Application of C1q complement protein in preparation of element for detecting Abeta42 protein, chip for detecting Abeta42 protein and application of chip

Abstract

The invention provides application of C1q complement protein in preparation of an element for detecting Abeta42 protein, a chip for detecting the Abeta42 protein and application of the chip. The C1q complement protein is used as an element for specifically recognizing the Abeta42 protein, the C1q complement protein can be fixed on the chip body, and the obtained chip can be used for preparing a specific element, such as a biosensor. Compared with the prior art, when the biosensor based on the dual-polarization interference technology is used for detecting the Abeta42 protein, synthesis of a complex nano material or design of a complex signal transduction pathway is not needed, and the biosensor has the advantages of being easy and convenient to operate, high in selectivity and high in renewability.

Description

Technical Field

[0001] The present invention belongs to the field of biosensor technology, and specifically relates to a C1q complement protein for the preparation and detection of Aβ 42 Application of protein components, a method for detecting Aβ 42 Protein chips and their applications. Background Art

[0002] Alzheimer's disease (AD) is a neurodegenerative disease characterized by progressive cognitive dysfunction and behavioral impairment, which seriously threatens the health and quality of life of the elderly. One of the main pathological characteristics of AD is the abnormal aggregation of amyloid β protein (Aβ) in the brain, forming neuritic plaques. Aβ is a polypeptide fragment produced by β-secretase and γ-secretase from amyloid precursor protein (APP), which is usually 39 to 43 amino acids in length. Among them, Aβ 40 and Aβ 42 are the two main forms. 40 The content in the brain is relatively high, but Aβ 42 Because it is more prone to aggregation and has higher toxicity, it is considered a key molecule in the pathogenesis of AD. 42 Proteins are relatively stable under physiological conditions, but when their concentration exceeds a certain threshold, they begin to aggregate to form oligomers, fibers and eventually inflammatory plaques. These aggregates not only interfere with the normal function of nerve cells, but also trigger a series of inflammatory reactions and oxidative stress, leading to neuronal damage and death, and then causing symptoms such as cognitive dysfunction and memory loss.

[0003] Currently, the diagnosis of AD mainly relies on clinical symptom assessment, neuroimaging examination and neuropsychological testing, but these methods can only make a diagnosis after the disease has progressed to a certain extent, which may lead to missing the best treatment opportunity. 42 The level of protein is of vital importance for the early diagnosis of AD, monitoring of disease progression and evaluation of treatment effects. 42 Protein concentration can also provide an important basis for studying the pathogenesis of AD, developing new treatments, and evaluating drug efficacy.

[0004] Currently, Aβ 42 Many detection methods have been reported, such as enzyme-linked immunosorbent assay, fluorescence spectroscopy, colorimetry, etc. However, most of these technologies are based on different signal transduction mechanisms and are constructed by labeling or introducing various signal probes, which requires the synthesis of complex nanomaterials or the design of complex signal transduction pathways. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide a C1q complement protein for the preparation and detection of Aβ 42 Application of protein components, a method for detecting Aβ 42 Protein chip and its application. The present invention uses C1q complement protein as a specific recognition element, does not require additional fluorophores or nanomaterials, and has the advantages of easy construction, strong reproducibility, low cost, and good selectivity.

[0006] To achieve this object, the present invention adopts the following technical solutions:

[0007] In the first aspect, the present invention provides a C1q complement protein for preparing and detecting Aβ 42 Application in protein components;

[0008] The C1q complement protein is a complex protein composed of six identical heterotrimers, each of which contains a C1qA chain (sequence shown in SEQ ID No: 1), a C1qB chain (sequence shown in SEQ ID No: 2) and a C1qC chain (sequence shown in SEQ ID No: 3). One C1qA chain, one C1qB chain and one C1qC chain constitute a heterotrimer.

[0009] In a second aspect, the present invention provides a method for detecting Aβ 42 A protein chip comprising a chip body and a C1q complement protein immobilized on the chip body;

[0010] The C1q complement protein is a complex protein composed of six identical heterotrimers, each of which contains a C1qA chain (sequence shown in SEQ ID No: 1), a C1qB chain (sequence shown in SEQ ID No: 2) and a C1qC chain (sequence shown in SEQ ID No: 3).

[0011] Preferably, the surface of the chip body contains functional groups, and the functional groups are amino groups.

[0012] Preferably, the reagent used for the functional group modification is selected from a silanization reagent with an amino group, such as (3-aminopropyl)trimethoxysilane, 3-aminopropyltriethoxysilane, N-(2-aminoethyl)-3-aminopropyltrimethoxysilane, N-(2-aminoethyl)-3-aminopropyltriethoxysilane, etc.

[0013] Preferably, the chip body and the C1q complement protein are connected via an imine bond (-C=N-). Specifically, a substance with two aldehyde groups, such as glutaraldehyde, can be used. The two aldehyde groups react with the amino groups on the surface of the amino-modified chip and the amino groups of the C1q complement protein to form a Schiff base reaction to form an imine bond (-C=N-).

[0014] Preferably, the ratio of the C1q complement protein to the chip body is 100-300 μL of 0.1-0.3 μmol / L complement protein C1q per chip body, preferably 200 μL of 0.1 μmol / L complement protein C1q per chip body.

[0015] In a third aspect, the present invention provides a chip for detecting Aβ 42 Application in protein components.

[0016] In a fourth aspect, the present invention provides a biosensor based on dual polarization interferometry technology, comprising a dual polarization interferometer instrument and a sensor chip mounted on the dual polarization interferometer instrument;

[0017] The sensor chip is the chip involved in the above technical solution.

[0018] In a fifth aspect, the present invention provides a method for preparing the above-mentioned biosensor, comprising the following steps:

[0019] S1: Perform baseline stabilization and calibration on the dual-polarization interferometer equipped with the chip body;

[0020] S2: Injecting a cross-linking agent and C1q complement protein into the surface of the chip body, and after a cross-linking reaction occurs, a sensor chip is obtained, and then a biosensor is obtained.

[0021] Preferably, the cross-linking agent is a glutaraldehyde solution; the solvent of the glutaraldehyde solution is a PBS buffer solution; the mass fraction of the glutaraldehyde solution is 0.1-1.0%, preferably 0.4%.

[0022] Preferably, after the cross-linking reaction is completed, a capping agent is introduced to the surface of the chip body; the capping agent is selected from ethanolamine, lysine or bovine serum albumin.

[0023] Preferably, the blocking agent is an ethanolamine solution with a pH of 7 to 10, preferably pH=8;

[0024] Alternatively, the capping agent is lysine at a pH of 8-10.

[0025] Alternatively, the blocking agent is bovine serum albumin (BSA) with a pH of 7-8.

[0026] It should be noted that within the above pH range, if the pH is too low, the free amino groups will be reduced and the blocking efficiency will be reduced; on the contrary, if the pH is too high, it may cause changes in protein conformation or changes in the surface charge of the chip, which may affect the experimental results. The present invention also provides a biosensor for label-free detection of Aβ 42Protein method, which can be for diagnostic or non-diagnostic purposes. If it is for non-diagnostic purposes, its purpose is to study the mechanism of action of proteins through molecular interactions. Specifically, the method comprises the following steps:

[0027] The sensor chip is used to detect the sample to be tested. After obtaining the signal, the Aβ content in the sample to be tested is obtained according to the standard curve method. 42 Protein concentration.

[0028] Preferably, after the sensor chip detects the sample to be tested, an eluent is used to remove Aβ 42 The protein is removed and the sensor chip is regenerated to continue detecting the sample to be tested.

[0029] Preferably, the eluent is selected from glycine solution, guanidine hydrochloride solution or urea solution.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] The present invention provides a C1q complement protein for preparing and detecting Aβ 42 Based on the application of the protein element, a method for detecting Aβ was developed 42 Protein chip, and corresponding detection element, which can be a biosensor based on dual polarization interference technology.

[0032] In the present invention, the biosensor can be used to detect amyloid β protein (Aβ 42 ), which specifically uses complement protein C1q as a specific recognition element and uses dual polarization interferometry technology to monitor the interaction between C1q complement protein and Aβ in real time 42 The specific binding events between proteins can obtain real-time data such as the mass, thickness, and density of the interface layer before, during, and after the binding, thereby obtaining the thickness of the interface layer and the relationship between the thickness of the interface layer and the Aβ 42 Linear regression equation of protein concentration, mass and Aβ 42 Linear regression equations for protein concentration, density and Aβ 42 The linear regression equation of protein concentration is calculated, and then the mass, thickness, density and other data of the interface layer obtained after the actual sample to be tested is substituted into the corresponding regression equation to finally obtain the actual Aβ content in the sample to be tested. 42 Protein concentration.

[0033] Compared with traditional Aβ 42 Compared with protein detection methods, this biosensor does not require additional material synthesis or labeling process, is simple and convenient to operate, low in cost, highly selective, and is reproducible in actual detection and can be reused. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 This is a graph showing the real-time mass, thickness, and density changes of the interface layer when glutaraldehyde, Clq, and ethanolamine are sequentially added to the surface of the sensor chip in Example 1;

[0035] Figure 2 Schematic diagram of the construction and detection principle of the label-free biosensor based on dual-polarization interferometry technology in the present invention;

[0036] Figure 3 Different concentrations of Aβ 42 Protein corresponding mass signal diagram and mass signal and target Aβ 42 Protein (Aβ 42 M) concentration relationship diagram;

[0037] in, Figure 3 A represents different concentrations of Aβ 42 Protein (Aβ 42 M) The corresponding quality signal diagram, Figure 3 B is the mass signal and target Aβ 42 Protein (Aβ 42 M) concentration, Figure 3 C is the target Aβ 42 Protein (Aβ 42 M) Standard calibration curve of the test;

[0038] Figure 4 Different concentrations of Aβ 42 Thickness signal diagram corresponding to the protein and thickness signal and target Aβ 42 Protein (Aβ 42 M) concentration relationship diagram;

[0039] in, Figure 4 A represents different concentrations of Aβ 42 Thickness signal diagram corresponding to the protein, Figure 4 B is the thickness signal and target Aβ 42 Protein (Aβ 42 M) concentration, Figure 4 C is the target Aβ 42 Protein (Aβ 42 M) Standard calibration curve of the test;

[0040] Figure 5 Different concentrations of Aβ 42 Density signal map corresponding to the protein and density signal and target Aβ 42 Protein (Aβ 42 M) concentration relationship diagram;

[0041] in, Figure 5 A represents different concentrations of Aβ 42 Density signal map corresponding to the protein, Figure 5B is the density signal and target Aβ 42 Protein (Aβ 42 M) concentration, Figure 5 C is the target Aβ 42 Protein (Aβ 42 M) Standard calibration curve of the test;

[0042] Figure 6 A result diagram of the selectivity test of the biosensor provided by the present invention;

[0043] Figure 7 This is a graph showing the test results of the reproducibility of the biosensor provided by the present invention. DETAILED DESCRIPTION

[0044] The following will clearly and completely describe the technical solutions of the present invention in conjunction with the embodiments of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0045] The present invention provides a C1q complement protein for preparing and detecting Aβ 42 Application of protein components.

[0046] The present invention also provides a method for detecting Aβ 42 A protein chip comprises a chip body and C1q complement protein immobilized on the chip body. The ratio of C1q complement protein to the chip body is 100-300 μL of 0.1-0.3 μmol / L complement protein C1q per chip body, preferably 200 μL of 0.1 μmol / L complement protein C1q per chip body.

[0047] In the present invention, the C1q complement protein is a complex protein composed of six identical heterotrimers, each of which contains a C1qA chain (sequence shown in SEQ ID No: 1), a C1qB chain (sequence shown in SEQ ID No: 2) and a C1qC chain (sequence shown in SEQ ID No: 3).

[0048] In the present invention, the surface of the chip body contains functionalized amino groups to facilitate cross-linking with the C1q complement protein. The functionalized groups are modified using reagents selected from amino-containing silanization reagents, such as (3-aminopropyl)trimethoxysilane, 3-aminopropyltriethoxysilane, N-(2-aminoethyl)-3-aminopropyltrimethoxysilane, and N-(2-aminoethyl)-3-aminopropyltriethoxysilane. The chip body and the C1q complement protein are preferably linked via an imine bond (-C=N-). Specifically, a substance with two aldehyde groups, such as glutaraldehyde, can be used. The two aldehyde groups react with the amino groups on the surface of the aminated chip and the amino groups of C1q, respectively, to form an imine bond (-C=N-).

[0049] The present invention also provides a method for detecting Aβ by using the above chip. 42 Application in protein instruments, wherein the instrument may be a biosensor.

[0050] It has been reported that Dual Polarization Interferometry (DPI), as an optical analysis technique, provides a new solution for the detection of biomolecular interactions. Based on the principle of Thomas-Young interference, DPI technology can monitor the binding and dissociation processes of biomolecules on the chip surface in real time and without labels by measuring the phase difference changes between two beams of polarized light during the interaction of biomolecules. Compared with traditional technologies such as surface plasmon resonance (SPR) and quartz crystal microbalance (QCM-D), DPI technology has higher sensitivity and resolution and can provide richer information, including the adsorption mass, thickness, density, and binding kinetic parameters of biomolecules. In addition, DPI technology has the advantages of simple operation, no need for complex labeling process, and low sample requirements, making it particularly suitable for detecting specific interactions between biomolecules.

[0051] The DPI instrument primarily consists of a helium-neon laser light source, a polarization conversion controller, an interference signal acquisition device, a microfluidic system, and a temperature control system. The microfluidic system primarily consists of a syringe pump, a six-port valve, and a dosing loop. The syringe pump ensures that the buffer solution is uniformly and continuously injected into the flow system and carries the sample added to the dosing loop into the sensor waveguide. The temperature control system maintains the system temperature at 20°C. The laser light source generates polarized light through a polarization conversion controller. This polarized light is incident on one end of the chip, propagates through the chip, and then exits, interfering with it, forming alternating light and dark interference fringes in the far field. These interference fringes are recorded in real time and processed by computer software to derive phase changes in the transverse magnetic field (TM) and transverse electric field (TE). Data processing can then be used to translate these phase changes into changes in the mass, thickness, and density of the chip surface. Figure 2A schematic diagram shows how a laser illuminates a chip, forms interference fringes in the far field, and obtains phase changes in the transverse magnetic field (TM) and transverse electric field (TE).

[0052] Currently, biosensors based on protein-specific interactions as recognition elements mostly utilize electrochemical, fluorescence, or colorimetric methods for detection, but these often require the synthesis of additional signaling molecules and labeling. In response to this, the present invention provides a biosensor based on dual-polarization interferometry technology, comprising a dual-polarization interferometer instrument and a sensor chip mounted on the instrument.

[0053] In this application, the dual-polarization interferometer (DPI) instrument is the AnaLightBio200 from Farfield Sensors, a UK company. This DPI instrument primarily includes a He-Ne laser source (wavelength: 632.8 nanometers), a sensor chip, a polarization conversion controller, an interference signal acquisition device, a microfluidics system, and a temperature control system.

[0054] In the present invention, the sensor chip is the chip described in the above-mentioned technical solution, comprising a chip body and C1q complement protein immobilized on the chip body, preferably comprising a functionalized chip body and C1q complement protein cross-linked to the surface of the functionalized chip body. The functionalized chip body and C1q complement protein undergo Schiff base reactions with amino groups on the surface of the amino-encapsulated chip and amino groups of C1q, respectively, via two aldehyde groups of glutaraldehyde, to form imine (-C=N-) crosslinks.

[0055] Among them, the functionalized chip body is preferably an amino chip body, which is specifically obtained by treating the chip body with an amino reagent; the amino reagent is selected from silane coupling agents such as (3-aminopropyl)trimethoxysilane, 3-aminopropyltriethoxysilane, N-(2-aminoethyl)-3-aminopropyltrimethoxysilane, and N-(2-aminoethyl)-3-aminopropyltriethoxysilane; the chip body is selected from silicon oxynitride chip or glass / silicon-based integrated chip.

[0056] In some embodiments of the present invention, the amino chip body is prepared according to the following method:

[0057] Immerse the chip in piranha acid solution (V 硫酸 :V 双氧水 =7:3) for 12 hours to remove surface impurities and activate the surface. After cleaning, the chip was immersed in 10% (v / v) (3-aminopropyl)trimethoxysilane (APTMS) at 37°C for 2 hours to functionalize the chip surface with amino groups. The chip was ultrasonically cleaned three times with ultrapure water and ethanol, then dried with nitrogen gas to obtain the amino-functionalized chip body.

[0058] The above parameters can be adjusted within a reasonable range.

[0059] The present invention also provides a method for preparing the above-mentioned biosensor, comprising the following steps:

[0060] S1: Perform baseline stabilization and calibration on the dual-polarization interferometer equipped with the chip body;

[0061] S2: Injecting a cross-linking agent and C1q complement protein into the surface of the chip body, and after a cross-linking reaction occurs, a sensor chip is obtained, and then a biosensor is obtained.

[0062] According to the present invention, a dual polarization interferometer is first provided, which is equipped with a chip body, preferably a functionalized chip body. The functionalized chip body is prepared according to the relevant contents of the above technical solution, which will not be repeated here.

[0063] In the present invention, it is preferred to perform baseline stabilization processing on the dual polarization interferometer.

[0064] For example, 10 mmol / L PBS (containing 150 mmol / L NaCl, pH = 7.4) is used as the buffer mobile phase solution for the entire process of the dual polarization interferometer instrument, and the buffer mobile phase solution is set to flow through the functionalized chip surface to quickly remove the air in the channel and achieve a stable baseline.

[0065] After the baseline stabilization process is completed, a calibration process is performed according to the present invention.

[0066] For example, an 80% (v / v) ethanol / water solution and pure water with known refractive index are injected onto the surface of the functionalized chip for calibration of the functionalized chip.

[0067] According to the present invention, after the calibration process is completed, a cross-linking agent and C1q complement protein are injected into the surface of the chip body (or functionalized chip body), and after a cross-linking reaction occurs, a sensor chip is obtained, and then a biosensor is obtained.

[0068] In some embodiments of the present invention, after calibration, a glutaraldehyde solution is preferably added to the surface of the chip (or functionalized chip) as a crosslinker. 0.1 μmol / L of C1q complement protein is then added to immobilize the protein on the functionalized chip surface via a Schiff base reaction. In the present invention, the solvent for the glutaraldehyde solution is PBS buffer, and the mass fraction of the glutaraldehyde solution is 0.1-1.0%, preferably 0.4%.

[0069] In some preferred embodiments of the present invention, after the reaction is completed, a capping agent is preferably introduced to the surface of the sensor chip to cap the unreacted aldehyde groups to obtain the sensor chip.

[0070] In the present invention, the capping agent is selected from ethanolamine, lysine or bovine serum albumin.

[0071] In some embodiments of the present invention, the capping agent is an ethanolamine solution with a pH of 7 to 10, preferably a pH of 8, 0.1 mol / L ethanolamine;

[0072] Alternatively, the capping agent is lysine at a pH of 9.0 to 9.6;

[0073] Alternatively, the blocking agent is bovine serum albumin (BSA) with a pH of 7.4-7.5.

[0074] Within the above pH range, if the pH is too low, the free amino groups will be reduced and the blocking efficiency will be reduced; conversely, if the pH is too high, it may cause changes in protein conformation or changes in chip surface charge, affecting the experimental results.

[0075] The present invention also provides a biosensor for label-free detection of amyloid β protein (Aβ 42 protein).

[0076] The present invention also provides a biosensor for label-free detection of amyloid β protein (Aβ 42 Protein) method, comprising the steps of:

[0077] The sensor chip is used to detect the sample to be tested. After obtaining the signal, the Aβ content in the sample to be tested is obtained according to the standard curve method. 42 Protein concentration.

[0078] In more detail, the following steps are included:

[0079] S1: Different concentrations of Aβ 42 The protein solution was injected into the surface of the sensor chip in the biosensor, and the interaction between the sensor chip and Aβ was monitored in real time by a dual polarization interferometer. 42 After the entire experiment, the data were analyzed using AnaLightBio200 analysis software, and it was found that the interface layer parameters (including the thickness, mass, and density of the interface layer) were significantly related to the target Aβ 42 The concentration of the protein is linearly related within a certain range, and the thickness of the chip surface interface layer and Aβ 42 Linear regression equation of protein concentration, mass and Aβ 42 Linear regression equations for protein concentration, density and Aβ 42 Linear regression equation for protein concentration;

[0080] S2: The sample to be tested is injected into the surface of the sensor chip in the biosensor, and the binding event between the sensor chip and the sample to be tested is monitored in real time by a dual polarization interferometer. After completion, the data is analyzed using software to obtain the thickness, mass, and density of the interface layer. According to the linear regression equation obtained in step S1, the Aβ content in the sample to be tested is determined. 42 Protein concentration.

[0081] The above-mentioned samples to be tested refer to the samples that actually need to be tested for Aβ 42 The concentration of protein in the solution.

[0082] After investigation, it was found that the biosensor provided by the present invention is sensitive to Aβ 42 Protein has excellent selectivity.

[0083] Furthermore, the biosensor provided by the present invention is used to detect Aβ in samples to be detected. 42 After the protein concentration is detected, Aβ is removed using an elution agent. 42 The protein is removed and the sensor chip is regenerated to continue detecting the sample to be tested.

[0084] Specifically, in some embodiments of the present invention, preferably after the above step S2 is completed, a glycine solution is injected into the surface of the sensor chip that has been tested with the sample to be tested, so that the surface of the sensor chip and the Aβ bound to the C1q complement protein are 42 The protein is eluted to regenerate the surface of the sensor chip, and then step S2 is repeated to perform subsequent detection of the sample to be detected. The glycine solution is preferably a 15 mmol / L glycine solution (pH=2.0).

[0085] After investigation, it was found that the regenerated chip can be used again for Aβ 42 Protein detection showed that the biosensor had good reproducibility.

[0086] In summary, compared with the existing Aβ 42 Compared with the protein detection method, the biosensor provided by the present invention does not require additional antibody labeling process and additional material synthesis process. The required raw materials are all commercial products. It has the advantages of simple operation, convenience, high selectivity, and can be eluted by glycine to bind Aβ. 42 Protein, thereby regenerating the sensing interface and making it reusable.

[0087] To further illustrate the present invention, the following examples are provided for detailed description. The dual polarization interferometer used in the following examples is an AnaLight Bio200 instrument from Farfield Sensors, UK.

[0088] Example 1

[0089] This embodiment provides a method for preparing a biosensor based on dual polarization interferometry technology, such as Figure 1 As shown, the specific steps include:

[0090] Step 1: Soak the silicon oxynitride chip in piranha acid solution (V 硫酸 :V 双氧水 =7:3) for 12 hours to remove surface impurities and activate the surface. After cleaning, the chip surface was functionalized with amino groups by soaking in 10% (v / v) (3-aminopropyl)trimethoxysilane (APTMS) at 37°C for 2 hours. The chip was ultrasonically cleaned three times with ultrapure water and ethanol, then dried with nitrogen gas to obtain the amino-functionalized chip.

[0091] Step 2: The amino-functionalized chip was mounted on a dual-polarization interferometer (AnaLight Bio200, Farfield). 10 mM PBS (containing 150 mM NaCl, pH 7.4) was used as the mobile phase throughout the analysis. The buffer solution was flowed over the chip at a rate of 50 μL / min to rapidly remove air from the channel and achieve baseline stability.

[0092] Step 3: Inject 200 μL of an 80% (v / v) ethanol / water solution over the chip surface. After 4 minutes, switch back to the buffer solution. Once the baseline stabilizes, repeat the above steps by injecting 200 μL of pure water over the chip surface. After 4 minutes, switch back to the buffer solution. Calibrate the instrument using the known refractive indices of the two solutions.

[0093] Step 4: After calibration, change the buffer flow rate to 20 μL / min. Inject 100 μL of 0.4% glutaraldehyde solution onto the amino-functionalized chip surface as a crosslinker. After the baseline stabilizes, add 200 μL of 0.1 μM complement protein C1q to immobilize it on the chip surface via a Schiff base reaction. Then, block the chip twice with 100 μL of 0.1 M ethanolamine (pH 8.0) to obtain a C1q-modified chip.

[0094] Figure 1 It is a curve diagram of the real-time changes in mass, thickness, and density when glutaraldehyde, C1q, and ethanolamine are added to the chip surface in sequence.

[0095] Example 2

[0096] In this example, the biosensor according to the embodiment is used for Aβ 42 Protein concentration detection includes the following steps:

[0097] Step 1: This step is the same as step 1 in Example 1.

[0098] Step 2: This step is the same as step 2 in Example 1.

[0099] Step 3: This step is the same as step 3 in Example 1.

[0100] Step 4: This step is the same as step 4 in Example 1.

[0101] Step 5: Add 200 μL of Aβ at different concentrations (0.5, 0.8, 1, 3, 5, 10, 15, 20, 25 μmol / L) 42 The protein solution was injected and flowed over the modified chip surface obtained in step 4 of Example 1. Its binding to C1q was monitored in real time using dual-polarization interferometry. After completion, the data were analyzed using AnaLight Bio200 analysis software. The resulting data revealed changes in the thickness, mass, and density of the interface layer over time.

[0102] The overall experimental process of Example 2 is as follows Figure 2 Each concentration of Aβ was injected 42 The initial conditions of the data of the interface layer parameters (mass, thickness and density) of the protein solution over time are reset to zero, that is, the time and interface layer parameters at the starting point all start from 0. Then different concentrations of Aβ are added. 42 The changes in chip interface layer parameters after protein solution are shown in one figure. Figure 3 A, 4A, and 5A. Arrows indicate the direction of Aβ 42 The protein concentration was increased from 0.5 to 25 μmol / L. The equilibrium binding data for 800 seconds after adding the sample was used as the interface layer signal of the detection result and Aβ 42 Protein concentration was plotted to obtain Figure 3 B, 4B and 5B. In the concentration range of 0.5 to 10 μmol / L, the interface layer signal showed a linear relationship with the protein concentration, and its standard calibration curve is shown in Figure 3 As shown in C~5C.

[0103] Example 3

[0104] This embodiment provides an Aβ based on dual polarization interferometry technology. 42 The specific steps for selective detection of protein biosensors are as follows:

[0105] Step 1: This step is the same as step 1 in Example 1.

[0106] Step 2: This step is the same as step 2 in Example 1.

[0107] Step 3: This step is the same as step 3 in Example 1.

[0108] Step 4: This step is the same as step 4 in Example 1.

[0109] Step 5: 200 μL of different proteins (Aβ 42 protein, Aβ 42 Solutions of oligomers (e.g., bovine serum albumin, human serum albumin, and β-lactoglobulin) and a 10 mM PBS buffer (containing 150 mM NaCl, pH 7.4) as a blank sample were injected and flowed over the modified chip surface. Binding events to C1q were monitored in real time using dual-polarization interferometry. Data were analyzed using AnaLight Bio200 analysis software. The resulting data revealed temporal changes in the thickness, mass, and density of the interface layer.

[0110] Take the equilibrium data 800 seconds after adding the protein sample as the test result and draw a bar graph to get Figure 6 Aβ 42 M, Aβ 42 O, BSA, HSA, BLG and Blank represent Aβ 42 protein, Aβ 42 oligomers, bovine serum albumin, human serum albumin, β-lactoglobulin and blank control. Figure 6 A~C are the changes in mass, thickness and density of the chip surface after adding different proteins at the same concentration (10 μmol / L), and the error bars are the standard deviations of three measurements. 42 After the protein was added, the quality, thickness and density of the chip surface changed significantly, indicating that the biosensor has good selectivity.

[0111] Example 4

[0112] This embodiment provides an Aβ based on dual polarization interferometry technology. 42 The reproducibility test of the protein biosensor includes the following steps:

[0113] Step 1: This step is the same as step 1 in Example 1.

[0114] Step 2: This step is the same as step 2 in Example 1.

[0115] Step 3: This step is the same as step 3 in Example 1.

[0116] Step 4: This step is the same as step 4 in Example 1.

[0117] Step 5: Add 200 μl of different concentrations of Aβ 42The protein solution was injected and flowed over the modified chip surface, and its binding event with C1q was monitored in real time using dual-polarization interferometry technology.

[0118] Step 6: Inject 100 μL of 15 mmol / L glycine solution (pH = 2.0) into the Aβ-tested 42 The chip surface of the protein is connected to the Aβ 42 The protein is eluted and the chip surface is regenerated.

[0119] The surface quality of the chip that has not been tested is compared with the surface quality of the chip that has been tested each time Aβ is injected. 42 The surface quality of the chip after protein and glycine is obtained by plotting the dot-line graph according to the experimental sequence. Figure 7 The vertical axis represents the chip surface quality under different states, and the horizontal axis represents the experimental order. The purple and brown dotted circles represent the initial state without detection and the state after adding glycine solution, respectively. The solid circles represent the state after adding 3 μmol / L (red), 5 μmol / L (green), and 10 μmol / L (blue) Aβ solution. 42 The error bars are the standard deviation of three measurements. 42 After the protein was added, the quality of the chip surface increased significantly. After adding glycine solution, the Aβ bound to the chip surface could be 42 The protein was eluted to restore it to the level before detection.

[0120] In this embodiment, the regenerated chip can be used again for Aβ 42 Protein detection showed that the biosensor had good reproducibility.

[0121] The above description of the disclosed embodiments will enable one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be readily apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention is not limited to the embodiments shown herein but is to be construed in the widest manner consistent with the principles and novel features disclosed herein.

Claims

1. C1q complement protein in the preparation of Aβ detection 42 Application in protein components; The C1q complement protein is a complex protein composed of six identical heterotrimers, each of which contains a C1qA chain, a C1qB chain, and a C1qC chain; in, The sequence of the C1qA chain is shown in SEQ ID No: 1; The sequence of the C1qB chain is shown in SEQ ID No: 2; The sequence of the C1qC chain is shown in SEQ ID No:

3.

2. A method for detecting Aβ 42 A protein chip, characterized in that It includes a chip body and a C1q complement protein fixed on the chip body; The C1q complement protein is a complex protein composed of six identical heterotrimers, each of which contains a C1qA chain, a C1qB chain, and a C1qC chain; Wherein, the sequence of the C1qA chain is shown as SEQ ID No: 1; The sequence of the C1qB chain is shown in SEQ ID No: 2; The sequence of the C1qC chain is shown in SEQ ID No:

3.

3. The chip according to claim 2, characterized in that The surface of the chip body contains functional groups, and the functional groups are amino groups; The reagent used for the functional group modification is selected from a silanization reagent with an amino group; The silanizing agent having an amino group is selected from (3-aminopropyl)trimethoxysilane, 3-aminopropyltriethoxysilane, N-(2-aminoethyl)-3-aminopropyltrimethoxysilane, and N-(2-aminoethyl)-3-aminopropyltriethoxysilane.

4. The chip according to claim 2 or 3, characterized in that: The chip body and the C1q complement protein are connected via -C=N-.

5. The chip according to any one of claims 2 to 4, characterized in that The ratio of the C1q complement protein to the chip body is 100-300 microliters of 0.1-0.3 micromol / L complement protein C1q per chip body.

6. The chip according to any one of claims 2 to 5 for detecting Aβ 42 Application in protein components.

7. A biosensor based on dual polarization interferometry technology, characterized in that: It includes a dual polarization interferometer and a sensor chip mounted on the dual polarization interferometer; The sensor chip is the chip according to any one of claims 2 to 5.

8. A method for preparing a biosensor according to claim 7, characterized in that: The following steps are involved: S1: Perform baseline stabilization and calibration on the dual-polarization interferometer equipped with the chip body; S2: Injecting a cross-linking agent and C1q complement protein into the surface of the chip body, and after a cross-linking reaction occurs, a sensor chip is obtained, and then a biosensor is obtained.

9. The preparation method according to claim 8, characterized in that The cross-linking agent is a glutaraldehyde solution; the solvent of the glutaraldehyde solution is a PBS buffer solution; the mass fraction of the glutaraldehyde solution is 0.1-1.0%; After the cross-linking reaction is completed, a capping agent is introduced to the surface of the chip body; the capping agent is selected from ethanolamine, lysine or bovine serum albumin.

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