Preparation method of biomolecule cross-linked compound and derivative of biomolecule cross-linked compound

Abstract

The invention discloses a preparation method of a biomolecule cross-linked compound. In particular, the method comprises the steps of reacting a plurality of monomers with a plurality of comonomers in the presence of ammonia in a first solution to form a compound; extracting the obtained compound from the first solution for cleaning; reacting the cleaned compound with a second solution, and connecting a crosslinking agent to the compound through a first amine functional group of the crosslinking agent; and adding a plurality of biomolecules into the second solution, and combining the biomolecules with the compound through a second amine functional group of the cross-linking agent to prepare the biomolecule cross-linked compound.

Description

Technical Field

[0001] The present invention relates to a method for preparing a compound capable of cross - linking biomolecules to form a cross - linked biomolecule. Specifically, the cross - link between the compound and the biomolecule is achieved by interaction with the amine functional group on the cross - linker. The present invention also relates to the cross - linked compound of the biomolecule and its derivatives. Background Art

[0002] The rapid development of nanomaterial technology has made significant contributions to the recent progress in the field of biosensors. 1 Nanoparticles (NP) are defined as small in size, and they usually exhibit unique properties due to their high surface - to - volume ratio, which is useful in biosensors. In principle, biosensors rely on the specific interaction between an analyte and a biorecognition element on the biosensor, such as enzyme - substrate or antigen - antibody. 2,3 Therefore, immobilizing biomolecules on nanoparticles has become an effective strategy to improve the sensitivity and selectivity of biosensors. 4,5 Currently, a variety of biomolecule - functionalized nanoparticles, such as carbon nanotubes, gold nanoparticles, and other metal nanoparticles, have been developed and applied in the field of biosensing. For example, glutaraldehyde is used as a cross - linker, which can achieve the coating of a large number of bioreceptors on the nanoparticle surface. It can bind to a variety of bioreceptors (such as proteins, genetic materials), but in an undesirable non - specific manner. U.S. Patent No. 7226794B2 discloses gold nanoparticles (AuNP) for detecting biomolecules using surface - enhanced Raman spectroscopy (SERS). In addition, European Patent Application No. 1794590A4 discloses a method for immobilizing biomolecules on single - wall carbon nanotubes using carbon nanotubes.

[0003] In addition, silica nanoparticles (SNP) are a class of inorganic nanomaterials for biomolecule immobilization that have received much attention. SNP has characteristics such as biocompatibility, mechanical stability, and tunable size and morphology. 9 SNP can form various morphologies and structures, such as conventional non - porous SNP, mesoporous silica nanoparticles 11,12 , hollow mesoporous silica nanoparticles 13 and core - shell structured silica 14 , thus showing different performances. Due to its multifunctional characteristics, SNP has been widely applied in biomedical fields such as drug delivery and theranostics. 9,15,16 Research shows that amino - functionalized SNP has biocompatibility and is suitable as a drug delivery carrier. (3 - Aminopropyl) triethoxysilane (APTES) is used to provide amino groups for the preparation of amino - functionalized silica nanoparticles (AFSNP). In traditional methods, APTES is modified on the SNP surface through a condensation reaction.18-21 However, there are some limitations in this method, such as uncontrollable number of functional groups, limited storage stability, and random molecular orientation, which lead to a decrease in the efficiency of biomolecule immobilization. This ultimately reduces the sensitivity of biosensing applications.

[0004] There are various immobilization strategies 22,23 ; however, they can be mainly divided into two categories: non-covalent binding and covalent binding. Non-covalent binding relies on electrostatic interactions, hydrophobic interactions, polar interactions, and affinity interactions. Affinity interactions such as biotin-avidin binding, histidine-metal chelation. Meanwhile, covalent binding involves chemical bonds formed between biomolecule functional groups and the material surface, such as amino groups, carboxyl groups, thiol groups, aldehyde groups, or alkynyl groups 24 To achieve the immobilization of biomolecules on silica nanoparticles (SNP), the surface of SNP needs to be functionalized first, and then a cross-linking agent between SNP and biomolecules is introduced. The most commonly used cross-linking agents for biomolecule immobilization are glutaraldehyde and 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide / N-hydroxysuccinimide (EDC / NHS) 25 However, the limitation of such cross-linking agents is that they can only achieve cross-linking between the N-terminus and the C-terminus. Therefore, there is an urgent need to develop a method that can interact through other functional groups and only cross-link between the N-terminus and the C-terminus to achieve the immobilization of biomolecules on the nanoparticle surface, so as to broaden the application potential of biosensors developed based on this principle. Summary of the Invention

[0005] The present invention relates to a method for preparing a compound capable of cross-linking biomolecules or a compound cross-linked with biomolecules. Specifically, the disclosed method uses a pair of compatible monomers and comonomers, and a silica nanoparticle (SNP) or a compound can be prepared through a one-step reaction, which has the characteristic of significant simplification compared with the traditional method for synthesizing compounds.

[0006] Another object of the present disclosure is to provide a method for preparing silica nanoparticles or their derivatives under relatively mild conditions, the temperature of which is in the range of about room temperature or slightly higher than room temperature.

[0007] A further object of the present invention is to provide a method for preparing a compound cross-linked with biomolecules or a silica nanoparticle cross-linked with biomolecules in a controlled manner. This method uses a specifically modified cross-linking agent, preferably through functionalized amine groups, rather than the traditional N-terminus and C-terminus interactions, to achieve the cross-linking of modified or unmodified biomolecules and the compound.

[0008] In addition, another object of the present invention is a compound cross-linked with biomolecules, wherein the biomolecules are cross-linked with the compound through a cross-linking agent having a pair of amine functional groups, and this pair of amine functional groups can bind the compound and the biomolecules.

[0009] The present invention wholly or partly achieves the foregoing at least one objective. One embodiment of the present invention is: a method for preparing a biomolecule-crosslinked compound. The method basically comprises the following steps: reacting a plurality of monomers with a plurality of comonomers in the presence of ammonia in a first solution to generate a compound; extracting the obtained compound from the first solution for washing; reacting the washed compound with a second solution to connect a crosslinking agent to the compound through a first amine functional group of the crosslinking agent; and adding a plurality of biomolecules to the second solution to bind the biomolecules to the compound through a second amine functional group of the crosslinking agent, thereby obtaining a biomolecule-crosslinked compound. Preferably, the monomer is tetrapropyl orthosilicate (TPOS), tetraethyl orthosilicate (TEOS), tetramethyl orthosilicate (TMOS), tetra-isopropyl orthosilicate or tetrabutyl orthosilicate. Preferably, the comonomer is selected from (3-aminopropyl)-triethoxysilane), (3-aminopropyl)-trimethoxysilane), (3-aminopropylmethyldiethoxysilane), (3-(dimethoxymethylsilyl)propylamine), (3-aminopropylsilanetriol), N-(2-aminoethyl)(3-aminopropyl)methyldimethoxysilane) or N-(2-aminoethyl)(3-aminopropyl)trimethoxysilane).

[0010] According to certain embodiments, the disclosed method further comprises: precipitating the prepared biomolecule-crosslinked compound and dispersing the precipitated biomolecule-crosslinked compound in distilled water.

[0011] According to certain embodiments, the molar ratio of the monomer to the comonomer is 1.3 - 4.0:0.4 - 3.0. More preferably, in other embodiments, the first solution contains ammonium hydroxide dissolved in a mixture of distilled water and ethanol, wherein the volume ratio of distilled water to ethanol is 1 - 10:20 - 30.

[0012] According to several embodiments, the second solution contains itaconic acid, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS), and the three are dispersed in the aqueous phase in a molar ratio of 15 - 35:60 - 90:30 - 60.

[0013] According to several embodiments, the reaction step is carried out within a temperature range of 20°C to 40°C.

[0014] According to certain embodiments, the biomolecule is any one of an antigen, an antibody, a peptide chain, DNA or RNA having an amino terminus capable of binding to the second amine functional group of the crosslinking agent.

[0015] On the other hand, the present invention discloses a biomolecule-crosslinked compound having the following chemical structure:

[0016] R1-X-R2

[0017] Wherein X is a crosslinking agent of the following chemical formula:

[0018] (i) -HN-C(=O)-CH2C(=CH2)-C(=O)-NH- or

[0019] (ii) -HN-C(=O)-CH2C(=CH2)-C-NH-.

[0020] Preferably, R1 is a silica nanoparticle and R2 is a biomolecule with an amino terminus capable of binding to the amine functional group of the crosslinking agent.

[0021] In some embodiments of the compounds of the present invention, the crosslinking agent is prepared by reacting itaconic acid, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), and N-hydroxysuccinimide (NHS) in an aqueous phase at a molar ratio of 15-35:60-90:30-60.

[0022] In some embodiments of the compounds of the present invention, the silica nanoparticles comprise monomers and comonomers prepared at a molar ratio of 1.3-4.0:0.4-3.0. Preferably, the monomer is tetrapropyl orthosilicate (TPOS), tetraethyl orthosilicate (TEOS), tetramethyl orthosilicate (TMOS), tetra-isopropyl orthosilicate, or tetrabutyl orthosilicate. Preferably, the comonomer is selected from (3-aminopropyl)triethoxysilane, (3-aminopropyl)trimethoxysilane, (3-aminopropyl)methyldiethoxysilane, (3-(dimethoxymethylsilyl)propylamine), (3-aminopropyl)silanetriol, (N-(2-aminoethyl)(3-aminopropyl)methyldimethoxysilane), or N-(2-aminoethyl)(3-aminopropyl)trimethoxysilane).

[0023] Another aspect of the present invention is a method for preparing amino-functionalized silica nanoparticles (AFSNP), comprising: reacting a plurality of monomers and a plurality of comonomers in the presence of ammonia in a first solution to form AFSNP; and extracting the resulting AFSNP from the first solution for washing. Preferably, the monomer is tetrapropyl orthosilicate (TPOS), tetraethyl orthosilicate (TEOS), tetramethyl orthosilicate (TMOS), tetra-isopropyl orthosilicate, or tetrabutyl orthosilicate, and the comonomer is selected from (3-aminopropyl)triethoxysilane, (3-aminopropyl)trimethoxysilane, (3-aminopropyl)methyldiethoxysilane, (3-(dimethoxymethylsilyl)propylamine), (3-aminopropyl)silanetriol, (N-(2-aminoethyl)(3-aminopropyl)methyldimethoxysilane), or N-(2-aminoethyl)(3-aminopropyl)trimethoxysilane).

[0024] In further embodiments of the method for preparing AFSNP, the step further comprises: reacting the washed AFSNP with a second solution to attach a crosslinker to the compound through a first amine functional group of the crosslinker. Preferably, the crosslinker has the following chemical formula:

[0025] (i) -HN-C(=O)-CH2C(=CH2)-C(=O)-NH-; and / or

[0026] (ii) -HN-C(=O)-CH2C(=CH2)-C-NH-. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Schematic diagrams showing the synthesis of amine-functionalized silica nanoparticles (AFSNP) by (A) the one-step synthesis process of the method of the present invention and (B) the conventional synthesis process;

[0028] Figure 2 Schematic diagram showing the immobilization of biomolecules on the surface of ASFNP using itaconic acid as a crosslinker;

[0029] Figure 3 (A) and (B) show the morphological characteristics of silica nanoparticles and amine-functionalized silica nanoparticles, and (C) and (D) show the particle size distributions of silica nanoparticles and amine-functionalized silica nanoparticles, respectively;

[0030] Figure 4 SEM images of AFSNP at different APTES concentrations, with APTES concentrations of (A) 30 mol%; (B) 50 mol%; (C) 70 mol%; and (D) the ζ-potential measurement results of AFSNP;

[0031] Figure 5 Graph showing the effect of APTES concentration on the antibody binding efficiency on the nanoparticle surface by ELISA technology;

[0032] Figure 6 Graph comparing the differences in the stability of biomolecule immobilization between AFSNP prepared by the traditional synthesis method of AFSNP and the one-step synthesis method of AFSNP;

[0033] Figure 7 Showing: (A) Schematic diagram of the role of AFSNP in improving the performance of traditional ELISA; (B) ELISA test results; (C) Graph showing the ELISA detection sensing performance in the presence and absence of AFSNP;

[0034] Figure 8To show the curve graph of the specificity of the anti-rabies virus detection ELISA test, where the anti-rabies virus antibody concentration is 2.5 IU / mL and the bovine serum albumin (BSA) concentration is 1 mg / mL. Detailed implementation manners

[0035] The present invention will be described below in conjunction with preferred embodiments and the accompanying drawings. It should be clear that the embodiments and the drawings are only used to illustrate the technical solutions of the present invention, and those skilled in the art can make various modifications within the scope of the appended claims.

[0036] As used herein, the term "in certain embodiments" means some but not all embodiments.

[0037] As used herein, the term "about" or "approximately" when describing component concentrations, conditions, or other measured values means within the range of ±5%, ±4%, ±3%, ±2%, ±1%, ±0.5%, or ±0% of the stated value.

[0038] The terms "amine-functionalized silica nanoparticles" (AFSNP) and "conjugate" are used interchangeably in this specification, and both refer to substances synthesized by a specified method and conditions using monomers and comonomers in the following proportions, which have functional terminal amino groups capable of binding to amine functional groups located on the crosslinker, and the crosslinker realizes the connection of the substance to the biomolecule.

[0039] As used herein, the term "biomolecule" refers to one or more compounds derived from an organism or a similar source, such as a virus. These biomolecules can be subjected to any known pretreatment and / or modification before binding to AFSNP to achieve at least one of the foregoing purposes.

[0040] Refer to Figure 1 A and Figure 2 , which shows a method for preparing a biomolecule-crosslinked compound. Basically, the method includes the following steps: reacting a plurality of monomers with a plurality of comonomers in the presence of ammonia in a first solution to form a compound; extracting the obtained compound from the first solution for cleaning; reacting the cleaned compound with a second solution to connect a crosslinker to the compound through a first amine functional group of the crosslinker; and adding a plurality of biomolecules to the second solution to bind the biomolecules to the compound through a second amine functional group of the crosslinker, thereby obtaining a biomolecule-crosslinked compound.

[0041] According to several embodiments of the method of the present invention, the first solution contains ammonium hydroxide dissolved in a mixture of distilled water and ethanol, where the volume ratio of distilled water to ethanol is 1-10:20-30. It should be noted that in other embodiments of the method of the present disclosure, other water-soluble ammonium salts, such as ammonium carbonate, ammonium chloride, ammonium nitrate, can be used to prepare the first solution instead of ammonium hydroxide. Similarly, as long as the ammonium salt can dissolve and ionize in the first solution, thereby promoting the simultaneous hydrolysis and condensation reactions of the monomer and the comonomer, other water-miscible alcohols, such as n-propanol, isopropanol, tert-butanol, etc., can be used to prepare the first solution.

[0042] In more embodiments, the monomer is tetrapropyl orthosilicate (TPOS), tetraethyl orthosilicate (TEOS), tetramethyl orthosilicate (TMOS), tetraisopropyl orthosilicate or tetrabutyl orthosilicate. Correspondingly, the comonomers that can react with the monomer in the disclosed method are selected from: (3-aminopropyl)triethoxysilane), (3-aminopropyl)trimethoxysilane), (3-aminopropylmethyldiethoxysilane), (3-(dimethoxymethylsilyl)propylamine), (3-aminopropylsilanetriol), (N-(2-aminoethyl)(3-aminopropyl)methyldimethoxysilane) or N-(2-aminoethyl)(3-aminopropyl)trimethoxysilane). The molar ratio of the monomer to the comonomer in the first solution is 1.3-4.0:0.4-3.0.

[0043] The inventors have found that the method is preferably carried out under relatively mild conditions, making the reaction process controllable and adjustable, and basically avoiding the generation of common by-products in traditional methods. Preferably, the reaction step is carried out at room temperature. However, the reaction step can also be carried out at a temperature slightly lower or slightly higher than room temperature. The temperature range of the reaction step is 20 to 45 °C. Preferably, by gently stirring the reactants for a period of time, preferably 2 to 24 hours, the specific duration depending on the amount of reactants in the reaction step, the simultaneous hydrolysis and condensation reactions in the first solution can be accelerated.

[0044] After the reaction step is completed, the produced or generated compound can be extracted from the first solution for cleaning to remove the unreacted reagents on the surface of the compound, and then the compound is reacted with the second solution and the reagents contained in the second solution. The produced compound can be extracted by filtration, centrifugation or other methods known in the art. This cleaning step or treatment can avoid cross-contamination of the reagents in the first solution to the second solution. The cleaned compound can also be kept in the form of freezing or freeze-dried powder for a period of time until the cleaned compound contacts and reacts with the second solution and the reagents contained in the second solution.

[0045] According to several preferred embodiments of the method of the present invention, the second solution contains itaconic acid, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), and N-hydroxysuccinimide (NHS), and the three are dispersed in the aqueous phase in a molar ratio of 15-35:60-90:30-60. It should be particularly noted that the itaconic acid is used as a cross-linking agent to connect biomolecules with compounds or AFSNPs. More preferably, the itaconic acid contains an amine functional group at each of its ends, where one end amine functional group is used to bind to AFSNP or a compound, and the other end amine functional group is specifically used to bind to a biomolecule. Still, in some embodiments, the itaconic acid can be modified to obtain a compound for biomolecule cross-linking with desired properties. In some embodiments of the disclosed method, the itaconic acid in the second solution can react with the compound and the biomolecule simultaneously, such that the functional groups at both ends of the itaconic acid have an equal chance to bind to either the biomolecule or the compound, ultimately forming a biomolecule-crosslinked compound. In such embodiments, the contacting step and the adding step of the disclosed method are carried out in a synchronous manner. Generally, sufficient reaction time is required for the compound, the biomolecule, and the itaconic acid, preferably about 15 to 120 minutes, which can be adjusted according to the reagent dosage, and then the formed biomolecule-crosslinked compound is extracted from the second solution. After adding the compound and the biomolecule, gentle stirring can be applied to uniformly mix the biomolecule and the compound in the second solution to achieve a better binding equilibrium state. In other embodiments, the contacting step and the adding step are carried out in a sequential order, that is, the biomolecule is added after the compound and the itaconic acid have reacted for a predetermined time, and vice versa.

[0046] According to other embodiments, the disclosed method further includes: precipitating the prepared biomolecule-crosslinked compound and dispersing the precipitated biomolecule-crosslinked compound in distilled water. The precipitation step can be achieved by centrifugation or filtration, and the dispersion step aims to remove the residual unreacted reagent contaminants on the surface of the biomolecule from the obtained biomolecule-crosslinked compound. These residual unreacted reagents may affect the efficiency of the produced biomolecule-crosslinked compound in detecting or determining other substances, preferably substances present in a sample when the biomolecule targets them, where the biomolecule is linked to AFSNP.

[0047] In some embodiments, the biomolecule is any one of an antigen, an antibody, a peptide chain, a hormone, DNA, or RNA with an amino terminus that can bind to the second amine functional group of the cross-linking agent. This biomolecule is specifically used to bind, conjugate, link, or hybridize with one or more analytes such as corresponding antigens, peptide chains, complementary DNA, complementary RNA, etc. that may be present in a sample obtained from an object to obtain a diagnostic conclusion or a detection result.

[0048] Another aspect of the present invention relates to a biomolecule-crosslinked compound having a chemical structure of R1-X-R2. Thus, X is a crosslinker represented by chemical formula (i) -HN-C(=O)-CH2C(=CH2)-C(=O)-NH- or (ii) -HN-C(=O)-CH2C(=CH2)-C-NH-; R1 is a silica nanoparticle, and R2 is a biomolecule having an amino terminus capable of binding to the amine functional group of the crosslinker. It should be specifically noted that the disclosed biomolecule-crosslinked compound can be synthesized by, but not limited to, the above methods.

[0049] The inventors of the present disclosure have found that the crosslinker can be prepared by dispersing itaconic acid, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), and N-hydroxysuccinimide (NHS) in an aqueous phase at a molar ratio of 15-35:60-90:30-60 and reacting at a temperature of 20°C to 40°C. Specifically, the carboxyl groups at both ends of itaconic acid react with EDC / NHS to form two amide bonds. One amino group is specifically used to bind biomolecules, such as proteins and amino-labeled nucleotides, and the other amino group binds to an amine-functionalized substrate including AFSNP. This N-terminal to N-terminal binding ability is particularly suitable for biosensor applications, especially amine-functionalized silicon-based platforms, such as silicon photonic chips, silicon nanowires, and optical fibers.

[0050] As described in the examples, the compound is preferably synthesized from multiple monomers and comonomers at a molar ratio of 1.3-4.0:0.4-3.0. More preferably, some embodiments of the compound of the present disclosure can be synthesized using tetrapropyl orthosilicate (TPOS), tetraethyl orthosilicate (TEOS), tetramethyl orthosilicate (TMOS), tetra-isopropyl orthosilicate, or tetrabutyl orthosilicate. Correspondingly, the comonomers are selected from (3-aminopropyl)triethoxysilane, (3-aminopropyl)trimethoxysilane, (3-aminopropylmethyldiethoxysilane), (3-(dimethoxymethylsilyl)propylamine), (3-aminopropylsilanetriol), N-(2-aminoethyl)(3-aminopropyl)methyldimethoxysilane, or N-(2-aminoethyl)(3-aminopropyl)trimethoxysilane for several embodiments of the compound of the present disclosure.

[0051] In some embodiments of the compound of the present disclosure, the biomolecule is any one of an antigen, an antibody, a peptide chain, DNA, or RNA having an amino terminus capable of binding to the second amine functional group of the crosslinker. This biomolecule is specifically used to bind, conjugate, link, or hybridize with one or more analytes such as corresponding antigens, peptide chains, complementary DNA, complementary RNA, etc. that may be present in a sample obtained from an object to obtain a diagnostic conclusion or a detection result.

[0052] The following examples are used to further illustrate the present invention, but should not be construed as limiting the specific embodiments of the present invention.

[0053] Example 1

[0054] Adopt Figure 1 The one-step synthesis method shown and the conventional synthesis method are used to prepare amino-functionalized silica nanoparticles (AFSNP). In the one-step synthesis method, silicon-based monomers of TEOS and APTES are used to produce or modify surface-functionalized nanoparticles ( Figure 1 A). The hydrolysis and condensation reaction is initiated by adding a basic catalyst to the mixed solution, while in the conventional AFSNP synthesis method, TEOS is first used as the silicon source, and the bare SNP is prepared by an improved method ( Figure 1 B). Then APTES is added to achieve amino-functionalization to cover the surface of the SNP. In particular, in this experiment, an improved reaction is used to synthesize AFSNP with uniform particle size in one step. In particular, TEOS and APTES are added to a mixed solvent of ethanol and distilled water, and ammonium hydroxide is used as the hydrolysis and condensation catalyst. The mixture is stirred at room temperature for 6 hours. After three centrifugation and redispersion cleaning processes. Finally, the freeze-dried product is collected for standby.

[0055] The particle size and surface charge of AFSNP in ethanol are measured using a laser particle size analysis system (NANO ZS, Malvern instruments Ltd., UK). A He-Ne laser is used as the light source. The incident wavelength is 633 nm, and the detection angle is 173°. The ζ potential test is completed using a highly diluted colloidal dispersion at 25 °C. The morphology of AFSNP is observed by scanning electron microscopy (SEM, JSM-IT800). An energy-dispersive X-ray spectroscopy (EDS) detector is used with the SEM to determine the elemental composition of the particles under study during electron microscopy measurement. When combined with the SEM for testing, the diluted colloidal dispersion is dropped onto the copper tape of an aluminum substrate (cleaned with ethanol) to produce well-fractioned particles on the surface. A Fourier transform infrared spectrometer (FTIR) is used to confirm the chemical bond structure of AFSNP. The spectrum in the wavenumber range of 500 - 4000 cm is recorded by an FTIR spectrometer (Thermo Scientific, U.S.A). The sample is scanned in the attenuated total reflection (ATR) mode.

[0056] Figure 3 The scanning electron microscopy (SEM) image of the nanoparticles is shown. SNP ( Figure 3 A) and AFSNP ( Figure 3B) Both exhibited typical spherical morphologies with smooth surfaces and uniform particle sizes. The particle size of AFSNP (about 161 ± 5.9 nm) was slightly smaller than that of bare SNP (about 164 ± 17.4 nm). The particle size distribution was estimated by dynamic light scattering (DLS). As Figure 3 C and Figure 3 D show, the histograms of the particle size distributions of SNP and AFSNP are presented, respectively. The histograms were fitted with log-normal distribution curves, and the peaks were taken as the average particle sizes of about 218 ± 2.5 nm (SNP) and 216 ± 1.2 nm (AFSNP), and the polydispersity index (PDI) was low, indicating a highly uniform particle size distribution, consistent with the SEM results. However, the particle sizes from different tests between SEM and DLS originated from non-uniform statistics in the weighting process, which was inevitable 26 .

[0057] Example 2

[0058] In addition, the inventors of the present disclosure synthesized AFSNPs by mixing different concentration ratios of TEOS and APTES, as Figure 4 (A - C) show. As the APTES concentration increased (30, 50, and 70 mol%), the morphological characteristics revealed that the particle size of AFSNP increased to 224 ± 8.4 nm, 279 ± 18.0 nm, and 384 ± 44.6 nm, respectively, and the surface roughness of the particles increased due to the incompletely hydrolyzed and condensed amino groups (-NH2). Figure 4 D shows the surface charge from ζ-potential tests. It can be seen that compared with the bare SNPs with negatively charged oxygen atoms on the surface, all AFSNPs under all conditions showed positive charges. This confirmed that surface amino-functionalization modification could be achieved by adding APTES to the system 18,21 . However, the similar charges in the range of +32 mV to +39 mV could not confirm the number of amino groups due to the difference in the number of particles in the solution.

[0059] Energy-dispersive spectroscopy (EDS) analysis of the elemental composition and mapping images of SNP had mapping images obtained using elemental K-line spectra. The inventors found that the components were non-uniformly distributed in the nanoparticles, and high-content regions of O and Si were present in both samples, while low-content regions of N were only seen in Figure 4 B. The results were consistent with the elemental components of O, Si, and N, where the atomic ratios of O, Si, and N were close to those of SNP (4.5:1:0) and AFSNP (3.1:1:0.3), respectively. This indicated that the elements were randomly distributed in the nanoparticles. Thus, this confirmed the structure of the silicon-based nanoparticles.

[0060] The Fourier transform infrared spectra (FTIR) of SNP and AFSNP (conventional method and one-step method) were in the range of 1049 cm -1 to 1100 cm -1and 795 cm -1 Peaks appear near, which are respectively attributed to the stretching vibrations of Si-O-Si bonds in the framework and free silanol groups 10 . An absorption peak at 939 cm -1 to 945 cm -1 indicates Si-OH vibration 27 . Compared with SNP and AFSNP (conventional method), AFSNP (one-step method) shows weaker absorption than SNP. This is because part of Si-OH is replaced by the bending vibration of -NH (primary amine) in APTES and a new band is formed at 694 cm -1 , and this peak does not appear in AFSNP (conventional method) because no crosslinking is formed between TEOS and APTES.

[0061] In addition, AFSNP (conventional method) shows broad peaks at 3260 cm -1 and 1628 cm -1 , corresponding to the stretching and bending of -NH2 29,30 . This indicates that the copolymer of TEOS and APTES successfully modifies the nanoparticle surface. It is worth noting that the stretching and bending bands of -NH2 in AFSNP (one-step method) shift to lower wavenumbers due to hydrogen bonding 31 , thus showing higher molecular reactivity and specific interactions. Comparing the spectra of AFSNP (one-step method) at different concentrations of added APTES, it is found that at the peak at 694 cm -1 , the intensity of 70 mol% APTES is higher than that of 50 mol% APTES.

[0062] Example 3

[0063] The crosslinking reaction was carried out in a 1.5 mL centrifuge tube. Itaconic acid, distilled water, EDC, and NHS were mixed and reacted at room temperature for 30 minutes. Subsequently, AFSNP and rabies virus antigen were added synchronously. AFSNP was incubated with rabies virus antigen (concentrations were 0.4 μg, 0.2 μg, 0.1 μg, 0.05 μg, 0.025 μg, and 0.0125 μg respectively) at 25 °C for 1 hour. After the functionalized nanoparticles were centrifuged at 4000 rpm for 10 minutes, the precipitated nanoparticles were resuspended three times with washing buffer to remove free antigen. The supernatant was collected for antigen quantification. As Figure 2 shown, the newly developed crosslinker realizes the connection of amino-functionalized biomolecules and amino-functionalized ASFNP through covalent bonds. The amino concentration efficiency ( Figure 6 ), the stability of biomolecule immobilization (see Figure 6) and AFSNP performance. Compare the signals of SNPs and AFSNPs synthesized at different APTES concentrations in the ELISA detection of rabies virus antibodies. It was found that the concentration of APTES added to the particle synthesis system affected the morphological characteristics of the particles such as particle size and surface roughness. It also affected the amine group content on the particle surface, thereby affecting antigen conjugation. Since the antigen is a protein, it requires primary amine groups for binding through the use of itaconic acid linkage and functional cross-linkers.

[0064] Example 4

[0065] Coat the antigen-immobilized AFSNP (0.01 mg / mL) on a 96-well ELISA plate (Immune; Nunc) and let it stand overnight at 4°C. Subsequently, after washing three times with the washing buffer, block it with the blocking buffer for 6 minutes. After removing the blocking buffer, add the antibody diluent (100 μL) at the desired concentrations (2.50, 1.25, 0.63, 0.31, 0.16, and 0.08 IU / mL) and incubate at room temperature for 1 hour. Then, after washing the well plate seven times with the washing buffer, add rabbit anti-horse IgG (whole molecule)-HRP (100 μL / well, optimized dilution ratio 1:3000) and incubate at room temperature for 1 hour. Wash the well plate seven times again with the washing buffer. After that, add the TMB substrate to each well and react at room temperature for 15 minutes. Finally, add the stop solution to terminate the reaction, and measure the absorbance of the product at wavelengths of 450 nm and 570 nm using a microplate reader ( M Plex, Tecan Trading AG, Switzerland).

[0066] As Figure 5 shown, the SNP with 0 mol% APTES did not show an ELISA signal, indicating that the antigen could not be immobilized on the surface of the SNP, while the AFSNP showed a higher ELISA signal with increasing antigen concentration. At the initial stage of the curve, the 30 mol% APTES with the smallest number showed the highest slope for antibody immobilization due to the smallest particle size and the largest specific surface area 34 . However, due to the limited number of available amine groups on the surface for antibody immobilization, even if the antibody concentration is increased after reaching the saturation point, there is not enough antigen to maintain antibody immobilization, resulting in the curve flattening out. For the APTES concentrations of 50 mol% and 70 mol%, due to the sufficient number of available amine groups on the surface, the immobilization efficiency is extended, causing the absorbance value to continue to rise with the increase in antibody capture. Based on this, the inventors of the present disclosure believe that the one-step synthesis technology can be used to design and quantify antibodies, antigens, polypeptides, etc. on the surface of a detection platform integrating functionalized AFSNPs.

[0067] In addition, the inventors of the present disclosure determined and compared the biomolecular binding capabilities of AFSNPs prepared by the conventional method and the one-step synthesis method, as Figure 1-2As shown, it was found that the one-step synthesis method of AFSNP showed higher ELISA signals in anti-rabies virus detection compared to traditional methods. In addition, this method has the advantage of using less APTES during synthesis. Although nanoparticles of similar particle sizes were used in this study, the ELISA signals of the one-step synthesized nanoparticles remained high due to the arrangement of amine (-NH2) structures in the particles, which facilitated the immobilization of nanoparticles and antigens. 35 . Figure 7 Showed a highly sensitive ELISA by using itaconic acid crosslinker in indirect ELISA based on AFSNP as a nanocarrier for anti-rabies virus antigen. This indirect ELISA technique employed a two-step detection method. 36 . Specifically, horse anti-rabies virus (primary antibody) specific to rabies virus antigen was bound to AFSNP, and then detected by anti-horse IgG-HRP (labeled secondary antibody) binding to the primary antibody, as shown in Figure 7 A. The anti-rabies virus detection performances of traditional ELISA and AFSNP-based ELISA were also compared. It was expected that when AFSNP was applied to ELISA, the amount of antigen immobilization and ELISA signals could increase. This was attributed to the increased surface area and functionalization characteristics of synthesized AFSNP. Figure 7 In B, the reaction product color of applying AFSNP to ELISA development showed significantly deeper yellow than traditional ELISA. The absorbance measured at 450 nm showed that the absorbance of AFSNP conjugated to ELISA showed higher signals than the traditional ELISA method, as shown in Figure 7 C. The limit of detection (LOD) of the conjugated AFSNP reached 0.05 IU, and the sensitivity was ten times higher than that of traditional ELISA (0.5 IU). This evidence demonstrated the advantage of ELISA in detection sensitivity through the functionalization efficiency of biological receptors. AFSNP could provide an increased surface area, which led to a higher biomolecule binding capacity. 37 . Thus, AFSNP could enhance the signal amplification and detection capabilities of ELISA technology, which was applicable to the early infection diagnosis of extremely low concentration biomarkers. 38 .

[0068] Figure 8 Showed the specificity of the said ELISA for anti-rabies virus detection. Another sample, bovine serum albumin (BSA), was recognized by AFSNP-rabies virus antigen. The microplate was first coated with AFSNP-rabies virus antigen, and the absorbance value of BSA was the same as that of the negative control group and did not cause color change. In addition, the negative control group without using itaconic acid crosslinker in the system could verify the absence of immobilized rabies antigen. The absorbance value of anti-rabies virus was 60 times higher than that of BSA, indicating that the enhanced ELISA based on AFSNP had high specificity for anti-rabies virus detection.

[0069] It should be understood that the present invention can be implemented in other specific forms and is not limited to the above single embodiment. However, the modification schemes and equivalent technical solutions that are easily conceivable by those skilled in the art should be included within the scope of the appended claims.

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Claims

1. A biomolecule-crosslinked compound, whose chemical structure is: R1-X-R2 wherein X is a crosslinking agent with the following chemical formula: (i) -HN-C(=O)-CH2C(=CH2)-C(=O)-NH- or (ii) -HN-C(=O)-CH2C(=CH2)-C-NH-; wherein R1 is a silica nanoparticle and R2 is a biomolecule with an amino terminus capable of binding to the amine functional group of the crosslinking agent.

2. The biomolecule-crosslinked compound according to claim 1, wherein, The crosslinking agent is prepared by dispersing itaconic acid, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), and N-hydroxysuccinimide (NHS) in an aqueous phase at a molar ratio of 15-35:60-90:30-60 and reacting them.

3. The biomolecule-crosslinked compound according to claim 1, wherein, The biomolecule is any one of an antigen, an antibody, a peptide chain, DNA, or RNA.

4. The biomolecule-crosslinked compound according to claim 1, wherein The silica nanoparticle contains monomers and comonomers with a molar ratio of 4.0:0.4 to 1.3:3.

0.

5. The biomolecule-crosslinked compound according to claim 4, wherein The monomer is tetrapropyl orthosilicate (TPOS), tetraethyl orthosilicate (TEOS), tetramethyl orthosilicate (TMOS), tetra-isopropyl orthosilicate, or tetrabutyl orthosilicate.

6. The biomolecule-crosslinked compound according to claim 4, wherein, The comonomer is selected from (3-aminopropyl)triethoxysilane, (3-aminopropyl)trimethoxysilane, (3-aminopropylmethyldiethoxysilane), (3-(dimethoxymethylsilyl)propylamine), (3-aminopropylsilanetriol), (N-(2-aminoethyl)(3-aminopropyl)methyldimethoxysilane), or N-(2-aminoethyl)(3-aminopropyl)trimethoxysilane).

7. A preparation method of a biomolecule-crosslinked compound, comprising the following steps: Reacting a variety of monomers and a variety of comonomers in the presence of ammonia in a first solution to generate a compound; Extracting the obtained compound from the first solution for cleaning; Reacting the cleaned compound with a second solution to connect the crosslinking agent to the compound through the first amine functional group of the crosslinking agent; and Adding a variety of biomolecules to the second solution to bind the biomolecules to the compound through the second amine functional group of the crosslinking agent, thereby obtaining the biomolecule-crosslinked compound, wherein the monomer is tetrapropyl orthosilicate (TPOS), tetraethyl orthosilicate (TEOS), tetramethyl orthosilicate (TMOS), tetra-isopropyl orthosilicate, or tetrabutyl orthosilicate, wherein the comonomer is selected from (3-aminopropyl)-triethoxysilane, (3-aminopropyl)-trimethoxysilane, (3-aminopropylmethyldiethoxysilane), (3-(dimethoxymethylsilyl)propylamine), (3-aminopropylsilanetriol), (N-(2-aminoethyl)(3-aminopropyl)methyldimethoxysilane), or N-(2-aminoethyl)(3-aminopropyl)trimethoxysilane).

8. The method according to claim 7, wherein The molar ratio of the monomer to the comonomer is 4.0:0.4 to 1.3:3.

0.

9. The method according to claim 7, wherein The reaction step is carried out within a temperature range of 20°C to 40°C.

10. The method according to claim 7, characterized in that The first solution contains ammonium hydroxide dissolved in a mixed solution of distilled water and ethanol, wherein the volume ratio of distilled water to ethanol is 1-10:20-30.

11. The method according to claim 7, characterized in that, The crosslinking agent is itaconic acid, which contains amine functional groups at both ends.

12. The method according to claim 7, wherein The second solution contains itaconic acid, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), and N-hydroxysuccinimide (NHS), and the three are dispersed in the aqueous phase at a molar ratio of 15-35:60-90:30-60.

13. The method according to claim 7, characterized in that The biomolecule is any one of an antigen, an antibody, a peptide chain, DNA, or RNA having an amino terminus with a second amine functional group capable of binding to a crosslinker.

14. A method for preparing amine-functionalized silica nanoparticles (AFSNP), comprising: reacting a plurality of monomers with a plurality of comonomers in the presence of ammonia in a first solution to produce AFSNP; and extracting the obtained AFSNP from the first solution for washing, wherein the monomer is tetrapropyl orthosilicate (TPOS), tetraethyl orthosilicate (TEOS), tetramethyl orthosilicate (TMOS), tetra-isopropyl orthosilicate, or tetrabutyl orthosilicate, and the comonomer is selected from (3-aminopropyl)triethoxysilane, (3-aminopropyl)trimethoxysilane, (3-aminopropylmethyldiethoxysilane), (3-(dimethoxymethylsilyl)propylamine), (3-aminopropylsilanetriol), (N-(2-aminoethyl)(3-aminopropyl)methyldimethoxysilane), or N-(2-aminoethyl)(3-aminopropyl)trimethoxysilane).

15. The method according to claim 14, further comprising reacting the washed AFSNP with a second solution to attach a crosslinker to the compound through a first amine functional group of the crosslinker, wherein the crosslinker has the following chemical formula: (i) -HN-C(=O)-CH2C(=CH2)-C(=O)-NH- or (ii) -HN-C(=O)-CH2C(=CH2)-C-NH-.

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