A gated mesoporous nanomaterial and its preparation method and kit

Abstract

The present invention discloses a gated mesoporous nanomaterial, its preparation method, and a kit, belonging to the field of detection technology. The preparation method of the present invention enables the gated mesoporous nanomaterial to have good sealing stability and a rapid signal release rate. Furthermore, based on the application of this nanomaterial in a rapid detection kit, the present invention achieves a rapid test strip signal amplification function, improves the test strip's detection sensitivity, and can simultaneously detect both large and small molecule targets using the same detection mechanism.

Description

Technical Field

[0001] The present invention belongs to the field of detection technology, and in particular relates to a gated mesoporous nanomaterial and a preparation method and a kit thereof. Background Art

[0002] Aptamer-gated mesoporous materials are widely used in the field of target detection. Mesoporous materials have a large internal volume, and their properties allow many signal molecules (such as fluorescent dyes, etc.) to be loaded inside the mesoporous materials. At the same time, the mesoporous materials have controllable pore sizes, and their pore sizes can be sealed by aptamers to block the small molecule materials loaded inside; then, the sealing materials will bind to external targets, resulting in structural changes in the sealing materials to open the blocked pores and release the signal molecules loaded inside the mesoporous materials, which is the "gating" effect. The advantage of using aptamer-gated mesoporous materials is that a small amount of the substance to be tested can stimulate the release of a large number of signal molecules, ultimately forming an amplification of the detection signal and improving the detection sensitivity.

[0003] The effectiveness of gating depends directly on the material sealing method. Traditional sealing methods are generally categorized into three types. The first is electrostatic adsorption, which utilizes the electrostatic attraction between the mesoporous material and the sealing material to coat the sealing material on the surface of the mesoporous material, thereby blocking the pores. This method is considered the simplest and most versatile, but its stability is poor because the electrostatic attraction is easily disrupted by changes in external conditions such as ion concentration, temperature, and pH, leading to leakage of internal signal molecules. The second method is the "plugging" method, in which a sealing aptamer is designed to act as a "plug" to block the pores of the mesoporous material. Specifically, the mesoporous material is covalently coupled to the aptamer, which is designed to form a hairpin structure. The hairpin structure acts as a "plug," blocking the pore and preventing leakage of the internal material. Subsequently, upon binding to the target, its secondary structure changes, opening the pore and releasing the internal signal molecule. However, this design often suffers from unstable sealing, which can lead to leakage of internal signal molecules and false positives during target detection. The third method is the "bridging" method, which uses a group of sealing aptamers to form a "bridge" to block the pore size of the mesoporous material. Specifically, the mesoporous material is covalently coupled to the arm-shaped ssDNA, and then the arm-shaped ssDNA is combined with the aptamer through complementary base pairing to form a "bridge" to seal the mesopore. Once the aptamer is coupled to the target, it can cause the aptamer to separate from the arm-shaped ssDNA, causing the pore to open and release the internal signal molecules. However, since the single-stranded size of the aptamer is smaller than the mesoporous pore (1nm vs. 3nm), there will also be leakage of internal signal molecules. If a "bridge" is formed in the form of a double-stranded DNA, the "bridge" bonding force is too strong, and the aptamer is not easily separated from the arm-shaped ssDNA, resulting in failure to release the signal molecule. Furthermore, both the "plugging" and "bridging" methods couple aptamers or arm-shaped ssDNA to the surface of the mesoporous material, which can affect the release of internal signal molecules. When the mesopores are relatively small (e.g., ~3 nm), the signal release is too slow, making it unsuitable for rapid detection applications. If large mesopores are used, this can lead to loose seals and leakage of signal molecules.

[0004] For these reasons, aptamer-gated mesoporous materials have not been used in the development of rapid test strips. This is because rapid test strips require ligand-gated mesoporous materials with good sealing stability to prevent signal molecule leakage and a fast signal release rate.

[0005] Rapid test strips are a type of on-site rapid detection and analysis method based on chromatography. Due to its significant cost-effectiveness, it has been widely used in many fields, such as diagnosis and food safety. Most test strips use a sandwich test format. In a positive test, the analyte in the sample binds to the gold nanoparticle-labeled primary antibody and moves along the paper strip by capillary action, and is captured by the target secondary antibody on the test line (T line), thereby forming a visible signal on the T line (i.e., aggregation of gold nanoparticles). To ensure the accuracy of the test results, a control line (C line) is usually set downstream of the T line. It is usually assembled from anti-primary antibody antibodies. Regardless of whether the test result is positive or negative, the C line should show a signal to ensure that the test is valid.

[0006] Although many test strips have been successfully commercialized, such as early pregnancy and ovulation test strips, their detection sensitivity is relatively low. This problem stems from the fact that when the concentration of the target analyte is low, the concentration of the target-gold-labeled antibody that can generate a signal is too low, so too little aggregation on the T line fails to generate a significant signal. At the same time, the test strips also lack a corresponding signal enhancement mechanism. Therefore, rapid test strips are usually only used when the concentration of the target analyte is high, such as early pregnancy tests where the concentration of human chorionic gonadotropin increases significantly during pregnancy. To improve the detection sensitivity of the paper strip detection platform, people have tried to use high-efficiency fluorescent quantum dots to replace gold nanoparticles as signal agents to enhance the signal, but the signal amplification function is limited and there is still much room for improvement.

[0007] Furthermore, when using rapid test strips to detect small molecules, sandwich assays are not suitable due to the limited binding sites of small molecule targets. Therefore, competitive assays are primarily used. The competitive assay relies on competition with colloidal gold-labeled antibodies to achieve detection. This method involves coupling a small amount of antibody with colloidal gold and coating it on the sample conjugation pad, while the antigen is coated at the T-line. During the test, when the target antigen is present in the sample, the antigen first binds to the gold-labeled antibody. Due to the low concentration of the gold-labeled antibody, the antigen at the T-line cannot bind to the gold-labeled antibody, resulting in no color development. If the sample is negative, the antigen at the T-line can bind to the gold-labeled antibody and develop color. However, competitive assays are not as sensitive as sandwich assays. Furthermore, at very low antigen concentrations, false negative results may occur if the antigen does not fully occupy the antibody at the conjugation pad. Summary of the Invention

[0008] To address the aforementioned issues of unstable sealing, slow signal release, and low sensitivity of rapid test strips associated with aptamer-gated mesoporous materials, the present invention provides a gated mesoporous nanomaterial based on dendrimer-bound aptamers and a method for preparing the material. This material exhibits excellent sealing stability and rapid signal release. Furthermore, a method for applying this nanomaterial in a rapid test kit is also provided, enabling rapid test strip signal amplification, improving the strip's sensitivity, and enabling the detection of both large and small molecule targets using the same detection mechanism.

[0009] To achieve the above purpose, the specific technical solutions of the present invention are as follows:

[0010] A method for preparing a gated mesoporous nanomaterial, wherein the gated mesoporous nanomaterial can be used to prepare a test strip, comprises the following steps:

[0011] (1) Signal molecules are loaded into mesoporous nanoparticles to obtain signal molecule-loaded mesoporous nanoparticles OSM@MSNs.

[0012] (2) coupling the target-specific aptamer to the OSM@MSNs obtained in step (1) to obtain the mesoporous nanoparticle surface-coupled aptamer OSM@MSNs-aptamer loaded with the signal molecule.

[0013] (3) coupling ssDNA with the nth generation carboxyl polyamidoamine dendrimer to obtain ssDNA-Gn, wherein n≥3.5,

[0014] The ssDNA is used to bind to the aptamer in step (2) and is competitively dissociated from the single-stranded DNA in the presence of the detection target,

[0015] (4) Assembling the ssDNA-Gn obtained in step (3) with the OSM@MSNs-aptamer obtained in step (2), and allowing the nth generation carboxyl polyamide-amine dendrimer to seal the mesoporous nanoparticles through the binding between the ssDNA and the aptamer.

[0016] In the present examples, a 3.5-generation carboxypolyamidoamine dendrimer is used as an example because its molecular size is just larger than the mesopore size. In principle, any macromolecule larger than the mesopore size will suffice. For example, 4th, 4.5th, 5th, and 5.5th-generation carboxypolyamidoamine dendrimers are all suitable.

[0017] Preferably, the signal molecule is at least one of rhodamine B, fluorescein isothiocyanate, Cy3, Cy5, and fluorescein. The mesoporous nanoparticles are mesoporous silica nanoparticles, mesoporous titanium dioxide nanoparticles, metal organic frameworks, mesoporous alumina, mesoporous magnetic beads, mesoporous ferrosoferric oxide, or mesoporous copper sulfide nanoparticles.

[0018] Specifically, in step (2), when the target-specific aptamer is coupled to the OSM@MSNs obtained in step (1), the N=C=O group is introduced by functionalizing the surface of the OSM@MSNs obtained in step (1), and the end group of the aptamer is modified with an amino group, and the aptamer is coupled through the reaction between the N=C=O group and the amino group;

[0019] Alternatively, the surface of the OSM@MSNs obtained in step (1) is functionalized to introduce amino groups, the terminal group of the aptamer is modified with a -SH group, and the aptamer is coupled with Na-maleimidoacetyl-oxysuccinimide ester.

[0020] Alternatively, -SH groups are introduced by functionalizing the surface of the OSM@MSNs obtained in step (1), and the end groups of the aptamer are modified with amino groups, and then coupled to the aptamer via Na-maleimidoacetyl-oxysuccinimide ester;

[0021] Alternatively, amino groups are introduced by functionalizing the surface of OSM@MSNs obtained in step (1), and the end groups of the aptamer are modified with amino groups, and then coupled with the amino-containing aptamer via glutaraldehyde or (bis(sulfosuccinimide) suberate);

[0022] In step (3), when ssDNA is coupled with the n-generation carboxyl polyamidoamine dendrimer to obtain ssDNA-Gn, the two undergo a coupling reaction mediated by EDC / NHS.

[0023] Preferably, the size of the mesoporous nanoparticles is 3 to 5 nm.

[0024] More preferably, the size of the mesoporous nanoparticles is 4 nm.

[0025] Traditional aptamer-based gated mesoporous nanomaterials typically use MCM-41 (with a pore size of approximately 3 nm) as a carrier to meet the needs of different aptamer structures (single-stranded about 1 nm, double-stranded about 2 nm) for blocking mesopores. However, the blocking effect is not ideal because the size of the blocking structure is much smaller than the mesopore size (as described in the background). The embodiment of the present invention uses an aptamer combined with G3.5 as a sealing material. The molecular size of G3.5 is approximately 4.5 nm, which is larger than the mesopore size (4 nm) used in the present invention. Therefore, the sealing effect is stable and the problem of signal molecule leakage will not occur.

[0026] The traditional gated mesoporous nanomaterial MCM-41, used for aptamers, has a pore size of approximately 3nm, resulting in a slow release rate of signal molecules, making it suitable for use in scenarios where detection rates are not critical. The mesopore size used in this invention is 4nm, allowing for faster signal molecule release; therefore, it can be used in rapid detection applications such as rapid test strips. Other similar mesoporous materials with pore sizes less than 3nm can achieve similar results, but ultimately have lower release rates.

[0027] The present invention also provides a gated mesoporous nanomaterial prepared by the preparation method.

[0028] The present invention also provides the use of the gated mesoporous nanomaterial in preparing a detection kit, wherein the kit includes a detection test strip, the detection test strip includes a conjugate pad, a detection line T line, and a quality control line C line, and the gated mesoporous nanomaterial is embedded in the conjugate pad.

[0029] The present invention also provides a rapid detection test paper kit based on the gated mesoporous nanomaterial, the kit comprising a test paper strip, the test paper strip comprising a conjugate pad, a detection line T line, and a quality control line C line, the conjugate pad being embedded with the gated mesoporous nanomaterial.

[0030] Preferably, the conjugate pad is further embedded with fluorescein, the detection line T is embedded with an antibody against the signal molecule, and the quality control line C is embedded with an anti-fluorescein antibody against the fluorescein.

[0031] To improve the sensitivity of test strips and enable the detection of both large and small molecule targets using the same detection mechanism, we have developed a test strip detection method based on gated mesoporous nanomaterials with dendrimer-bound aptamers. This method provides a signal amplification mechanism within the test strip detection platform, enhancing detection sensitivity. Furthermore, this detection mechanism can be used to detect both large and small molecule targets.

[0032] Its structure and detection mechanism are as follows Figure 2 As shown, porous silica nanoparticles gated by aptamer / G3.5 are embedded on the conjugate pad to load rhodamine B (used as a fluorescent signal molecule on the T line); fluorescein is also embedded on the conjugate pad to serve as a fluorescent signal molecule on the C line. In addition, the T line and C line of the paper strip are respectively embedded with anti-rhodamine B and anti-fluorescein antibodies, which serve to specifically capture these two fluorescent signal molecules.

[0033] When the sample is positive, the target binds to the aptamer on the surface of the porous silica nanoparticles, disrupting the bond between the aptamer and the ssDNA, dissociating the G3.5 molecules used to seal the nanoparticles, and releasing a large amount of rhodamine B signal molecules. The released rhodamine B signal molecules are captured by antibodies at the T line along the direction of water flow, resulting in a red color. The fluorescein signal molecules are captured at the C line and, as a quality control, appear green.

[0034] When the sample is negative, the Rhodamine B signal molecule will not be released, so the T line will not show color, but the C line will still appear green.

[0035] The specific production plan of the test strips is as follows:

[0036] (1) Sample pad preparation

[0037] The sample pad was made of glass fiber, pre-soaked in blocking buffer for 12 hours and then air-dried. The blocking buffer consisted of 1X PBS (pH 7.4), 5% bovine serum albumin, 5% sucrose, 0.01% Tween-20, 0.02% Triton X-100, and 0.01% Proclin 300.

[0038] (2) Bonding pad production

[0039] The conjugate pad is made of polyester membrane, which is pre-soaked in blocking buffer for 12 hours and then air-dried. 1 mg / mL OSM@MSNs-aptamer / G3.5 (where the signaling molecule is rhodamine B) and 10 μg / mL fluorescein are then sprayed on the membrane and air-dried. The blocking buffer consists of 1X PBS (pH 7.4), 5% bovine serum albumin, 5% sucrose, 0.01% Tween-20, 0.02% Triton X-100, and 0.01% proclin 300.

[0040] (3) T-line and C-line

[0041] 1 mg / mL anti-rhodamine B and anti-fluorescein antibodies were sprayed on the T and C lines of the nitrocellulose membrane, respectively, with a 4 mm interval between the T and C lines, and then dried at 37°C.

[0042] (4) Assembly

[0043] The test strip consists of an overlapping assembly of a sample pad, conjugate pad, nitrocellulose membrane, and adsorbent pad assembled on a PVC card. First, the conjugate pad is slightly overlapped on the nitrocellulose membrane, and then the sample pad is placed so that it overlaps the conjugate pad. The conjugate and sample pads are placed at the ends of the nitrocellulose membrane. On the other side of the membrane, the adsorbent pad overlaps the nitrocellulose membrane. All overlaps are 2 mm in length. The assembled card is then cut into 3 mm strips and stored in a desiccator until use.

[0044] Beneficial effects of the present invention:

[0045] 1. The gated mesoporous nanomaterial of the present invention has a stable seal, does not leak signal molecules and has a fast release rate.

[0046] 2. The gated mesoporous nanomaterial of the present invention has a strong ability to prevent interference from nonspecific targets in the matrix. G3.5 has excellent surface antifouling properties. When used as a sealing material, it can prevent interference from nonspecific targets in the matrix with surface aptamers, reducing false positives caused by nonspecific adsorption or increased background noise.

[0047] 3. This invention provides a signal amplification solution for test strips, improving detection sensitivity. Traditional signal amplification solutions use nanoparticles with stronger signals (such as quantum dots and fluorescent latex microspheres) instead of gold nanoparticles, which essentially improves signal intensity. This invention uses a small amount of target to stimulate a large number of signal molecules, amplifying the detection signal in terms of signal quantity.

[0048] 4. The present invention can detect both large and small molecule targets using the same detection mechanism. Unlike traditional colloidal gold test strips, which require a sandwich assay for large target substances and a competitive assay for small molecule substances, the present invention uses aptamers to bind to the target, opening the G3.5 seal and releasing signal molecules. Therefore, the present invention provides a universal test strip detection solution that can detect both large and small molecule targets without changing the detection mechanism. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 Flow chart of the method for preparing gated mesoporous nanomaterials based on dendrimer-binding aptamers.

[0050] Figure 2 Schematic diagram of the paper strip structure and the detection principle.

[0051] Figure 3 Figure 2 is a fluorescence intensity diagram showing the coupling of aptamers to mesoporous nanomaterials using fluorescently labeled aptamers.

[0052] Figure 4 The fluorescence intensity graph is a graph showing the coupling of ssDNA and G3.5 by using fluorescently labeled ssDNA.

[0053] Figure 5 The particle size changes during the synthesis steps.

[0054] Figure 6 This is a test diagram of gating performance and signal molecule release rate.

[0055] Figure 7 The effect of milk matrix on seal stability.

[0056] Figure 8 A rapid test strip based on gated mesoporous nanomaterials with dendrimer-binding aptamers was used to test ochratoxin A in real milk samples.

[0057] Figure 9 Silica nanoparticles produced by the "plugging method".

[0058] Figure 10 Silica nanoparticles produced by the "bridge method". DETAILED DESCRIPTION

[0059] The technical solutions of gated mesoporous nanomaterials based on dendrimers combined with aptamers are as follows: Figure 1 As shown, the following steps are included:

[0060] (1) Signal molecules are loaded into mesoporous nanoparticles to obtain signal molecule-loaded mesoporous nanoparticles OSM@MSNs.

[0061] (2) coupling the target-specific aptamer to the OSM@MSNs obtained in step (1) to obtain the mesoporous nanoparticle surface-coupled aptamer OSM@MSNs-aptamer loaded with the signal molecule.

[0062] (3) coupling ssDNA with the nth generation carboxyl polyamidoamine dendrimer to obtain ssDNA-Gn, wherein n≥3.5,

[0063] The ssDNA is used to bind to the aptamer in step (2) and is competitively dissociated from the single-stranded DNA in the presence of the detection target,

[0064] (4) Assembling the ssDNA-Gn obtained in step (3) with the OSM@MSNs-aptamer obtained in step (2), and allowing the nth generation carboxyl polyamide-amine dendrimer to seal the mesoporous nanoparticles through the binding between the ssDNA and the aptamer.

[0065] The signal molecule can be one of rhodamine B, fluorescein isothiocyanate, Cy3, Cy5 and fluorescein. In the present invention, rhodamine B is selected as the signal molecule as an example.

[0066] The mesoporous nanoparticles can be silica mesoporous nanoparticles, mesoporous titania nanoparticles, metal organic frameworks, mesoporous alumina, mesoporous magnetic beads, mesoporous ferrosoferric oxide or mesoporous copper sulfide nanoparticles. In the present invention, silica mesoporous nanoparticles are used as the mesoporous nanoparticles as an example.

[0067] Specifically, in step (2), when the target-specific aptamer is coupled to the OSM@MSNs obtained in step (1), the N=C=O group is introduced by functionalizing the surface of the OSM@MSNs obtained in step (1), and the end group of the aptamer is modified with an amino group, and the aptamer is coupled through the reaction between the N=C=O group and the amino group;

[0068] Alternatively, amino groups are introduced by functionalizing the surface of OSM@MSNs obtained in step (1), and the terminal groups of the aptamer are modified with -SH groups, and coupled to the aptamer via N-α-maleimidoacetyl-oxysuccinimide ester.

[0069] Alternatively, -SH groups are introduced by functionalizing the surface of the OSM@MSNs obtained in step (1), and the end groups of the aptamer are modified with amino groups, and the aptamer is coupled to the aptamer via N-α-maleimidoacetyl-oxysuccinimide ester;

[0070] Alternatively, amino groups are introduced by functionalizing the surface of OSM@MSNs obtained in step (1), and the end groups of the aptamer are modified with amino groups, and then coupled with the amino-containing aptamer via glutaraldehyde or (bis(sulfosuccinimide) suberate);

[0071] In step (3), when ssDNA is coupled with the n-generation carboxyl polyamidoamine dendrimer to obtain ssDNA-Gn, the two undergo a coupling reaction mediated by EDC / NHS.

[0072] The mesoporous nanoparticles have a mesopore size of 3 to 5 nm.

[0073] The technical solution of the present invention is described below with reference to specific embodiments.

[0074] Example 1

[0075] A method for preparing a gated mesoporous nanomaterial based on dendrimer-binding aptamers comprises the following steps:

[0076] (1) Rhodamine B loaded into silica mesoporous nanoparticles (RhB@MSNs)

[0077] 100 mg of mesoporous silica nanoparticles (MSNs, Sigma, Cat. No. 748161) (particle size 200 nm, pore size 4 nm) were mixed with 76.64 mg of rhodamine B. 10 mL of acetonitrile was then added to the mixture and allowed to react for 24 h to fully load the rhodamine B into the mesoporous material.

[0078] (2) Functionalization of the microsphere surface, introduction of N=C=O groups

[0079] 247.6 μL of isocyanatepropyltriethoxysilane was added to the reaction solution in step (1) and the mixture was shaken at room temperature for 5.5 h. Subsequently, the mesoporous particles were filtered using filter paper and washed once with 5 mL of acetonitrile. Finally, the particles were dried at 38°C for 18 h before use.

[0080] (3) Surface coupling of mesoporous nanoparticles loaded with rhodamine B and aptamers (i.e., RhB@MSNs-aptamer)

[0081] First, prepare a 1mM Rhodamine B acetonitrile solution; then take 1mg of the nanoparticles prepared in step (2) and mix them with 700μL of the Rhodamine B acetonitrile solution prepared in step (2) and 2μL of triethylamine, and then add 100μL of the aptamer solution with amino group modification at the end (its initial concentration is 10μM). After the mixed solution is shaken for 3h, centrifuged at 3000g for 3min, the precipitate is collected, and then washed twice with Tris-HCl buffer (pH=7.5) and freeze-dried. The aptamer sequence used in this experiment is It is an ochratoxin A-specific aptamer.

[0082] (4) Preparation of the 3.5th generation carboxyl polyamide-amine dendrimer (i.e., G3.5-COOH) and ssDNA conjugate (i.e., ssDNA-G3.5)

[0083] A reaction mixture containing 1 μM amino-modified ssDNA (i.e., NH2-ssDNA, wherein the ssDNA can base-pair with the aptamer), 1 nM G3.5-COOH, 5 mM N-hydroxysulfosuccinimide (NHS), and 2 mM 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) was reacted in 0.1 M 2-morpholinoethanesulfonic acid (MES) solution (pH 6.0) at room temperature for 2 hours. The resulting mixture was then filtered using an Amicon Ultra centrifugal filter (cutoff 10 kDa) to collect the ssDNA-G3.5 conjugate (Mw > 10 kDa) and dissolved in 0.1 mL of Tris-HCl buffer (pH = 7.5) to obtain a mixture for later use.

[0084] (5) ssDNA-G3.5 is coupled with MSNs-aptamer to seal MSNs

[0085] 1 mg of the MSNs-aptamer nanoparticles obtained in step (3) was mixed with 0.1 mL of the mixture obtained in step (4), reacted at room temperature for 30 min, and then centrifuged at 3000 g for 3 min to collect the precipitate. Finally, 0.1 mL of Tris-HCl buffer (pH = 7.5) was added to obtain a gated mesoporous nanomaterial of dendrimer-bound aptamers, wherein the signal molecule was rhodamine B (i.e., RhB@MSNs-aptamer / G3.5). The ssDNA sequence used in the experiment was

[0086] (6) Sample pad preparation

[0087] The sample pad was made of glass fiber, pre-soaked in blocking buffer for 12 hours and then air-dried. The blocking buffer consisted of 1X PBS (pH 7.4), 5% bovine serum albumin, 5% sucrose, 0.01% Tween-20, 0.02% Triton X-100, and 0.01% Proclin 300.

[0088] (7) Bonding pad production

[0089] The conjugate pad was made of a polyester membrane, which was pre-soaked in blocking buffer for 12 hours and then dried. 1 mg / mL of RhB@MSNs-aptamer / G3.5 prepared in step (5) and 10 μg / mL of fluorescein were then sprayed on the membrane and allowed to dry. The blocking buffer consisted of 1X PBS (pH 7.4), 5% bovine serum albumin, 5% sucrose, 0.01% Tween-20, 0.02% Triton X-100, and 0.01% proclin 300.

[0090] (3) T-line and C-line

[0091] 1 mg / mL anti-rhodamine B and anti-fluorescein antibodies were sprayed on the T and C lines of the nitrocellulose membrane, respectively, with a spacing of 4 mm between the T and C lines, and then dried at 37°C.

[0092] (4) Assembly

[0093] The test strip consists of an overlapping assembly of a sample pad, conjugate pad, nitrocellulose membrane, and adsorbent pad assembled on a PVC card. First, the conjugate pad is slightly overlapped on the nitrocellulose membrane, and then the sample pad is placed so that it overlaps the conjugate pad. The conjugate and sample pads are placed at the ends of the nitrocellulose membrane. On the other side of the membrane, the adsorbent pad overlaps the nitrocellulose membrane. All overlaps are 2 mm in length. The assembled card is then cut into 3 mm strips and stored in a desiccator until use.

[0094] The schematic diagram of the prepared test strip structure and the detection principle is as follows Figure 2 shown.

[0095] Example 2 Characterization

[0096] 1. Aptamer characterization: Coupling of aptamers and mesoporous nanomaterials

[0097] By using fluorescently labeled aptamers, the aptamers were coupled to the surface of the mesoporous nanoparticles according to step (3) in Example 1 to characterize the success of the coupling of the aptamers to the surface of the mesoporous nanoparticles. Figure 3 As shown, the number of aptamers on the surface of the aptamer chemical conjugation group was significantly higher than that of the no-aptamer group and the aptamer physical adsorption group, demonstrating successful aptamer conjugation to the mesoporous nanoparticle surface. The value for the aptamer chemical conjugation group was 2338±861, indicating that 2338±861 pmol of aptamer could be conjugated per mg of mesoporous nanoparticle surface. The aptamer sequence used in this experiment is:

[0098] 2. Coupling of ssDNA and G3.5

[0099] Similarly, by step (4) using fluorescently labeled ssDNA (i.e. ) was coupled to G3.5 to characterize ssDNA-G3.5. Figure 4 As shown, the fluorescence intensity of G3.5+ssDNA (chemical coupling group) was significantly higher than that of G3.5 group and G3.5+ssDNA (physical adsorption group), which proved that ssDNA was successfully coupled to G3.5. Subsequently, the number of ssDNA coupled to the G3.5 surface was calculated using a standard curve. The results showed that one G3.5 molecule surface could couple 44±6 ssDNAs (3 repeated experiments).

[0100] 3. Particle size

[0101] In addition, the success of the synthesis of steps (1) to (5) was confirmed again by comparing the particle size of the synthesized particles. Figure 5 As shown, the original G3.5 molecule size was approximately 4.1 nm. After coupling with ssDNA, its size increased to 9.9 nm, indicating successful coupling. Furthermore, the original MSN particle size was 205.3 nm. After coupling with the aptamer (i.e., MSN-aptamer), its particle size increased to 255.8 nm, confirming successful coupling. Subsequently, after the MSN-aptamer was re-coupled with ssDNA-G3.5, its particle size increased again to 291.5 nm, indicating the successful synthesis of MSN-aptamer / G3.5.

[0102] Example 3 Gating Performance Test

[0103] The gating performance and release rate of the signaling molecule were verified by adding ochratoxin A to trigger the gated opening of the material of the present invention (i.e., RhB@MSNs-aptamer / G3.5), releasing the signaling molecule Rhodamine B. The "blocking" and "bridging" methods were also used for comparison.

[0104] Among them, the plugging method: the plugging method structure is as follows Figure 9 As shown, silica nanoparticles MCM-41 (pore size 3 nm, Sigma, catalog number 926876) were selected and N=C=O groups were introduced on their surface according to step 2 of Example 1. Then, according to step 3, rhodamine B was loaded on them and they were coupled with a blocking aptamer with an amino terminal group to complete the preparation of silica nanoparticles by the "plugging method". The sequence is:

[0105] Bridge method: The structure of bridge method is as follows Figure 10 As shown, silica nanoparticles MCM-41 (with a pore size of 3 nm, Sigma, product number 926876) were selected to introduce N=C=O groups on their surface through step 2 in Example 1. Then, through step 3, they were loaded with rhodamine B and coupled with the arm single-stranded DNA with an amino terminal group. Subsequently, the product was subjected to base complementary pairing with the sealing aptamer. Specifically, 100 μL of 10 μM sealing aptamer was added to 1 mg of the obtained product, reacted at room temperature for 30 minutes, and then centrifuged for 3 min × 3000 g, the precipitate was taken, and finally 0.1 mL of Tris-HCL buffer (pH = 7.5) was added to obtain the silica nanoparticles prepared by the "bridge method". The arm single-stranded DNA is: The sealing aptamer sequence is:

[0106] like Figure 6 As shown in Figure A, positive samples can all cause the mesoporous nanoparticles sealed using the three different methods to open the gate and release signal molecules. However, in the present invention, the signal molecule release performance of RhB@MSNs-aptamer / G3.5 is superior to the other two methods. Observations show that the signal molecule release rate of the material of the present invention reached its maximum value at 6 minutes, while the signal molecule release rate of the other two methods was still showing an upward trend at 30 minutes. The results prove that the gating in the present invention can quickly respond to the target substance, allowing the rapid release of signal molecules, which can reach the maximum value within 6 minutes, meeting the application requirements on the test strip.

[0107] In addition, under negative sample test ( Figure 6B) shows that the signal molecules in the gated mesoporous material of the present invention are almost not released, and the value shows a stable trend, proving that the seal is stable and does not leak signal molecules. In contrast, the signal molecules in the mesoporous materials of the other two sealing methods are continuously released, and the values show a continuous upward trend, proving that the seal is unstable, signal molecules leak, and cannot meet the requirements of application on test strips.

[0108] In addition, in order to test the effect of milk matrix on the sealing stability of gated mesoporous materials, mesoporous nanoparticles sealed by three different methods were immersed in negative milk matrix, and the release of signal molecules was observed at the same time. Figure 7 As shown, the gated mesoporous material of the present invention releases almost no signal molecules, demonstrating the stability of its seal. This is likely due to the antifouling function of G3.5, which gives the nanomaterial excellent resistance to nonspecific adsorption. In contrast, the mesoporous particles sealed with the other two methods showed a significant and rapid increase in signal, demonstrating that other substances in milk can open the gate and release signal molecules, making them unsuitable for use in rapid test strips.

[0109] Example 4 Real sample detection

[0110] A rapid test strip based on gated mesoporous nanoparticles with dendrimer-binding aptamers was prepared by steps (6) to (9) and tested on real milk samples spiked with different concentrations of ochratoxin A (50, 100, 200, 500 fg / mL and 1, 10, 100 ng / mL). Figure 8 As shown, the T line develops color at ochratoxin A concentrations of 50 fg / mL and above, demonstrating a visual detection limit of 50 fg / mL, which is superior to other reported test strips. Furthermore, when tested with negative samples (see 0 fg / mL), the T line does not develop color, demonstrating that the test strip has no false positives.

Claims

1. A method for preparing a gated mesoporous nanomaterial, wherein the gated mesoporous nanomaterial can be used to prepare a test strip, characterized in that: The following steps are involved: (1) Signal molecules are loaded into mesoporous nanoparticles to obtain signal molecule-loaded mesoporous nanoparticles, namely OSM@MSNs. (2) The target-specific aptamer is coupled to the OSM@MSNs obtained in step (1) to obtain a surface-coupled aptamer of mesoporous nanoparticles loaded with signal molecules, namely, OSM@MSNs-aptamer. (3) ssDNA is coupled with the nth generation carboxyl polyamide-amine dendrimer to obtain ssDNA-Gn, The ssDNA is used to bind to the aptamer in step (2) and is competitively dissociated in the presence of the detection target, wherein n=3.5, (4) assembling the ssDNA-Gn obtained in step (3) with the OSM@MSNs-aptamer obtained in step (2), and allowing the nth generation carboxyl polyamide-amine dendrimer to seal the mesoporous nanoparticles through the binding between the ssDNA and the aptamer; The mesoporous nanoparticles have a mesopore size of 4 nm.

2. The method for preparing gated mesoporous nanomaterials according to claim 1, characterized in that: The signal molecule is at least one of rhodamine B, fluorescein isothiocyanate, Cy3, Cy5 and fluorescein.

3. The method for preparing gated mesoporous nanomaterials according to claim 1, characterized in that: The mesoporous nanoparticles are mesoporous silicon dioxide nanoparticles, mesoporous titanium dioxide nanoparticles, metal organic frameworks, mesoporous aluminum oxide, mesoporous magnetic beads, mesoporous ferrosoferric oxide or mesoporous copper sulfide nanoparticles.

4. The method for preparing gated mesoporous nanomaterials according to claim 1, characterized in that: In step (2), when the target-specific aptamer is coupled to the OSM@MSNs obtained in step (1), the N=C=O group is introduced by functionalizing the surface of the OSM@MSNs obtained in step (1), and the end group of the aptamer is modified with an amino group, and the aptamer is coupled by reaction between the N=C=O group and the amino group; Alternatively, amino groups are introduced by functionalizing the surface of OSM@MSNs obtained in step (1), and the terminal groups of the aptamer are modified with -SH groups, and coupled to the aptamer via N-α-maleimidoacetyl-oxysuccinimide ester. Alternatively, -SH groups are introduced into the surface of the OSM@MSNs obtained in step (1), and the end groups of the aptamer are modified with amino groups, and the aptamer is coupled to the aptamer via N-α-maleimidoacetyl-oxysuccinimide ester; Alternatively, amino groups are introduced by functionalizing the surface of the OSM@MSNs obtained in step (1), and the end groups of the aptamer are modified with amino groups, and then coupled with the amino-containing aptamer via glutaraldehyde or (bis(sulfosuccinimide) suberate); In step (3), when ssDNA is coupled with the nth generation carboxyl polyamide-amine dendrimer to obtain ssDNA-G3.5, the two undergo a coupling reaction mediated by EDC / NHS.

5. The gated mesoporous nanomaterial prepared by the preparation method according to any one of claims 1 to 4.

6. Use of the gated mesoporous nanomaterial according to claim 5 in preparing a detection kit, wherein the kit comprises a detection test strip, the detection test strip comprises a conjugate pad, a detection line T line, and a quality control line C line, and the gated mesoporous nanomaterial is embedded in the conjugate pad.

7. A detection kit comprising a test strip, wherein the test strip comprises a conjugate pad, a detection line T line, and a quality control line C line, characterized in that: The conjugate pad is embedded with the gated mesoporous nanomaterial according to claim 5.

8. The detection kit according to claim 7, characterized in that The conjugate pad is further embedded with fluorescein, the detection line T is embedded with an antibody against the signal molecule, and the quality control line C is embedded with an anti-fluorescein antibody against the fluorescein.

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