Preparation of fluorescent nanoparticles and their conjugates for in vitro and in vivo diagnostics

Abstract

This invention provides fluorescent nanoparticles and their conjugates, as well as methods for using them in in vivo and in vitro diagnostics and other applications. In some embodiments, the invention provides fluorescent nanoparticles with high solid-state absolute quantum yield. In some embodiments, the invention provides methods for manufacturing such nanoparticles. The nanoparticles may comprise monomers, such as styrene, and fluorophores, such as AIEgen. TM Bright green.

Description

Technical Field

[0001] This invention relates to polymer nanoparticles that can be prepared using various methods for use as signaling molecules in in vitro and in vivo diagnostics. Background Technology

[0002] Nanomaterials can be linked with biorecognition molecules to create specialized molecular diagnostic probes. Recent advancements in nanotechnology have yielded a series of unique nanosensing platforms that enhance the detection capabilities, sensitivity, ease of operation, and portability of biosensor components, revolutionizing diagnostic techniques in healthcare. This novel nanodiagnostic approach can further develop point-of-care diagnostic and detection technologies.

[0003] Immunolabeling is a well-established technique for detecting and locating target antigens (e.g., proteins). It is widely used in various fields, including molecular biology, biochemistry, and the in vitro diagnostics industry. There are two main methods of immunolabeling: direct and indirect. In the direct method, the target antigen is detected by a primary antibody conjugated to a fluorescent tag or enzyme. In the indirect method, the target antigen is first recognized by the primary antibody and then detected by a labeled secondary antibody that specifically binds to the primary antibody. Both direct and indirect methods can be applied to immunoblotting, immunohistochemistry, immunofluorescence, enzyme-linked immunosorbent assay (ELISA), and fluorescence-activated cell screening (FACS). Generally, the direct method minimizes potential species cross-reactivity caused by the secondary antibody. However, the sensitivity of direct immunolabeling is significantly lower than that of the indirect method because the primary antibody can bind to several labeled secondary antibodies, leading to signal amplification.

[0004] Lateral flask assay (LFA) works on a principle similar to ELISA. LFA is widely used in hospitals and clinical laboratories, as well as in veterinary drug testing, environmental assessment, and food production processes for safety testing. This assay is affordable, sensitive, specific, user-friendly, rapid, and stable. Results are obtained within minutes after adding a sample (e.g., urine and blood), requiring minimal or no additional equipment.

[0005] Signal tags are crucial for lateral chromatography assays. These particles or molecules, possessing color or other detectable properties, can be coupled to binding / detection molecules. Gold nanoparticles have been widely used in lateral chromatography assays. These particles are characterized by ease of preparation and functionalization, good biocompatibility, and cost-effectiveness. Gold nanoparticles generate readings that do not require any visualization equipment. [1] However, this method is usually qualitative or semi-quantitative, with low signal strength and poor sensitivity.

[0006] The search for novel labels with improved analytical performance remains an ongoing challenge. While fluorescent labels have been reported to facilitate the sensitive and quantitative detection of trace analytes, most of them have too low a luminescence quantum efficiency for application in lateral chromatography assays. Furthermore, given the mass conditions per unit volume, typically (nanograms per milliliter or milligrams per milliliter) or (milli-international units per milliliter), current fluorescent lateral chromatography assays offer a narrow detection range (10-1). 0 -10 2 ) [1] .

[0007] In addition, fluorescence intensity quenching is a common drawback of fluorescent materials. [2] Therefore, there is a great need to find a material that exhibits non-fluorescence quenching and can be encapsulated into nanoparticles or nanoclusters. Signal labels that do not exhibit fluorescence intensity quenching effects can effectively improve detection sensitivity and enable quantitative analysis, while retaining the simplicity, speed, and portability of conventional lateral chromatography immunoassays.

[0008] One of the most advantageous fluorescent labels is quantum dots (QDs). They possess a variety of unique physicochemical properties, including high quantum yield, tunable emission wavelength, and strong photostability. However, QDs have drawbacks such as high toxicity, roughness, complex synthesis procedures, and colloidal instability. CdSe (cadmium selenide)-based quantum dots are highly toxic and require a stable polymer shell. Furthermore, current technology makes it difficult to achieve large-scale production of more than one kilogram of protein-functionalized quantum dots. [3] Although they have been shown to exhibit good fluorescence properties when fabricated into nanoparticles, they are affected by emission quenching at higher concentrations and in the solid state. Furthermore, according to previous results, quantum dots have a very narrow detection range. [4-6]

[0009] Upconversion fluorescent materials are a class of rare-earth-containing crystalline particles that can upconvert photons of lower-energy infrared light into higher-energy visible light. However, these materials have low luminescence quantum yields. Lanthanide-labeled materials, such as Eu, typically emit in the red region and have an absolute quantum yield efficiency of around 40%. [7]

[0010] Aggregation-induced emission (AIE) materials can also be used as fluorescent signal output materials. [8]

[0011] The fluorescent nanoparticles reported here possess unique properties, such as a luminescence quantum yield of at least 20%, tunable emission color, and uniform size distribution. More importantly, the large-scale production of these fluorescent nanoparticles is relatively easy and inexpensive. In appropriate combinations, functionalized nanoparticle probes can be used for quantitative single and multiplex detection in compact point-of-care platforms. AIE nanoparticles can also replace conventional antibody-conjugated tags in immunolabeling. AIE-conjugated antibodies can address the low sensitivity of direct immunolabeling methods and further improve the sensitivity of indirect immunolabeling methods. Therefore, the nanoparticles we report can be applied to applications such as early disease detection, genomic technologies, in vivo diagnostics, and personalized and predictive medicine. Summary of the Invention

[0012] In some embodiments, the present invention provides unique fluorescent nanoparticles and functionalized nanoparticles exhibiting a high solid-state absolute quantum yield of not less than 20%. In some embodiments, the emission wavelength of the fluorescent core can be ultraviolet (200-420 nm), visible (420-780 nm), or infrared (780-1200 nm). Appropriate emission wavelengths can be selected for different diagnostic purposes. In some embodiments, the detection range is 10... 0 -10 4 (milli-International Units / mL), two orders of magnitude wider than conventional products.

[0013] Preparation of fluorescent particles

[0014] Fluorophores can be mixed with monomers and then polymerized, or they can be directly encapsulated by macromolecules. These methods allow for maintaining high fluorescence quantum yields in aggregated, clustered, or high-concentration states. For example, fluorophores can be polymerized with monomers such as styrene, methyl methacrylate (MMA), and acrylic acid. Fluorescent nanoparticles can also be fabricated by other methods, such as microfluidics and ultrasonic methods, with or without chemical reactions. For example, fluorescent nanoparticles can be fabricated using typical microfluidic flow focusing devices that involve chemical reactions. Figure 1 ).

[0015] Special characteristics of fluorescent particles

[0016] The fluorescent particles provided by this invention cover a wide range of sizes. The diameter of the fluorescent particles of this invention can be as small as a few millimeters (10⁻⁶). -3 From nanometers (milliparticles) to micrometers (10) -6 From meters (microparticles) to nanometers (10 nanoparticles) -9 Within the range of nanoparticles (meters), it exhibits good uniformity in size, shape, and surface properties. Microparticles with suitable properties can also be used.

[0017] As is well known, the appropriate size and good uniformity of nanoparticles are important in biomedical applications. The size of nanoparticles needs to be optimized to suit their function for different biomedical applications, as the chemical and physical properties of nanoparticles depend on their size. Furthermore, nanoparticles should have a narrow size distribution to avoid any discrepancies in detection. For example, the size of nanoparticles used in lateral flow immunoassays should be as small as possible. Binding molecules and antibodies typically have a size of 5-15 nanometers, and due to their small size, they are more easily released from the conjugated pads than the complex conjugates of binding molecules immobilized on the surface of nanoparticles. This makes background fluorescence easily removed by filters. Moreover, the fluorescent particles of this invention are not affected by quenching effects at high concentrations or in aggregated states. [8]

[0018] Nanoparticles functionalized for different diagnostic purposes

[0019] To leverage the unique physical properties of nanoparticles for diverse diagnostic purposes, further functionalization with sensing molecules is required. These molecules can be small molecules (e.g., biotin), aptamers, analytes, or biomacromolecules such as RNA, DNA, or proteins (e.g., antibodies or enzymes). The functionalization of nanoparticles with sensing molecules has been achieved through (i) electrostatic interactions, (ii) chemisorption (e.g., via thiols or amine groups), (iii) covalent binding, and (iv) affinity-based systems. For example, molecules such as antibodies or other proteins can be covalently bound to nanoparticles using crosslinking agents such as 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) or EDC / N-hydroxysuccinimide (NHS).

[0020] A fluorescent nanoparticle with a core-shell structure has a hydrophilic surface with functional groups (e.g., carboxyl, amino, thiol, etc.) for biomodification. For example, proteins (e.g., antibodies) can be attached to the nanoparticle by linking the functional groups attached to the nanoparticle surface to the N-terminal or C-terminal amino acids of the protein. The antibody-linked fluorescent nanoparticle can be used for immunoassays. Figure 2 A schematic diagram depicting one embodiment of fluorescent nanoparticles is shown. Those skilled in the art can select them based on the specific needs of proteins and other sensing molecules. Attached Figure Description

[0021] Figure 1An embodiment of a microfluidic device for fabricating fluorescent nanoparticles is depicted. The following abbreviations are used: methyl methacrylate (MMA), acrylic acid (AA), sodium dodecylbenzenesulfonate (SDBS), ethanol ethoxylate (AEO, with repeating units 3 / 5 / 7), sodium dodecyl sulfate (SDS), ammonium persulfate (APS), 2,2'-azobisisobutyronitrile (AIBN), and benzoyl peroxide (BPO).

[0022] Figure 2 Nanoparticles with a core-shell structure are shown, which have functional groups for biomodification.

[0023] Figure 3 The illustration shows a side view of a lateral chromatography immunoassay strip representing some embodiments of the present invention.

[0024] Figure 4 The illustration shows a top view of a lateral chromatography immunoassay strip representing some embodiments of the present invention.

[0025] Figure 5 Photographs of hCG test strips at different concentrations of 0, 2, 5, 5, 103, 25, 100, 200, 500, 1000, and 10000 mlU / mL are shown. The photographs were taken using the same camera settings under light excitation at 365 nm on the first day of the experiment (left image) and six months later (right image).

[0026] Figure 6 The photoluminescence of fluorescent nanoparticles prepared as described in Example 1 is shown under natural light and 365 nm UV excitation.

[0027] Figure 7 The images show hCG (left) and LH test strips (right) at different concentrations of hCG (0, 20, 200, 2000, 10000 mlU / mL) and LH (0, 20, 200, 2000 mlU / mL), with a sample volume of 100 μL and 365 nm UV excitation.

[0028] Figure 8 The images show the effects of AIEgen under natural light (left) and 365 nm UV excitation (right). TM A photograph of red fluorescent nanoparticles.

[0029] Figure 9 Photographs of paper strips with two test lines under 365 nm excitation are shown, where T1 and T2 are used for LH and hCG detection, respectively.

[0030] Figure 10The performance tests of the AIE-conjugated antibody are shown; (A) Direct detection of 7 ng LH protein on immunoblot using 1:5000 AIE-conjugated mouse anti-β-LH antibody (arrow); (B) Indirect detection of 7 ng LH protein using 1:5000 unbound mouse anti-β-LH antibody (arrow) and 1:5000 FITC-conjugated goat anti-mouse secondary antibody (arrow); The images in Figures A and B were captured under the same conditions. Detailed Implementation

[0031] Applications of fluorescent nanoparticle conjugates

[0032] [1] Fluorescent nanoparticle conjugates can be used as indirect or direct indicators in Western blotting (WB), in immunohistochemistry (IHC), in immunocytochemistry / immunofluorescence (ICC / IF), in flow cytometry (FC), and in lateral chromatography assays (LFA).

[0033] [2] In some embodiments, the present invention can effectively improve the detection sensitivity of lateral chromatography assays and enable quantitative analysis. Such assays can be applied, for example, to health monitoring, medical diagnosis in emergency and resource-scarce environments, food quality control of foodborne diseases, and water monitoring for harmful ion contamination.

[0034] [3] One embodiment of the present invention is a one-step lateral chromatography immunoassay strip comprising a sample pad, a conjugation pad, a reaction membrane (e.g., a nitrocellulose membrane (NC)), an adsorption pad, and at least one fluorescent conjugate that does not exhibit emission quenching at a higher concentration dispensed on the conjugation pad. All these components are mounted on a back card. In some embodiments of the present invention, a solid support matrix may be used instead of a back card. Figure 3 and 4 Some examples of lateral chromatography assays are described.

[0035] [4] One embodiment consists of overlapping membranes mounted on a backing or solid support matrix in the following order: sample pad, conjugation pad, reaction membrane, and adsorption pad. The conjugation pad consists of at least one fluorescent nanoparticle conjugate specific to the target analyte, which does not exhibit emission quenching at higher concentrations. The sample is applied to the sample pad and then moves along the membrane by capillary forces to the conjugation pad conjugate containing the fluorescent nanoparticles, where it binds to the biorecognition molecule. The sample then reaches the reaction membrane containing the test line and control line of the target analyte, and finally reaches the adsorption pad, which retains waste. Different capture molecules may be immobilized on the test line or control line of the strip, depending on the nature of the target analyte. For example, the test line and control line comprise a primary antibody and a secondary antibody, respectively, for a lateral chromatography test strip based on a double-antibody sandwich method. The primary antibody specifically binds to the second antigenic site of the target analyte, while the secondary antibody specifically binds to the labeled antibody.

[0036] [5] This invention can be applied to the detection of antigens, such as human chorionic gonadotropin (hCG) and luteinizing hormone (LH), in lateral chromatography immunoassay. Body fluids, such as blood, serum, urea, saliva, nasal secretions, and tears, can be applied to a sample pad and migrate through a conjugate pad containing a fluorescent antibody-conjugate specific to the target analyte. The sample, along with the antibody-conjugate bound to the target analyte, moves along a strip to the detection zone of an NC membrane, where a specific antibody is immobilized in the line and reacts with the analyte bound to the antibody in the conjugate (sandwich assay). Recognition of the sample analyte results in an appropriate fluorescence signal response on the test line, while a response on the control line indicates that liquid has flowed through the test strip. Readings are represented by lines of varying intensities and can be evaluated using a dedicated analyzer. The fluorescence intensity tested is used to determine the amount of the analyte in the sample.

[0037] [6] Depending on the purpose, various analyzers may be used in different embodiments of the present invention. For example, in some embodiments, a complementary metal-oxide-semiconductor (CMOS) or charge-coupled device (CCD) may be used to record fluorescence images onto a color matrix of (x, y, z) values, where x represents red, y represents green, and z represents blue. The image is then processed using an image processing algorithm that determines analyte concentration test lines and control lines and their intensity ratios based on fluorescence intensity. In some embodiments, fluorescence signals are collected and converted into electronic signals by a photoelectric converter (e.g., a photodiode). For both methods, the analyzer should include: (i) an excitation light source, such as a light-emitting diode (LED); (ii) an electronic power source or power driver for keeping the LED lit during CMOS / photodiode capture of the fluorescence signal; (iii) a transmission module for transmitting data to a smart platform such as a smartphone or personal computer; (iv) a memory for data storage; (v) a printed circuit board fully integrated with all components; and (vi) optionally, fiber optic / waveguide glass / filters.

[0038] [7] In some embodiments, the present invention has a broad detection range for a variety of detectable proteins. For example, for hCG, the detection range can be from 10 0 Up to 10 4 mIU / mL. For LH, the detection range can be 10 mIU / mL. 0 ~10 3 mIU / mL. In some embodiments, no significant degradation of the fluorescent bands was observed after six months of use. Figure 5 This indicates that the fluorescent antibody conjugate has excellent photostability, allowing it to preserve test records for a long time.

[0039] [8] In some embodiments, a luminescent source that does not exhibit quenching at higher concentrations or in aggregated states may be used instead of a fluorophore.

[0040] [9] Nanoparticles can be crosslinked with biodetection molecules via a "click" reaction between azido and alkynyl groups or via a reaction of thiols with bromides and a metal-free catalyst. Other suitable crosslinking methods can be used.

[0041]

[10] In some embodiments, the analyzer used is capable of detecting electroluminescence, mechanoluminescence, triboluminescence, chemiluminescence, piezoluminescence and mechanochromic effects.

[0042]

[11] In some embodiments, the luminescent group may comprise a fluorophore. In some embodiments, the fluorophore may have the following skeletal structure:

[0043]

[0044] Each of R1, R2, R3, R4, R5, and R6 is independently selected from hydrogen, alkyl, unsaturated alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl; each of X is independently selected from oxygen, sulfur, selenium, tellurium, carbon, silicon, germanium, phosphorus, arsenic, and antimony, and X may be further substituted with hydrogen, alkyl, unsaturated alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl. Those skilled in the art will understand that, depending on the valence of X, it can have 0-3 substituents. For example, if X is chosen from a Group 4 element (e.g., Si), it may be combined with two protons, or two other substituents (e.g., alkyl, unsaturated alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl), or a proton and another substituent.

[0045]

[12] In some embodiments, nanoparticles having a luminescence quantum yield of more than 20% comprise luminescent clusters entangled in a polymer network, wherein the amount of luminescent clusters in the polymer network can be any value in the range of 1-50 w / w%.

[0046] The process of preparing fluorescent nanoparticles through polymerization reaction

[0047]

[13] In some embodiments of the present invention, fluorescent nanoparticles may be prepared according to the following process.

[0048]

[14] Preferably, all monomers should be purified, for example by passing through an alumina (Al2O3) column, and oxygen should be removed from deionized (DI) water, for example by bubbling with nitrogen for 30 minutes.

[0049]

[15] Assemble the apparatus consisting of a two-necked flask, a mechanical stirrer, and an oil bath. Immerse the two-necked flask in the oil bath. The two-necked flask should not be shaken while the mechanical stirrer is in operation. Then, add 0.01-0.2 g of sodium dodecylbenzenesulfonate (SDBS), 0.05-0.5 g of ammonium bicarbonate (NH4HCO3), and 5-20 mL of deionized water to the two-necked flask. Turn on the mechanical stirrer at a speed of 100-1000 rpm and raise the oil bath temperature to 60-80 degrees Celsius.

[0050]

[16] An initiator aqueous solution was prepared by dissolving 0.01-0.1 g of ammonium persulfate [(NH4)2S2O8] in deionized water.

[0051]

[17] A monomer mixture was prepared by dissolving 4,4'-(1,2-bis(dibenzo[b,d]thiophene-2-yl)ethylene-1,2-diyl)bis(N,N-diphenylaniline) (commercial name AIEgen™ Bright Green, formerly known as NSTPE) in 0.1-900 mL of [styrene(St) / methyl methacrylate (MMA) / acrylic acid (AA) (volume fraction ratio 1:0.02-1:0.02-1)].

[0052]

[18] When the reaction mixture becomes clear, 3-5% by volume of the prepared monomer mixture is added to the reaction mixture for pre-emulsification. In some embodiments, 1 to <100% by volume of the prepared monomer mixture may be added to the reaction. After 10-60 minutes, the initiator aqueous solution and the remaining monomer mixture are added simultaneously to the flask at a rate of 2-10 drops over several minutes, slightly later than the monomer mixture. The reaction mixture is kept heated at 70-90 degrees Celsius until it becomes milky white. The crude product is then purified after the reaction mixture is cooled.

[0053]

[19] In some embodiments, the surfactant used to prepare the reaction mixture may be one or more of the following: sodium decylbenzenesulfonate, sodium dodecyl sulfate, sodium docusate, perfluorooctane sulfonic acid, perfluorobutane sulfonic acid. Sodium stearate, quaternary ammonium salt ammonium cation The R group can be one or more alkyl or aryl groups [e.g., benzalkonium chloride and distearate dimethyl ammonium chloride], alkyl polysaccharide glycosides, where m can vary between 1 and 100 [e.g., decyl glucoside], glyceryl monostearate, nonylphenoxy polyethoxyethanol, where n can vary between 1 and 100, etc.

[0054]

[20] In some embodiments, fluorophores that are completely soluble in the monomer solution are used to achieve small size and good uniformity of the nanoparticles. As shown in Table 1, the solubility of the fluorophores in the monomer solution decreases from feed 1 to feed 3. If the fluorophores are insoluble in the monomer solution (feed 3), polymerization does not occur. Although polymerization occurs when the fluorophores are partially soluble in the monomer solution (feed 2), small size and good uniformity of the nanoparticles cannot be achieved. Only when the fluorophores are completely soluble in the monomer solution do the nanoparticles have very small size (<50 nm) and good uniformity.

[0055] Table 1. Solubility of some fluorophores in some monomers:

[0056]

[0057]

[0058] The process of fabricating fluorescent nanoparticles using microfluidics

[0059]

[21] In some embodiments, typical microfluidic flow focusing devices can be used to fabricate fluorescent nanoparticles ( Figure 1 Microfluidic devices can be fabricated from polydimethylsiloxane (PDMS) using standard microforming methods. Alternatively, all microchannels of a specific size can be etched into a silicon wafer using deep reactive ion etching or wet etching methods. The etched silicon wafer can then be bonded to a glass substrate using anodic bonding. A heater (platinum) fabricated on the glass substrate provides heat to the microchannels. A temperature sensor (e.g., a platinum sensor) fabricated on the glass substrate can record the temperature within the microchannels. The particle size of the fluorescent nanoparticles can be adjusted by changing the flow parameters.

[0060] Purification process of fluorescent nanoparticles

[0061]

[22] In some embodiments of the present invention, nanoparticles can be purified according to the following process.

[0062]

[23] Centrifuge the crude product at 5-15 rpm for 3-30 minutes to remove free AIEgen. TM The liquid is bright green. Transfer the supernatant to a test tube and add ethanol at a volume ratio of 1:0.1-10 to break the emulsion. A clear precipitate will be observed after several hours. Then, centrifuge the mixture at 5-15 rpm for 3-30 minutes to obtain a solid product. The separated product is then redispersed in 1X phosphate-buffered saline (PBS) with the aid of an ultrasonic device. The final solution is stored at room temperature for use.

[0063] Labeling reaction process

[0064]

[24] In some embodiments, the labeling reaction may be carried out as described below.

[0065]

[25] 146 μL of 2-ethanesulfonic acid (MES) buffer (pH 6.0), 50 μL of nanoparticle (<200 nm) solution, 2 μL of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) solution (20 mg / mL) and 2 μL of N-hydroxysulfosuccinimide (sulfonyl-NHS) solution (55 mg / mL) were added to a 1.5 mL test tube. The EDC and sulfonyl-NHS solutions were prepared with MES buffer (pH 6.0) before use. The reaction mixture was shaken at 100–1000 rpm (Eppendorf centrifuge 5424R) for 15–60 min at room temperature, and then 1.4 μL of 2-mercaptoethanol was added to the tube to inactivate the EDC. The reaction mixture was then centrifuged at 5–15 krpm for 3–30 min. The solid product was collected and washed several times with 200 μL of MES buffer.

[0066]

[26] The purified solid product was redispersed in 600 μL of 1X PBS buffer (pH 7.4) to prepare an activated fluorescent nanoparticle solution. 200 μL of the activated fluorescent nanoparticle solution and 200 μL of antibody were transferred to a 1.5 mL tube. The reaction mixture was kept at room temperature and shaken at 100–1000 rpm for 1–4 hours. The mixture was then centrifuged at 0–4°C and 5–15 krpm for 1–60 minutes to collect the solid product. The solid product was redispersed in 200 μL of 1% bovine serum albumin (BSA) solution (in 1X PBS buffer) and the mixture was shaken at 0–4°C and 100–1000 rpm. After 1–24 hours, the mixture was centrifuged at 0–4°C and 5–15 krpm for 3–30 minutes to collect the solid product. With the aid of an ultrasonic machine, the solid product was redispersed in 200 μL of triethanolamine buffered saline (Tween-20 0.05%) to obtain the nanoparticle antibody conjugate. The labeled nanoparticles were stored at 4–8 degrees Celsius for use.

[0067] Lateral chromatography test paper preparation process

[0068]

[27] In some embodiments of the present invention, the lateral chromatography test strip can be prepared according to the following procedure.

[0069]

[28] (i) Preparation of the binding pad:

[0070] Labeled nanoparticles were diluted with TBS-T buffer (pH 7.4) containing 0.1–1 mol / L sodium chloride (NaCl), 1–20 mmol / L EDTA, 1–10% (w / v) BSA, 1–10% (w / v) sucrose, and 0.01–0.1% (w / v) sodium azide (NaNs) to produce conjugate solutions with a final concentration of 1–100 μg / mL. 5–25 μL of the conjugate solution was added to 3 x 6 mm binding pads and then dried at 37 °C for 3 hours.

[0071]

[29] (ii) Fixation of the capturing reagent:

[0072] 0.1–2 mg / mL of mouse anti-human alpha-LH-mAB (or mouse anti-human alpha-hCG-mAB for hCG test strips) and 0.1–2 mg / mL of goat anti-mouse IgG (LH and hCG detection) were applied as test and control lines, respectively, onto a nitrocellulose membrane. Both test and control lines were positioned 5 mm from the center of the membrane. The capture reagent was added in 9 μL... -1 cm -1 The solution is applied to the membrane and dried at 37°C for 1-2 hours. Finally, the membrane is sealed with 0.1-5% (w / v) bovine serum albumin, dried, and then sealed for storage.

[0073]

[30] (iii) Preparation of sample and absorbent pad:

[0074] The samples and absorbent pads were made of nonwoven fabric and 100% pure cellulose fiber (Millipore). 15 x 300 mm sample pads were saturated with pH 8.0 buffer containing 0.5–5.0% (w / v) bovine serum albumin, 0.5–5.0% (w / v) sucrose, 2–20 mmol / L sodium borate, and 0.01–0.1% (w / v) sodium azide, and then dried for storage. The absorbent pads were cut into 40 × 300 mm pieces.

[0075]

[31] (iv) Assembly of lateral chromatography test strips:

[0076] Assemble the sample pad, conjugate pad, NC membrane, and adsorption pad sequentially onto a plastic backing plate, overlap them with a 1-2 mm moving coil, cover both ends with colored film, and then cut them into 3 mm wide test strips using a CM4000 cutter (Bio-Dot). Seal the test strips in a plastic bag in the form of desiccant gel and store them at 4 degrees Celsius.

[0077] Example

[0078] Example 1. Preparation of fluorescent nanoparticles

[0079]

[32] Table 2 summarizes a range of chemicals and reaction conditions used in the polymer. Table 3 lists the nanoparticle parameters measured by dynamic light scattering (DLS) machine.

[0080]

[33] An apparatus consisting of a double-necked flask, a mechanical stirrer, and an oil bath was assembled. The double-necked flask, immersed in the oil bath, should not be shaken during mechanical stirring. Then, 0.2 g of SDBS, 0.5 g of NH4HCO3, and 20 mL of deionized water were added to the double-necked flask. The mechanical stirrer was turned on at 1000 rpm, and the oil bath temperature was raised to 60 degrees Celsius.

[0081]

[34] Then add 0.2 g SDBS, 0.5 g NH4HCO3 and 20 mL deionized water to the flask, turn on the mechanical stirrer at 1000 rpm, and raise the oil bath temperature to 60 degrees Celsius. Alternatively, use a cell disruptor (SCIENTZ-95E) instead of a mechanical stirrer to stir at 600 W for 3 minutes (specified as P5 in Table 3).

[0082]

[35] An initiator aqueous solution was prepared by dissolving 0.01 g ammonium persulfate [(NH4)2S2O8] in 1 mL of DI water. An initiator aqueous solution was prepared by dissolving 0.001 g AIEgen... TM Bright Green was dissolved in 0.1 mL of St / MMA / AA (v / v) to prepare monomer mixtures with the following volume ratios: 1:0.1:0.1 (designated as P2 in Table 3); 1:1:1 (designated as P3 in Table 3); 1:0.02:0.18 (designated as P4 in Table 3); 1:0.1:0.1 (designated as P5 in Table 3). After heating the mixture at 60°C for 5 minutes, the fluorophore completely dissolved in the monomer solution, and the solution became clear. Then, 3% by volume of the prepared monomer mixture was added to the reaction mixture for pre-emulsification. After 10 minutes, the initiator aqueous solution and the remaining monomer mixture were simultaneously added to a two-necked reaction flask at a rate of 10 drops per minute. Notably, the initiator was added to the reaction mixture slightly later than the monomer mixture. The reaction mixture was kept heated at 70°C until it turned milky white. After cooling the reaction mixture, the crude product was purified.

[0083] Example 2. Preparation of fluorescent nanoparticles

[0084]

[36] Nanoparticles P6, P7 and P8 were prepared using the same process as P2-P4, except that the following pure monomer solutions were used: P6: styrene only; P7: MMA only; P8: AA only. The particle size of P6 was 80 nm and the PDI was 0.035; the particle size of P7 was 98 nm and the PDI was 0.05; the particle size of P8 was 103 nm and the PDI was 0.045.

[0085] Table 2. Reactants and reaction conditions for polymerization reactions

[0086]

[0087] Example 3. Preparation of fluorescent nanoparticles.

[0088]

[37] Table 4 summarizes a range of chemicals and reaction conditions for the polymer. Table 3 lists the nanoparticle parameters measured by a dynamic light scattering (DLS) machine. 0.1 g SDBS, 0.5 g NH4HCO3 and 15 mL DI water were added to a two-necked flask. The flask was placed in an oil bath and the temperature was raised to 70°C to dissolve the solids. The flask was removed and the reaction mixture was cooled. An aqueous initiator solution [(NH4)2S2O8] was prepared by dissolving 0.02 g ammonium persulfate in 1 mL deionized water. 0.01 g AIEgen was used to prepare the initiator solution. TM Red was dissolved in 0.6 mL of St / MMA / AA (v / v) to prepare a monomer mixture at a volume ratio of 1:0.1:0.1 (specified as P93 in the table). The prepared monomer mixture was added to the reaction mixture and pre-emulsified for 0.5 hours at 500 W using a cell disruptor (SCIENTZ-95E). An apparatus consisting of a two-necked flask, a mechanical stirrer, and an oil bath was assembled. The two-necked flask was immersed in a 70°C oil bath and mechanically stirred at 300 rpm. The apparatus should not be shaken during mechanical stirring. After stirring for 10 minutes, the initiator aqueous solution was added to the two-necked reaction flask at a rate of 5 drops per minute. The reaction mixture was then heated at 70°C for 1 hour. The crude product was then purified after the reaction mixture was cooled.

[0089] Example 3. Purification of fluorescent nanoparticles

[0090]

[38] The crude product obtained from Example 1 was centrifuged at 3 krcf for 3 minutes to remove free AIEgen. TM BrightGreen. The supernatant was transferred to a tube and ethanol was added at a volume ratio of 1:0.1 to demulsify. A clear precipitation was observed after several hours. The mixture was then centrifuged at 5 krpm for 3 minutes to obtain a solid product. Subsequently, the separated product was redispersed in 1X phosphate-buffered saline (PBS) under ultrasonication (Guanboshi GS1530P-30L-900W) for 1 minute at 900 W. The final solution was stored at room temperature for further use. Figure 6 The photoluminescence of fluorescent nanoparticles under natural light and 365 nm ultraviolet excitation was demonstrated.

[0091] Table 3. Particle size of selected examples

[0092]

[0093]

[0094] a Ultrasonic pre-emulsification for 10 minutes

[0095] Table 4. Chemical composition and reaction conditions of the polymerization reaction

[0096]

[0097] Table 5. Antibody labeling reaction formulations for selected examples

[0098]

[0099]

[0100] Example 4. Purification of fluorescent nanoparticles

[0101]

[39] The crude product obtained from Example 1 was centrifuged at 3 kJ / cf for 3 min to remove free AIEgen. TM The supernatant was bright green. The supernatant was transferred to a tube and ethanol was added at a volume ratio of 1:0.1 to demulsify. A clear precipitation was observed after several hours. The mixture was then centrifuged at 5 krpm for 3 minutes to obtain a solid product. The separated product was then redispersed in 1X phosphate-buffered saline (PBS) with the aid of an ultrasonic machine (Guanboshi GS1530P-30L-900W) at 900 W for 1 minute. The final solution was stored at room temperature for further use. Figure 8 The photoluminescence effect of fluorescent nanoparticles under natural light and 365nm ultraviolet excitation was demonstrated.

[0102] Example 5. Purification of fluorescent nanoparticles

[0103]

[40] The crude product obtained in Example 3 was centrifuged twice at 3 kJrcf for 10 minutes each time to remove free AIEgen. TM Red. Transfer the upper layer to a tube and add ethanol (crude product: EtOH, v / v) at a volume ratio of 1:4 for demulsification. Then centrifuge the mixture at 6600 rcf for 50 min to obtain a solid product. Redisperse the solid product in DDI water by brief sonication. After centrifugation at 21 krcf for 30 min, redisperse the obtained solid product again in DDI water. After centrifugation at 21 krcf for 30 min again, redisperse the purified product in 0.1 M 2-(N-morpholine) ethanesulfonic acid (MES, pH 6.0) at 900 W for 20 min with the aid of an ultrasonic machine (Guanboshi GS1530P-30L-900W). The final dispersion is stored at room temperature or 4°C for further use. Figure 8The photoluminescence effect of fluorescent nanoparticles under natural light and 365nm ultraviolet excitation was demonstrated.

[0104] Example 6. The process of labeling NL10 and LH3

[0105]

[41] 146 μL of 2-ethanesulfonic acid (MES) buffer (pH 6.0), 50 μL of P2 nanoparticle solution, 2 μL of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) solution (20 mg / mL), and 2 μL of hydroxysulfosuccinimide (sulfonyl-NHS) solution (55 mg / mL) were added to a 1.5 mL test tube. The EDC and sulfonyl-NHS solutions were prepared with MES buffer (pH 6.0) before use. The mixture was shaken at 300 rpm (Eppendorf 5424R) at room temperature for 15 minutes, 1.4 μL of 2-mercaptoethanol was added to inactivate the EDC, and then the mixture was centrifuged at 5 krpm for 5 minutes. The solid product was collected and washed several times with 200 μL of MES buffer. Table 5 lists examples of antibody labeling.

[0106]

[42] The purified solid product was redispersed in 600 μL of 1XPBS buffer (pH 7.4) to prepare an activated fluorescent nanoparticle solution. 200 μL of the activated fluorescent nanoparticle solution and 200 μL of antibody (NL10 with β-hCG-mAb or LH3 with β-LH-mAb as shown in Table 5) were transferred to a 1.5 mL tube. The reaction mixture was continuously shaken at 150 rpm for 3 hours at room temperature. Then, it was centrifuged at 0°C and 6 krpm for 8 minutes and the solid product was collected. The solid product was redispersed in 200 μL of 1% bovine serum albumin (BSA) solution (in 1XPBS buffer) and the mixture was shaken at 240 rpm at 0–4°C. After 3 hours, the mixture was centrifuged at 0–4°C and 6 krpm for 4 minutes and the solid product was collected. With the aid of a micro-ultrasound machine, the solid product was redispersed in 200 μL of triethanolamine buffered saline (Tween-20 0.05%) to obtain the nanoparticle antibody conjugate. The labeled nanoparticle solution was stored at 8°C for later use.

[0107] Example 7. Preparation of lateral chromatography test strips

[0108]

[43] (i) Preparation of the binding pad:

[0109] The labeled nanoparticles were diluted with a diluent containing 0.1 mol / L sodium chloride, 20 mmol / L EDTA, 1% (w / v) bovine serum albumin, 1% (w / v) sucrose, and 0.02% (w / v) sodium azide to prepare a coupling solution with a final concentration of 50 μg / mL. 20 μL of the coupling solution was added to a 3 x 6 mm binding pad and then dried at 37°C for 3 hours.

[0110]

[44] (ii) Fixation of the capture reagent:

[0111] 0.1 mg / mL of mouse anti-human alpha-LH-mAB (or mouse anti-human alpha-hCG-mAB for hCG test strips) and 0.5 mg / mL of goat anti-mouse IgG (for both LH and hCG detection) were applied to a nitrocellulose membrane as the test line and control line, respectively. Both the test line and control line were positioned 5 mm from the center of the membrane. The capture reagent was added in 9 μL... -1 cm -1 The coating was applied to the membrane and dried at 37°C for 1 hour. Finally, the membrane was sealed with 5% (w / v) bovine serum albumin, dried, and then sealed for storage.

[0112]

[45] (iii) Preparation of sample pads and absorbent pads:

[0113] The samples and absorbent pads were made of nonwoven fabric and 100% pure cellulose fiber (Millipore). 15 x 300 mm sample pads were saturated with pH 8.0 buffer containing 0.5% (w / v) bovine serum albumin, 0.5% (w / v) sucrose, 2 mmol / L sodium borate, and 0.01% (w / v) sodium azide, and then dried for storage. The absorbent pads were cut into 40 × 300 mm pieces.

[0114]

[46] (iv) Assembly of lateral chromatography test strips:

[0115] Assemble the sample pad, conjugation pad, NC membrane, and adsorption pad sequentially onto a plastic backing plate, overlapping them with a 2mm moving coil. Cover both ends with colored film and then cut the mother card into 3mm wide test strips using a CM4000 cutter (Bio-Dot). Seal the test strips in a dry gel form in a plastic bag and store at 4 degrees Celsius for later use.

[0116] Example 8. Detection procedure for protein standard solutions

[0117]

[47] Tests were performed using Example 5. LH standard solutions of varying concentrations (0 to 200 mL U / mL) were prepared by dilution in triethanolamine buffered aqueous solution (pH 7.4). 100 μL of sample was loaded onto the sample pad of the test strip, and after 10–60 minutes, the strip was inserted into an analyzer containing a photodiode. All experiments were performed in triplicate, and the average of the three samples was analyzed. Detection of hCG standard solutions with concentrations ranging from 0 to 1 k mL U / mL was similar. Figure 7 The test strips show different concentrations of hCG and LH under 365 nm UV excitation.

[0118] Example 9. The process of labeling α-hCG-mAb reaction

[0119]

[48] ​​130 mL of 2-ethanesulfonic acid (MES) buffer (pH 7.0), 60 mL of P2 nanoparticle solution, 1–4 μL of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) solution (15–25 mg / mL), and 1 μL of N-hydroxysulfosuccinimide (sulfonyl-NHS) solution (50 mg / mL) were added to a 1.5 mL test tube. The EDC and sulfonyl-NHS solutions were prepared with MES buffer (pH 6.0) before use. The mixture was shaken at 200 rpm (Eppendorf 5424R) at room temperature for 10 min, 1 μL of 2-mercaptoethanol was added to inactivate the EDC, and then the mixture was centrifuged at 5 krpm for 5 min. The solid product was collected and washed several times with 2 μL of MES buffer. The purified solid product was redispersed in 1000 μL of 1XPBS buffer to prepare an activated fluorescent nanoparticle solution. 200 μL of activated fluorescent nanoparticle solution and 700 μL of antibody (α-hCG-mAB) were transferred to a 1.5 mL tube. The reaction mixture was shaken at 150 rpm for 3 hours at room temperature, centrifuged at 6 krpm for 5 minutes, and the solid product was collected. The solid product was redispersed in 700 μL of 2% bovine serum albumin solution (in 1X PBS buffer), and the mixture was shaken at 20 rpm at 0 °C. After 3 hours, the mixture was centrifuged at 6 krpm for 3 minutes, and the solid product was collected. The solid product was redispersed by sonication in 200 μL of triethanolamine buffer (Tween-20 0.05%) to obtain the nanoparticle antibody conjugate. The labeled nanoparticle solution was stored at 4 °C for later use.

[0120] Example 10. Preparation of a lateral chromatography assay strip for simultaneous detection of two proteins.

[0121]

[49] Preparation of conjugate pads: The conjugate solution was prepared by diluting the labeled nanoparticles to a final concentration of 50 μg / mL. The diluent was a triethanolamine buffered aqueous solution containing 0.1 mol / L sodium chloride, 20 mmol / L EDTA, 1% (w / v) bovine serum albumin, 1% (w / v) sucrose and 0.02% (w / v) sodium azide. 20 μL of the conjugate solution was added to a 3 x 6 mm conjugate pad and dried at 37°C for 3 hours. (ii) Immobilization of capture reagents: 0.1 mg / mL mouse-anti-human alpha-LH monoclonal antibody for LH detection, mouse-anti-human beta-hCG monoclonal antibody for hCG detection, and 0.5 mg / mL goat-anti-mouse IgG (for both LH and hCG detection) were added to a nitrocellulose membrane to prepare detection line 1, detection line 2 and control line, respectively. The test line and control line were set in the middle of the nitrocellulose membrane, spaced 4 mm apart. These reagents were sprayed onto the membrane at a rate of 9 μL / cm and dried at 37°C for 1 hour. Finally, the membrane was blocked with 5% (w / v) bovine serum albumin and dried under sealed conditions for later use. (iii) Preparation of sample and absorbent pad: The sample and absorbent pad were made of nonwoven fabric and 100% pure cellulose fiber (Millipore). A 15 x 300 mm sample pad was saturated with pH 8.0 buffer containing 0.5% (w / v) bovine serum albumin, 0.5% (w / v) sucrose, 2 mmol / L sodium borate, and 0.01% (w / v) NaN3, and then dried for storage. The absorbent pad was cut into 40 × 300 mm sizes. (iv) Assembly of lateral chromatography test strip: The sample pad, conjugation pad, NC membrane, and absorbent pad were sequentially assembled on a plastic backing plate, overlapped with a 2 mm moving coil, and both ends covered with colored film. The master card was cut into 3 mm wide strips using a CM4000 Bio-Dot cutter. The test strips were then sealed in plastic bags with a desiccant gel and stored at 4 degrees Celsius.

[0122] Example 11. Procedure for simultaneous detection of two protein standard solutions

[0123]

[50] Tests were performed using Example 8. Different combinations of LH and hCG standard solutions were prepared at concentrations of 0–100 mL U / mL and 0–1000 mL U / mL, diluted with PBS buffer (pH 7.4). 100 μL of sample was loaded onto the sample pad of the test strip and inserted into the analyzer and photodiode detector after 10–60 minutes. All experiments were performed in duplicate, and the average value copy was used for analysis. Figure 9 Test strips with different protein concentrations are shown under 365nm UV excitation.

[0124] Example 12. Labeling reaction process of COVID-19 nucleocapsid protein

[0125] Add 130 μL of 2-ethanesulfonic acid (MES) buffer (pH 6.0), 50 μL of P2 nanoparticle solution, 1–4 μL of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) solution (15–25 mg / mL), and 1 μL of N-hydroxysulfosuccinimide (sulfonyl-NHS) solution (50–60 mg / mL) to a 1.5 mL test tube. Prepare the EDC and sulfonyl-NHS solutions with MES buffer (pH 6.0) before use. After shaking the reaction mixture at 200 rpm (Eppendorf centrifuge 5424R) for 10 minutes at room temperature, add 1 μL of 2-mercaptoethanol to the tube to inactivate the EDC. Then, centrifuge the reaction mixture at 5 krpm for 5 minutes. Collect the solid product and wash several times with 200 μL of MES buffer. The purified solid product was redispersed in 500-1000 μL of 1X PBS buffer (pH 7.4) to prepare an activated fluorescent nanoparticle solution. 200-700 μL of the activated fluorescent nanoparticle solution and 200-700 μL of the protein (2019-nCov nucleocapsid protein) were transferred to a 1.5 mL tube. The reaction mixture was vortexed at 150-300 rpm for 1-3 hours at room temperature. Then, the mixture was centrifuged at 6-8 krpm for 5-10 minutes, and the solid product was collected. The solid product was redispersed in 200-700 μL of 0.5-2% bovine serum albumin (BSA) solution (in 1X PBS buffer), and the mixture was vortexed at 200-300 rpm at 0-4°C. After 1-3 hours, the mixture was centrifuged at 6-8 krpm for 3-10 minutes, and the solid product was collected. The solid product was redispersed in 200-700 μL of TBS-T buffer (Tween-200.05-1%) with the aid of an ultrasonic machine to produce nanoparticle antibody conjugates. The labeled nanoparticle solutions were stored at 4-8 °C for further use.

[0126] Example 13. Preparation of lateral flow chromatography test strips for detecting 2019nCov nucleocapsid protein IgM and IgG

[0127]

[51] Preparation of conjugate pads: The conjugate solution was prepared by diluting a solution containing 0.1 mol / L sodium chloride, 20 mmol / L EDTA, 1% (w / v) bovine serum albumin, 1% (w / v) sucrose and 0.02% (w / v) sodium azide to a concentration of 50 μg / mL. 20 μL of the conjugate solution was added to a 3 x 6 mm conjugate pad and then dried at 37°C for 3 h. (ii) Fixation of capture reagents: 0.1 mg / mL mouse anti-human IgM-mAB for IgM detection, 0.1 mg / mL mouse anti-human IgG-mAB for IgG detection, and 0.5 mg / mL mouse anti-nCov nucleocapsid monoclonal antibody were applied separately as test line 1, test line 2, and control line, respectively, onto a nitrocellulose membrane. Both the test line and control line were positioned 4 mm from the center of the membrane. These reagents were applied to the membrane in dots at a density of 9 μL per centimeter and dried at 37°C for 1 hour. Finally, the membrane was sealed with 5% (w / v) bovine serum albumin, dried, and sealed for storage. (iii) Preparation of samples and absorbent pads:

[0128]

[52] The sample and absorbent pads were made of nonwoven fabric and 100% pure cellulose fiber (Millipore). The 15x300 mm sample pads were saturated with pH 8.0 buffer containing 0.5% (w / v) bovine serum albumin, 0.5% (w / v) sucrose, 2 mmol / L sodium borate and 0.01% (w / v) sodium azide, and then dried for storage. The absorbent pads were cut into 40×300 mm sizes. (iv) Assembly of lateral chromatography test strips: The sample pad, conjugation pad, NC membrane and absorbent pad were assembled in sequence on a plastic backing plate with a 2 mm moving coil overlap and both ends covered with colored film. The master card was cut into 3 mm wide strips using a CM4000 cutter (Bio-Dot). The test strips were then sealed in plastic bags in the form of desiccant gel and stored at 4 degrees Celsius.

[0129] Example 14. Detection procedure for IgM and IgG against 2019-nCoV nucleocapsid protein

[0130]

[53] Tests were performed using Example 11. Positive serum samples from patients or negative serum samples from healthy individuals were loaded onto the sample pad of the test strip, and then inserted into the analyzer and photodiode transducer for 10–60 minutes. The test line intensity ratio was calculated. Table 6 shows the results for positive and negative serum samples.

[0131]

[54] Table 6

[0132]

[0133]

[0134] Example 15. Application of AIE conjugate antibodies in immunoblotting

[0135]

[55] AIE-conjugate antibodies can improve the sensitivity of immunolabeling. Immunoblotting performance was tested between direct immunolabeling with AIE-conjugate antibodies and indirect immunolabeling with traditional fluorescent conjugates (such as FITC) antibodies. In this experiment, equal amounts of protein were imprinted on two energy fiber membranes. Proteins on one membrane (A) were detected by binding to AIE-conjugate primary antibody; while proteins on the other membrane (B) were detected by binding to equal amounts of unconjugate primary antibody and FITC-conjugate secondary antibody. Under the same conditions, the signal intensity of the target protein was detected using the direct method. The AIE-conjugate antibody on the immunoblot showed significantly stronger signal intensity than the indirect detection result using the FITC-conjugate secondary antibody. Figure 10 ).

[0136] References

[0137] [1]S.Thalhammer,E.Linares,Novel cluster for the detection of ananalyte,2016,PCT / EP20 15 / 068 193.

[0138] [2]JSLee,S.Lee,MHChoi,H.Soo,SKKim,JYKim,Enhanced Infrared RayAbsorbing / Emitting Nanoparticles and On-Site Diagnosis Kit Using Same,2018,PCT / KR2018 / 002616.

[0139] [3]JWMoon,TJPhelps,CLFitzgerald,Jr.,RFLind,JGElkins,GGJang,PCJoshi,M.Kidder,BLArmstrong,TRWatkins,INIvanov,DEGraham,Appl Microbiol Biotechnol2016,100,7921.

[0140] [4]S.Wang,N.Mamedova,NAKotov,W.Chen,J.Studer,Nano Letters2002,2,817.

[0141] [5]W.J.Parak,D.Gerion,T.Pellegrino,D.Zanchet,C.Micheel,S.C.Williams,R.Boudreau,M.A.L.Gros,C.A.Larabell,A.P.Alivisatos,Nanotechnology2003,14,R15.

[0142] [6]G.M.Whitesides,Nature Biotechnology2003,21,1161.

[0143] [7]A.Care,N.Sayyadi,R.Connally,A.Try,P.L.Bergquist,A.Sunna,Luminescent biomolecmlar complex and use thereof,2016,PCT / AU2016 / 000263.

[0144] [8]J.Luo,Z.Xie,J.W.Y.Lam,L.Cheng,B.Z.Tang,H.Chen,C.Qiu,H.S.Kwok,X.Zhan,Y.Liu,D.Zhu,Chemical Communications2001,1740.

Claims

1. A solid-state luminescent nanoparticle with a quantum yield higher than 20%, comprising luminescent groups entangled in a polymer network, said luminescent group being 4,4'-(1,2-bis(dibenzo[b,d]thiophene-2-yl)). Ethylene-1,2-diyl)-bis(N,N-diphenylaniline) is obtained by copolymerizing styrene, methyl methacrylate, and acrylic acid.

2. The nanoparticles as described in claim 1, characterized in that, The emission wavelength of the luminescent group is adjustable from 400 nm to 850 nm, and the excitation wavelength is from 220 nm to 800 nm.

3. The nanoparticles as described in claim 1, characterized in that, The diameter of the nanoparticles ranges from 20 to 1000 nm.

4. The nanoparticles as described in claim 1, characterized in that, The nanoparticles can be conjugated to one or more antibodies.

5. A method for preparing solid-state luminescent nanoparticles with a quantum yield higher than 20% according to claim 1, comprising the following steps: a. Dissolving the luminescent group in one or more monomers to form a monomer mixture, said monomer mixture containing 1-500 g / L of the luminescent group; b. Dissolve the initiator in deionized water to form an initiator solution; c. Dissolve the surfactant in deionized water to form a reaction mixture; d. Add the monomer mixture and the initiator solution to the reaction mixture; and e. Heat the total mixture from step (d) until it turns milky white, in which the nanoparticles are formed.

6. The method as described in claim 5, characterized in that, Following step (c), a portion of the monomer mixture from step (a) is added to the reaction mixture under a relative centrifugal force of 100 to 4000, wherein the remaining monomer mixture is used in step (d).

7. The method as described in claim 6, characterized in that, The portion constitutes ≥1% to <99% of the volume of the monomer mixture.

8. The method as claimed in claim 5, characterized in that, The monomer mixture and the initiator solution are added simultaneously to the reaction mixture.

9. The method as claimed in claim 5, characterized in that, Following step (c), a portion of the monomer mixture is added to the reaction mixture under ultrasonic treatment at 400 to 2000 watts microliters.

10. The method as described in claim 5, characterized in that, A microfluidic device is used in step (d).

11. The method as described in claim 5, characterized in that, The initiator is selected from one of ammonium persulfate, ammonium persulfate (APS), 2,2'-azobis(isobutyronitrile) (AIBN), benzoyl peroxide (BPO), potassium persulfate, and 4,4'-azobis(4-cyanopentanoic acid).

12. The method of claim 5, characterized in that, The concentration of the initiator in the initiator solution is from 0.1% to 1%.

13. The method of claim 5, characterized in that, The surfactant is selected from one of sodium decylbenzenesulfonate, sodium dodecyl sulfate, sodium docusate, perfluorooctane sulfonic acid, perfluorobutane sulfonic acid, sodium stearate, quaternary ammonium cation, alkyl polyglycoside, glyceryl monostearate, or nonylphenoxypolyethoxyethanol.

14. The method as described in claim 5, characterized in that, The concentration of the surfactant in the reaction mixture is 3-12 g / L.

15. The method as described in claim 5, characterized in that, The heating temperature in step (e) is 70 to 90°C.

16. The method as described in claim 5, characterized in that, Step (e) also includes a purification process.

17. The method as described in claim 5, characterized in that, The polydispersity index of the nanoparticles ranges from 0.009 to 1.

18. A lateral chromatography test strip, comprising a sample pad, a binding pad, a reaction membrane, and an adsorption pad, characterized in that, The bonding pad is configured with at least one nanoparticle according to claim 1.

19. The lateral chromatography test strip according to claim 18, characterized in that, The lateral chromatography test paper emits emission at a wavelength of 400 nm to 800 nm.

20. The lateral chromatography test strip according to claim 18, characterized in that, The lateral chromatography test strip has one or more test lines on a single strip.

21. The lateral chromatography test strip according to claim 18, characterized in that, The emission intensity of the lateral chromatography test paper is measured to obtain a quantitative reading for analyzing the concentration.

22. An antibody test kit comprising the nanoparticles of claim 1.

23. A cell labeling kit comprising the nanoparticles of claim 1.

Images