Giant magnetoresistance biosensor for detecting protease as well as preparation method and application of giant magnetoresistance biosensor

Abstract

The invention belongs to the technical field of biosensing, and particularly relates to a giant magnetoresistance biosensor for detecting protease and a preparation method and application thereof, the giant magnetoresistance biosensor comprises a giant magnetoresistance sensor, a glass sheet plated with a gold film, a functional polypeptide and a metal ion chelating magnetic bead; wherein the glass sheet plated with the gold film is fixed on the giant magnetoresistive sensor, one end of a peptide chain of the functional polypeptide is connected with the glass sheet plated with the gold film, and the other end of the peptide chain of the functional polypeptide is connected with the metal ion chelating magnetic bead; the giant magnetoresistance biosensor provided by the invention can be used for monitoring the activity of protease, is high in sensitivity, strong in anti-interference capability, stable in performance and easy to integrate, so that the giant magnetoresistance biosensor can be developed into portable detection equipment for large-scale popularization, and has a wide application prospect in the fields of diagnosis and treatment of serious diseases and development of related medicines.

Description

Technical Field

[0001] The invention belongs to the technical field of biosensors, and in particular relates to a giant magnetoresistance biosensor for detecting proteases, and a preparation method and application thereof. Background Art

[0002] Protease is a general term for a class of enzymes that hydrolyze peptide bonds in proteins or polypeptides, including β-secretase, trypsin and matrix metalloproteinases. It plays an important role in life activities such as metabolism, vascular function, immune function and cell apoptosis. Proteases are widely distributed in the human body and participate in many physiological functions of the human body. Abnormal changes in the content of some proteases in the human body are directly or indirectly related to the development of many diseases, for example,

[0003] Abnormal expression of β-secretase is associated with Alzheimer's disease; abnormal expression of trypsin is associated with pancreatic diseases such as acute pancreatitis and pancreatic cancer; abnormal expression of matrix metalloproteinase-9 is associated with chronic obstructive pulmonary disease; abnormal expression of matrix metalloproteinase-2 is associated with female pelvic floor dysfunction; therefore, monitoring of protease activity is of great significance in the biomedical field.

[0004] At present, the main methods for detecting protease activity are high performance liquid chromatography, mass spectrometry, enzyme-linked immunosorbent assay (ELISA) and fluorescence resonance energy transfer (FRET) probe method. High performance liquid chromatography and mass spectrometry are highly sensitive and have strong universality, but they require sophisticated equipment, complex operations and long-term analysis by professionals, and the analysis cost is high, which is not suitable for the needs of rapid detection. Although ELISA and FRET probe methods are also effective, they both have certain difficulties in practical applications. For example, ELISA has the disadvantages of expensive antibodies, poor repeatability, cumbersome operation and long analysis cycle. FRET fluorescent probe method is formed by connecting the fluorescent donor and the fluorescent acceptor to the two ends of the enzyme substrate peptide by chemical bonding. However, the preparation process of the fluorescent donor and the quenching acceptor is very complicated, the synthesis cost is high, and the labeling steps are cumbersome and the cycle is long. In addition, since the excitation wavelength of some easily labeled fluorescent groups often coincides with the absorption wavelength of many drug small molecules, the selection requirements of the fluorescent donor and the acceptor of the FRET probe are high. These factors limit the actual application of FRET fluorescent probes. Therefore, in order to meet the needs of practical applications, there is an urgent need to develop new methods that are versatile, sensitive, simple and rapid to monitor protease activity.

[0005] In recent years, the giant magnetoresistance (GMR) biosensor, which is developed by combining the giant magnetoresistance (GMR) sensor with magnetic labeling technology and biosensing technology, has attracted much attention in the field of biosensing technology due to its advantages of high sensitivity, fast response, integration and miniaturization. GMR biosensor is a kind of biological probe that detects biological samples through magnetic labeling. Since the magnetic label is very stable, not affected by photobleaching, and does not have strong environmental noise, it also has unique advantages in stability and anti-interference ability. Currently, researchers have used GMR biosensors to detect liver cancer genotyping (Xiao Zhi et al. Lab Chip, 2012, 12, 741-745), DNA mutations (G. Rizzi et al. ACS Nano, 2017, 11, 8864-8870.), influenza activation genes (N. Ravi et al. Biosensors and Bioelectronics, 2022, 205, 114086-9), anti-SARS-CoV-2 neutralizing antibodies (E. Ng et al. Sensors and Actuators B: Chemical, 2023, 387, 133773-6), C-reactive protein (Meng-Zhe Tsai et al. Analyst, 2018, 143, 503-510) and osteosarcoma cells (D. Su et al. Sensors and Actuators A, 2023, 1, 114115-9). These research works are mainly based on nucleic acid hybridization reactions between DNA molecules or immune reactions between antigens and antibodies to achieve recognition between biological molecules, thereby establishing biosensing methods. Therefore, the biological recognition elements of the sensor are DNA probes or antibodies and antigens. However, research on establishing biosensing methods using the interaction between polypeptides and enzymes has rarely been reported.

[0006] In fact, as a biorecognition element of a biosensor, peptides are stable, reliable, low-cost, easy to synthesize and modify, and have advantages such as high affinity and strong biological activity compared to antibodies, and have unique advantages in biomolecule detection (Y. Jia et al. Chem. Res. Chinese Universities, 2021, 37 (4), 1130-6.). Therefore, if functional peptides can be used as biorecognition elements to construct GMR biosensors for the detection of proteases, the advantages of the fusion of magnetic labels, peptide probes, and GMR sensors can be greatly utilized, providing a new tool and means for protease detection and its inhibitor screening.

[0007] In view of the above technical background, the present invention provides a giant magnetoresistance biosensor for detecting proteases, and its preparation method and application, and uses the sensor to achieve simple and sensitive detection of proteases, which has broad application prospects in the diagnosis and treatment of major diseases and related drug development. Summary of the invention

[0008] The object of the present invention is to provide a giant magnetoresistance biosensor for detecting proteases, comprising a giant magnetoresistance biosensor, a glass sheet coated with a gold film, a functional polypeptide and metal ion chelated magnetic beads;

[0009] The glass sheet coated with gold film is fixed on a giant magnetoresistance sensor, the amino acid sequence of the functional polypeptide contains a recognition peptide segment of a protease in the middle, a cysteine ​​residue containing a thiol group at the carbon end or the nitrogen end, and a His tag at the other end, the functional polypeptide is connected to the glass sheet coated with gold film via the thiol group on the cysteine ​​residue, and the functional polypeptide is connected to the metal ion chelated magnetic beads via the His tag.

[0010] Preferably, the metal ion in the metal ion chelated magnetic beads is Co 2+ or Ni 2+ .

[0011] Preferably, the protease recognition peptide contained in the functional polypeptide includes any one of the recognition peptide of trypsin, the recognition peptide of matrix metalloproteinase-2, the recognition peptide of matrix metalloproteinase-7, the recognition peptide of matrix metalloproteinase-9 and the recognition peptide of β-secretase.

[0012] Preferably, the amino acid sequence of the functional polypeptide recognizing β-secretase is any one of: A. Cys-Leu--Gly-Gly-Glu-Val-Asn-Leu-Asp-Ala-Phe-Gly-Gly--Leu-His-His-His-His-His, B. Cys-Asp-Asp-Asp-Glu-Val-Asn-Leu-Asp-Ala-Phe-Asp-Asp-Asp-His-His-His-His-His-His, C. Cys-Arg-Pro-Pro-Val-Asn-Leu-Asp-Ala-Phe-Pro-Pro-Lys-His-His-His-His-His-His;

[0013] Preferably, the amino acid sequence of the functional polypeptide that recognizes trypsin is any one of A. Cys-Leu-Gly-Gly-Lys-Gly-Ala-Lys-Gly-Tyr-Gly-Gly--His-His-His-His-His-His, B. Cys-Asp-Asp-Arg-Cys-Phe-Arg-Gly-Gly-Asp-Asp-His-His-His-His-His-His;

[0014] Preferably, the amino acid sequence of the functional polypeptide that recognizes matrix metalloproteinase-2 is any one of A. Cys-Leu-Gly-Pro-Leu-Gly-Val-Arg-Gly-Gly-Gly-Gly-Leu-His-His-His-His-His-His, B. Cys-Gly-Pro-Pro-Gly-Val-Val-Gly-Glu-Lys-Gly-Glu-Gln-His-His-His-His-His-His;

[0015] Preferably, the amino acid sequence of the functional polypeptide that recognizes matrix metalloproteinase-7 is any one of A. Cys-Leu-Lys-Gly-Met-Thr-Leu-Ser-Leu-Pro-Val-His-His-His-His-His-His, B. Cys-Leu-Gly-Val-Pro-Leu-Ser-Leu-Thr-Met-Gly--His-His-His-His-His-His;

[0016] Preferably, the amino acid sequence of the functional polypeptide that recognizes matrix metalloproteinase-9 is any one of A. Cys-Leu-Gly-Gly-Lys-Gly-Pro-Leu-Gly-Leu-Pro-Gly-Gly-Leu-His-His-His-His-His-His, B. Cys-Leu-Lys-Gly-Pro-Arg-Ser-Leu-Ser-Gly-Lys-Leu-His-His-His-His-His-His;

[0017] Preferably, the glass sheet coated with the gold film has a thickness of less than 0.15 mm, and the thickness of the gold film is 80-100 nm.

[0018] The second object of the present invention is to provide a method for preparing the giant magnetoresistive biosensor, comprising the following steps:

[0019] (1) Designing and synthesizing a functional polypeptide, and then dissolving it in a phosphate buffered saline solution for standby use;

[0020] (2) immersing the glass slide coated with the gold film into the phosphate buffered saline solution containing the functional polypeptide obtained in step (1) to react, immobilizing the functional polypeptide on the glass slide coated with the gold film, and blocking with hexanethiol and bovine serum albumin;

[0021] (3) immersing the gold-coated glass sheet obtained in step (2) in a phosphate buffered saline solution dispersed with metal ion chelating magnetic beads for coupling reaction, and utilizing the chelation between the His tag and the metal ions to connect the metal ion chelating magnetic beads to the functional polypeptide, thereby obtaining a glass sheet with fixed magnetic beads;

[0022] (4) placing the glass sheet obtained in step (3) on the surface of the giant magnetoresistive biosensor and fixing it so that the gold film area covers the detection area of ​​the giant magnetoresistive biosensor, thereby obtaining a giant magnetoresistive biosensor for detecting protease.

[0023] Preferably, the phosphate buffered saline solution in step (2) has a pH of 7.4, contains 10 mmol / L PBS and 0.1% Tween 20, and the concentration of the functional polypeptide in the solution is 1 μmol / L to 6 μmol / L.

[0024] Preferably, the concentration of the metal ion chelated magnetic beads in step (3) is 0.005 mg / mL to 0.05 mg / mL, the average particle size is 200 nm to 1 μm, and the reaction time with the polypeptide is 20 to 60 min.

[0025] The third object of the present invention is to provide a method for using the giant magnetoresistance biosensor or the giant magnetoresistance biosensor prepared by the preparation method in detecting proteases, comprising the following steps:

[0026] (1) Applying a bias magnetic field of a certain strength through the Helmholtz coil to magnetize the magnetic beads fixed on the gold film in the detection area of ​​the giant magnetoresistance biosensor, and measuring the output voltage of the giant magnetoresistance biosensor, which is recorded as V0;

[0027] (2) taking out the glass slide in the detection area of ​​the giant magnetoresistance biosensor, dripping the acetate buffer solution containing the hydrolytic protease to be tested onto the gold film of the glass slide, incubating, reacting, and then washing the glass slide with the acetate buffer solution to remove the magnetic beads detached after the enzyme cleavage reaction, blowing dry with N2, placing the glass slide on the surface of the giant magnetoresistance biosensor and fixing it so that the gold film area covers the detection area of ​​the giant magnetoresistance biosensor for testing;

[0028] (3) Apply a bias magnetic field of a certain strength through the Helmholtz coil to magnetize the remaining magnetic beads on the detection area of ​​the GMR biosensor obtained in step (2), and measure the output voltage of the GMR sensor at this time, which is recorded as V X ;

[0029] (4) Calculate the output voltage V0 obtained in step (1) and the output voltage V obtained in step (3) X The difference ΔV = V0-V X , the content of BACE1 was obtained according to the linear regression equation between the difference in output voltage of the giant magnetoresistance biosensor and the concentration of hydrolyzing protease.

[0030] Preferably, when the giant magnetoresistance biosensor detects β-secretase, the external bias magnetic field strength in step (1) is 5 to 60 Oe, the acetate buffer solution in step (2) is 0.1 mol / L, the pH value is 4.0 to 5.8, the enzyme cleavage reaction temperature is 35°C to 45°C, and the reaction time is 1h to 3h.

[0031] The fourth object of the present invention is to provide the application of the giant magnetoresistance biosensor or the giant magnetoresistance biosensor prepared by the preparation method in any one of the following (1) to (3):

[0032] (1) Qualitative or quantitative detection of proteases;

[0033] (2) Screening of protease inhibitors;

[0034] (3) Quantitative comparison or efficacy evaluation of protease inhibitors.

[0035] Preferably, the linear range of the giant magnetoresistance biosensor for detecting β-secretase activity is 0.5 U / mL-80.0 U / mL, and the detection limit is 0.1 U / mL.

[0036] The fifth object of the present invention is to provide a portable protease detection device, wherein the detection device comprises the giant magnetoresistance biosensor.

[0037] The beneficial effects of the present invention are:

[0038] (1) The present invention provides a giant magnetoresistance biosensor for detecting protease, comprising a giant magnetoresistance sensor, a glass sheet coated with a gold film, a functional polypeptide and metal ion chelate magnetic beads; the glass sheet coated with a gold film is fixed on the giant magnetoresistance sensor, one end of the peptide chain of the functional polypeptide is connected to the glass sheet coated with a gold film via the thiol group on the cysteine ​​residue, and the other end is connected to the metal ion chelate magnetic beads via the His tag; the polypeptide probe of the biosensor adopts a magnetic label, which is more stable than the traditional fluorescent label, is not affected by photobleaching, and does not have strong environmental noise. The giant magnetoresistance biosensor has high sensitivity, strong anti-interference ability, fast response speed and stable performance. The linear range of its detection of β-secretase activity is 0.5U / mL-80.0U / mL, and the detection limit is 0.1U / mL.

[0039] (2) Compared with the existing methods for detecting proteases, the giant magnetoresistive biosensor described in the present invention is used to detect proteases. It does not require the aid of large-scale instruments, and can quickly and accurately perform quantitative analysis of protease activity. It is also easy to integrate and can be developed into a portable detection device for large-scale promotion. It has a good practical application prospect in the diagnosis of major diseases and the development of related new drugs.

[0040] (3) The GMR biosensor provided by the present invention does not use DNA or antigens and antibodies as the biorecognition elements of the sensor for the recognition of the biological molecules to be detected, but uses polypeptides that are easy to design and synthesize as the biorecognition elements of the sensor. By changing the sequence of the polypeptide probe, the GMR biosensor can be designed for monitoring the activity of different types of proteases. The sensing principle is cleverly designed and has a certain degree of versatility. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 Flow chart of the preparation of the giant magnetoresistance (GMR) biosensor for detecting β-secretase in Example 1;

[0042] Figure 2 In Example 1, Ni 2+ Scanning electron microscope (SEM) image of chelated magnetic beads;

[0043] Figure 3 In Example 1, Ni 2+ X-ray energy dispersive spectrometry (EDS) diagram of chelated magnetic beads;

[0044] Figure 4 In Example 1, Ni on the Au film on the surface of the GMR biosensor 2 Microscope image of the dispersion of chelated magnetic beads;

[0045] Figure 5 In Example 5, Co 2+ Scanning electron microscope (SEM) image of chelated magnetic beads;

[0046] Figure 6 In Example 5, Co 2+ X-ray energy dispersive spectrometry (EDS) diagram of chelated magnetic beads;

[0047] Figure 7 Schematic diagram of the principle of GMR biosensor for detecting β-secretase;

[0048] Figure 8 Response graph of the GMR biosensor detecting β-secretase in Example 6;

[0049] Fig. 9 The linear curve of the GMR biosensor detecting β-secretase in Example 6;

[0050] Fig.10 A graph showing the anti-interference ability of the GMR biosensor in detecting β-secretase in Example 7. DETAILED DESCRIPTION

[0051] In order to make the technical means, creative features, purpose and effect of the present invention easy to understand, the present invention is further described below in conjunction with specific embodiments. The following examples are only used to illustrate the technical solution of the present invention but not to limit it. Although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solution of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solution of the present invention, which should be included in the scope of the claims of the present invention.

[0052] It should be noted that the methods used in the following examples are conventional methods unless otherwise specified.

[0053] In the following examples, the reagents used, unless otherwise specified, can be purchased from the market.

[0054] In the following examples, the functional polypeptide sequence used in the preparation of the giant magnetoresistive biosensor for detecting β-secretase is as follows:

[0055] A.Cys-Leu--Gly-Gly-Glu-Val-Asn-Leu-Asp-Ala-Phe-Gly-Gly--Leu-His-His-His-His-His-His;

[0056] B.Cys-Asp-Asp-Asp-Glu-Val-Asn-Leu-Asp-Ala-Phe-Asp-Asp-Asp-His-His-His-His-His-His;

[0057] C.Cys-Arg-Pro-Pro-Val-Asn-Leu-Asp-Ala-Phe-Pro-Pro-Lys-His-His-His-His-His-His;

[0058] In the following examples, the functional polypeptide sequence used in the preparation of the giant magnetoresistance biosensor for detecting trypsin is as follows:

[0059] A.Cys-Leu-Gly-Gly-Lys-Gly-Ala-Lys-Gly-Tyr-Gly-Gly--His-His-His-His-His-His;

[0060] B.Cys-Asp-Asp-Arg-Cys-Phe-Arg-Gly-Gly-Asp-Asp-His-His-His-His-His-His;

[0061] In the following examples, the functional polypeptide sequences used in the preparation of giant magnetoresistance biosensor for detecting matrix metalloproteinase-2 are as follows: A. Cys-Leu-Gly-Pro-Leu-Gly-Val-Arg-Gly-Gly-Gly-Gly-Leu-His-His-His-His-His-His;

[0062] B.Cys-Gly-Pro-Pro-Gly-Val-Val-Gly-Glu-Lys-Gly-Glu-Gln-His-His-His-His-His-His;

[0063] In the following examples, the functional polypeptide sequences used in the preparation of giant magnetoresistance biosensor for detecting matrix metalloproteinase-7 are as follows: A. Cys-Leu-Lys-Gly-Met-Thr-Leu-Ser-Leu-Pro-Val-His-His-His-His-His-His;

[0064] B.Cys-Leu-Gly-Val-Pro-Leu-Ser-Leu-Thr-Met-Gly--His-His-His-His-His-His;

[0065] In the following examples, the functional polypeptide sequences used in the preparation of the giant magnetoresistance biosensor for detecting matrix metalloproteinase-9 are as follows: A. Cys-Leu-Gly-Gly-Lys-Gly-Pro-Leu-Gly-Leu-Pro-Gly-Gly-Leu-His-His-His-His-His;

[0066] B.Cys-Leu-Lys-Gly-Pro-Arg-Ser-Leu-Ser-Gly-Lys-Leu-His-His-His-His-His-His;

[0067] Example 1 Preparation method of a giant magnetoresistance (GMR) biosensor for detecting β-secretase

[0068] (1) Design a functional polypeptide and contact a biological company for synthesis. The specific amino acid sequence is as follows: Cys-Leu--Gly-Gly-Glu-Val-Asn-Leu-Asp-Ala-Phe-Gly-Gly--Leu-His-His-His-His-His, and its purity is greater than 95%. Dissolve the synthesized functional polypeptide in phosphate buffered saline (10 mmol / L PBS, pH = 7.4, containing 0.1% Tween20) to form a 5 μmol / L functional polypeptide solution for standby use.

[0069] (2) First, immerse the glass slide coated with the Au film in the phosphate buffered saline solution containing the functional polypeptide obtained in step (1), incubate the reaction at room temperature for 20 minutes, then gradually add a high concentration of sodium chloride (NaCl) solution to the mixed solution until the NaCl concentration in the mixed solution reaches 300 mmol / L and incubate at 4°C for 24 hours to fix the functional polypeptide on the Au film of the glass slide, and finally block it with 0.1 mmol / L hexanethiol and 1% bovine serum albumin;

[0070] (3) First, 0.02 mg / mL Ni 2+ The chelated magnetic beads are uniformly dispersed in a phosphate buffered saline solution (20 mmol / L PBS, pH=7.4, containing 500 mmol / L NaCl), and the glass slide obtained in step (2) is immersed in the solution, incubated with shaking to perform a magnetic bead coupling reaction, and then the glass slide is taken out, rinsed three times with a phosphate buffered saline solution, and blown dry with N2, so that the magnetic beads can be fixed on the Au film of the glass slide through the functional polypeptide;

[0071] (4) The glass sheet obtained in step (3) is placed on the surface of the GMR sensor and fixed so that the Au film area covers the detection area of ​​the GMR sensor, thereby obtaining a GMR biosensor for detecting β-secretase.

[0072] Figure 1 Flow chart of the preparation of the giant magnetoresistance (GMR) biosensor for detecting β-secretase in Example 1; as can be seen from the figure, the functional polypeptide is fixed on the area coated with Au film on the glass sheet by using the interaction between thiol (-SH) and Au, and the magnetic beads are fixed by using Ni 2+ The chelation between the His tag on the peptide is linked to the functional peptide, thereby realizing the magnetic labeling of the peptide probe.

[0073] Figure 2 and Figure 3 The Ni used in Example 1 is 2+ Scanning electron microscope (SEM) and X-ray energy dispersive spectrometer (EDS) images of chelated nanomagnetic beads. Figure 2It can be seen that the magnetic beads have uniform particle size, with an average particle size of about 200 nm. Figure 3 It can be seen that the magnetic beads contain Si, O, Fe and Ni elements, which indicates that Ni 2+ It exists on the surface of magnetic beads and provides a good chelating site for 6His-tagged functional polypeptides.

[0074] Figure 4 Ni on the surface of the GMR biosensor made in Example 1 2 Microscope image of the dispersion of chelated magnetic beads. Figure 4 It can be seen that Ni 2+ The chelated magnetic beads can be evenly fixed on the Au film on the surface of the detection area of ​​the GMR sensor.

[0075] Since the GMR biosensor prepared in Example 1 has good performance, the inventors changed the type, concentration and reaction condition parameters of the raw materials based on Example 1, and then observed the performance of the prepared GMR biosensor, and obtained the following examples:

[0076] Example 2 Preparation method of a giant magnetoresistance (GMR) biosensor for detecting β-secretase

[0077] The preparation steps of the giant magnetoresistance (GMR) biosensor for detecting β-secretase in this embodiment are the same as those in Example 1, except that: the concentration of the functional polypeptide is 1 μmol / L, Ni 2+ The concentration of chelated magnetic beads was 0.005 mg / mL, and the reaction time between the functional polypeptide and the magnetic beads was 20 min.

[0078] Example 3 Preparation method of a giant magnetoresistance (GMR) biosensor for detecting β-secretase

[0079] The preparation steps of the giant magnetoresistance (GMR) biosensor for detecting β-secretase in this embodiment are the same as those in Example 1, except that: the concentration of the functional polypeptide is 6 μmol / L, Ni 2+ The concentration of chelated magnetic beads was 0.05 mg / mL, and the reaction time between the functional polypeptide and the magnetic beads was 60 min.

[0080] Example 4 Preparation method of a giant magnetoresistance (GMR) biosensor for detecting β-secretase

[0081] The preparation steps of the giant magnetoresistance (GMR) biosensor for detecting β-secretase involved in this embodiment are the same as those in Example 1, except that the amino acid sequence of the functional polypeptide used is: Cys-Arg-Pro-Pro-Val-Asn-Leu-Asp-Ala-Phe-Pro-Pro-Lys-His-His-His-His-His-His, the concentration of the functional polypeptide is 5 μmol / L, and Ni2+ The concentration of chelated magnetic beads was 0.01 mg / mL, and the reaction time between the functional polypeptide and the magnetic beads was 40 min.

[0082] Example 5 Preparation method of a giant magnetoresistance (GMR) biosensor for detecting β-secretase

[0083] The preparation steps of the giant magnetoresistance (GMR) biosensor for detecting β-secretase involved in this embodiment are the same as those in Example 1, except that the amino acid sequence of the functional polypeptide used is: Cys-Asp-Asp-Asp-Glu-Val-Asn-Leu-Asp-Ala-Phe-Asp-Asp-Asp-His-His-His-His-His-His, the concentration of the functional polypeptide is 5 μmol / L, and the magnetic beads are Co 2+ Chelated magnetic beads, the concentration is 0.01 mg / mL, and the reaction time between functional peptide and magnetic beads is 40 min. 2+ Transmission electron microscopy characterization of chelating magnetic beads Figure 5 As shown, the magnetic beads have uniform particle size, with an average particle size of about 1 μm. Figure 6 For Co 2+ The X-ray energy spectrum analysis (EDS) of the chelated nanoparticles shows that the magnetic beads contain Si, O, Fe and Co elements, which indicates that Co 2+ exists on the surface of magnetic beads, so Co 2+ The chelating magnetic beads and the 6His-tagged functional polypeptide are linked through chelation.

[0084] Application of the giant magnetoresistance (GMR) biosensor described in Example 6 to detect β-secretase

[0085] The test system for detecting β-secretase using the GMR biosensor prepared in Example 1 includes a Holm Hertz coil, a current source and a nanovoltmeter, and its specific functions are as follows: the Holm Hertz coil is used to provide an external bias magnetic field, and the magnitude of the field strength is controlled by changing the current output of the DC power supply. The constant current source provides a constant excitation current for the GMR sensor, and the nanovoltmeter is used to reflect the resistance change of the GMR sensor during the detection process. By calculating the change in the output voltage caused by different concentrations of magnetic beads on the sensor surface, the influence of the test object on the GMR sensor is analyzed.

[0086] The specific testing process includes the following steps:

[0087] (1) A bias magnetic field of 5 Oe is applied through a Helmholtz coil to magnetize the magnetic beads fixed on the Au film in the detection area of ​​the GMR biosensor, and the output voltage of the GMR sensor at this time is measured and recorded as V0;

[0088] (2) First, take out the glass slide of the detection area of ​​the GMR biosensor, and drip 20 μL of the acetate buffer solution of the sample to be tested onto the Au film, wherein the concentration of the acetate buffer solution is 0.1 mol / L and the pH value is 4.5; then, incubate the glass slide with the sample to be tested at 37°C for 1 hour for enzyme cleavage reaction, then rinse the glass slide three times with the buffer solution to remove the magnetic beads detached after the enzyme cleavage reaction, and blow dry with N2, finally, place the glass slide on the surface of the GMR sensor and fix it so that the Au film area covers the detection area of ​​the GMR sensor for testing;

[0089] (3) Apply an external bias magnetic field of 5 Oe through the Helmholtz coil to magnetize the remaining magnetic beads on the Au film in the detection area of ​​the biosensor obtained in step (2), and measure the output voltage of the GMR sensor at this time, which is recorded as V X ;

[0090] (4) Calculate the output voltage V0 obtained in step (1) and the output voltage V obtained in step (3) X The difference ΔV = V0-V X , according to the linear regression equation between the difference in the output voltage of the GMR sensor and the concentration of β-secretase, the content of β-secretase was obtained.

[0091] Figure 7 The schematic diagram of the principle of the GMR biosensor of the present invention for detecting β-secretase. As shown in the figure, when the β-secretase to be detected is not dripped on the sensor surface, a large number of magnetic beads are fixed on its surface. When a certain strength of external bias magnetic field is applied to magnetize the magnetic beads, the resistance of the GMR sensor will change significantly, so a higher voltage value is output. When the target β-secretase is dripped onto the surface of the GMR sensor, it can specifically hydrolyze and shear the magnetically labeled polypeptide probe under certain conditions, and the magnetic beads will be released from the surface of the GMR sensor. After the buffer solution is rinsed, the magnetic beads are removed from the sensor surface, and the voltage value output by the sensor will decrease. The change in the voltage value detected by the sensor is negatively correlated with the change in the concentration of the β-secretase to be detected, so that the β-secretase to be detected can be quantitatively analyzed.

[0092] Figure 8 This is a graph showing the change in output voltage of the detection system after different concentrations of β-secretase react with the peptide probe on the surface of the GMR sensor. Fig. 9 The linear plot is the logarithmic value of β-secretase concentration and the sensor response voltage.

[0093] from Figure 8 It can be seen that when the concentration of β-secretase gradually increases in the range of 0-120.0 U / mL, the change in the output voltage of the detection system also increases, which indicates that the sensor has a good response to changes in the concentration of β-secretase.

[0094] from Fig. 9 The data show that the concentration change of β-secretase has a good linear relationship with the output voltage value of the detection system. The linear range of β-secretase detection of β-secretase activity is 0.5U / mL-80.0U / mL, the detection limit is 0.1U / mL, and its linear equation is y=0.06425x+0.06175. The correlation coefficient R 2 =0.99616.

[0095] Anti-interference ability of giant magnetoresistance (GMR) biosensor in detecting β-secretase described in Example 7

[0096] This example is to verify the anti-interference ability of the GMR biosensor prepared in Example 1 to detect the magnetism of β-secretase. The detection steps are the same as those in Example 6, except that: the target β-secretase in the detection system is replaced by glucose oxidase (GOX), trypsin, matrix metalloproteinase-2 (MMP-2), bovine serum albumin (BSA), and human immunoglobulin (IgG) which may exist in the actual sample, respectively, and the output voltage of the detection system changes. The results are shown in FIG. Fig.10 shown.

[0097] from Fig.10 It can be seen that when anti-interference substances that may coexist with β-secretase are present in the analyte, only β-secretase can obviously cause changes in the output voltage of the detection system, while other interfering substances do not have obvious changes, which indicates that the GMR biosensor of the present invention has strong anti-interference ability and is suitable for the detection of β-secretase in complex environments.

[0098] In summary, the present invention provides a giant magnetoresistance biosensor for detecting protease, comprising a giant magnetoresistance sensor, a glass sheet coated with a gold film, a functional polypeptide and metal ion chelate magnetic beads; the glass sheet coated with a gold film is fixed on the giant magnetoresistance sensor, one end of the peptide chain of the functional polypeptide is connected to the glass sheet coated with a gold film through the thiol group on the cysteine ​​residue, and the other end is connected to the metal ion chelate magnetic beads through the His tag; the polypeptide probe of the biosensor adopts a magnetic label, which is more stable than the traditional fluorescent label, is not affected by photobleaching, and does not have strong environmental noise. The giant magnetoresistance biosensor has high sensitivity, strong anti-interference ability, fast response speed and stable performance. The linear range of its detection of β-secretase activity is 0.5U / mL-80.0U / mL, and the detection limit is 0.1U / mL. Compared with the existing methods for detecting proteases, the giant magnetoresistance biosensor of the present invention is used to detect proteases. It can quickly and accurately perform quantitative analysis on protease activity without the aid of large instruments, and is easy to integrate, so that it can be developed into a portable detection device for large-scale promotion, and has a good practical application prospect in the diagnosis of major diseases and the development of related new drugs. The GMR biosensor provided by the present invention does not use DNA or antigens and antibodies as the biorecognition element of the sensor for the recognition of the biological molecules to be detected, but uses polypeptides that are easy to design and synthesize as the biorecognition element of the sensor. The GMR biosensor can be designed by changing the sequence of the polypeptide probe to monitor the activity of different types of proteases. The sensing principle is cleverly designed and has a certain versatility.

[0099] Finally, it should be noted that the above is only a preferred embodiment of the present invention and does not limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for those skilled in the art to modify the technical solutions described in the aforementioned embodiments, or to make equivalent substitutions for some of the technical features therein. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A giant magnetoresistance biosensor for detecting proteases, characterized in that: It includes a giant magnetoresistance sensor, a glass sheet coated with a gold film, a functional peptide and metal ion chelating magnetic beads; The glass sheet coated with gold film is fixed on a giant magnetoresistance sensor, the amino acid sequence of the functional polypeptide contains a recognition peptide segment of a protease in the middle, a cysteine ​​residue containing a thiol group at the carbon end or the nitrogen end, and a His tag at the other end, the functional polypeptide is connected to the glass sheet coated with gold film via the thiol group on the cysteine ​​residue, and the functional polypeptide is connected to the metal ion chelated magnetic beads via the His tag.

2. The giant magnetoresistance biosensor according to claim 1, characterized in that: The recognition peptide segment of the protease includes any one of the recognition peptide segment of β-secretase, the recognition peptide segment of trypsin, the recognition peptide segment of matrix metalloproteinase-2, the recognition peptide segment of matrix metalloproteinase-7 and the recognition peptide segment of matrix metalloproteinase-9.

3. The giant magnetoresistive biosensor according to claim 1, characterized in that: The metal ion in the metal ion chelated magnetic beads is Co 2+ or Ni 2+ .

4. The method for preparing the giant magnetoresistive biosensor according to any one of claims 1 to 3, characterized in that: The steps include: (1) Designing and synthesizing a functional polypeptide, and then dissolving it in a phosphate buffered saline solution for standby use; (2) immersing the glass slide coated with the gold film into the phosphate buffered saline solution containing the functional polypeptide obtained in step (1) to react, immobilizing the functional polypeptide on the glass slide coated with the gold film, and blocking with hexanethiol and bovine serum albumin; (3) immersing the gold-coated glass sheet obtained in step (2) in a phosphate buffered saline solution dispersed with metal ion chelating magnetic beads for coupling reaction, and utilizing the chelation between the His tag and the metal ions to connect the metal ion chelating magnetic beads to the functional polypeptide, thereby obtaining a glass sheet with fixed magnetic beads; (4) placing the glass sheet obtained in step (3) on the surface of the giant magnetoresistive sensor and fixing it so that the gold film area covers the detection area of ​​the giant magnetoresistive sensor, thereby obtaining a giant magnetoresistive biosensor for detecting proteases.

5. The preparation method according to claim 4, characterized in that: The pH of the phosphate buffered saline solution in step (2) is 7.4, and it contains 10 mmol / L PBS and 0.1% Tween-20. The concentration of the functional polypeptide in the solution is 1 μmol / L to 6 μmol / L.

6. The preparation method according to claim 4, characterized in that: The concentration of the metal ion chelated magnetic beads in step (3) is 0.005 mg / mL to 0.05 mg / mL, the average particle size is 200 nm to 1 μm, and the reaction time with the polypeptide is 20 to 60 min.

7. The method for detecting protease using the giant magnetoresistance biosensor according to any one of claims 1 to 3, characterized in that: The following steps are involved: (1) Applying a bias magnetic field of a certain strength through the Helmholtz coil to magnetize the magnetic beads fixed on the gold film in the detection area of ​​the giant magnetoresistance biosensor, and measuring the output voltage of the giant magnetoresistance biosensor, which is recorded as V0; (2) taking out the glass slide in the detection area of ​​the giant magnetoresistance biosensor, dropping a buffer solution containing the protease to be tested onto the gold film of the glass slide, incubating, reacting, and then washing the glass slide with a buffer solution to remove the magnetic beads detached after the enzyme cleavage reaction, blowing dry with N2, placing the glass slide on the surface of the giant magnetoresistance biosensor and fixing it so that the gold film area covers the detection area of ​​the giant magnetoresistance biosensor for testing; (3) Applying an external bias magnetic field of a certain strength through the Helmholtz coil to magnetize the magnetic beads remaining on the detection area of ​​the giant magnetoresistive biosensor obtained in step (2), and measuring the output voltage of the giant magnetoresistive sensor at this time, which is recorded as V X ; (4) Calculate the output voltage V0 obtained in step (1) and the output voltage V obtained in step (3) X The difference ΔV = V0-V X , according to the linear regression equation between the difference in output voltage of the giant magnetoresistance biosensor and the protease concentration, the protease content was obtained.

8. Use of the giant magnetoresistance biosensor according to any one of claims 1 to 3 or the giant magnetoresistance biosensor prepared by the preparation method according to any one of claims 4 to 6 in any one of the following (1) to (3): (1) Qualitative or quantitative detection of proteases; (2) Screening of protease inhibitors; (3) Quantitative comparison or efficacy evaluation of protease inhibitors.

9. A portable protease detection device, characterized in that: The detection device comprises the giant magnetoresistance biosensor according to any one of claims 1-3.

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