glycoprotein includes an antibody.
In an embodiment of the disclosure, releasing the organic boronate ester is performed through polyols.
The antibody chip of the disclosure uses the oriented and covalent method for immobilizing the glycoprotein.
Based on the above, the oriented and covalent method for immobilizing the glycoprotein of the disclosure uses the boronic acid tosyl probe, which not only enables the alcohol group of the glycan of the glycoprotein or the glycan chain of the Fc fragment and the boronic acid group of the boronic acid tosyl probe to form organic boronate ester, but also enables the nucleophilic residue on the glycoprotein to be replaced with the tosyl group in the boronic acid tosyl probe by the SN2 reaction to form an oriented irreversible covalent bond. In addition, the oriented and covalent method for immobilizing the glycoprotein of the disclosure uses the silver-coated solid surface disposed with the cuprous oxide nanoparticles. The silver-coated solid surface has surface resonance to enhance a fluorescence signal (metal-enhanced fluorescence, MEF), and the cuprous oxide nanoparticles enable the boronic acid tosyl probe to be bound by self-catalyzed azide-alkyne cycloaddition (SAAC) reaction. Therefore, different from the conventional Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reaction, the use of additional copper ions is not required, and the issue of degraded boronate reactivity caused by Cu(I) can be solved. In this way, the activity can be effectively improved, the binding force can be strengthened, and the detection sensitivity can be increased.
Hereinafter, embodiments of the disclosure will be described in detail. However, the embodiments are illustrative, and the disclosure is not limited thereto.
In the disclosure, the range represented by “a value to another value” is a general way to avoid listing all values in the range one by one in the specification. Therefore, the recitation of a specific value range covers any value in the value range and a smaller value range defined by any value in the value range, which is the same as writing the arbitrary value and the smaller value range in the specification.
Please refer to
Please refer to
where in Formula (A), n is a positive integer from 5 to 15, and in Formula (B), x is a positive integer from 1 to 15, and y is a positive integer from 1 to 15.
In the embodiment, adding thiol represented by Formula (C) may adjust the degree of alkynylation of a surface. The molar ratio of the added amount of alkyne thiol represented by Formula (A) or Formula (B) to thiol represented by Formula (C) is, for example, 0.01 to 100. In
Please refer to
In the embodiment, the structure of the boronic acid tosyl probe may be represented by Formula (1) or Formula (1A):
where in Formula (1) and Formula (1A), R1 is a boron-containing group, and the structure of an aromatic group with R1 group may be represented by Formula (2), Formula (3), or Formula (4):
where X1 is NH,
m is, for example, a positive integer from 1 to 8, a is, for example, a positive integer from 2 to 10, b is, for example, a positive integer from 2 to 10, c is, for example, a positive integer from 1 to 15, and d is, for example, a positive integer from 1 to 15.
In
The specific structure of the boronic acid tosyl probe may be represented by Formula (1-1), Formula (1-2), or Formula (1-3):
Please refer to
Next, please refer to
Polyols may include glycerol, sorbitol, mannitol, or polyethylene glycol, but the disclosure is not limited thereto. In this way, by the oriented and covalent method for immobilizing the glycoprotein of the disclosure, when the glycoprotein is, for example, the antibody, the binding force of the antibody can be improved, and the dissociation of the antibody caused by the substance in a complex specimen sample can be avoided. Therefore, the oriented and covalent method for immobilizing the glycoprotein of the disclosure is suitable for further detection of an antigen in a blood sample, which can effectively improve the sensitivity of subsequent analysis of the antibody.
The disclosure also provides an antibody chip using the oriented and covalent method for immobilizing the glycoprotein.
Hereinafter, the oriented and covalent method for immobilizing the glycoprotein of the above embodiment will be explained in detail through an experimental example. However, the following experimental example is not intended to limit the disclosure.
In order to prove that the oriented and covalent method for immobilizing the glycoprotein proposed by the disclosure can effectively improve the activity, strengthen the binding force, and increase the detection sensitivity, the following experimental example is provided.
It should be noted that since the oriented and covalent method for immobilizing the glycoprotein has been described in detail above, the following description of the oriented and covalent method for immobilizing the glycoprotein is omitted for convenience of explanation.
The oriented and covalent method for immobilizing the glycoprotein of the disclosure was used to measure two single biomarkers SAP and C-RP. Different concentrations of SAP (0.01 μ/mL, 0.1 μg/mL, 1 μg/mL, 5 μg/mL, and 10 μg/mL) were reacted together with an anti-SAP antibody produced using the oriented and covalent method for immobilizing the glycoprotein of the disclosure. Then, biotinylated anti-SAP pAb (1 μg/mL) was used to assess the presence of a binding protein, and fluorescent Cy3-labeled streptavidin (10 μg/mL) was then used.
B, C, and D of
As shown in
In summary, the oriented and covalent method for immobilizing the glycoprotein of the disclosure uses the boronic acid tosyl probe, which not only enables the alcohol group of the glycan of the glycoprotein or the glycan chain of the Fc fragment and the boronic acid group of the boronic acid tosyl probe to form organic boronate ester, but also enables the nucleophilic residue on the glycoprotein to be replaced with the tosyl group in the boronic acid tosyl probe by the SN2 reaction to form an oriented irreversible covalent bond and have higher binding specificity. In addition, the oriented and covalent method for immobilizing the glycoprotein of the disclosure uses the silver-coated solid surface disposed with the cuprous oxide nanoparticles. The silver-coated solid surface has surface resonance to enhance a fluorescence signal (metal-enhanced fluorescence, MEF), and the cuprous oxide nanoparticles enable the boronic acid tosyl probe to be bound by self-catalyzed azide-alkyne cycloaddition (SAAC) reaction. Therefore, different from the conventional Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reaction, the use of additional copper ions is not required, and the issue of degraded boronate reactivity caused by Cu(I) can be solved. In this way, the glycoprotein can resist dissociation in a complex sample (for example, a blood sample) while effectively improving the activity, strengthening the binding force, and increasing the detection sensitivity, and the orientation can also be taken into account.
Number | Date | Country | Kind |
---|---|---|---|
110132354 | Aug 2021 | TW | national |