A biomacromolecule-linked colloidal gold-labeled ultrasensitive nanoparticle and its preparation method and application

Abstract

The present invention discloses a biomacromolecule-connected colloidal gold-labeled ultrasensitive nanoparticle and a preparation method and application thereof, including a nanocore, nanocolloidal gold particles and biomacromolecules, wherein the nanocolloidal gold particles are connected and evenly distributed on the surface of the nanocore, and the biomacromolecules are connected to the nanocolloidal gold particles. The present invention is used for the preparation of in vitro diagnostic reagents, especially lateral flow chromatography reagents, which can greatly improve the sensitivity of lateral flow chromatography in vitro diagnostic reagents, especially improve the sensitivity of lateral flow chromatography detection reagents for coronavirus antigens by more than 100 times, and the effective detection concentration reaches CT=36, which is basically comparable to nucleic acid detection, and greatly exceeds all similar products already on sale in the global market. In some usage scenarios, the present invention can completely replace nucleic acid detection for use.

Description

Technical Field

[0001] The invention belongs to the technical field of in vitro diagnosis, and specifically relates to a biomacromolecule-connected colloidal gold-labeled ultrasensitive nanoparticle, and a preparation method and application thereof. Background Art

[0002] The transmission capacity of the currently known strong mutant strains of the coronavirus has increased from an average of 2-3 people infected by one infected person to an average of 8-9 people infected by one infected person, which has greatly increased; the viral load has also increased significantly, resulting in an increase in the proportion of severe and critical illness and an earlier onset of time; the immune escape situation has worsened, and a large number of people who have received two doses of the new crown vaccine have been infected.

[0003] Therefore, the global anti-epidemic situation will place increasingly higher demands on rapid and accurate diagnosis of coronavirus infection.

[0004] In response to the tense epidemic, the research and development of coronavirus antigen detection reagents has accelerated. Driven by the accelerated research and development of enterprises and the continued release of market demand, the global coronavirus antigen detection market size will further expand, and the demand for improved sensitivity of antigen reagents has received more and more attention from global diagnostic reagent manufacturers.

[0005] In May 2021, the Paul Ehrlich Institute (PEI) of Germany, a world-renowned evaluation agency, published the "Comparative sensitivity evaluation for 122 CE-marked SARS-CoV-2antigen rapid tests". This study evaluated the sensitivity of 122 coronavirus antigen rapid test reagents that have obtained CE certification worldwide. The research objects of the report basically include almost all antigen rapid test products with relatively good market share and brand awareness in the international market, including colloidal gold and fluorescence technology platforms, including internationally renowned diagnostic reagent manufacturers such as Roche, Abbott, and Siemens. Therefore, this authoritative report can basically represent the highest level of new crown antigen rapid test products in the world in May 2021. Among them, the top five results of domestic manufacturers' reagents are as follows:

[0006]

[0007]

[0008] The top five results of foreign manufacturers' reagents are as follows:

[0009]

[0010] On the whole, the technical levels in the field of rapid coronavirus antigen detection reagents at home and abroad are not much different. The highest level on the market is the effective detection of samples with CT < 30, and the overall compliance rate of CT > 30 is very poor. In the existing technology, the PCR reagents for coronavirus use 40 cycles for extended detection. It is generally believed that CT = 36 is the critical value for effective PCR detection. The specificity is very good within 36 cycles (CT < 36), and the sensitivity is further improved at 36-40 cycles (CT 36-40), but the specificity will decrease, and there is a certain probability of false positives. In addition, the sensitivity of rapid coronavirus antigen detection reagents is significantly different from that of PCR reagents, with a gap of 2 6 times (64 times) or more Summary of the invention

[0011] The purpose of the present invention is to overcome the defects of the prior art and provide a biomacromolecule-connected colloidal gold-labeled ultra-sensitive nanoparticle.

[0012] Another object of the present invention is to provide the application of the above-mentioned biomacromolecule-connected colloidal gold-labeled ultra-sensitive nanoparticles.

[0013] One of the technical solutions of the present invention is as follows:

[0014] A biomacromolecule-connected colloidal gold-labeled ultrasensitive nanoparticle comprises a nanocore, nanocolloidal gold particles and biomacromolecules, wherein the nanocolloidal gold particles are connected and evenly distributed on the surface of the nanocore, the biomacromolecules are connected to the nanocolloidal gold particles, the particle size of the nanocore is 100-500nm, and the particle size of the nanocolloidal gold particles is 15-100nm, wherein:

[0015] The nano core has activated -NH2, the nano colloidal gold particles have -COOH activated by thiol compounds, and the biomacromolecules have activated -NH2. The activated -COOH on the nano colloidal gold particles reacts with the activated -NH2 on the nano core and the biomacromolecules to form amides, thereby realizing the chemical connection between the nano colloidal gold particles, the nano core and the biomacromolecules.

[0016] In a preferred embodiment of the present invention, the nano core is a nano silicon core or a latex microsphere core.

[0017] In a preferred embodiment of the present invention, the thiol compound is 1-mercaptohexanoic acid, thioctic acid, 1-mercaptoundecanoic acid, 1-mercaptododecanoic acid, 1-mercaptohexadecanoic acid or mercaptosuccinic acid.

[0018] The application of the above-mentioned biomacromolecule-connected colloidal gold-labeled ultrasensitive nanoparticles in the preparation of in vitro diagnostic reagents.

[0019] The second technical solution of the present invention is as follows:

[0020] A biomacromolecule-connected colloidal gold-labeled ultrasensitive nanoparticle comprises a nanocore, nanocolloidal gold particles and biomacromolecules, wherein the nanocolloidal gold particles are connected and evenly distributed on the surface of the nanocore, the biomacromolecules are connected to the nanocolloidal gold particles, the particle size of the nanocore is 100-500nm, and the particle size of the nanocolloidal gold particles is 15-100nm, wherein:

[0021] The nano core has activated -COOH, the nano colloidal gold particles have -NH2 activated by a thiol compound, and the biomacromolecule has activated -NH2. The activated -NH2 on the nano colloidal gold particles reacts with the activated -COOH on the nano core to form amides, and the activated -NH2 on the biomacromolecule reacts with the -NH2 on the nano colloidal gold particles through a bridging molecule to form amides, thereby achieving chemical connection between the nano colloidal gold particles, the nano core, and the biomacromolecule.

[0022] The molecular chain of the above-mentioned bridging molecule has one at each end.

[0023] In a preferred embodiment of the present invention, the nano core is a nano silicon core or a latex microsphere core.

[0024] In a preferred embodiment of the present invention, the thiol compound is cysteamine or 1-mercaptoundecanamine.

[0025] In a preferred embodiment of the present invention, the bridging molecule is BS(PEG)5, whose structural formula is

[0026] The application of the above-mentioned biomacromolecule-connected colloidal gold-labeled ultrasensitive nanoparticles in the preparation of in vitro diagnostic reagents.

[0027] The third technical solution of the present invention is as follows:

[0028] A biomacromolecule-connected colloidal gold-labeled ultrasensitive nanoparticle comprises a nanocore, nanocolloidal gold particles and biomacromolecules, wherein the nanocolloidal gold particles are connected and evenly distributed on the surface of the nanocore, the biomacromolecules are connected to the nanocolloidal gold particles, the particle size of the nanocore is 100-500nm, and the particle size of the nanocolloidal gold particles is 15-100nm, wherein:

[0029] The nano core has -NH2 activated by a bridging molecule, the nano colloidal gold particles have -NH2 activated by a thiol compound, and the biomacromolecule has activated -NH2. The activated -NH2 on the nano colloidal gold particles reacts with the activated -NH2 on the nano core through the bridging molecule to form an amide, and the activated -NH2 on the biomacromolecule reacts with the activated -NH2 on the nano colloidal gold particles through the bridging molecule to form an amide, thereby achieving chemical connection between the nano colloidal gold particles, the nano core, and the biomacromolecule.

[0030] The molecular chain of the above-mentioned bridging molecule has one at each end.

[0031] In a preferred embodiment of the present invention, the nano core is a nano silicon core or a latex microsphere core.

[0032] In a preferred embodiment of the present invention, the thiol compound is cysteamine or 1-mercaptoundecanamine.

[0033] In a preferred embodiment of the present invention, the bridging molecule is BS(PEG)5, whose structural formula is

[0034] The application of the above-mentioned biomacromolecule-connected colloidal gold-labeled ultrasensitive nanoparticles in the preparation of in vitro diagnostic reagents.

[0035] The beneficial effects of the present invention are as follows: the present invention is used for the preparation of in vitro diagnostic reagents, especially lateral flow chromatography reagents, which can greatly improve the sensitivity of lateral flow chromatography in vitro diagnostic reagents, especially the sensitivity of lateral flow chromatography detection reagents for coronavirus antigens can be increased by more than 100 times, and the effective detection concentration of coronavirus reaches CT=36, which is basically comparable to nucleic acid detection and greatly exceeds all similar products already on sale in the global market. In some usage scenarios, the present invention can completely replace nucleic acid detection. DETAILED DESCRIPTION

[0036] The technical solution of the present invention is further illustrated and described below through specific implementation methods.

[0037] Example 1

[0038] (1) Heat 100 ml of 0.01% chloroauric acid solution to boiling and quickly add 0.5 ml of 1% trisodium citrate. 3+ After reduction, it can be polymerized into 80nm colloidal gold nanoparticles to obtain 80nm colloidal gold solution;

[0039] (2) adjusting the pH of the colloidal gold solution to 9.0, adding 0.8 ml of 1% 1-mercaptoundecanoic acid, and shaking at room temperature for 3 h to carboxylate the surface of the colloidal gold nanoparticles to obtain carboxylated colloidal gold nanoparticles with a particle size of 80 nm;

[0040] (3) Centrifuge at 8000 rpm for 10 min at 4°C to remove excess 1-mercaptoundecanoic acid in the material obtained in step (2), reconstitute with pure water and wash the carboxyl colloidal gold nanoparticles by centrifugation, reconstitute with 20 mM pH 6.5 PBS, add 100 ul 1% EDC and 150 ul 1% NHS and shake at room temperature for 1 h, centrifuge at 8000 rpm for 10 min at 4°C to remove excess EDC / NHS, reconstitute with 20 mM pH 7.4 PBS, add 100 ul 1% 200 nm amino silicon particles, shake at 10°C for 3 h to chemically cross-link the activated carboxyl colloidal gold nanoparticles to the surface of the amino silicon particles, centrifuge at 3000 rpm for 5 min at 4°C to remove unconnected carboxyl colloidal gold nanoparticles, and reconstitute the coupled nanoparticles in 20 mM pH 6.5 PBS.

[0041] (4) Add 100ul 1% EDC and 150ul 1% NHS to the material obtained in step (3), shake at room temperature for 1h, centrifuge at 5000rpm and 4℃ for 10min to remove excess EDC / NHS, re-dissolve with 20mM pH7.4PBS, add 0.5mg coronavirus antibody (with -NH2), chemically cross-link the coronavirus antibody to the surface of the synthesized nanoparticles, and shake at 4℃ overnight. Centrifuge at 5000rpm and 4℃ for 10min to remove the supernatant, wash with pure water re-dissolve and centrifuge, re-dissolve with 20mM pH9.0 boric acid solution, then add 1g BSA to block non-specific sites on the surface, shake at room temperature for 4h, and obtain a material containing the biomacromolecule-linked colloidal gold-labeled ultra-sensitive nanoparticles;

[0042] (5) The material obtained in step (4) was centrifuged at 5000 rpm and 4°C for 10 min to remove the supernatant, washed twice by reconstitution and centrifugation with pure water, reconstituted with 5 ml of gold pad coating solution, and then coated on the colloidal gold release pad, and dried at 50°C for 45 min;

[0043] (6) A colloidal gold in vitro diagnostic lateral flow device is prepared using a sample loading pad, a colloidal gold release pad pre-coated with colloidal gold-labeled ultrasensitive nanoparticles connected to biomacromolecules, a nitrocellulose membrane pre-coated with binding proteins, absorbent filter paper, a substrate and other materials.

[0044] The coronavirus lateral flow reagent of this example was used to test coronavirus clinical samples at the International Center for Diarrheal Disease Research in Bangladesh ICDDR, B, one of the 13 clinical reference laboratories certified by the World Health Organization. The results are as follows:

[0045]

[0046]

[0047] Compared with traditional colloidal gold particles, the biomacromolecule-linked colloidal gold-labeled ultrasensitive nanoparticles of this embodiment have a sensitivity that is 40-60 times higher, are not affected by surfactants and salt concentrations, and are not easy to fall off in a dry state. The proportion of colloidal gold particles participating in the reaction on the test line exceeds 50%, and compared with the second-generation carboxyl colloidal gold particles, the sensitivity is increased by 20 times. Compared with Ultra-Bright Gold Nanoshell Reporters of nanocomposix (see Increasing the Sensitivity of Lateral Flow Diagnostic Assays with Ultra-Bright Gold Nanoshell Reporters.), its wavelength consistency is more stable, the absorption peak is in the visible light range, and the sensitivity is increased by 2 times.

[0048] Example 2

[0049] (1) Heat 100 ml of 0.01% chloroauric acid solution to boiling and quickly add 0.85 ml of 1% trisodium citrate. 3+ After reduction, it can be polymerized into 40nm colloidal gold nanoparticles to obtain a 40nm colloidal gold solution;

[0050] (2) adjusting the pH of the colloidal gold solution to 9.0, adding 0.8 ml of 1% 1-mercaptohexanoic acid, and shaking at room temperature for 3 h to carboxylate the surface of the colloidal gold nanoparticles to obtain carboxyl colloidal gold nanoparticles with a particle size of 40 nm;

[0051] (3) Centrifuge at 10000 rpm and 4°C for 10 min to remove excess 1-mercaptohexanoic acid in the material obtained in step (2), reconstitute with pure water and wash the carboxyl colloidal gold nanoparticles by centrifugation, reconstitute with 20 mM pH 6.5 PBS, add 100 ul 1% EDC and 150 ul 1% NHS and shake at room temperature for 1 h, centrifuge at 10000 rpm and 4°C for 10 min to remove excess EDC / NHS, reconstitute with 20 mM pH 7.4 PBS, add 100 ul 1% 200 nm amino latex microspheres, shake at 10°C for 3 h to chemically crosslink the activated carboxyl colloidal gold nanoparticles to the surface of the amino latex microspheres, centrifuge at 5000 rpm and 4°C for 5 min to remove unconnected carboxyl colloidal gold nanoparticles, and reconstitute the coupled nanoparticles in 20 mM pH 6.5 PBS.

[0052] (4) Add 100ul 1% EDC and 150ul 1% NHS to the material obtained in step (3), shake at room temperature for 1h, centrifuge at 7000rpm and 4℃ for 10min to remove excess EDC / NHS, re-dissolve with 20mM pH7.4 PBS, add 0.5mg recombinant anti-Flu A virus antibody (with -NH2), chemically cross-link the recombinant anti-Flu A virus antibody to the surface of the synthesized nanoparticles, and shake at 4℃ overnight. Centrifuge at 7000rpm and 4℃ for 10min to remove the supernatant, wash with pure water re-dissolve and centrifuge, re-dissolve with 20mM pH9.0 boric acid solution, then add 1g BSA to block non-specific sites on the surface, shake at room temperature for 4h, and obtain a material containing the biomacromolecule-linked colloidal gold-labeled ultra-sensitive nanoparticles;

[0053] (5) The material obtained in step (4) was centrifuged at 7000 rpm and 4°C for 10 min to remove the supernatant, washed twice by reconstitution and centrifugation with pure water, reconstituted with 5 ml of gold pad coating solution, and then coated on the colloidal gold release pad, and dried at 50°C for 45 min;

[0054] (6) A colloidal gold in vitro diagnostic lateral flow device is prepared using a sample loading pad, a colloidal gold release pad pre-coated with colloidal gold-labeled ultrasensitive nanoparticles connected to biomacromolecules, a nitrocellulose membrane pre-coated with binding proteins, absorbent filter paper, a substrate and other materials.

[0055] The colloidal gold in vitro diagnostic lateral flow device for detecting FluA was prepared by the method of this embodiment, wherein the colloidal gold particles had a particle size of 40 nm, the nanocore was a latex microsphere core with a particle size of 200 nm, and the 1-mercaptoundecanoic acid in step (2) was replaced with 1-mercaptohexanoic acid. The colloidal gold in vitro diagnostic lateral flow device of this embodiment was used for detection, and its overall chromaticity was improved by 4-5 chromaticities, and the sensitivity was improved by about 16 times. The specific results are shown in the following table:

[0056]

[0057] Among them, the ultrasensitive reagent corresponds to the colloidal gold in vitro diagnostic lateral chromatography device of this embodiment, and the control reagent is the influenza A virus antigen detection kit (colloidal gold method) produced by Innovent (National Medical Device Registration No. 20143401923). It can be seen that compared with the parallel test of the control reagent, the sensitivity of this embodiment is significantly higher than that of the control reagent, and there is no obvious difference in specificity.

[0058] Example 3

[0059] (1) Heat 100 ml of 0.01% chloroauric acid solution to boiling and quickly add 0.85 ml of 1% trisodium citrate. 3+ After reduction, it can be polymerized into 40nm colloidal gold nanoparticles to obtain a 40nm colloidal gold solution;

[0060] (2) adjusting the pH of the colloidal gold solution to 9.0, adding 0.8 ml of 1% 1-mercaptoundecanoic acid, and shaking at room temperature for 3 h to carboxylate the surface of the colloidal gold nanoparticles to obtain carboxylated colloidal gold nanoparticles with a particle size of 40 nm;

[0061] (3) Centrifuge at 10000 rpm and 4°C for 10 min to remove excess 1-mercaptoundecanoic acid in the material obtained in step (2), reconstitute with pure water and wash the carboxyl colloidal gold nanoparticles by centrifugation, reconstitute with 20 mM pH 6.5 PBS, add 100 ul 1% EDC and 150 ul 1% NHS and shake at room temperature for 1 h, centrifuge at 10000 rpm and 4°C for 10 min to remove excess EDC / NHS, reconstitute with 20 mM pH 7.4 PBS, add 100 ul 1% 120 nm amino silicon particles, shake at 10°C for 3 h to chemically cross-link the activated carboxyl colloidal gold nanoparticles to the surface of the amino silicon particles, centrifuge at 4500 rpm and 4°C for 5 min to remove unconnected carboxyl colloidal gold nanoparticles, and reconstitute the coupled nanoparticles in 20 mM pH 6.5 PBS.

[0062] (4) Add 100ul 1% EDC and 150ul 1% NHS to the material obtained in step (3), shake at room temperature for 1h, centrifuge at 6000rpm and 4℃ for 10min to remove excess EDC / NHS, re-dissolve with 20mM pH7.4 PBS, add 0.5mg recombinant anti-Flu B virus antibody (with -NH2), chemically cross-link the recombinant anti-Flu B virus antibody to the surface of the synthesized nanoparticles, and shake at 4℃ overnight. Centrifuge at 6000rpm and 4℃ for 10min to remove the supernatant, wash with pure water re-dissolve and centrifuge, re-dissolve with 20mM pH9.0 boric acid solution, then add 1g BSA to block non-specific sites on the surface, shake at room temperature for 4h, and obtain a material containing the biomacromolecule-linked colloidal gold-labeled ultra-sensitive nanoparticles;

[0063] (5) The material obtained in step (4) was centrifuged at 6000 rpm and 4°C for 10 min to remove the supernatant, washed twice by reconstitution and centrifugation with pure water, reconstituted with 5 ml of gold pad coating solution, and then coated on the colloidal gold release pad, and dried at 50°C for 45 min;

[0064] (6) A colloidal gold in vitro diagnostic lateral flow device is prepared using a sample loading pad, a colloidal gold release pad pre-coated with colloidal gold-labeled ultrasensitive nanoparticles connected to biomacromolecules, a nitrocellulose membrane pre-coated with binding proteins, absorbent filter paper, a substrate and other materials.

[0065] The colloidal gold in vitro diagnostic lateral flow device for detecting Flu B was prepared by the method of this embodiment, wherein the colloidal gold particles had a particle size of 40 nm, the nanocore was a nanosilicon core with a particle size of 120 nm, and the 1-mercaptoundecanoic acid in step (2) was replaced with 1-mercaptohexanoic acid. The colloidal gold in vitro diagnostic lateral flow device of this embodiment was used for detection, and its overall chromaticity was improved by 4-5 chromaticities, and the sensitivity was improved by about 60 times. The specific results are shown in the following table:

[0066]

[0067]

[0068] Among them, the ultrasensitive reagent corresponds to the colloidal gold in vitro diagnostic lateral chromatography device of this embodiment, and the control reagent is the influenza B virus antigen detection kit (colloidal gold method) produced by Innovent (National Medical Device Registration No. 20143401924). It can be seen that compared with the parallel test of the control reagent, the sensitivity of this embodiment is significantly higher than that of the control reagent.

[0069] The above description is only a preferred embodiment of the present invention, and therefore cannot be used to limit the scope of the present invention. That is, equivalent changes and modifications made according to the patent scope of the present invention and the contents of the specification should still fall within the scope of the present invention.

Claims

1. A method for preparing ultrasensitive nanoparticles labeled with colloidal gold connected to biomacromolecules for immunochromatographic detection, characterized in that: The steps include: (1) Heat 100 ml of 0.01% chloroauric acid solution to boiling and quickly add 0.85 ml of 1% trisodium citrate. 3+ After reduction, it can be polymerized into 40nm colloidal gold nanoparticles to obtain a 40nm colloidal gold solution; (2) The pH of the colloidal gold solution was adjusted to 9.0, 0.8 ml of 1% 1-mercaptoundecanoic acid was added, and the solution was shaken at room temperature for 3 h to carboxylate the surface of the colloidal gold nanoparticles to obtain carboxylated colloidal gold nanoparticles with a particle size of 40 nm; (3) Centrifuge at 10000 rpm for 10 min at 4°C to remove excess 1-mercaptoundecanoic acid in the material obtained in step (2), re-dissolve the carboxyl colloidal gold nanoparticles in pure water by centrifugation, re-dissolve in 20 mM pH 6.5 PBS, add 100 ul 1% EDC and 150 ul 1% NHS and shake at room temperature for 1 h, centrifuge at 10000 rpm for 10 min at 4°C to remove excess EDC / NHS, re-dissolve in 20 mM pH 7.4 PBS, add 100 ul 1% 120 nm amino silica particles, shake at 10°C for 3 h to chemically cross-link the activated carboxyl colloidal gold nanoparticles to the surface of the amino silica particles, centrifuge at 4500 rpm for 5 min at 4°C to remove unconnected carboxyl colloidal gold nanoparticles, and re-dissolve the coupled nanoparticles in 20 mM pH 6.5 PBS; (4) Add 100ul 1% EDC and 150ul 1% NHS to the material obtained in step (3), shake at room temperature for 1h, centrifuge at 6000rpm at 4℃ for 10min to remove excess EDC / NHS, reconstitute with 20mM pH7.4 PBS, add 0.5mg recombinant anti-Flu B virus antibody, chemically cross-link the recombinant anti-Flu B virus antibody to the surface of the synthesized nanoparticles, shake at 4℃ overnight, centrifuge at 6000rpm at 4℃ for 10min to remove the supernatant, reconstitute with pure water and wash by centrifugation, reconstitute with 20mM pH9.0 boric acid solution, then add 1g BSA to block nonspecific sites on the surface, shake at room temperature for 4h to obtain a material containing the biomacromolecule-linked colloidal gold-labeled ultrasensitive nanoparticles.

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