An in situ method for identification and quantification of components of a multilayer protein corona on a nanoparticle

Abstract

The present application relates to the technical field of analytical detection, and particularly relates to a method for in-situ identification and quantification of components of a multilayer protein corona on the surface of nanoparticles. The method comprises: fixing the nanoparticles to be detected on the surface of a biosensor; incubating the nanoparticles to be detected fixed on the surface of the biosensor in a biological sample solution; monitoring the formation of the protein corona on the surface of the nanoparticles by using a biological membrane interference technology; after the formation of the protein corona, eluting the soft protein corona and the hard protein corona on the surface of the nanoparticles with an eluent; and identifying the components of the protein corona by using a mass spectrometry technology. The method can realize controllable detection and identification of the multilayer protein corona formed on the surface of the nanoparticles in time and space, and has a wide application prospect in real-time dynamic and accurate analysis of the soft protein corona, and provides an effective method for further exploring the interaction between the nanoparticles and the organisms and the rational design of functional nanodrugs.

Description

Technical Field

[0001] This invention relates to the field of analytical detection technology, specifically to an in-situ method for the identification and quantification of multilayer protein crown components on the surface of nanoparticles. Background Technology

[0002] In recent years, nanoparticles have shown promising applications in the biomedical field due to their unique physicochemical properties. Once nanoparticles enter a living organism, they inevitably come into contact with various bodily fluids. During this process, different types of proteins adsorb onto the surface of the nanoparticles, forming "protein crowns." These protein crowns may alter the original surface properties of the nanoparticles, thereby affecting their subsequent interaction with the organism. The formation of protein crowns may influence the blood circulation time, targeting, biodistribution, immune response, and toxicity of nanomedicines. Therefore, research on the composition, structure, and function of protein crowns is crucial for a deeper and more comprehensive understanding of the biological processes involved in nanoparticles / nanomedicines and their resulting biological effects.

[0003] Proteins with high affinity and slow dissociation rates form "hard protein crowns" on the surface of nanoparticles, while proteins with low affinity and fast dissociation rates form "soft protein crowns". Studies have found that different protein components forming scleroprotein coronas play different roles in cellular uptake of nanoparticles and the inflammatory response induced by nanoparticles (D. Baimanov, J. G. Wu, R. R. Chu, R. Cai, B. Wang, M. J. Cao, Y. Tao, J. M. Liu, MY. Guo, J. Wang, X. Yuan, CD. Ji, Y. L. Zhao, W. Y. Feng*, L. L. Wang*, CY. Chen*. Immunological responses induced by blood protein coronas on two-dimensional MoS2 nanosheets. ACS Nano, 2020, 14(5), 5529-5542.; R. Cai, J. R. Ren, Y. L. Ji, Y. L. Wang, Y. Liu, Z. Q. Chen, Z. F. Sabet, X. C. Wu, I. Lynch, CY. Chen*. Corona of thorns: the surface chemistry-mediated protein corona perturbs the recognition and immune response of macrophages. ACS Appl.Mater.Interfaces,2020,12(2),1997-2008.).Studies have found that the loss of targeting ability of various targeted nanomedicines applied in vivo is also due to the formation and interference of multilayer protein corona on the surface (R. Gaspar*. Nanoparticles pushed off target with proteins. Nat. Nanotechnol., 2013, 8(2), 79-80.; A. Salvati, ASPitek, MP Monopoli, K. Prapainop, FBBombelli, D. D. Hristov, PM Kelly, C. Aberg, E. Mahon*, K. A. Awson*. Transferrin-functionalized nanoparticles lose their targeting capabilities when a biomolecule corona adsorbs on the surface. Nat. Nanotechnol., 2013, 8(2), 137-143.).

[0004] Current methods for studying the protein crown components on the surface of nanoparticles mainly rely on centrifugation. Specifically, nanoparticles are incubated with a biological liquid to form stable protein crowns on their surface. Then, the nanoparticle-protein complex is separated from the biological liquid by centrifugation, yielding a precipitate of the nanoparticle-protein complex. The nanoparticles are washed twice. A 5% sodium dodecyl sulfate aqueous solution is added, and the mixture is heated at 95°C for 15 minutes to denature and peel off the proteins on the nanoparticle surface. The nanoparticles are then separated again by centrifugation, and the protein crown components are collected. Finally, mass spectrometry is used for detection. This centrifugation method, due to the need for repeated high-speed centrifugation and continuous elution, typically retains the hard protein crowns on the nanoparticle surface and cannot obtain the loosely attached soft protein crowns. Furthermore, for very small, low-density nanoparticles, it is difficult to separate and elute them using centrifugation. In addition, centrifugation is time-consuming (typically from several minutes to several hours). Currently, there is no universally applicable method for detecting soft protein crown components. Therefore, there is an urgent need to develop new, rapid, in-situ, and real-time methods to accurately analyze the composition of multilayer protein crowns on the surface of nanoparticles. This would enable more realistic and direct detection of the formation, composition, and changes of protein crowns, providing effective methods for further exploring the interaction between nanoparticles and organisms, as well as the rational design of functional nanomedicines. Summary of the Invention

[0005] One of the objectives of this invention is to provide a method for detecting the interaction between nanoparticles and proteins.

[0006] Another objective of this invention is to provide an in-situ method for the identification and quantification of multilayer protein crown components on the surface of nanoparticles.

[0007] Specifically, the present invention provides the following technical solutions:

[0008] In a first aspect, the present invention provides a method for detecting the interaction between nanoparticles and proteins, the method comprising: immobilizing the nanoparticles to be tested on the surface of a biosensor, incubating the nanoparticles immobilized on the surface of the biosensor in a solution of a protein to be tested, and detecting the binding of the nanoparticles and proteins using biomembrane interferometry.

[0009] Preferably, the fixation method involves first modifying the surface of the biosensor with immunoglobulins, then sealing the surface of the immunoglobulin-modified biosensor with a blocking solution, and finally co-incubating it with nanoparticles.

[0010] This invention does not impose any particular restrictions on the types of biosensors. All biosensors capable of being used for biomembrane interferometry detection can be used, including commercially available or self-made aminopropylsilane sensors (APS sensors), second-generation amino-coupled sensors (AR2G sensors), streptavidin sensors (SA sensors), super streptavidin sensors (SSA sensors), Protein A sensors (ProA sensors), NTA sensors, etc.

[0011] In some embodiments of the present invention, the surface of the biosensor contains carboxyl groups. The surface is first activated with EDC / NHS, then immunoglobulins are fixed by amino-carboxyl group reaction, and then nanoparticles are adsorbed by immunoglobulins, thereby fixing the nanoparticles on the surface of the biosensor.

[0012] The present invention does not have any particular limitations on the types of nanoparticles to be tested, including inorganic nanomaterials (carbon nanomaterials and carbon nanomaterial derivatives doped with other elements, silicon dioxide nanomaterials), metal and metal oxide nanomaterials, metal sulfide nanomaterials, organic nanomaterials (organic polymers, liposomes, exosomes, viral vectors), organic-inorganic hybrid composite nanomaterials, and various nanomedicines, etc.

[0013] In the above method, the immunoglobulins include IgG, IgA, IgM, IgD, and IgE.

[0014] In some embodiments of the present invention, the immunoglobulin is IgG, IgM or IgA.

[0015] In the above method, in order to reduce the non-specific adsorption of proteins and immunoglobulins on the biosensor without affecting the interaction between nanoparticles and proteins, the biosensor needs to be pre-blocked with a blocking solution before immobilizing the nanoparticles and contacting them with the protein solution to be tested.

[0016] Preferably, the blocking solution is a phosphate buffer comprising the following components: 0.03-0.08% Tween-20 and 0.03-0.08% polyethylene glycol. The pH of the blocking solution is preferably 7.2-7.5, more preferably 7.4-7.5. Using the above blocking solution can effectively reduce the non-specific adsorption of proteins to the biosensor without adversely affecting the interaction between nanoparticles and proteins.

[0017] In the above-mentioned test protein solution, the concentration of the test protein is preferably 0.1-10 mg / mL, and the pH is 0.5-9.

[0018] In the above method, it is preferable to prepare the test protein into different concentration gradients (usually at least 4 concentrations) as analytes to detect the affinity between nanoparticles and test proteins.

[0019] In the above methods, the detection using biomembrane interferometry can be achieved using a biomembrane interferometer analyzer.

[0020] Secondly, this invention develops a method for detecting multilayer protein crown components on the surface of nanoparticles based on biomembrane interferometry. Compared with existing detection methods, the biomembrane interferometry-based "fishing" detection method of this invention can achieve real-time dynamic monitoring of the binding of nanoparticles to proteins and the formation, exchange, and dissociation of protein crowns on the nanoparticle surface, thereby enabling rapid, real-time, and in-situ detection of nanoparticle-protein interactions and protein crowns on the nanoparticle surface. When detecting protein crown components, this method supports in-situ separation of multilayer protein crown components and can detect their dynamic changes over time. It can detect both hard and soft protein crowns with high accuracy, breaking through the current technical bottleneck for the detection of soft protein crown components.

[0021] Specifically, the present invention provides an in-situ identification and quantification method for multilayer protein crown components on the surface of nanoparticles. The method includes: immobilizing the nanoparticles to be tested on the surface of a biosensor; placing the nanoparticles immobilized on the surface of the biosensor in a biological sample solution for incubation; monitoring the formation of protein crowns on the surface of the nanoparticles using biomembrane interferometry; after the protein crowns have formed, eluting the soft and hard protein crowns on the surface of the nanoparticles with an elution buffer; and identifying the protein crown components using mass spectrometry.

[0022] The flowchart of the above method is as follows: Figure 1 As shown.

[0023] In the above method, the fixation method involves first modifying the surface of the biosensor with immunoglobulins, then sealing the surface of the immunoglobulin-modified biosensor with a blocking solution, and then co-incubating it with nanoparticles.

[0024] To better integrate nanoparticles with biosensors and prevent them from being eluted during protein elution, this invention explores and optimizes methods for immobilizing nanoparticles on biosensors. This invention discovers that, unlike proteins and small molecule compounds, nanoparticles cannot directly bind to biosensors, and stable binding is difficult to achieve even with common modifications. Therefore, it is challenging to stably bind nanoparticles to the biosensor surface and prevent them from being affected by protein elution. Through extensive experimentation, this invention unexpectedly discovered that using immunoglobulins (such as IgG, IgM, and IgA) as bridging molecules, through chemical coupling, allows for stable binding to the biosensor surface. These immunoglobulins also adsorb nanoparticles with high affinity, resulting in very strong and stable immobilization of nanoparticles on the biosensor surface. During protein elution, the nanoparticles remain intact on the biosensor, enabling repeated detection in multiple cycles as needed.

[0025] In the above method, the immunoglobulin is preferably IgG, IgM or IgA.

[0026] Biological samples derived from living organisms, their lysates, or physiological environment simulation solutions have highly complex compositions. Proteins and other components of the biological samples being tested can easily undergo non-specific adsorption with the biosensor itself, interfering with protein binding and detection. To reduce non-specific adsorption without affecting the interaction between nanoparticles and the protein solution, this invention discovers that pre-blocking the immunoglobulin-modified biosensor in a blocking solution before contacting it with nanoparticles and the biological sample solution can significantly reduce non-specific adsorption and achieve accurate detection of the interaction between proteins and nanoparticles in complex biological samples.

[0027] This invention explores the composition of the blocking solution. Preferably, the blocking solution is a phosphate buffer containing the following components: 0.03-0.08% Tween-20 and 0.03-0.08% polyethylene glycol. The pH of the blocking solution is preferably 7.2-7.5. Using the above blocking solution can effectively reduce the non-specific adsorption of proteins to biosensors without adversely affecting the interaction between nanoparticles and proteins.

[0028] The sealing process is preferably performed for 30-120 seconds.

[0029] During the co-incubation of nanoparticles with immunoglobulin-modified biosensors, the nanoparticles and immunoglobulins bind efficiently and are thus immobilized on the surface of the biosensors.

[0030] This invention does not impose any particular restrictions on the types of biosensors. All biosensors capable of being used for biomembrane interferometry detection can be used, including commercially available or self-made aminopropylsilane sensors (APS sensors), second-generation amino-coupled sensors (AR2G sensors), streptavidin sensors (SA sensors), super streptavidin sensors (SSA sensors), Protein A sensors (ProA sensors), NTA sensors, etc.

[0031] In some embodiments of the present invention, the surface of the biosensor contains carboxyl groups. The surface of the biosensor is first activated with EDC and NHS, then immunoglobulins are fixed by amino-carboxyl group reaction, and then nanoparticles are adsorbed by immunoglobulins to fix the nanoparticles on the surface of the biosensor.

[0032] The present invention does not have any particular limitations on the types of nanoparticles to be tested, including inorganic nanomaterials (carbon nanomaterials and carbon nanomaterial derivatives doped with other elements, silicon dioxide nanomaterials), metal and metal oxide nanomaterials, metal sulfide nanomaterials, organic nanomaterials (organic polymers, liposomes, exosomes, viral vectors), organic-inorganic hybrid composite nanomaterials, and various nanomedicines, etc.

[0033] In some embodiments of the present invention, the method for immobilizing nanoparticles on the surface of a biosensor is as follows:

[0034] (1) Modification of the biosensor surface with immunoglobulin: The biosensor containing carboxyl groups on its surface was pre-wetted for 10-15 minutes, and then activated for 250-350 seconds by mixing 80-120 mM 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and 40-60 mM N-hydroxysuccinimide (NHS) in a 1:1 ratio; the immunoglobulin was diluted with 8-12 mM sodium acetate solution at pH 3.8-4.2 to obtain an immunoglobulin solution with a concentration of 40-60 μg / mL; the biosensor was immersed in the immunoglobulin solution for 250-350 seconds; and then blocked with 0.8-1.2 M ethanolamine aqueous solution at pH 8-8.5 for 250-350 seconds.

[0035] (2) The surface of the immunoglobulin-modified biosensor obtained in step (1) is blocked with a blocking solution; the blocking solution is a phosphate buffer containing the following components: 0.03-0.08% Tween-20, 0.03-0.08% polyethylene glycol, pH 7.2-7.5; the blocking time is 30-120 seconds;

[0036] (3) Immobilize nanoparticles on the surface of the biosensor: Immerse the biosensor coated with immunoglobulin and blocked in step (2) into an aqueous solution of nanoparticles and incubate for 250-350 seconds to immobilize the nanoparticles on the surface of the biosensor.

[0037] Furthermore, to better elute the hard and soft protein crowns in a gradient manner, i.e., to elute the multi-layered protein crown components sequentially from the outside to the inside, this invention has explored the composition and concentration of the eluent used to elute the protein crowns. Preferably, the eluent is an aqueous solution containing one or more of the following components: sodium dodecyl sulfate, sodium hydroxide, trifluoroacetic acid, formic acid, acetic acid, hydrochloric acid, sodium chloride, and magnesium chloride.

[0038] Preferably, the eluent used to elute the soft protein crown is an aqueous solution containing the following components: 0.001-0.05% trifluoroacetic acid aqueous solution.

[0039] The eluent used to elute scleroprotein crowns is an aqueous solution containing the following components: 0.1-1% aqueous trifluoroacetic acid.

[0040] This invention discovers that by using the different eluents described above, the outer soft protein crown and the inner hard protein crown of nanoparticles can be eluted simply and efficiently, achieving a relatively accurate distinction and identification of the soft protein crown and hard protein crown components.

[0041] During the elution process, the eluted protein solution is collected for subsequent mass spectrometry identification of protein components.

[0042] For the biological sample solution to be tested, the concentration of total protein is preferably 0.1-10 mg / mL, and the pH is 0.5-9.

[0043] Controlling the total protein concentration in the biological sample solution within the above range is beneficial for further reducing non-specific adsorption.

[0044] The biological samples described above can be bodily fluids or their processed solutions derived directly from organisms, or physiological environment simulation solutions. Preferably, the biological samples include cell lysis buffers, lysosomal lysis buffers, mitochondrial lysis buffers, tissue lysis buffers, and various bodily fluids including serum, plasma, urine, cerebrospinal fluid, saliva, lymph, gastric simulation solution, intestinal simulation solution, etc.

[0045] In the above method, the incubation time for the biosensor with immobilized nanoparticles and the biological sample solution is monitored in real time using biomembrane interferometry to dynamically monitor the formation of the protein crown, and the incubation time is determined based on the monitoring results.

[0046] Typically, incubation time can range from 1 second to 7 days. Considering the protein crown formation time and protein stability, the preferred incubation time is 1 second to 48 hours.

[0047] In the above method, the incubation temperature can be adjusted from 6 to 40°C. Considering the consistency with human body temperature, an incubation temperature of 37°C is preferred.

[0048] During incubation, in order to ensure that the biological sample and the nanoparticles are in full contact, the sample plate can be set to vibration mode with a vibration speed adjustable from 0 to 5000 rpm, preferably 500 to 2000 rpm.

[0049] To further improve detection accuracy, an unfixed nanoparticle biosensor was incubated with the target biological sample as a blank control. In the above method, the incubation and protein crown elution steps can be considered as a cycle. To collect enough protein for mass spectrometry detection, this cycle can be repeated 1-100 times. Considering the sensor's reusability and time efficiency, a cycle count of 1-30 times is preferred.

[0050] Of the methods described above, LC-MS / MS is preferred for mass spectrometry identification of the protein corona components. Before mass spectrometry identification, the collected protein corona elution solution usually needs to be concentrated and the solution replaced. Concentration and solution replacement methods include, but are not limited to, using ultrafiltration tubes, desalting columns, freeze-drying, and polyacrylamide gel electrophoresis (SDS-PAGE).

[0051] In some embodiments of the present invention, the protein crown solution is replaced with ultrapure water for subsequent mass spectrometry identification.

[0052] Preferably, the in-situ identification and quantification method for the multilayer protein crown components on the surface of nanoparticles described above further includes: after identifying the protein crown components using mass spectrometry, using the above-described method for detecting the interaction between nanoparticles and proteins to detect the affinity between the hard protein crown components and the nanoparticles and / or the affinity between the hard protein crown and the soft protein crown components.

[0053] Specifically, after mass spectrometry identification, based on the mass spectrometry identification results, pure proteins of the protein components whose affinity is to be tested are prepared. Biomembrane interference technology is used to detect the affinity between the scleroprotein crown component (inner layer protein) and nanoparticles, and between the scleroprotein crown component (inner layer protein) and the soft protein crown component (outer layer protein), thereby verifying and evaluating the binding strength (affinity) between nanoparticles and protein crowns.

[0054] When detecting the affinity between the scleroprotein crown component and the soft protein crown component, the above-mentioned biosensor with immobilized nanoparticles is first co-incubated with the biological sample to form a scleroprotein crown, and then incubated with the soft protein crown component to be tested to detect the binding between the scleroprotein crown component and the soft protein crown component.

[0055] In the above methods, the detection using biomembrane interferometry can be achieved using a biomembrane interferometer analyzer.

[0056] The beneficial effects of this invention are as follows:

[0057] (1) The detection method provided by this invention fills the gap in the real-time dynamic, in-situ detection and selective, efficient and rapid layer separation of multilayer protein crowns on the surface of nanoparticles. It realizes controllable detection and identification in time and space. In particular, it has a very broad application prospect in the real-time, dynamic and accurate analysis of soft protein crowns. It provides an effective method for further exploring the interaction between nanoparticles and organisms (including cell recognition, cytotoxicity and transport of nanomaterials in organisms) and the rational design of functional nanomedicines.

[0058] (2) In the detection method of the present invention, the biosensor with nanoparticles fixed can be reused multiple times after the protein crown is eluted, which saves detection costs and improves material use and detection efficiency.

[0059] (3) The detection method of the present invention can be applied to complex biological samples or physiological environment simulation solutions;

[0060] (4) The detection method of the present invention also has the advantages of being simple and easy to operate, fast and convenient, and highly scalable. Attached Figure Description

[0061] Figure 1 This is a schematic flowchart of the in-situ identification and quantification method for the multilayer protein crown components on the surface of nanoparticles according to the present invention.

[0062] Figure 2 This is a schematic diagram of the preparation method of Cu2S nanoparticles in Example 1 of the present invention.

[0063] Figure 3 This is a transmission electron microscope (TEM) image of Cu2S nanoparticles in Example 1 of the present invention, where the scale bar is 10 nm.

[0064] Figure 4 This is a schematic flowchart of the experimental method for detecting and identifying the protein crown components on the surface of Cu2S nanoparticles using biomembrane interferometry in Example 2 of the present invention.

[0065] Figure 5In Example 2 of this invention, different types of biosensors are used to immobilize nanoparticles.

[0066] Figure 6 In Example 2 of this invention, Cu2S nanoparticles are immobilized on the surface of a biosensor by modification with immunoglobulins (IgG, IgM, and IgA).

[0067] Figure 7 In Example 2 of this invention, different types of nanoparticles are immobilized on the surface of a biosensor by IgG modification.

[0068] Figure 8 The formation of the protein crown in Embodiment 2 and Embodiment 3 of the present invention, and the use of eluents with different components for eluting the protein crown.

[0069] Figure 9 The composition of the sealing solution before the nanoparticles and analytes are introduced is optimized in Example 2 of this invention.

[0070] Figure 10 This invention provides an example of further research on the use of eluents with different components and concentrations for eluting protein crowns in Example 3.

[0071] Figure 11 In Example 3 of this invention, selective elution of soft and hard protein crowns is achieved by controlling the composition and concentration of the eluent.

[0072] Figure 12 This is a graph showing the change in the composition and proportion of the soft protein crown after incubation of nanoparticles with 10% fetal bovine serum over time in Example 4 of the present invention.

[0073] Figure 13 This is a graph showing the change in the composition and proportion of the scleroprotein crown after incubation of nanoparticles with 10% fetal bovine serum over time in Example 5 of the present invention.

[0074] Figure 14 This study investigates the affinity between nanoparticles (NPs) and a representative protein (Tf) in scleroprotein crowns in Example 6 of this invention, where the Tf concentrations are 100, 50, 25, and 12.5 nM.

[0075] Figure 15 This study investigates the affinity between the hard protein crown (HC) on the surface of nanoparticles and the representative protein (Tf) in the soft protein crown in Example 7 of the present invention, wherein the Tf concentrations are 100, 50, 25, and 12.5 nM.

[0076] Figure 16 This is a graph showing the changes in the composition and proportion of the soft protein crown after incubation of nanoparticles with 10% mouse serum over time in Example 8 of the present invention.

[0077] Figure 17This is a graph showing the changes in the composition and proportion of the scleroprotein crown after incubation of nanoparticles with 10% mouse serum over time in Example 9 of the present invention. Detailed Implementation

[0078] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention.

[0079] Example 1: Preparation of Nanoparticles

[0080] Cu₂S nanoparticles were used as a nanomaterial / nanomedicine model to detect and identify the protein crown components adsorbed on their surface. This example illustrates the preparation of Cu₂S nanoparticles.

[0081] Add 95 mL of ultrapure water to a round-bottom flask, then add 1 mmol CuCl2·2H2O and 2 mmol cysteine ​​hydrochloride monohydrate, and mix and dissolve at 30 °C for about 30 minutes. Take 10 mL of the reaction solution and add it to 90 mL of ultrapure water, and mix at 50 °C for 10 minutes. Then adjust the pH with 1 M NaOH until the color turns transparent yellow or brown. Finally, add 0.5 mmol thioacetamide to the reaction solution, and stir vigorously for 30-40 minutes under constant N2 atmosphere. After cooling to room temperature, mix the reactants with isopropanol at a 1:1 (v / v) ratio, centrifuge at 9500 rpm for 5 minutes, collect the precipitate and resuspend it in ultrapure water to prepare Cu2S nanoparticles. Figure 2 ).

[0082] The transmission electron microscopy (TEM) results of the nanoparticles prepared above are as follows: Figure 3 As shown, the Cu2S nanoparticles are spherical, uniformly dispersed, and have an average particle size of about 4 nm.

[0083] Example 2: In-situ identification and quantification of multilayer protein crown components on the surface of nanoparticles based on biomembrane interferometry.

[0084] This embodiment provides a method for in-situ identification and quantification of multilayer protein crown components on the surface of nanoparticles based on biomembrane interferometry, specifically including the following steps ( Figure 4 ):

[0085] 1) Modifying immunoglobulin IgG on biosensors:

[0086] like Figure 5As shown, taking Cu2S nanoparticles as an example, the nanoparticles cannot directly bind to AR2G sensors with surface carboxyl groups, APS sensors with hydrophobic surfaces, or sensors with surface-modified streptavidin proteins. Through extensive experimentation, this invention discovered that using immunoglobulins as bridging molecules can adsorb Cu2S nanoparticles with high affinity, thereby very firmly and stably immobilizing Cu2S nanoparticles on the surface of the biosensor.

[0087] Specifically, the AR2G biosensor was immersed in a pre-wetted plate for 10 minutes, then activated for 300 seconds using a 1:1 mixture of 100 mM 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and 50 mM N-hydroxysuccinimide (NHS). Immunoglobulin IgG, IgM, and IgA solutions were prepared at a concentration of 50 μg / mL using 10 mM sodium acetate at pH 4.0. The AR2G biosensor was then immersed in each of these solutions for 300 seconds. Finally, it was blocked for 300 seconds using a 1 M ethanolamine aqueous solution at pH 8.5. Figure 6 The process of modifying IgG, IgM and IgA onto the biosensor is shown.

[0088] 2) Immobilizing nanoparticles onto biosensors:

[0089] like Figure 6 As shown, AR2G biosensors coated with IgG, IgM, and IgA were blocked with blocking solution (pH 7.4 phosphate buffer of 0.05% Tween 20 and 0.05% polyethylene glycol) for 60 seconds, and then immersed in Cu2S nanoparticle aqueous solution for 300 seconds to immobilize the nanoparticles on the biosensors.

[0090] Furthermore, the immunoglobulin-coated AR2G sensor constructed in this invention is universally applicable to different types of nanoparticles. For example... Figure 7 As shown, in addition to Cu2S nanoparticles, MoS2 nanosheets (purchased from Jiangsu Xianfeng Nanomaterials Technology Co., Ltd., item number: 103294) and graphene (purchased from Jiangsu Xianfeng Nanomaterials Technology Co., Ltd., item number: 100393) can also be stably immobilized on IgG-coated AR2G sensors.

[0091] 3) Formation of protein crowns on the surface of nanoparticles:

[0092] The biosensor carrying Cu2S nanoparticles was incubated with 10% fetal bovine serum solution (diluted with pH 7.4 phosphate buffer as a protein crown formation solution) at 37℃ and 1000 rpm. The formation of the protein crown was monitored in real time using a biofilm interferometer. Figure 8The pH 7.4 phosphate buffer solution consists of 137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4, and 1.4 mM KH2PO4.

[0093] It is worth noting that, in order to reduce the non-specific adsorption of the protein crown-forming solution to the IgG-coated AR2G biosensor itself, while not affecting the interaction between the nanoparticles and the protein crown-forming solution, such as... Figure 9 As shown, pre-blocking the biosensor with a pH 7.4 phosphate buffer containing both 0.05% Tween 20 and 0.05% polyethylene glycol before binding nanoparticles effectively reduces non-specific adsorption. However, using only a pH 7.4 phosphate buffer containing 0.05% polyethylene glycol or only a pH 7.4 phosphate buffer containing 0.05% Tween 20 as the blocking solution still results in strong non-specific adsorption of the protein crown-forming solution and the IgG-coated AR2G biosensor itself.

[0094] 4) Elution and collection of protein crowns: Soft and hard protein crowns on the surface of Cu2S nanoparticles were eluted sequentially using different eluents and collected in 96-well plates. The protein crown eluent was concentrated using a Milipore ultrafiltration tube (0.5 mL, molecular weight cutoff 3 kDa) and the solution was replaced with ultrapure water.

[0095] 5) Identification of protein crowns: The components of the soft and hard protein crowns eluted by gradient elution were identified by LC-MS / MS mass spectrometry.

[0096] In the above method, the elution method in step 4) is as follows: first, a portion of the protein crown (weakly bound protein crown, i.e., soft protein crown) is eluted from the nanoparticles using an aqueous solution containing 0.005% trifluoroacetic acid, and then all the protein crown is eluted from the Cu2S nanoparticles using an aqueous solution containing 0.1% trifluoroacetic acid (after elution, the baseline just returns to the initial baseline before the formation of the protein crown).

[0097] Example 3: Analysis of the elution effect of different eluents

[0098] like Figure 8As shown, the elution effects of different eluent compositions on the protein crown on the surface of Cu2S nanoparticles were investigated. The biosensor carrying Cu2S nanoparticles was incubated with 10% fetal bovine serum solution (diluted with pH 7.4 phosphate buffer) to allow stable protein crowns to form on the nanoparticle surface. Then, aqueous solutions of 0.01% formic acid (FA), 0.5% sodium dodecyl sulfate (SDS), 0.01% trifluoroacetic acid (TFA), and 5 mM sodium hydroxide (NaOH) were used as eluents. It was found that all these eluents could elute some of the protein crown components from the nanoparticles.

[0099] This embodiment further investigated the elution effect of different concentrations of TFA aqueous solutions on protein corona. For example... Figure 10 As shown, an aqueous solution containing 0.005-0.05% TFA can elute some of the protein corona from the Cu2S nanoparticles. With increasing TFA concentration, more and more protein corona can be eluted. When using an aqueous solution containing 0.1% TFA, all the protein corona can be eluted from the nanoparticles (the eluted baseline exactly returns to the initial baseline before the protein corona formation). Since the eluted baseline is not lower than the initial baseline, this indicates that the nanoparticles on the biosensor surface have not been eluted; that is, the nanoparticles remain on the biosensor, allowing for repeated experiments as needed. As a control, water itself cannot elute the protein corona from the nanoparticles.

[0100] like Figure 11 As shown, the soft and hard protein crowns on the surface of Cu2S nanoparticles can be eluted by sequentially using aqueous solutions containing 0.005% TFA and 0.1% TFA, respectively, and collected in a 96-well plate.

[0101] Example 4: Detection and identification of the soft protein crown component on the surface of nanoparticles (analyte: 10% fetal bovine serum)

[0102] The soft protein crown component on the surface of nanoparticles was detected and identified using the method in Example 2:

[0103] The biosensor carrying Cu2S nanoparticles was incubated with 10% fetal bovine serum solution (diluted with pH 7.4 phosphate buffer) for 3 min and 30 min under vibration at 37 °C and 1000 rpm. The soft protein crowns on the nanoparticle surface were then eluted and collected with an aqueous solution containing 0.005% TFA. The soft protein crown components were detected by LC-MS / MS mass spectrometry. The soft protein crown components were identified by manually searching the bovine protein database on the UniProt website.

[0104] like Figure 12The results showed that albumin had the highest content of the soft protein crown component in Cu2S nanoparticles. With increasing time (from 3 minutes to 30 minutes), the contents of transport proteins, immunoglobulins, and complement proteins decreased, with transport proteins showing the most significant decrease, while the contents of albumin and other proteins increased. This indicates that the soft protein crown on the surface of nanoparticles is a complex and dynamic process involving continuous exchange and substitution between different proteins.

[0105] Example 5: Detection and identification of the scleroprotein crown component on the surface of nanoparticles (analyte: 10% fetal bovine serum).

[0106] The scleroprotein crown component on the surface of nanoparticles was detected and identified using the method in Example 2:

[0107] The biosensor carrying Cu2S nanoparticles was incubated with 10% fetal bovine serum solution (diluted with pH 7.4 phosphate buffer) for 3 minutes and 30 minutes under vibration conditions of 37°C and 1000 rpm. The soft protein crown on the nanoparticle surface was first eluted with 0.005% TFA aqueous solution, followed by elution and collection of the hard protein crown on the nanoparticle surface with an aqueous solution containing 0.1% TFA. The hard protein crown components were detected by LC-MS / MS mass spectrometry. The hard protein crown components were identified by manually searching the bovine protein database on the UniProt website.

[0108] like Figure 13 As shown, the main components of the scleroprotein crown are other proteins, followed by albumin and transport proteins. As the incubation time increases (from 3 minutes to 30 minutes), the contents of transport proteins, immunoglobulins and complement proteins decrease, with the decrease in transport proteins being the most significant. Meanwhile, the contents of albumin and other proteins increase, consistent with the trend of the soft protein crown over time.

[0109] Example 6: Affinity detection of nanoparticles with representative proteins in scleroprotein crowns

[0110] To investigate the binding of protein components in scleroprotein crowns to nanoparticles, a biomembrane interferometry analyzer was used to detect the affinity between Cu2S nanoparticles and a representative protein (transferrin Tf) present in high concentrations in scleroprotein crowns. The specific steps included:

[0111] 1) Immobilizing immunoglobulin IgG onto a biosensor:

[0112] The AR2G biosensor was immersed in a pre-wetted plate for 10 minutes, then activated for 300 seconds using a 1:1 mixture of 100 mM 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and 50 mM N-hydroxysuccinimide (NHS). An IgG protein solution of 50 μg / mL was prepared using 10 mM sodium acetate at pH 4.0. The AR2G biosensor was immersed in the IgG protein solution for 300 seconds. It was then blocked with a 1 M ethanolamine aqueous solution at pH 8.5 for 300 seconds. Finally, it was equilibrated for 60 seconds in a pH 7.4 phosphate buffer containing 0.05% Tween 20 and 0.05% polyethylene glycol (average molecular weight 600).

[0113] 2) Immobilizing Cu2S nanoparticles onto biosensors:

[0114] The IgG-coated AR2G biosensor was immersed in an aqueous solution of Cu2S nanoparticles for 300 seconds to immobilize the Cu2S nanoparticles onto the biosensor.

[0115] 3) Detecting the affinity between Cu2S nanoparticles and representative proteins:

[0116] Transferrin (Tf) was prepared into four concentration gradients (100, 50, 25, and 12.5 nM) using pH 7.4 phosphate buffer. Under conditions of 37°C and vibration at 1000 rpm, it was bound to a biosensor immobilized with Cu₂S nanoparticles for 300 seconds and then dissociated for 300 seconds. The affinity K was calculated using data analysis software from a biomembrane interferometer. D The results showed that Cu₂S nanoparticles have a very strong binding affinity to Tf, with an affinity of 1.0 nM ( ). Figure 14 ).

[0117] Example 7: Affinity detection of representative proteins in hard and soft protein crowns on the surface of nanoparticles.

[0118] To investigate the binding of scleroprotein and leucinogen on the surface of nanoparticles, a biomembrane interferometry analyzer was used to detect the affinity between scleroprotein-coated Cu2S nanoparticles and a representative protein (transferrin Tf) present in high concentrations in the leucinogen. The specific steps included:

[0119] 1) Immobilizing immunoglobulin IgG onto a biosensor:

[0120] The AR2G biosensor was immersed in a pre-wetted plate for 10 minutes, then activated for 300 seconds using a 1:1 mixture of 100 mM 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and 50 mM N-hydroxysuccinimide (NHS). An IgG protein solution of 50 μg / mL was prepared using 10 mM sodium acetate at pH 4.0. The AR2G biosensor was immersed in the IgG protein solution for 300 seconds. It was then blocked with a 1 M ethanolamine aqueous solution at pH 8.5 for 300 seconds. Finally, it was equilibrated for 60 seconds in a pH 7.4 phosphate buffer containing 0.05% Tween 20 and 0.05% polyethylene glycol (average molecular weight 600).

[0121] 2) Immobilizing Cu2S nanoparticles onto biosensors:

[0122] The IgG-coated AR2G biosensor was immersed in an aqueous solution of Cu2S nanoparticles for 300 seconds to immobilize the Cu2S nanoparticles onto the biosensor.

[0123] 3) Formation of hard protein crowns on the surface of Cu2S nanoparticles:

[0124] Under conditions of 37°C and 1000 rpm vibration, the biosensor carrying Cu2S nanoparticles was incubated with 10% fetal bovine serum solution (diluted with pH 7.4 phosphate buffer) for a short time (60 seconds) to make the surface of Cu2S nanoparticles mainly consist of strongly bound scleroprotein crowns.

[0125] 4) Detecting the affinity between Cu2S nanoparticles coated with scleroprotein crowns and representative proteins in soft protein crowns:

[0126] A representative protein from the scleroprotein crown (transferrin Tf) was prepared into four concentration gradients (100, 50, 25, and 12.5 nM) using pH 7.4 phosphate buffer. These gradients were then used to bind to Cu₂S nanoparticles coated with the scleroprotein crown for 300 seconds and dissociate for 300 seconds, respectively. The affinity K was calculated using data analysis software from a biomembrane interferometer. D The results showed that the binding strength between the Cu2S nanoparticles coated with scleroprotein crown and Tf was moderate, with an affinity of 36.7 nM ( ). Figure 15 This result indicates that there is indeed an interaction between the scleroprotein crown and the soft scleroprotein crown.

[0127] Example 8: Detection and identification of the soft protein crown component on the surface of nanoparticles (analyte: mouse serum)

[0128] To demonstrate that the method of this invention can be used for the detection of various complex biological samples, this embodiment uses mouse serum as the analyte and Cu2S nanoparticles as an example to detect and identify the soft protein crown component on the surface of the nanoparticles using the method of Example 2:

[0129] Blood was collected from the orbital cavity of mice, allowed to coagulate at room temperature for 30 minutes, and then centrifuged at 4°C and 2800 rpm for 10 minutes to obtain serum. The serum was then purified by centrifugation at 10500 rpm for 5 minutes to remove insoluble fragments and aggregated proteins. The final mouse serum was stored at -80°C for subsequent experiments.

[0130] The biosensor carrying Cu2S nanoparticles was incubated with 10% mouse serum solution (diluted with phosphate buffer at pH 7.4) for 3 min and 30 min under vibration at 37 °C and 1000 rpm. The soft protein crowns on the nanoparticle surface were then eluted and collected with an aqueous solution containing 0.005% TFA. The soft protein crown components were detected by LC-MS / MS mass spectrometry. The soft protein crown components were identified by manually searching the bovine protein database on the UniProt website.

[0131] like Figure 16 The results showed that at 3 minutes, the coronal components of Cu2S nanoparticles with relatively high levels of albumin and immunoglobulins were: as time progressed, at 30 minutes, the acute phase protein content increased significantly, the immunoglobulin and complement protein content decreased significantly, and the albumin and apolipoprotein content did not change much.

[0132] Example 9: Detection and identification of the scleroprotein crown component on the surface of nanoparticles (analyte: mouse serum)

[0133] To demonstrate that the method of this invention can be used for the detection of complex biological samples, this embodiment uses mouse serum as the analyte and Cu2S nanoparticles as an example to detect and identify the scleroprotein crown component on the surface of the nanoparticles using the method of Example 2:

[0134] Blood was collected from the orbital cavity of mice, allowed to coagulate at room temperature for 30 minutes, and then centrifuged at 4°C and 2800 rpm for 10 minutes to obtain serum. The serum was then purified by centrifugation at 10500 rpm for 5 minutes to remove insoluble fragments and aggregated proteins. The final mouse serum was stored at -80°C for subsequent experiments.

[0135] The biosensor carrying Cu2S nanoparticles was incubated with 10% mouse serum solution (diluted with pH 7.4 phosphate buffer) for 3 minutes and 30 minutes under vibration conditions of 37°C and 1000 rpm. The soft protein crown on the nanoparticle surface was first eluted with 0.005% TFA aqueous solution, followed by elution and collection of the hard protein crown on the nanoparticle surface with an aqueous solution containing 0.1% TFA. The hard protein crown components were detected by LC-MS / MS mass spectrometry. The hard protein crown components were identified by manually searching the bovine protein database on the UniProt website.

[0136] like Figure 17 As shown, after 3 minutes of incubation, the main components of the scleroprotein crown on the surface of Cu2S nanoparticles were other proteins, followed by albumin. With an incubation time extended to 30 minutes, the contents of immunoglobulins and acute-phase proteins increased significantly, while the contents of albumin, apolipoproteins, complement proteins, and other proteins decreased significantly. Therefore, the method of this invention can be used to rapidly and in real-time identify changes in the composition and content of the surface protein crown of nanoparticles after contact with complex biological samples.

[0137] In summary, the method of the present invention can be used to quickly and conveniently monitor and analyze the time-dependent adsorption and exchange of soft and hard protein crowns on the surface of nanoparticles.

[0138] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A method for in situ identification and quantification of components of a multilayer protein corona on the surface of a nanoparticle, characterized in that, The method comprises: fixing the to-be-tested nanoparticles on a biosensor surface, incubating the to-be-tested nanoparticles fixed on the biosensor surface in a biological sample solution, and monitoring the formation of a protein corona on the surface of the nanoparticles by using a biological membrane interference technology; after the protein corona is formed, eluting the soft protein corona and the hard protein corona on the surface of the nanoparticles by using an eluent; and identifying the components of the protein corona by using a mass spectrometry technology. The fixing method comprises: first, performing immunoglobulin modification on the biosensor surface; then, performing blocking treatment on the biosensor surface modified by the immunoglobulin by using a blocking solution; and finally, co-incubating the nanoparticles.

2. The method for in situ identification and quantification of nanoparticulate surface multilayer protein corona components according to claim 1, characterized in that, The immunoglobulin is IgG, IgM or IgA.

3. The method for in situ identification and quantification of nanoparticulate surface multilayer protein corona constituents according to claim 1, characterized in that, The blocking solution is a phosphate buffer solution comprising 0.03-0.08% Tween-20 and 0.03-0.08% polyethylene glycol.

4. The method for in situ identification and quantification of nanoparticulate surface multilayer protein corona constituents according to claim 1, characterized in that, The pH of the blocking solution is 7.2-7.

5.

5. The method for in situ identification and quantification of the components of a nanoparticulate surface multilayer protein corona according to any one of claims 1 to 4, characterized in that, The eluent is an aqueous solution comprising one or more of the following components: sodium dodecyl sulfate, sodium hydroxide, trifluoroacetic acid, formic acid, acetic acid, hydrochloric acid, sodium chloride and magnesium chloride.

6. The method for in situ identification and quantification of nanoparticulate surface multilayer protein corona components according to claim 5, characterized in that, The eluent for eluting the soft protein corona is an aqueous solution of 0.001-0.05% trifluoroacetic acid.

7. The method for in situ identification and quantification of nanoparticulate surface multilayer protein corona components according to claim 5, characterized in that, The eluent for eluting the hard protein corona is an aqueous solution of 0.1-1% trifluoroacetic acid.

8. The method for in situ identification and quantification of nanoparticulate surface multilayer protein corona components according to claim 6, characterized in that, The eluent for eluting the hard protein corona is an aqueous solution of 0.1-1% trifluoroacetic acid.

9. The method for in situ identification and quantification of the components of a protein nanoparticle surface multilayer corona according to any one of claims 1 to 4, characterized in that, In the biological sample solution, the concentration of total protein is 0.1-10 mg / mL, and the pH is 0.5-9.

10. The method for in situ identification and quantification of the components of a protein nanoparticle surface multilayer corona according to any one of claims 1 to 4, characterized in that, The method further comprises: after identifying the components of the protein corona by using the mass spectrometry technology, detecting the affinity between the components of the hard protein corona and the nanoparticles and / or the affinity between the components of the hard protein corona and the soft protein corona. The method further comprises: after identifying the components of the protein corona by using the mass spectrometry technology, detecting the affinity between the components of the hard protein corona and the nanoparticles and / or the affinity between the components of the hard protein corona and the soft protein corona.

11. The method of claim 10, wherein, The fixing method comprises: first, performing immunoglobulin modification on the biosensor surface; then, performing blocking treatment on the biosensor surface modified by the immunoglobulin by using a blocking solution; and finally, co-incubating the nanoparticles.

12. The method of claim 11, wherein, The immunoglobulin is IgG, IgM or IgA.

13. The method according to claim 11 or 12, characterized in that, The blocking solution is a phosphate buffer solution comprising 0.03-0.08% Tween-20 and 0.03-0.08% polyethylene glycol.

14. The method of claim 11 or 12, wherein, The pH of the blocking solution is 7.2-7.

5.

15. The method of claim 10, wherein, In the to-be-tested protein solution, the concentration of the to-be-tested protein is 0.1-10 mg / mL, and the pH is 0.5-9.

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