A rapid in-situ detection technology based on the interaction between photocatalyst nanoparticles and proteins

Abstract

The present application relates to a kind of rapid in-situ detection technology based on the interaction between photocatalysis-based nanoparticles and protein, a method for separating and extracting protein interacting with nanoparticles, the method steps are as follows: preparation contains photocatalysis probe and labeled reaction substrate nanoparticles, or preparation contains photocatalysis probe nanoparticles and labeled reaction substrate is added to the sample containing protein;2) the nanoparticles of step 1 and the sample containing protein are contacted to form the interaction between nanoparticles and protein;the labeled reaction substrate in nanoparticles is activated by the laser irradiation of the wavelength corresponding to photocatalysis probe, and the protein interacting with nanoparticles is obtained;3) the labeled protein interacting with nanoparticles is separated using streptavidin-coated magnetic beads.

Description

Technical Field

[0001] This invention relates to a method for separating and extracting proteins that interact with nanoparticles, and particularly to a rapid in-situ detection technique based on photocatalysis of the interaction between nanoparticles and proteins. Background Technology

[0002] The in vivo and in vitro fate of nanoparticles depends not only on their composition and structure but also on the environment. Due to their nanoscale size, nanoparticles possess high surface activity. Upon contact with biological fluids, proteins in the biological environment rapidly interact with the nanoparticles, adsorbing onto their surfaces to form a coating structure known as a "protein crown." Current methods for separating and extracting proteins interacting with nanoparticles are mostly label-free, with centrifugation being the most widely used. These methods, however, lead to the loss of proteins with weaker interactions with the nanoparticles and the introduction of impurities such as protein complexes with a certain particle size during the separation and extraction of protein crowns. Furthermore, label-free protein crown extraction and separation methods are typically time-consuming, thus often used to detect interactions between nanoparticles and proteins in equilibrium states, and cannot meet the requirements for detecting interactions between nanoparticles and proteins in non-equilibrium states. Although label-free protein crown separation and extraction methods are significant for studying nanoparticle-protein interactions, their temporal resolution is currently limited to a maximum of 30 seconds due to the inherent limitations of their separation principle, significantly restricting further improvements in temporal and spatial resolution. Summary of the Invention

[0003] This invention provides a rapid in-situ detection technology for the interaction between nanoparticles and proteins based on photocatalysis, which improves the spatiotemporal resolution and accuracy of the detection of the interaction between nanoparticles and proteins. Using this technology, the dynamic interaction spectrum between nanoparticles and proteins and other substances in complex biological samples can be revealed over the entire time period.

[0004] To achieve the above objectives, the present invention provides a method for separating and extracting proteins that interact with nanoparticles, the method comprising the following steps:

[0005] 1) Prepare nanoparticles containing photocatalytic probes and labeled reaction substrates, or prepare nanoparticles containing photocatalytic probes and add labeled reaction substrates to samples containing proteins;

[0006] 2) The nanoparticles from step 1 are brought into contact with a protein-containing sample to form an interaction between the nanoparticles and the protein; the labeled reaction substrate in the nanoparticles is activated by laser irradiation with a laser of the corresponding wavelength of the photocatalytic probe to obtain the labeled protein that interacts with the nanoparticles.

[0007] 3) Use streptavidin-coated magnetic beads to separate labeled proteins that interact with nanoparticles.

[0008] The nanoparticles include: liposomes, solid lipid nanoparticles, polymer nanoparticles, nanomicelles, dendritic macromolecules, nanocrystalline particles, albumin nanoparticles, ferritin nanoparticles, and fat emulsions.

[0009] The photocatalytic probe of this invention comprises portions containing thioxanthone, phenothiazine, flavin, phenothiazine, benzo[a]phenothiazine, coumarin, acetophenone, benzophenone, triarylmethane groups, rose red, porphyrin, dihydroporphyrin, chlorophyll, methylene blue, acridine dyes, xanthones dyes, and arylmethane dyes; it may also include ruthenium complex catalysts and their series of modifications, iridium complex catalysts and their series of modifications, platinum complex catalysts and their series of modifications, copper complex catalysts and their series of modifications, zirconium complex catalysts and their series of modifications, and iron complex catalysts and their series of modifications.

[0010] The preferred photocatalytic probe is dihydroporphyrin e6 (Ce6).

[0011] The active reaction portion of the labeling reaction substrate described in this invention includes diaziridine, azide, phenol, alkynyl, amino, glycosyl, carboxyl, and biotin. The labeling detection portion of the labeling reaction substrate includes biotin, desulfobiotin, alkynyl, azide, FLAG tag, fluorophore, and chloroalkyl functional group.

[0012] The preferred substrate for the labeling reaction is biotin-phenol (BP).

[0013] The protein-containing samples described in this invention include blood (plasma, serum), urine, cerebrospinal fluid, synovial fluid, tears, saliva, whole blood, milk, nipple aspirate, catheter lavage fluid, vaginal fluid, nasal fluid, ear fluid, gastric juice, pancreatic juice, trabecular fluid, pulmonary lavage fluid, sweat, groin fluid, semen, prostatic fluid, sputum, feces, bronchial lavage fluid, fluids from swabs, bronchial aspirate, fluidized solids, fine needle aspirate samples, tissue homogenates, lymph, cell culture samples, or any combination thereof.

[0014] Blood is the preferred protein-containing sample.

[0015] As a preferred technical solution, step 1) of this invention first prepares a core-shell nanoparticle (NPs@CeBP) loaded with Ce6 and BP. The preparation principle is as follows: Ce6 is stably positioned within the core of the nanoparticle through coordination interactions with calcium ions, while BP is distributed within the outer lipid bilayer. Therefore, step 1) of this invention can specifically employ the following steps:

[0016] Add dropwise the aqueous phase containing 0.1 μM–0.1 mM photocatalytic probe Ce6 to 1 mL–1 L of oil phase (1.5 M n-hexane and 0.6 M Triton X-100 mixed in cyclohexane) and stir for 15–60 minutes until homogeneous. Add dropwise the aqueous phase containing 10 μM–10 mM CaCl2 to 1 mL–1 L of oil phase and stir for 15–60 minutes until homogeneous. Then add dropwise the mixed solution containing Ce6 to the mixed solution containing CaCl2 and stir for 15–60 minutes. Add dropwise the aqueous phase containing 1 μM–1 mM Na2HPO4 and the chloroform solution containing 1 μM–1 mM DOPA to 2 mL–2 L of oil phase (1.5 M n-hexane and 0.6 M Triton X-100 mixed in cyclohexane) and stir for 15–60 minutes until homogeneous. Finally, add dropwise the mixed solution containing Na2HPO4 and DOPA to the solution containing Ce6. In a solution of CaCl2, stir for 0.5-4 hours to form granules, then add 8 mL-8 L of isopropanol for solidification. Centrifuge to collect the granules, discard the supernatant, wash the granules with isopropanol and centrifuge again. Repeat this washing process three times. The resulting granules are then redispersed in chloroform for later use.

[0017] Preparation of NPs(-)@CeBP: 1.1 μM-1.1 mM DOPC, 27.4 nM-27.4 μM DSPE-PEG2000, 289.7 nM-289.7 μM cholesterol and 275.1 nM-275.1 μM BP were dissolved in an organic solvent in a certain proportion and then mixed with particles dispersed in chloroform. The mixture was then subjected to rotary evaporation under reduced pressure to form a thin film. The film was hydrated at 60 °C and dispersed by ultrasonication at 70 W-120 W for 10-30 minutes to obtain NPs(-)@CeBP nanoparticles.

[0018] Preparation of NPs(+)@CeBP: 992.3 nM-992.3 μM DOPC, 24.7 nM-24.7 μM DSPE-PEG2000, 131.9 nM-131.9 μM DOTAP, 260.7 nM-260.7 μM cholesterol, and 275.1 nM-275.1 μM BP were dissolved in an organic solvent in a certain proportion and then mixed with particles dispersed in chloroform. The mixture was then subjected to rotary evaporation under reduced pressure to form a thin film. The film was hydrated at 60 °C and dispersed by ultrasonication at 70 W-120 W for 10-30 minutes to obtain NPs(+)@CeBP nanoparticles.

[0019] As a preferred technical solution, step 2) of this invention involves contacting nanoparticles with a protein-containing sample to form an interaction between the nanoparticles and the protein; then, the labeled reaction substrate in the nanoparticles is activated by laser irradiation with a laser of a corresponding wavelength of a photocatalytic probe, resulting in the labeled protein that interacts with the nanoparticles. The laser wavelength ranges from 200 to 2500 nanometers, with a preferred wavelength of 660 nanometers. Specifically, the following steps can be employed:

[0020] Take a protein-containing sample, either undiluted or diluted with PBS buffer to 0.1-20 mg / mL, mix the prepared nanoparticles NPs(-)@CeBP or NPs(+)@CeBP with the protein-containing sample, and incubate for 1 second to 1 hour. Irradiate the mixed solution with a laser of a specific wavelength for 1 second to 1 hour. The labeling reaction stops when the light is removed.

[0021] As a preferred technical solution, step 3) of the present invention involves separating the labeled protein that interacts with the nanoparticles using streptavidin-coated magnetic beads. Therefore, step 3) of the present invention can specifically employ the following steps:

[0022] The amount of biotin-labeled protein in samples extracted with or without streptavidin-coated magnetic beads was separated using SDS-PAGE and detected by Western blotting, as follows:

[0023] After thorough separation by SDS-PAGE, the samples were completely transferred to a PVDF membrane and blocked with sufficient blocking buffer at 37°C for 0.5–1 hour. The PVDF membrane was then gently shaken overnight in a horseradish peroxidase-streptavidin solution at 4°C. The PVDF membrane was washed six times with TBST solution (10 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1‰ Tween-20) for 5 minutes each time. The horseradish peroxidase chemiluminescent substrate was dropped onto the PVDF membrane, and the amount of biotin-labeled protein was detected under a gel imaging system.

[0024] The labeled proteins that interact with the nanoparticles were extracted and enriched using streptavidin-coated magnetic beads, as follows:

[0025] Centrifuge the sample in a 10K ultrafiltration tube at 14,000 rpm for 30 minutes. Resuspend in an equal volume of PBS and centrifuge again at 14,000 rpm for 30 minutes. Repeat this washing process three times. Finally, resuspend in an equal volume of PBS and transfer to a protein-low adsorption centrifuge tube. Take an excess of streptavidin-coated magnetic beads, wash three times with PBS, resuspend in PBS, and add to the sample. Invert and mix at 4°C overnight or at room temperature for 2 hours. After mixing, place the centrifuge tube on a magnetic rack. The magnetic beads will be enriched on the inner wall of the centrifuge tube by the rack. Discard the supernatant. Wash the magnetic beads 3-6 times with PBS, 6 times with washing buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Triton-X100), and 3-6 times with PBS. Boil the magnetic beads in protein loading buffer containing 2 mM biotin for 10 minutes to obtain the final product.

[0026] The amount of biotin-labeled protein was separated using SDS-PAGE and detected by a silver staining kit, as follows:

[0027] After thorough separation using SDS-PAGE, the gel was placed in approximately 100 ml of fixative (50 ml ethanol, 10 ml acetic acid, and 40 ml Milli-Q purified water) and shaken overnight at room temperature for 40 minutes at 60-70 rpm. The fixative was discarded, and 100 ml of 30% ethanol was added. The mixture was shaken for 10 minutes at room temperature at 60-70 rpm. The 30% ethanol was discarded, and 200 ml of Milli-Q purified water was added. The mixture was shaken for 10-60 minutes at room temperature at 60-70 rpm. The water was discarded, and 100 ml of silver staining sensitization solution (1X) was added. The mixture was shaken for 2 minutes at room temperature at 60-70 rpm. The original solution was discarded, and 200 ml of Milli-Q purified water was added. The mixture was shaken for 1 minute at room temperature at 60-70 rpm. The mixture was washed twice. Discard the water, add 100 ml of silver solution (1X), and shake on a shaker at room temperature for 10 minutes at a speed of 60-70 rpm. Discard the original solution, add 100 ml of Milli-Q purified water, and shake on a shaker at room temperature for 1-1.5 minutes at a speed of 60-70 rpm. Discard the water, add 100 ml of silver staining developer, and shake on a shaker at room temperature for 3-10 minutes until the desired protein band appears, shaking at a speed of 60-70 rpm. Discard the silver staining developer, add 100 ml of silver staining stop solution (1X), and shake on a shaker at room temperature for 10 minutes at a speed of 60-70 rpm. Discard the silver staining stop solution, add 100 ml of Milli-Q purified water, and shake on a shaker at room temperature for 2-5 minutes at a speed of 60-70 rpm. Store in Milli-Q purified water. Detect the amount of biotin-labeled protein using a gel imaging system.

[0028] The plasma proteins that interacted with nanoparticles at different incubation times were separated using SDS-PAGE and quantitatively detected using liquid chromatography-mass spectrometry. The method is as follows:

[0029] Samples were thoroughly separated using SDS-PAGE. After staining with Coomassie Brilliant Blue, protein bands were excised and placed in low-absorption centrifuge tubes. The gel was destained with 50 mM NH4HCO3:acetonitrile (1:1 v / v) at 37°C, washed twice with acetonitrile, and dried. 5 mM DTT was added and incubated at 45°C for 30 minutes, washed twice with acetonitrile, and dried. 11 mM IAM was added and incubated for 20 minutes, washed twice with acetonitrile, and dried. A precisely weighed amount of trypsin was added and incubated at 4°C for 1 hour, then at 37°C overnight. 10% trifluoroacetic acid was added. Peptides were extracted twice with 50% acetonitrile:0.1% trifluoroacetic acid (1:1 v / v) and then lyophilized. The lyophilized samples were redissolved in 0.1% trifluoroacetic acid solution. The resulting samples were separated by liquid chromatography and quantitatively detected by mass spectrometry. The results were searched using Proteome Discovery software and the UniProt database. All samples were subjected to three or more independent replicates. Trypsin was used as an internal standard protein in the samples. The strength of each protein in the sample was determined by the average strength of its top three most ionized peptides. Since the precise molar amount of trypsin was known, the approximate absolute molar amounts of all other detected proteins could be calculated.

[0030] In the preferred embodiment, this invention employs a photocatalytic probe, dihydroporphyrin e6 (Ce6), which, upon irradiation, activates the labeled substrate molecule biotin-phenol (BP) via free radical transfer. The activated BP reacts directly with surrounding proteins, thus tagging them with biotin. Due to the extremely short half-life of activated BP molecules, they can only label proteins within a range of tens of nanometers surrounding the nanoparticles, ensuring the high spatiotemporal resolution of this labeling technique. These biotin-tagged proteins are separated using streptavidin-coated magnetic beads and identified via liquid chromatography-mass spectrometry (LC-MS).

[0031] Compared to label-free existing technologies, this invention, based on photocatalytic proximity labeling, enables real-time, in-situ analysis of protein crowns at high spatiotemporal resolution. This technology can reveal the dynamic interaction patterns between nanoparticles and proteins and other substances in complex biological samples over a full time period. The method of this invention can be used for in-situ, short-term, and precise detection of interactions between nanoparticles and proteins. This invention can be used to determine 1 to 20,000 proteins.

[0032] The following is an explanation of the abbreviations and terms used in this invention specification.

[0033] Photocatalysis refers to the process by which a photochemical reaction is accelerated by a catalyst. A catalyst that can accelerate a photochemical reaction is called a photocatalyst.

[0034] Nanoparticles: refer to microscopic particles at the nanometer scale.

[0035] Proximity labeling: a biotechnology that labels biomolecules, typically proteins or RNA, that are close to a target.

[0036] Protein corolla: A dynamic coating layer of biomolecules (usually proteins) that spontaneously forms on the surface of nanoparticles when exposed to biological media.

[0037] Interaction map: refers to the physical interactions between molecules, and can also describe a series of indirect interactions between genes.

[0038] Ce6: dihydroporphyrin e6

[0039] DOPA: Dioleoylphosphatidylcholine

[0040] DSPE-PEG2000: Distearylphosphatidylethanolamine-polyethylene glycol 2000

[0041] DOPC: Dioleoyl lecithin

[0042] DOTAP: (2,3-Dioleoyl-propyl)-methylamine

[0043] BP: Biotin-phenol

[0044] DMPO: 5,5-Dimethyl-1-pyrrololine-N-oxide

[0045] BSA: Bovine serum albumin

[0046] TEMED: Tetramethylethylenediamine

[0047] DTT: Dithiothreitol

[0048] IAM: Iodoacetamide Detailed Implementation

[0049] The present invention will be further described in detail below with reference to the specific embodiments and accompanying drawings. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods not specifically described in the embodiments are generally performed under conventional conditions in the art or as recommended by the manufacturer.

[0050] The present invention will be further illustrated by the following examples.

[0051] Example 1: Preparation of two types of nanoparticles, NPs(-)@CeBP and NPs(+)@CeBP

[0052] Aqueous phase containing 1 μM photocatalytic probe Ce6 was added dropwise to 20 mL of oil phase (1.5 M n-hexane and 0.6 M Triton X-100 mixed in cyclohexane) and stirred for 15 minutes to mix. Aqueous phase containing 100 μM CaCl2 was added dropwise to 20 mL of oil phase and stirred for 15 minutes to mix. Then, mixed solution containing Ce6 photocatalytic probe was added dropwise to mixed solution containing CaCl2 and stirred for 15 minutes. Next, chloroform solution containing 10 μM Na2HPO4 and 10 μM DOPA was added dropwise to 40 mL of oil phase and stirred for 15 minutes to mix. Mixed solution containing Na2HPO4 and DOPA was added dropwise to solution containing Ce6 and CaCl2 and stirred for 0.5 hours to form particles. Then, 160 mL of isopropanol was added to solidify for 1 hour. The particles were collected by centrifugation, the supernatant was discarded, and the particles were washed with isopropanol and collected by centrifugation. This washing process was repeated three times. The resulting particles were redispersed in chloroform for later use;

[0053] Preparation of NPs(-)@CeBP: 11 μM DOPC, 274 nM DSPE-PEG2000, 2897 nM cholesterol and 2751 nM MBP were dissolved in an organic solvent and mixed with particles dispersed in chloroform. The mixture was then subjected to rotary evaporation under reduced pressure to form a thin film. The film was hydrated at 60 °C and dispersed by ultrasonication at 80 W for 15 minutes to obtain NPs(-)@CeBP nanoparticles.

[0054] Preparation of NPs(+)@CeBP: 9923 nM DOPC, 247 nM DSPE-PEG2000, 1319 nM DOTAP, 2607 nM cholesterol and 2751 nM BP were dissolved in an organic solvent and mixed with particles dispersed in chloroform. The mixture was then subjected to rotary evaporation under reduced pressure to form a thin film. The film was hydrated at 60 °C and dispersed by ultrasonication at 80 W for 15 minutes to obtain NPs(+)@CeBP nanoparticles.

[0055] The hydration radius and surface potential of nanoparticles were determined using dynamic light scattering. The morphology of the nanoparticles was examined using transmission electron microscopy (TEM). The elemental distribution of the nanoparticles was determined using a high-resolution TEM equipped with an energy dispersive spectroscopy (EDS) module. The types of free radicals generated during the photocatalytic process of the nanoparticles were determined using electron paramagnetic resonance (EPR). The covalent coupling between BP and tyrosine molecules was detected using triple quadrupole mass spectrometry (TPS). The effect of nanoparticles on the diffusion coefficient of Cy3-BSA was determined using fluorescence correlation spectroscopy (FCR).

[0056] Example 2: BSA labeling verification of the free photocatalytic probe Ce6

[0057] The labeling ability experiment was divided into two phases: illumination (660 nm laser, power density 200 mW / cm²) and illumination (660 nm laser, power density 200 mW / cm²). 2The experiment was divided into two groups: one with 5 minutes of illumination and the other without illumination. The three component parameters involved were Ce6 (20 μg / mL), BP (50 μg / mL), and BSA (0.5 mg / mL). The study of factors affecting labeling efficacy used a controlled variable approach, including four factors: the concentrations of Ce6 (5, 10, 15, 20 μg / mL) and BSA (0.25, 0.5, 1, 1.5 mg / mL), and the light intensity (50, 100, 200, 300 mW / cm²). 2 The parameters were set as follows: Ce6 concentration (10 μg / mL), BSA concentration (0.5 mg / mL), and light intensity (200 mW / cm²). When the factor was not an experimental condition, the parameters were fixed as follows: Ce6 concentration (10 μg / mL), BSA concentration (0.5 mg / mL), and light intensity (200 mW / cm²). 2 The illumination time was 10 seconds. The amount of biotin-labeled BSA in the samples obtained from both experiments was detected using Western blotting.

[0058] Example 3: Validation of BSA labeling in nanoparticles

[0059] The labeling ability experiment was divided into two phases: illumination (660 nm laser, power density 200 mW / cm²) and illumination (660 nm laser, power density 200 mW / cm²). 2 The experiment involved two main groups: one group with 5 minutes of illumination and the other without illumination. The three types of particles were NPs(-)@CeBP simultaneously coated with Ce6 and BP, NPs(-)@Ce6 coated with only Ce6, and NPs(-)@BP coated with only BP. The study of factors affecting labeling efficacy used a controlled variable approach, including four factors: BSA concentration (0.25, 0.5, 1, 1.5 mg / mL), light intensity (50, 100, 200, 300 mW / cm²), and light intensity. 2 The parameters were: BSA concentration (0.5 mg / mL) and time (0, 1, 5, 10, 20, 30, 60 seconds), and temperature (4, 10, 20, 30, 40, 50, 60℃). When the factor was not an experimental condition under investigation, the parameters were fixed as BSA concentration (0.5 mg / mL) and light intensity (200 mW / cm²). 2 The light exposure time was 10 seconds, and the ambient temperature was room temperature. The biotin-labeled BSA levels in the samples from both experiments were detected using Western blotting.

[0060] Example 4: Measurement of the distance of photocatalytic labeling of nanoparticles

[0061] Mix 270 μL of 30% acrylamide / methylene acrylamide (29:1), 250 μL of 1.5M Tris-HCl (pH 8.8), 460 μL of NPs(-)@CeBP, and 10 μL of AF488-BSA (1 mg / mL) thoroughly. Add 10 μL of 10% ammonium persulfate as a coagulant and 1 μL of TEMED as a coagulant accelerator. Allow to stand at room temperature to form a gel. Wash the gel three times with PBS and then perform photocatalytic labeling (660 nm laser, power density 200 mW / cm²). 2 After labeling (illumination time 10 seconds), the nanoparticles were washed three times with TBST and three times with PBS. Then, AF555-streptavidin was added to bind to the biotin tag on the protein. The nanoparticles were observed under a high-resolution fluorescence microscope. The results were deconvolved using Huygens software, and the closest distance from the nanoparticles to the protein was statistically analyzed.

[0062] Example 5: Morphology Detection of Protein Crowns Formed on the Surface of Nanoparticles

[0063] Purchased mouse plasma was removed from a -80°C freezer and slowly thawed on ice. After complete thawing, protein concentration was determined using a BCA kit, and the mouse plasma was diluted to 2 mg / mL with PBS buffer. The prepared nanoparticles were mixed with mouse plasma and incubated for 5, 10, 30, 60, 300, and 600 seconds. The mixture was then transferred to a protein hypoadsorption centrifuge tube containing 0.7 M sucrose solution and centrifuged at 14,000 rpm for 20 minutes at 4°C. The supernatant was discarded, and the resulting nanoparticles and protein crowns were washed with PBS and transferred to a new protein hypoadsorption centrifuge tube. This process was repeated three times. Finally, the obtained protein crowns were redispersed with deionized water and their morphology was observed using a transmission electron microscope.

[0064] Example 6: Preparation and Identification of Photocatalytic Proximity Labeled Biotinylated Plasma Proteins

[0065] After incubating the prepared nanoparticles and plasma proteins for different times, the proteins adsorbed on the surface of the nanoparticles were photocatalytically labeled. The method involved removing purchased mouse plasma from a -80°C freezer and slowly thawing it on ice. After complete thawing, the protein concentration was determined using a BCA kit, and the mouse plasma was diluted to 2 mg / mL with PBS buffer. The prepared nanoparticles and mouse plasma were mixed and incubated for 5, 10, 20, 30, 60, 300, 600, 1200, and 1800 seconds. The resulting samples, without treatment with streptavidin-coated magnetic beads, were analyzed using Western blotting to detect the biotinylated protein crown. The method involved thoroughly separating the samples using SDS-PAGE and completely transferring them to a PVDF membrane. The membrane was blocked with sufficient blocking buffer at 37°C for 0.5 hours, and then slowly shaken overnight in a horseradish peroxidase-streptavidin solution at 4°C. The PVDF membrane was washed six times with TBST solution for 5 minutes each time. Horseradish peroxidase chemiluminescent substrate was dropped onto a PVDF membrane and placed under a gel imaging system to detect the amount of biotin-labeled protein.

[0066] The obtained samples were extracted and enriched using streptavidin-coated magnetic beads, separated by SDS-PAGE, and the amount of biotin-labeled protein was detected by silver staining using a kit. The method was as follows: After thorough separation by SDS-PAGE, the gel was placed in approximately 100 ml of fixative (50 ml ethanol, 10 ml acetic acid, and 40 ml Milli-Q purified water) and shaken at room temperature for 40 minutes at 60 rpm. The fixative was discarded, and 100 ml of 30% ethanol was added. The mixture was shaken at room temperature for 10 minutes at 60 rpm. The 30% ethanol was discarded, and 200 ml of Milli-Q purified water was added. The mixture was shaken at room temperature for 10 minutes at 60 rpm. The water was discarded, and 100 ml of silver staining sensitization solution (1X) was added. The mixture was shaken at room temperature for 2 minutes at 60 rpm. Discard the original solution, add 200 ml of Milli-Q grade pure water, and shake on a shaker at room temperature for 1 minute at 60 rpm. Wash twice. Discard the water, add 100 ml of silver solution (1X), and shake on a shaker at room temperature for 10 minutes at 60 rpm. Discard the original solution, add 100 ml of Milli-Q grade pure water, and shake on a shaker at room temperature for 1 minute at 60 rpm. Discard the water, add 100 ml of silver staining developer, and shake on a shaker at room temperature for 10 minutes at 60 rpm. Discard the silver staining developer, add 100 ml of silver staining stop solution (1X), and shake on a shaker at room temperature for 10 minutes at 60 rpm. Discard the silver staining stop solution, add 100 ml of Milli-Q grade pure water, and shake on a shaker at room temperature for 5 minutes at 60 rpm. Store in Milli-Q grade pure water. Detect the amount of biotin-labeled protein using a gel imaging system.

[0067] The obtained samples were extracted and enriched using streptavidin-coated magnetic beads, separated by SDS-PAGE, and quantitatively detected by liquid chromatography-mass spectrometry (LC-MS) to identify the types and molar amounts of plasma proteins interacting with nanoparticles at different incubation times. The method was as follows: Samples were thoroughly separated by SDS-PAGE, stained with Coomassie Brilliant Blue, and protein bands were excised and placed in protein-low adsorption centrifuge tubes. The gel was decolorized with 50 mM NH4HCO3:acetonitrile (v / v) at 37 °C, washed twice with acetonitrile, and dried. 5 mM DTT was added and incubated at 45 °C for 30 min, washed twice with acetonitrile, and dried. 11 mM IAM was added and incubated for 20 min, washed twice with acetonitrile, and dried. A precisely weighed amount of trypsin was added and incubated at 4 °C for 1 hour, then incubated at 37 °C overnight. 10% trifluoroacetic acid was added. Peptides were extracted twice with 50% acetonitrile:0.1% trifluoroacetic acid (v / v) and then lyophilized. Lyophilized samples were redissolved in 0.1% trifluoroacetic acid solution. The resulting samples were separated by liquid chromatography and quantitatively detected by mass spectrometry. Results were searched using Proteome Discovery software and the UniProt database. All samples were performed in triplicate or at least repeatedly. Trypsin was used as an internal standard protein. The intensity of all proteins in the sample was determined by the average intensity of their top three most ionized peptides. Since the precise molar amount of trypsin was known, the approximate absolute molar amounts of all other detected proteins could be calculated.

[0068] Example 7: Preparation and Identification of Biotinylated Plasma Proteins Obtained by Centrifugation

[0069] Purchased mouse plasma was removed from a -80°C freezer and slowly thawed on ice. After complete thawing, protein concentration was determined using a BCA kit, and the mouse plasma was diluted to 2 mg / mL with PBS buffer. The prepared nanoparticles were mixed with mouse plasma and incubated for 5, 10, 30, 60, 300, and 600 seconds. The mixture was then transferred to a protein hypoadsorption centrifuge tube containing 0.7 M sucrose solution and centrifuged at 14,000 rpm for 20 minutes at 4°C for 20 minutes at 5300 g. The supernatant was discarded, and the resulting nanoparticles and protein crowns were washed with PBS and transferred to a new protein hypoadsorption centrifuge tube. The mixture was then centrifuged at 14,000 rpm for 20 minutes at 4°C for 20 minutes. This process was repeated three times. The resulting samples were separated using SDS-PAGE and the protein crowns were detected using a silver staining kit, following the same method as the relevant experimental section in Example 6. Alternatively, the resulting samples were separated using SDS-PAGE and quantitatively analyzed using liquid chromatography-mass spectrometry (LC-MS) to identify the types and molar amounts of plasma proteins interacting with the nanoparticles at different incubation times, following the same method as the relevant experimental section in Example 6.

[0070] Example 8: Detection of the interaction between nanoparticles and J774A.1 cells based on photocatalytic proximity labeling technology

[0071] Before use, prepare 9×10 6 J774A.1 cells were incubated for 30 minutes in complete medium containing 500 μM BP at 37°C. Afterwards, the culture medium was aspirated, and 9 × 10⁶ cells were cultured. 6 J774A.1 cells were washed with PBS and dispersed in nine centrifuge tubes for later use. Purchased mouse plasma was removed from a -80°C freezer and slowly thawed on ice. After complete thawing, protein concentration was determined using a BCA kit, and the mouse plasma was diluted to 2 mg / mL with PBS buffer. The prepared NPs(-)@Ce6 and NPs(+)@Ce6 nanoparticles were mixed with plasma proteins and incubated for 5, 10, 30, 60, 300, and 600 seconds. Then, BP-rich J774A.1 cells were added and rapidly mixed for 5 seconds, followed by photocatalytic labeling (660 nm laser, power density 200 mW / cm²). 2 (Illumination time: 10 seconds). After labeling, cells were collected, fixed with 4% paraformaldehyde at room temperature for 20 minutes, stained with AF488-streptavidin at 37°C for 1 hour, and stained with TRITC-phalloidin at 37°C for 0.5 hours. The stained cells were observed using a confocal laser scanning microscope. Attached Figure Description

[0072] Figure 1 The results showed that 660 nm laser irradiation of the photocatalytic probe Ce6 induced a large amount of BSA protein to be biotin-tagged. The absence of any component in the system resulted in the inactivation of the BP-tagged protein.

[0073] Figure 2 The labeling efficacy of BP was shown to be positively correlated with the concentrations of Ce6 and BSA, light intensity, and time.

[0074] Figure 3 The results showed that when BP and Ce6 coexist in solution, mass spectrometry analysis confirmed that laser irradiation can cause covalent coupling between BP and tyrosine molecules.

[0075] Figure 4 A shows that the surfaces of the two prepared nanoparticles (NPs(+)@CeBP and NPs(-)@CeBP) are positively and negatively charged, respectively. Figure 4 B shows that the two types of nanoparticles prepared have the same size distribution.

[0076] Figure 5 The two types of nanoparticles prepared were shown to have core-shell structure characteristics.

[0077] Figure 6 The results show that hydroxyl radicals and alkane radicals can be detected simultaneously when the nanoparticles prepared are irradiated with a 660 nm laser. * indicates the characteristic peak of hydroxyl radicals, ※ indicates the characteristic peak of alkane radicals.

[0078] Figure 7 The study showed that only the NPs(-)@CeBP nanoparticle group under 660 nm laser light exhibited significant protein biotinylation modification.

[0079] Figure 8 The labeling efficiency of the prepared nanoparticles was shown to be positively correlated with the concentration of BSA, light intensity and time, and exhibited a peak-shaped change with temperature.

[0080] Figure 9 The labeling distances detected using STED-based high-resolution fluorescence imaging are shown. Red dots represent the locations of nanoparticles, and yellow dots represent the locations of biotinylated proteins. Scale bars are 100 nm. Compared to the distance statistics in the unilluminated group due to the free diffusion of BP, the photocatalytic labeling group induced more biotinylation labeling of BSA molecules within 60 nm of the nanoparticles. The average labeling distance was 29 nm.

[0081] Figure 10 The images show the protein crowns that formed on the surface of the prepared nanoparticles after they were mixed with plasma for different durations. Scale bars are all 50 nm.

[0082] Figure 11 A and B show the Western blotting results of in situ biotin labeling of proteins adsorbed on the surface of the nanoparticles using the method disclosed in this invention after the prepared nanoparticles were mixed with plasma for different times. Figure 11 C and D are correct. Figure 11 Gray-scale analysis results of A and B.

[0083] Figure 12 The results show the SDS-PAGE of the nanoparticles after they were mixed with plasma for different times, the in situ biotin labeling of the proteins adsorbed on the surface of the nanoparticles using the method disclosed in this invention, and the extraction using streptavidin-coated magnetic beads.

[0084] Figure 13 The results of SDS-PAGE extraction of adsorbed proteins on the surface of nanoparticles were shown after the prepared nanoparticles were mixed with plasma for different times, using centrifugation.

[0085] Figure 14 The variation trend of the BSA diffusion coefficient when the prepared nanoparticles are mixed with Cy3-BSA for different times is shown.

[0086] Figure 15 The diagram shows the number of protein types identified at different times when two types of nanoparticles interacted with plasma proteins using the photocatalytic nanoparticle-protein interaction detection technology disclosed in this invention.

[0087] Figure 16The study shows the trends in similar protein changes detected by liquid chromatography-mass spectrometry and Western blotting.

[0088] Figure 17 This paper demonstrates the use of the photocatalytic nanoparticle-protein interaction detection technique disclosed in this invention to determine the interaction strength between the prepared nanoparticles and cells. Scale bars are all in 20 micrometers. 0.0001 < ***p < 0.001, *****p < 0.00001.

Claims

1. A method for isolating proteins that interact with nanoparticles, comprising, in combination: The method steps are as follows: 1) preparing nanoparticles containing photocatalytic probes and labeled reaction substrates, or preparing nanoparticles containing photocatalytic probes and adding labeled reaction substrates to the sample containing proteins; 2) contacting the nanoparticles of step 1 with the sample containing proteins to form an interaction between the nanoparticles and the proteins; activating the labeled reaction substrate biotin phenol in the nanoparticles by laser irradiation at the corresponding wavelength of the photocatalytic probe chlorin e6 to obtain labeled proteins interacting with the nanoparticles; 3) separating the labeled proteins interacting with the nanoparticles using streptavidin-coated magnetic beads.

2. The method of claim 1, wherein, Wherein, The nanoparticles include liposomes, solid lipid nanoparticles, polymer nanoparticles, nanomicelles, nanocrystal particles, albumin nanoparticles, ferritin nanoparticles, and fat emulsion; The sample containing proteins includes blood, urine, cerebrospinal fluid, synovial fluid, tears, saliva, milk, vaginal fluid, nasal fluid, ear fluid, gastric juice, pancreatic juice, trabecular fluid, sweat, semen, prostatic fluid, sputum, fecal matter, bronchial lavage, tissue homogenate, lymph, cell culture sample, or any combination thereof.

3. The method of claim 1, wherein, The sample containing proteins is blood.

4. The method of claim 1, wherein, The method of step 1) is as follows: The water phase containing 0.1 μM-0.1 mM photocatalytic probe chlorin e6 is added dropwise to 1 mL-1 L oil phase and stirred for 15-60 minutes to mix, the water phase containing 10 μM-10 mM CaCl2 is added dropwise to 1 mL-1 L oil phase and stirred for 15-60 minutes to mix, then the mixed solution containing photocatalytic probe chlorin e6 is added dropwise to the mixed solution containing CaCl2 and stirred for 15-60 minutes, the water phase containing 1 μM-1 mM Na2HPO4 and the chloroform solution containing 1 μM-1 mM DOPA are added dropwise to 2 mL-2 L oil phase and stirred for 15-60 minutes to mix, the mixed solution containing Na2HPO4 and DOPA is added dropwise to the solution containing chlorin e6 and CaCl2, stirred for 0.5-4 hours to form particles, then 8 mL-8 L isopropyl alcohol is added for solidification, the particles are collected by centrifugation, the supernatant is discarded, the particles are washed with isopropyl alcohol and collected by centrifugation, and the washing is repeated three times, and the obtained particles are redispersed in chloroform for standby; Wherein, the oil phase is prepared by mixing 1.5 M n-hexane and 0.6 M Triton X-100 in cyclohexane; Preparation of NPs(-)@CeBP: 1.1 μM-1.1 mM DOPC, 27.4 nM-27.4 μM DSPE-PEG2000, 289.7 nM-289.7 μM cholesterol, and 275.1 nM-275.1 μM biotin phenol are dissolved in an organic solvent in a certain proportion, then mixed with the particles dispersed in chloroform, vacuum rotary evaporation to form a thin film, hydration of the thin film at 60°C, and dispersion by ultrasonic wave with a power of 70 W-120 W for 10-30 minutes to obtain NPs(-)@CeBP nanoparticles; Preparation of NPs(+)@CeBP: 992.3 nM-992.3 μM DOPC, 24.7 nM-24.7 μM DSPE-PEG2000, 131.9 nM-131.9 μM DOTAP, 260.7 nM-260.7 μM Cholesterol and 275.1 nM-275.1 μM Biotin phenol were dissolved in organic solvent in proportion, mixed with particles dispersed in chloroform, and a thin film was formed by rotary evaporation under reduced pressure. The thin film was hydrated at 60°C and dispersed by ultrasonic wave of 70W-120W for 10-30 minutes to obtain NPs(+)@CeBP nanoparticles.

5. The method of claim 4, wherein, The method of step 2) is as follows: the sample containing the protein is not diluted or diluted with PBS buffer to 0.1-20 mg / mL, the prepared nanoparticles NPs(-)@CeBP or NPs(+)@CeBP are mixed with the sample containing the protein and incubated for 1 second-1 hour, then the mixed solution is irradiated with a specific wavelength laser for 1 second-1 hour, and the labeling reaction stops after stopping the irradiation, After the sample is fully separated by SDS-PAGE and completely transferred to the PVDF membrane, a sufficient amount of blocking solution is used to block at 37°C for 0.5-1 hour, the PVDF membrane is slowly shaken in horseradish peroxidase-streptavidin solution at 4°C overnight, the PVDF membrane is washed with TBST solution six times, each for 5 minutes, the horseradish peroxidase chemiluminescence substrate is dropped on the PVDF membrane, and the amount of biotin-labeled protein is detected under the gel imaging instrument, Preparation of TBST solution: 10 mM Tris-HCl, 150 mM NaCl, 1‰ Tween-20 with pH value of 7.

5.

6. The method of claim 4, wherein, The method of step 3) is as follows: The sample is placed in a 10K ultrafiltration tube and centrifuged at 14000 rpm for 30 minutes, resuspended with PBS equal to the volume of the sample, and then centrifuged at 14000 rpm for 30 minutes. This washing is repeated three times, and finally the sample is resuspended with an equal volume of PBS and transferred to a protein low adsorption centrifuge tube. The excess streptavidin-coated magnetic beads are washed three times with PBS, resuspended with PBS, and added to the sample. The two are mixed at 4°C overnight or at room temperature for 2 hours. After the mixing is completed, the centrifuge tube is placed on the magnetic rack, and the magnetic beads are enriched on the inner wall of the centrifuge tube. The supernatant is discarded, and the magnetic beads are washed with PBS for 3-6 times, washed with the washing solution for 6 times, and washed with PBS for 3-6 times. The magnetic beads are boiled in protein loading buffer containing 2 mM biotin for 10 minutes to obtain the product. Preparation of washing solution: 50 mM Tris-HCl, 150 mM Nacl, and 1% Triton-X100 with pH value of 7.

5.

7. The method of claim 4, wherein, The sample obtained after the three steps is separated by SDS-PAGE and the amount of biotin-labeled protein is detected by silver staining kit, and the method is as follows: After the sample is fully separated by SDS-PAGE, the gel is placed in about 100 ml of fixative and shaken on a shaker at room temperature for 40 minutes to overnight at a shaking speed of 60-70 rpm. The fixative is discarded, 100 ml of 30% ethanol is added, and shaken on a shaker at room temperature for 10 minutes at a shaking speed of 60-70 rpm. The 30% ethanol is discarded, 200 ml of Milli-Q grade water is added, and shaken on a shaker at room temperature for 10-60 minutes at a shaking speed of 60-70 rpm. The water is discarded, 100 ml of silver stain sensitizing solution, IX, is added, and shaken on a shaker at room temperature for 2 minutes at a shaking speed of 60-70 rpm. The original solution is discarded, 200 ml of Milli-Q grade water is added, and shaken on a shaker at room temperature for 1 minute at a shaking speed of 60-70 rpm. The water is discarded, 100 ml of silver solution, IX, is added, and shaken on a shaker at room temperature for 10 minutes at a shaking speed of 60-70 rpm. The original solution is discarded, 100 ml of Milli-Q grade water is added, and shaken on a shaker at room temperature for 1-1.5 minutes at a shaking speed of 60-70 rpm. The water is discarded, 100 ml of silver stain developing solution is added, and shaken on a shaker at room temperature for 3-10 minutes until a relatively ideal expected protein band appears at a shaking speed of 60-70 rpm. The silver stain developing solution is discarded, 100 ml of silver stain stop solution, IX, is added, and shaken on a shaker at room temperature for 10 minutes at a shaking speed of 60-70 rpm. The silver stain stop solution is discarded, 100 ml of Milli-Q grade water is added, and shaken on a shaker at room temperature for 2-5 minutes at a shaking speed of 60-70 rpm. It is stored in Milli-Q grade water and placed under a gel imager to detect the amount of biotin-labeled protein, The fixative is composed of 50 ml of ethanol, 10 ml of acetic acid, and 40 ml of Milli-Q grade water. The shaking speed of 60-70 rpm is the number of revolutions per minute.

8. The method of claim 4, wherein, The samples obtained after the three steps are separated by SDS-PAGE and quantitatively detected by liquid chromatography for identifying the types and molar amounts of plasma proteins interacting with nanoparticles at different incubation times, as follows: Samples were fully resolved by SDS-PAGE, after staining by Coomassie blue, protein bands were excised and placed in protein low adsorption microfuge tubes, gel was destained by 50 mM NH4HCO3:acetonitrile 1:1 by volume at 37°C, washed twice by acetonitrile and dried, 5 mM DTT was added and incubated at 45°C for 30 minutes, washed twice by acetonitrile and dried, 11 mM IAM was added and incubated for 20 minutes, washed twice by acetonitrile and dried, enough trypsin was added with accurate weight and placed at 4°C for 1 hour, then placed at 37°C overnight, 10% trifluoroacetic acid was added, peptides were extracted twice by 50% acetonitrile:0.1% trifluoroacetic acid 1:1 by volume, then lyophilized, lyophilized samples were re-dissolved by 0.1% trifluoroacetic acid solution, the obtained samples were separated by liquid phase and quantitatively detected by mass spectrometry, the results were searched by Proteome Discovery software and UniProt database, all samples were independently repeated for three times or more, trypsin was used as an internal standard protein, the average intensity of the first three optimal ionization peptides of all proteins in the sample was used as the intensity of the protein, the accurate molar amount of trypsin was known, so the approximate absolute molar amount of other detected proteins could be calculated.

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