Identification and quantification method for nanoparticle surface binding cell membrane receptor

Abstract

The invention relates to the technical field of analysis and detection, in particular to an identification and quantification method for a nano-particle surface binding cell membrane receptor. The method comprises the following steps: fixing to-be-detected nanoparticles on the surface of a biosensor, and incubating the to-be-detected nanoparticles fixed on the surface of the biosensor in a cell / cell membrane lysis solution containing sodium alginate, or incubating the to-be-detected nanoparticles fixed on the surface of the biosensor with body fluid such as plasma to form a'protein crown ', then incubating the'protein crown' with the cell / cell membrane lysis solution containing the sodium alginate, and finally incubating the'protein crown 'with the cell / cell membrane lysis solution containing the sodium alginate. The combination of the nanoparticles and the protein is monitored by adopting a biological membrane layer interference technology, the protein is eluted, a cell membrane receptor component in the protein is identified, and the affinity of the cell membrane receptor component and the nanoparticles is detected. According to the method provided by the invention, a blocking component (sodium alginate) is added into the cell / cell membrane lysis solution, and non-specific adsorption of various impurity components in the sensor and the lysis solution can be greatly reduced, so that interference is reduced, and the accuracy of a detection result is improved.

Description

Technical Field

[0001] The present invention relates to the technical field of analysis and detection, and in particular to an identification and quantitative method of nanoparticle surface-bound cell membrane receptors. Background Art

[0002] Nanomaterials have shown great application potential in biomedical fields such as drug delivery, disease diagnosis and treatment, tissue engineering, and vaccine development due to their unique physical and chemical properties. However, after nanoparticles enter the body, the interaction mechanism with the biological system is complex and diverse, involving multiple levels such as body fluids, organs, cells, and organelles. Among them, the interaction between nanoparticles and cell membrane receptors directly determines the efficiency of nanoparticle uptake by the biological system. Therefore, studying the interaction between nanoparticles and cell membrane receptors is crucial to optimizing the design of nanomaterials and improving the effectiveness and safety of their clinical applications.

[0003] At present, the experimental methods for studying the interaction between nanoparticles and cell membrane receptors require the specific cell membrane receptor proteins that are known to interact. The co-localization of nanoparticles and cell membrane receptors can be confirmed by fluorescent labeling and confocal microscopy imaging. The affinity of nanoparticles and cell membrane receptors can be calculated using technologies such as surface plasmon resonance (SPR), microthermophoresis (MST), and isothermal titration calorimetry (ITC).

[0004] For unknown cell membrane receptor proteins that interact with nanoparticles, the mixture of nanoparticles and cell / cell membrane lysate incubated can be separated by centrifugation or size exclusion chromatography to obtain nanoparticle-protein complexes, and sodium dodecyl sulfate solution is added to denature and strip the proteins on the surface of the nanoparticles. The nanoparticles are then separated by centrifugation, and the protein components are collected and identified by protein spectrum. However, for small-sized and low-density nanoparticles, it is difficult to separate and elute by centrifugation; the size exclusion chromatography method requires a large amount of sample and takes a very long time (one sample takes several hours). In addition, to identify cell membrane receptors that interact with nanoparticles, the gene screening library method can be used to study specific receptors involved in nanoparticle uptake. However, due to the high cost, time-consuming and complexity of gene editing tools and screening technologies, this method still has high challenges. Therefore, it is urgent to develop more efficient, accurate, in situ and real-time monitoring techniques to study the interaction between nanoparticles and cell membrane receptors.

[0005] In view of this, the present invention is proposed. Summary of the invention

[0006] In order to solve the above technical problems, the present invention provides a method for identifying and quantifying cell membrane receptors bound to the surface of nanoparticles.

[0007] Specifically, the technical solution of the present invention is as follows: First, the present invention provides a method for detecting the interaction between nanoparticles and cell membrane receptors, comprising: fixing the nanoparticles to be tested on the surface of a biosensor; placing the nanoparticles to be tested fixed on the surface of the biosensor in a cell membrane receptor solution to be tested for incubation; using biomembrane layer interference technology to detect the interaction between the nanoparticles and the cell membrane receptors; the cell membrane receptor solution to be tested contains 0.01-1 mg / mL of sodium alginate.

[0008] Preferably, the fixing method is selected from at least one of the following: (1) Direct adsorption of nanoparticles based on the carboxyl groups on the surface of the biosensor.

[0009] (2) Use EDC / NHS to activate the carboxyl groups on the biosensor surface, immobilize the nanoparticles by chemical reaction with the activated carboxyl groups, and then block them with ethanolamine.

[0010] (3) The nanoparticles are fixed on the biosensor surface through electrostatic, van der Waals forces, hydrogen bonding, π-π or hydrophobic interactions.

[0011] Preferably, the cell membrane receptor to be tested is prepared into a solution using PBS.

[0012] Further preferably, the concentration of the cell membrane receptor solution to be tested is preferably 1 nM-5 uM, and the pH is preferably 1-9.

[0013] Preferably, the cell membrane receptor to be tested is prepared into different concentration gradients as an analyte, and the affinity between the nanoparticles and the cell membrane receptor to be tested is detected.

[0014] More preferably, at least four analytes with concentration gradients are configured.

[0015] Preferably, the interaction between the nanoparticle-protein corona complex and the cell membrane receptor can also be studied by continuous sampling using a surface plasmon resonance (SPR) instrument.

[0016] The present invention also provides an in-situ identification and quantification method for surface cell membrane receptor components of nanoparticles, comprising: fixing the nanoparticles to be tested on the surface of a biosensor; placing the nanoparticles to be tested fixed on the surface of the biosensor in a cell / cell membrane lysis solution for incubation; using a biomembrane layer interference technique to monitor the binding of the nanoparticles and proteins, then eluting the proteins with an eluent, and identifying the cell membrane receptor components using a mass spectrometry technique; the cell / cell membrane lysis solution contains 0.01-1 mg / mL of sodium alginate.

[0017] Preferably, the fixing method is selected from at least one of the following: (1) Direct adsorption of nanoparticles based on the carboxyl groups on the surface of the biosensor.

[0018] (2) Use EDC / NHS to activate the carboxyl groups on the biosensor surface, immobilize the nanoparticles by chemical reaction with the activated carboxyl groups, and then block them with ethanolamine.

[0019] (3) The nanoparticles are fixed on the biosensor surface through electrostatic, van der Waals forces, hydrogen bonding, π-π or hydrophobic interactions.

[0020] Preferably, the concentration of the cell / cell membrane lysate is 0.1-5 mg / mL.

[0021] Further preferably, the cell lysate is obtained by lysing cells with NP-40 or RIPA solution and then taking the supernatant by centrifugation; and / or, the cell membrane lysate is obtained by extracting with a GPCR extraction and stabilization reagent and then taking the supernatant by centrifugation.

[0022] Preferably, the nanomaterials immobilized on the biosensor directly interact with the cell membrane receptors; alternatively, the nanomaterials immobilized on the biosensor first interact with body fluids to form a protein corona, and then interact with the cell membrane receptors; the dilution multiple of the body fluid is 5-20 times; the body fluid may be plasma or lymph.

[0023] Preferably, the eluent is trifluoroacetic acid of different concentrations; further preferably, the eluent is a 0.001%-0.5% trifluoroacetic acid aqueous solution; wherein 0.001-0.1% trifluoroacetic acid aqueous solution is used to elute proteins that are weakly bound to the surface of the nanomaterial, and 0.1-0.5% trifluoroacetic acid aqueous solution is used to elute proteins that are strongly bound to the surface of the nanomaterial.

[0024] Preferably, after the cell membrane receptor components are identified by mass spectrometry, the affinity between the cell membrane receptor and the nanoparticle is detected by the aforementioned method for detecting the interaction between the nanoparticle and the cell membrane receptor. Preferably, the interaction between the nanoparticle-protein corona complex and the cell membrane receptor can also be studied by continuous sampling of a surface plasmon resonance (SPR). Finally, the biological function of the cell membrane receptor is verified by detecting the nanoparticle uptake by cells after gene knockdown of the cell membrane receptor by flow cytometry.

[0025] Beneficial effects: The present invention provides a method for identifying and quantifying cell membrane receptors bound to the surface of nanoparticles. The method first fixes the nanoparticles to be tested on the surface of a biosensor; then the nanoparticles to be tested fixed on the surface of the biosensor are placed in a cell / cell membrane lysate for incubation; finally, the binding of the nanoparticles to the protein is monitored by biomembrane interferometry, and then the protein is eluted with an eluent, and the cell membrane receptor components therein are identified by mass spectrometry; finally, the interaction between the nanoparticles or nanoparticle-protein corona complex and the cell membrane receptor is detected by biomembrane interferometry and surface plasmon resonance; the cell / cell membrane lysate contains 0.01-1 mg / mL of sodium alginate. The method provided by the present invention can greatly reduce the nonspecific adsorption of various impurity components in the sensor and the lysate by adding a blocking component (sodium alginate) to the cell / cell membrane lysate, thereby reducing interference and improving the accuracy of the detection results. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the drawings required for use in the embodiments or the description of the prior art will be described below.

[0027] Figure 1 This is the method of fixing lipid nanoparticles by carboxyl reaction in Example 1 of the present invention.

[0028] Figure 2 is the P before and after the reaction between LNP and the modified component sodium alginate on the surface of AR2G sensor 31 NMR spectrum.

[0029] Figure 3 This is a scanning electron micrograph after LNP fixation.

[0030] Figure 4 This is the case in Example 1 of the present invention where the gold nanorods are fixed by electrostatic interactions or the like.

[0031] Figure 5 In Example 1 of the present invention, the black phosphorus nanosheet material is fixed by hydrogen bonding, van der Waals force, π-π, hydrophobic and other interactions.

[0032] Figure 6 The biosensor with immobilized lipid nanoparticles is incubated with cell lysate and then eluted in Example 2 of the present invention.

[0033] Figure 7 In Example 2 of the present invention, the biosensor with immobilized lipid nanoparticles is first incubated with plasma, and then incubated with cell membrane lysate and eluted.

[0034] Figure 8The figure is a comparison of the effects of different blocking components in Example 3 of the present invention on reducing the nonspecific adsorption of cell lysate to the empty sensor.

[0035] Fig. 9 The addition of sodium alginate in Example 3 of the present invention significantly reduces the nonspecific adsorption of cell lysate and the empty sensor.

[0036] Fig.10 This is the data result of proteomics in Example 4 of the present invention.

[0037] Fig.11 This is to detect the affinity of lipid nanoparticles to cell membrane receptors in Example 5 of the present invention.

[0038] Fig.12 This is to detect the interaction between the lipid nanoparticle-protein corona complex and the cell membrane receptor in Example 6 of the present invention.

[0039] Fig.13 The nanoparticle uptake by cells after knocking down the cell membrane receptor gene in Example 7 of the present invention is significantly reduced. DETAILED DESCRIPTION

[0040] The present invention provides a method for detecting the interaction between nanoparticles and cell membrane receptors, and an in-situ identification and quantification method for cell membrane receptor components on the surface of nanoparticles.

[0041] First, the present invention provides a method for detecting the interaction between nanoparticles and cell membrane receptors, the method comprising: fixing the nanoparticles to be detected on the surface of a biosensor, placing the nanoparticles to be detected fixed on the surface of the biosensor in a cell membrane receptor solution to be detected for incubation, and using biolayer interferometry (BLI) technology to detect the binding of the nanoparticles to the cell membrane receptors; wherein the cell receptor solution to be detected contains 0.01-1 mg / mL of sodium alginate.

[0042] The present invention has no particular limitation on the types of biosensors, and all biosensors that can be used for biofilm interferometry detection can be used, including commercial or homemade aminopropylsilane sensors (APS sensors), second-generation amino-coupled sensors (AR2G sensors), streptavidin sensors (SA sensors), super streptavidin sensors (SSA sensors), ProteinA sensors (ProA sensors), NTA sensors, etc.

[0043] In some embodiments of the present invention, the surface of the biosensor contains carboxyl groups, and the nanoparticles can be directly adsorbed, or the surface is first activated with EDC / NHS, and then the nanoparticles are fixed by chemical reaction with the activated carboxyl groups, and then blocked with ethanolamine, so that the nanoparticles are fixed on the surface of the biosensor. In addition, the nanoparticles can also be fixed on the surface of the biosensor through electrostatic, hydrogen bonding, van der Waals force, π-π, hydrophobic and other interactions.

[0044] The present invention has no particular limitation on the types of nanoparticles to be tested, including inorganic nanomaterials, metal and metal oxide nanomaterials, metal sulfide nanomaterials, organic nanomaterials, organic-inorganic hybrid composite nanomaterials, and various nanomedicines.

[0045] In the above method, the cell membrane receptor to be tested is prepared with PBS, preferably with a concentration of 1 nM-5 uM and a pH of 1-9.

[0046] In the above method, the cell membrane receptors to be tested are preferably prepared into different concentration gradients (usually at least 4 concentrations) as analytes to detect the affinity between the nanoparticles and the cell membrane receptors to be tested.

[0047] In the above method, the detection using biofilm layer interference technology can be achieved through a biofilm layer interference analyzer.

[0048] In the above method, the addition of sodium alginate can significantly reduce the nonspecific adsorption of the sensor and the cell receptor solution, thereby reducing interference and improving the accuracy of the detection results. The concentration of sodium alginate is preferably 0.01-1 mg / mL, more preferably 0.05 mg / mL.

[0049] Furthermore, the interaction between nanoparticle-protein corona complexes and cell membrane receptors can be studied by continuous sampling using a surface plasmon resonance (SPR) instrument.

[0050] Furthermore, the present invention develops an in-situ identification and quantification method for cell membrane receptor components on the surface of nanoparticles based on biomembrane interferometry. Compared with existing detection methods, the detection method based on biomembrane interferometry of the present invention can achieve in-situ enrichment, elution and collection of cell membrane receptors, and has the advantages of high throughput, small sample usage, rapid and real-time detection.

[0051] Specifically, the present invention provides an in situ identification and quantification method for cell membrane receptor components on the surface of nanoparticles, the method comprising: fixing the nanoparticles to be tested on the surface of a biosensor, placing the nanoparticles to be tested fixed on the surface of the biosensor in a cell / cell membrane lysis solution for incubation, using a biomembrane layer interference technique to monitor the binding of the nanoparticles and proteins, then eluting the proteins with an eluent, and identifying the cell membrane receptor components using a mass spectrometry technique; the cell / cell membrane lysis solution contains 0.01-1 mg / mL of sodium alginate.

[0052] The cell lysate can be obtained by lysing cells with a solution such as NP-40, RIPA, etc., and then taking the supernatant by centrifugation. The cell membrane lysate can be extracted with a GPCR extraction and stabilization reagent, and then the supernatant is obtained by centrifugation. The centrifugation conditions are 4°C, 16000 g centrifugation for 20 minutes. The obtained cell / cell membrane lysate is stored at -20°C for no more than 2 weeks.

[0053] Considering that after entering the body, nanoparticles first interact with body fluids such as plasma to form a "protein corona" and then interact with cell membrane receptors, the nanoparticles to be tested can be first fixed on the surface of the biosensor, and the nanoparticles to be tested fixed on the surface of the biosensor can be incubated in body fluids such as plasma, and then incubated in cell / cell membrane lysis buffer, and then the protein can be eluted with an elution buffer, and the cell membrane receptor components can be identified using mass spectrometry technology.

[0054] The concentration of the cell / cell membrane lysate to be tested is preferably 0.1-5 mg / mL, and the dilution multiple of the plasma or other body fluid is 5-20 times.

[0055] The present invention explores the composition of the blocking component and finds that the addition of sodium alginate can significantly reduce the nonspecific adsorption of the sensor and other impurity components in the cell / cell membrane lysate. When 0.01-1 mg / mL of sodium alginate (preferably 0.05 mg / mL of sodium alginate) is used as the blocking component, the nonspecific adsorption of proteins and the biosensor can be effectively reduced without adversely affecting the interaction between the nanoparticles and the cell membrane receptors, thereby improving the accuracy of the detection results.

[0056] The present invention has no particular limitation on the types of biosensors, and all biosensors that can be used for biomembrane interferometry detection can be used, including commercial or homemade 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.

[0057] In some embodiments of the present invention, the surface of the biosensor contains carboxyl groups, and the nanoparticles can be directly adsorbed, or the surface is first activated with EDC / NHS, and then the nanoparticles are fixed by chemical reaction with the activated carboxyl groups, and then blocked with ethanolamine, so that the nanoparticles are fixed on the surface of the biosensor. In addition, the nanoparticles can also be fixed on the surface of the biosensor through electrostatic, hydrogen bonding, van der Waals force, π-π, hydrophobic and other interactions.

[0058] The present invention has no particular limitation on the types of nanoparticles to be tested, including inorganic nanomaterials, metal and metal oxide nanomaterials, metal sulfide nanomaterials, organic nanomaterials, organic-inorganic hybrid composite nanomaterials, and various nanomedicines.

[0059] Furthermore, in order to better analyze the proteins bound to the surface of the nanoparticles, the present invention explores the composition and concentration of the eluent used to elute the proteins. Preferably, the eluent is a 0.001%-0.5% trifluoroacetic acid aqueous solution, wherein a 0.001-0.1% trifluoroacetic acid aqueous solution is used to elute proteins that are weakly bound to the surface of the nanomaterial (soft protein corona), and a 0.1-0.5% trifluoroacetic acid aqueous solution is used to elute proteins that are strongly bound to the surface of the nanomaterial (hard protein corona).

[0060] The present invention finds that the use of the above-mentioned eluents of different concentrations can simply and efficiently elute the outer layer protein and the inner layer protein of the nanoparticle surface, respectively, to achieve relatively accurate distinction and identification of the soft protein corona and the hard protein corona components.

[0061] During the elution process, the eluted protein solution is collected for subsequent mass spectrometry identification of protein components.

[0062] For the biological sample solution to be tested, the total protein concentration is preferably 0.1-10 mg / mL and the pH is 1-9.

[0063] Controlling the concentration of total protein in the biological sample solution within the above range is beneficial to further reduce nonspecific adsorption.

[0064] In the above method, the biosensor with fixed nanoparticles is incubated with the biological sample solution for a time period, and the binding protein is dynamically monitored in real time using biomembrane interferometry technology, and the incubation time is determined based on the monitoring results.

[0065] Generally, the incubation time can be from 1 second to 7 days. Considering the protein corona formation time and protein stability, the preferred incubation time is from 1 second to 48 hours.

[0066] In the above method, the incubation temperature can be adjusted from 6-40° C. From the perspective of consistency with human body temperature, the preferred incubation temperature is 37° C.

[0067] During the incubation process, in order to make the biological sample fully contact with the nanoparticles, the sample plate can be set to a vibration mode, and the vibration speed is adjustable from 0-5000 rpm, preferably 500-2000 rpm.

[0068] In the above method, for mass spectrometry identification of protein corona components, LC-MS / MS method is preferably used. Before mass spectrometry identification, the collected protein corona elution solution usually needs to be concentrated and the solution replaced. The concentration and solution replacement methods include but are not limited to the use of ultrafiltration tubes, desalting columns, freeze drying, polyacrylamide gel electrophoresis (SDS-PAGE), etc.

[0069] Preferably, the above-mentioned in situ identification and quantification method of cell membrane receptor components on the surface of nanoparticles also includes: after identifying the cell membrane receptor components by mass spectrometry, using the above-mentioned method for detecting the interaction between nanoparticles and cell membrane receptors to prepare cell membrane receptor proteins to be detected for affinity, using biomembrane layer interferometry technology to detect the affinity between cell membrane receptor components and nanoparticles, and also studying the interaction between nanoparticle-protein corona complexes and cell membrane receptors by continuous sampling using a surface plasmon resonance (SPR) instrument, thereby verifying and evaluating the binding of nanoparticles to cell membrane receptors.

[0070] The detection method provided by the present invention has the advantages of high throughput and small sample usage, filling the gap in methods for real-time dynamic, in situ detection and efficient and rapid separation of nanoparticles and cell membrane receptors, and has a very broad application prospect. It provides an effective method for further exploring the interaction between nanoparticles and organisms (including cell recognition, cell uptake, cytotoxicity, etc.) and the rational design of functional nanomedicines.

[0071] The detection method of the present invention has the technical advantage of high throughput and can analyze at least 8 groups of samples simultaneously, thereby improving detection efficiency.

[0072] The detection method of the present invention is applicable to complex biological samples, including cells and cell membrane lysates, and requires a small amount of sample.

[0073] The detection method of the present invention also has the advantages of being simple and easy to operate, rapid and convenient, and having strong scalability.

[0074] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0075] The endpoints and any values ​​of the ranges disclosed in this specification are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0076] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "specific implementations", or "some specific implementations" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the embodiment of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

[0077] In the examples provided in this specification, if no specific techniques or conditions are specified, the techniques or conditions described in the literature in this field or the product instructions are used. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased through regular channels.

[0078] Example 1 This example provides three methods for immobilizing nanomaterials on biosensors.

[0079] (1) Immobilize lipid nanoparticles through carboxyl reaction.

[0080] Lipid nanoparticles (LNP) were prepared using a microfluidic system or ultrasound. The AR2G biosensor was placed in a pre-wetted plate and soaked for 10 minutes, and then activated for 300 seconds using a 100 mM 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and 50 mM N-hydroxysuccinimide (NHS) mixture in a 1:1 ratio. LNP was prepared with 10 mM sodium acetate at pH 4.5 at a concentration of 0.1 mg / mL. The AR2G biosensor was immersed in the LNP solution for 300 seconds. It was then blocked with a 1 M ethanolamine aqueous solution at pH 8.5 for 300 seconds. Figure 1 The process of immobilizing LNPs on the biosensor is shown. Figure 2 is the P before and after the reaction of LNP and sodium alginate 31 NMR spectrum. The surface component of AR2G sensor is sodium alginate, which has many carboxyl groups. 31 The changes in the NMR spectrum proved that the P element on the LNP had a chemical reaction with the AR2G sensor, that is, the phosphate group had an esterification reaction with sodium alginate. Figure 3 This is a scanning electron microscope image after LNP was fixed. The results show that LNP is fixed on the sensor in a uniform and well-dispersed state.

[0081] (2) Fix the gold nanorod material through electrostatic and other interactions.

[0082] Gold nanorods modified with trimethyl hexadecyl ammonium bromide (Cat. No. YM-NM-002) were purchased from Changsha Yimo Biotechnology Co., Ltd. The AR2G biosensor was placed in a pre-wetted plate and soaked for 10 minutes. The gold nanorod nanomaterial was prepared with ultrapure water at a concentration of 20 ug / mL. The AR2G biosensor was immersed in ultrapure water for 60 s, then immersed in the gold nanorod solution for 200 s, and then immersed in ultrapure water to wash away the unbound gold nanorods. Figure 4 The process of immobilizing gold nanorods on the biosensor is shown.

[0083] (3) Fix black phosphorus nanosheets through hydrogen bonds, van der Waals forces, π-π, hydrophobic and other interactions.

[0084] Black phosphorus nanosheet dispersion (CAS No.: 7723-14-0) was purchased from Nanjing Xianfeng Nanomaterial Technology Co., Ltd. The SA biosensor was placed in a pre-wetted plate and soaked for 10 minutes. The black phosphorus nanosheet material was prepared with water at a concentration of 50 ug / mL. The SA biosensor was first immersed in water for 60 s, then immersed in the black phosphorus nanosheet solution for 200 seconds, and then immersed in water to wash away the unbound black phosphorus nanosheet material. Figure 5The process of immobilizing black phosphorus nanosheets on the biosensor is shown.

[0085] Example 2 This embodiment provides a method for detecting the interaction between lipid nanoparticles and cells / cell membrane lysate based on biomembrane interferometry technology.

[0086] The method provided in this embodiment specifically includes the following steps: 1) Immobilization of lipid nanoparticles on biosensors: According to the method of Example 1 (1), LNP nanoparticles were immobilized on the surface of AR2G biosensor ( Figure 1 ).

[0087] 2) Extraction of cell membrane proteins: Neutrophils (HL-60; 1×10 7 ), washed twice with ice-cold PBS, and then 1 mL of cold GPCR Extraction and Stabilization Reagent (Cat. No. A43436) was added to the cell pellet. After obtaining a homogenous cell suspension using a Dounce homogenizer (25-50 strokes), the cells were incubated at 4°C with constant mixing for 60 minutes. Then, the cells were centrifuged at 16,000 g for 20 minutes at 4°C, and the supernatant was used as the cell membrane lysate.

[0088] 3) Incubate the biosensor with cell lysate: like Figure 6 As shown, the LNP-immobilized AR2G biosensor was immersed in a 0.05 mg / mL sodium alginate aqueous solution for 60 seconds and then incubated in a cell membrane lysate solution with a concentration of 1 mg / mL and 0.05 mg / mL sodium alginate for 1800 seconds. The soft protein corona and hard protein corona bound to the LNP surface were then eluted using 0.005% and 0.5% trifluoroacetic acid, respectively.

[0089] In addition, the LNP-immobilized AR2G biosensor can also be pre-incubated with plasma and then combined with cells / cell membrane lysate. Figure 7 As shown, the LNP-immobilized AR2G biosensor was immersed in a 0.05 mg / mL sodium alginate aqueous solution for 60 s, then incubated in 10% plasma (containing 0.05 mg / mL sodium alginate) for 120 s, and then incubated with a cell membrane lysate for another 120 s, where the cell membrane lysate concentration was 1 mg / mL and contained 0.05 mg / mL sodium alginate. The soft protein corona and hard protein corona bound to the LNP surface were then eluted using 0.001% and 0.5% trifluoroacetic acid, respectively.

[0090] 4) Perform protein spectrum detection on the collected protein samples: The components of the soft protein corona and hard protein corona eluted by gradient were identified by LC-MS / MS mass spectrometry.

[0091] Example 3 This embodiment provides a method for reducing nonspecific adsorption of cell lysate to an empty sensor, which is achieved by adding sodium alginate.

[0092] First, the effects of different chemical compositions on reducing the nonspecific adsorption of cell lysate to the empty sensor were compared. Figure 8 As shown in the figure, (1) is the non-specific adsorption of the cell lysate itself to the AR2G sensor; (2) adding 0.05% Tween 20 to the cell lysate, (3) adding 0.5 mg / mL polyethylene glycol (molecular weight 2000 Da) to the cell lysate, neither can reduce the non-specific adsorption of the cell lysate itself to the AR2G sensor; (4) adding 0.01 mg / mL sodium alginate to the cell lysate can effectively reduce the non-specific adsorption of the cell lysate to the AR2G sensor.

[0093] The present invention further optimizes the concentration of sodium alginate. Fig. 9 As shown: (1) The cell lysate itself has a certain non-specific adsorption with the empty AR2G sensor. By adding (2) 0.001 mg / mL sodium alginate, (3) 0.01 mg / ml sodium alginate, and (4) 0.05 mg / mL sodium alginate to the cell lysate, as the concentration of sodium alginate increases, the non-specific adsorption becomes smaller and smaller, among which 0.05 mg / mL sodium alginate has the best effect.

[0094] Example 4 In this example, cell membrane receptors on the surface of nanoparticles are detected and identified.

[0095] The protein samples collected in Example 2 were subjected to protein spectrum detection. The experimental results are shown in Fig.10 As shown, by plotting p-value against logFc, combined with the biological function of the protein, the cell membrane receptor CSF2RB was identified.

[0096] Example 5 This example provides an affinity detection method and results for verifying the binding of nanoparticles to cell membrane receptor proteins.

[0097] In order to verify the binding of cell membrane receptors to nanoparticles, the affinity of LNP to cell membrane receptor CSF2RB was detected using a biomembrane interferometer, which specifically included the following steps: 1) Immobilization of LNPs on the biosensor: According to the method of Example 1 (1), LNP nanoparticles were immobilized on the surface of AR2G biosensor ( Figure 1 ).

[0098] 2) Prepare the cell membrane receptor CSF2RB solution The cell membrane receptor CSF2RB was prepared into different concentration gradients with PBS: 50 nM, 100 nM, 200 nM, 400 nM. 0.05 mg / mL sodium alginate was added to PBS.

[0099] 3) Use biofilm interferometry to detect the affinity of LNP to CSF2RB: The affinity of LNP to CSF2RB was detected by double subtraction method and biofilm interferometry. Fig.11 As shown in Figure 2, the binding of LNP to CSF2RB has obvious kinetic characteristics and strong binding ability, with an affinity of K D is 20 nM, R 2 It is 0.9917.

[0100] Example 6 This example provides a method and result for detecting the interaction between nanoparticle-complex and cell membrane receptor protein.

[0101] First, lipid nanoparticles were fixed on the surface plasmon resonance CM5 chip by carboxyl reaction. Specifically, 100 mM 1-ethyl-(3-dimethylaminopropyl) carbodiimide (EDC) and 50 mM N-hydroxysuccinimide (NHS) were mixed in a 1:1 ratio and the CM5 chip was activated for 1200 seconds. LNPs were prepared with 10 mM sodium acetate at pH 4.5 at a concentration of 0.1 mg / mL and injected for 300 seconds. Then, 1 M ethanolamine aqueous solution at pH 8.5 was used for blocking for 1200 seconds.

[0102] Then, by continuous injection, 200 nM of the protein corona component anti-PEG-IgG was first flowed over the chip surface, and then 200 nM of the cell membrane receptor CSF2RB was flowed over the chip surface. The surface plasmon resonance was used to detect the binding of lipid nanoparticles to anti-PEG-IgG to form a protein corona and then to the cell membrane receptor CSF2RB ( Fig.12 ).

[0103] Example 7 This example provides the effect of knocking down the cell membrane receptor gene on nanoparticle uptake in cells to verify the biological function of the cell membrane receptor.

[0104] The cell membrane receptor CSF2RB gene was knocked down in HL-60 cells. Then, DiR dye-labeled lipid nanoparticles were prepared. DiR dye-labeled lipid nanoparticles were incubated with HL-60 cells for 1.5 hours, and then the uptake of lipid nanoparticles by HL-60 cells was detected by flow cytometry. Fig.13 The uptake of lipid nanoparticles by HL-60 cells after CSF2RB gene knockdown was significantly reduced compared with wild-type HL-60 cells, indicating that the cell membrane receptor CSF2RB does play an important role in the cellular uptake of lipid nanoparticles.

[0105] In summary, the method of the present invention can be used to quickly and conveniently detect, identify and verify cell membrane receptor proteins bound to the surface of nanoparticles.

[0106] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for detecting the interaction between nanoparticles and cell membrane receptors, characterized in that: include: Immobilizing the nanoparticles to be tested on the surface of the biosensor; The nanoparticles to be tested fixed on the surface of the biosensor are placed in a cell membrane receptor solution to be tested for incubation; the interaction between the nanoparticles and the cell membrane receptors is detected using biomembrane layer interference technology; the cell membrane receptor solution to be tested contains 0.01-1 mg / mL of sodium alginate.

2. The method for detecting the interaction between nanoparticles and cell membrane receptors according to claim 1, characterized in that: The fixing method is selected from at least one of the following: (1) Direct adsorption of nanoparticles based on the carboxyl groups on the surface of the biosensor; (2) Using EDC / NHS to activate the carboxyl groups on the biosensor surface, immobilizing the nanoparticles by chemically reacting with the activated carboxyl groups, and then blocking with ethanolamine; (3) The nanoparticles are fixed on the biosensor surface through electrostatic, van der Waals forces, hydrogen bonding, π-π or hydrophobic interactions.

3. The method for detecting the interaction between nanoparticles and cell membrane receptors according to claim 1 or 2, characterized in that: The cell membrane receptor to be tested is prepared into a solution with PBS; Preferably, the concentration of the cell membrane receptor solution to be tested is preferably 1 nM-5 uM, and the pH is preferably 1-9.

4. The method for detecting the interaction between nanoparticles and cell membrane receptors according to any one of claims 1 to 3, characterized in that: The cell membrane receptor to be tested is prepared into different concentration gradients as an analyte, and the affinity between the nanoparticles and the cell membrane receptor to be tested is detected; Preferably, at least four analytes with concentration gradients are configured.

5. An in situ identification and quantification method of surface cell membrane receptor components of nanoparticles, characterized in that: include: Immobilizing the nanoparticles to be tested on the surface of the biosensor; The nanoparticles to be tested fixed on the surface of the biosensor are placed in a cell / cell membrane lysis solution for incubation; the binding of the nanoparticles to the protein is monitored using a biomembrane interferometry technique, and then the protein is eluted with an elution solution, and the cell membrane receptor components are identified using a mass spectrometry technique; the cell / cell membrane lysis solution contains 0.01-1 mg / mL of sodium alginate.

6. The in situ identification and quantification method of surface cell membrane receptor components of nanoparticles according to claim 5, characterized in that: The fixing method is selected from at least one of the following: (1) Direct adsorption of nanoparticles based on the carboxyl groups on the surface of the biosensor; (2) Using EDC / NHS to activate the carboxyl groups on the biosensor surface, immobilizing the nanoparticles by chemically reacting with the activated carboxyl groups, and then blocking with ethanolamine; (3) The nanoparticles are fixed on the biosensor surface through electrostatic, van der Waals forces, hydrogen bonding, π-π or hydrophobic interactions.

7. The in situ identification and quantification method of surface cell membrane receptor components of nanoparticles according to claim 5 or 6, characterized in that: The concentration of cell / cell membrane lysate is 0.1-5 mg / mL; Preferably, the cell lysate is obtained by lysing cells with NP-40 or RIPA solution and then taking the supernatant by centrifugation; and / or, the cell membrane lysate is obtained by extracting with a GPCR extraction and stabilization reagent and then taking the supernatant by centrifugation.

8. The in situ identification and quantification method of surface cell membrane receptor components of nanoparticles according to any one of claims 5 to 7, characterized in that: The nanomaterials fixed on the biosensor directly interact with the cell membrane receptors; or, the nanomaterials fixed on the biosensor first interact with the body fluid to form a protein corona, and then interact with the cell membrane receptors; the dilution multiple of the body fluid is 5-20 times; the body fluid can be selected from plasma and lymph.

9. The in situ identification and quantification method of surface cell membrane receptor components of nanoparticles according to any one of claims 5 to 8, characterized in that: The eluent is trifluoroacetic acid of different concentrations; preferably, the eluent is a 0.001%-0.5% trifluoroacetic acid aqueous solution; wherein 0.001-0.1% trifluoroacetic acid aqueous solution is used to elute proteins that are weakly bound to the surface of the nanomaterial, and 0.1-0.5% trifluoroacetic acid aqueous solution is used to elute proteins that are strongly bound to the surface of the nanomaterial.

10. The in situ identification and quantification method of surface cell membrane receptor components of nanoparticles according to any one of claims 5 to 9, characterized in that: After the cell membrane receptor components are identified by mass spectrometry, the affinity between the cell membrane receptors and the nanoparticles is detected by the method for detecting the interaction between the nanoparticles and the cell membrane receptors according to any one of claims 1 to 4.

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