A giant magnetoresistance biosensor for detecting protease and a preparation method and application thereof

Abstract

The application belongs to the technical field of biosensing, and particularly relates to a giant magnetoresistance biosensor for detecting protease, a preparation method and application thereof, which comprises a giant magnetoresistance sensor, a glass sheet plated with a gold film, a functional polypeptide and metal ion chelated magnetic beads; wherein the glass sheet plated with the gold film is fixed on the giant magnetoresistance 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 is connected with the metal ion chelated magnetic beads; the giant magnetoresistance biosensor provided by the application can realize monitoring of protease activity, has high sensitivity, strong anti-interference ability, stable performance, and is easy to integrate, so that the giant magnetoresistance biosensor can develop into a portable detection device for large-scale promotion, and has wide application prospects in the fields of diagnosis, treatment and related drug development of major diseases.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biosensing, and particularly relates to a giant magnetoresistance biosensor for detecting protease, a preparation method and application thereof. BACKGROUND

[0002] Protease is a general term for a class of enzymes that hydrolyze peptide bonds in proteins or polypeptides, including beta-secretase, trypsin and matrix metalloproteinase, etc. Protease plays an important role in life activities such as metabolism, vascular function, immune function and cell apoptosis. Protease is widely distributed in the human body and participates 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 beta-secretase is related to Alzheimer's disease; abnormal expression of trypsin is related to pancreatic diseases such as acute pancreatitis and pancreatic cancer; abnormal expression of matrix metalloproteinase-9 is related to chronic obstructive pulmonary disease; abnormal expression of matrix metalloproteinase-2 is related to female pelvic floor dysfunction; therefore, monitoring of protease activity has important significance in the biomedical field.

[0004] At present, the methods for detecting protease activity mainly include 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 have high sensitivity and strong universality, but require precise equipment, complex operation and long-time analysis by professional personnel, and have high analysis cost, which is not suitable for rapid detection requirements. ELISA and FRET probe method are also effective, but there are certain difficulties in practical application, such as the disadvantages of ELISA method, such as high price of antibody, poor repeatability, complicated operation and long analysis period, and FRET fluorescent probe method is formed by connecting fluorescent donor and fluorescent acceptor at both ends of enzyme substrate peptide through chemical bonding method, but the preparation process of fluorescent donor and quencher is very complex, the synthesis cost is high, the labeling step is complicated and the period is long, in addition, since the excitation wavelength of some easily labeled fluorescent groups often coincides with the absorption wavelength of many small drug molecules, the selection of fluorescent donor and acceptor of FRET probe is required to be high, and these factors limit the practical application of FRET fluorescent probe. Therefore, in order to meet the requirements of practical application, it is urgent to develop a new method for monitoring protease activity with strong universality, high sensitivity, simplicity and rapidness.

[0005] In recent years, giant magnetoresistance (GMR) biosensors, which combine giant magnetoresistance (GMR) sensors with magnetic labeling technology and biosensing technology, have attracted much attention in the field of biosensing technology due to their high sensitivity, rapid response, and integration and miniaturization. GMR biosensors are a kind of biosensor that detects biological samples by magnetic labeling of biological probes. Because magnetic labeling is very stable, it is not affected by light bleaching and has no strong environmental noise, it has unique advantages in stability and anti-interference ability. At present, 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-activated 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 between DNA molecules or immune reactions between antigens and antibodies to achieve biological molecular recognition, thereby establishing a biosensing method. Therefore, the biological recognition element of the sensor is DNA probe or antibody and antigen, and there are few reports on using the interaction between polypeptides and enzymes to establish a biosensing method.

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

[0007] In view of the above technical background, the present application provides a giant magnetoresistance biosensor for detecting protease, a preparation method and application thereof, and realizes simple and sensitive detection of protease by using the sensor, which has broad application prospects in the fields of diagnosis, treatment of major diseases and related drug development. SUMMARY

[0008] The present application aims to provide a giant magnetoresistance biosensor for detecting protease, which comprises a giant magnetoresistance biosensor, a glass sheet plated with a gold film, a functional polypeptide and a metal ion chelated magnetic bead.

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

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

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

[0012] Preferably, the amino acid sequence of the functional polypeptide for recognizing beta-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-His, B.Cys-Asp-Asp-Asp-Glu-Val-Asn-Leu-Asp-Ala-Phe-Asp-Asp-Asp-His-His-His-His-His-His, and C.Cys-Arg-Pro-Pro-Val-Asn-Leu-Asp-Ala-Phe-Pro-Pro-Lys-His-His-His-His-His-His.

[0013] Preferably, the functional polypeptide amino acid sequence for recognizing 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 functional polypeptide amino acid sequence for recognizing 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 functional polypeptide amino acid sequence for recognizing 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 functional polypeptide amino acid sequence for recognizing 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 plated with gold film has a thickness less than 0.15 mm, and the gold film has a thickness of 80-100 nm.

[0018] The second object of the present application is to provide a preparation method of the giant magnetoresistance biosensor, comprising the following steps:

[0019] (1) designing and synthesizing a functional polypeptide, and dissolving the functional polypeptide in a phosphate buffered saline solution for later use;

[0020] (2) the gold film plated glass sheet is immersed into the functional polypeptide containing phosphate buffered saline solution obtained in step (1) to react, and the functional polypeptide is fixed on the gold film plated glass sheet, and hexanethiol and bovine serum albumin are used for blocking;

[0021] (3) the gold film plated glass sheet obtained in step (2) is immersed into the phosphate buffered saline solution in which metal ion chelated magnetic beads are dispersed to perform coupling reaction, and the metal ion chelated magnetic beads are connected to the functional polypeptide by chelation between the His tag and the metal ion, so that the glass sheet on which the magnetic beads are fixed is obtained;

[0022] (4) the glass sheet obtained in step (3) is placed on the surface of the giant magnetoresistance biosensor and fixed, so that the gold film region covers the detection area of the giant magnetoresistance biosensor, and the giant magnetoresistance biosensor for detecting hydrolytic protease is obtained.

[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-6 μmol / L.

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

[0025] The third object of the present application is to provide a use method of the giant magnetoresistance biosensor or the giant magnetoresistance biosensor prepared by the preparation method in detecting protease, which comprises the following steps:

[0026] (1) a certain intensity of external bias magnetic field is applied by a Helmholtz coil to magnetize the magnetic beads fixed on the gold film in the detection area of the giant magnetoresistance biosensor, and the output voltage of the giant magnetoresistance biosensor is measured, which is recorded as V0;

[0027] (2) the glass sheet in the detection area of the giant magnetoresistance biosensor is taken out, an acetate buffer solution containing the hydrolytic protease to be detected is added to the gold film of the glass sheet, and incubation, reaction and washing of the glass sheet with the acetate buffer solution are performed to remove the magnetic beads separated after the enzyme cutting reaction, N2 is blown to dry, and the glass sheet is placed on the surface of the giant magnetoresistance biosensor and fixed, so that the gold film region covers the detection area of the giant magnetoresistance biosensor and is ready for detection;

[0028] (3) a certain intensity of external bias magnetic field is applied by a Helmholtz coil to magnetize the remaining magnetic beads in the detection area of the GMR biosensor obtained in step (2), and the output voltage of the GMR biosensor at this time is measured, which is recorded as V X ;

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

[0030] Preferably, when detecting the beta-secretase, the bias magnetic field strength in step (1) is 5-60 Oe, the acetate buffer solution in step (2) is 0.1 mol / L, the pH value is 4.0-5.8, the enzyme cutting reaction temperature is 35-45℃, and the reaction time is 1-3 hours.

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

[0032] (1) qualitative or quantitative detection of protease;

[0033] (2) screening of protease inhibitors;

[0034] (3) quantitative comparison or effect evaluation of protease inhibitors.

[0035] Preferably, the linear range of the giant magnetoresistance biosensor for detecting the activity of beta-secretase 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 application is to provide a portable protease detection device, wherein the detection device comprises the giant magnetoresistance biosensor.

[0037] The present application has the following advantages:

[0038] (1) The present application provides a giant magnetoresistance biosensor for detecting protease, which comprises a giant magnetoresistance sensor, a glass sheet plated with a gold film, a functional polypeptide and a metal ion chelated magnetic bead; the glass sheet plated 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 plated with a gold film through the sulfhydryl group on the cysteine residue, and the other end is connected to the metal ion chelated magnetic bead through a His tag; the polypeptide probe of the biosensor is magnetically labeled, which has higher stability than traditional fluorescent labeling, is not affected by light bleaching, and has no strong environmental noise; the giant magnetoresistance biosensor has high sensitivity, strong anti-interference ability, fast response speed and stable performance, and has a linear range of 0.5 U / mL-80.0 U / mL for detecting the activity of beta-secretase and a detection limit of 0.1 U / mL.

[0039] (2) Compared with existing methods for detecting proteases, the giant magnetoresistive biosensor described in this invention can quickly and accurately quantify protease activity without the need for large instruments. It is also easy to integrate, thus enabling it to be developed into a portable detection device for large-scale promotion. It has great practical application prospects 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 biorecognition elements of the sensor for the recognition of biomolecules to be tested. Instead, it uses peptides that are easy to design and synthesize as biorecognition elements of the sensor. The GMR biosensor can be designed to monitor the activity of different types of proteases by changing the sequence of the peptide probe. The sensing principle is ingeniously designed and has a certain degree of versatility. Attached Figure Description

[0041] Figure 1 Flowchart of the fabrication process 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 spectroscopy (EDS) analysis of chelated magnetic beads;

[0044] Figure 4 Ni on the Au film on the surface of the GMR biosensor in Example 1 2 Microscopic 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 spectroscopy (EDS) analysis of chelated magnetic beads;

[0047] Figure 7 A schematic diagram illustrating the principle of GMR biosensor detection of β-secretase;

[0048] Figure 8 The response diagram of the GMR biosensor to detect β-secretase in Example 6;

[0049] Figure 9 Linear curve of β-secretase detection by GMR biosensor in Example 6;

[0050] Figure 10 Figure 7 shows the anti-interference ability of the GMR biosensor in Example 7 in detecting β-secretase. DETAILED DESCRIPTION

[0051] In order to make the technical means, creative features, purposes and effects of the present application easy to understand, the present application is further described below in conjunction with specific embodiments. The following embodiments are only used to illustrate the technical solutions of the present application and not to limit the same. Although the present application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present application can be modified or replaced equivalently without departing from the purpose and scope of the present application, and all should be covered in the scope of the claims of the present application.

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

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

[0054] In the following embodiments, the functional polypeptide sequence in the giant magnetoresistance 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 embodiments, the functional polypeptide sequence in 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-Gly-Pro-Pro-Gly-Val-Val-Gly-Glu-Lys-Gly-Glu-Gln-His-His-His-His-His-His;

[0061] In the following example, the functional polypeptide sequence for use in a giant magnetoresistance biosensor for detecting matrix metalloproteinase-2 is 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 example, the functional polypeptide sequence for use in a giant magnetoresistance biosensor for detecting matrix metalloproteinase-7 is 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 example, the functional polypeptide sequence for use in a giant magnetoresistance biosensor for detecting matrix metalloproteinase-9 is as follows: A. Cys-Leu-Gly-Gly-Lys-Gly-Pro-Leu-Gly-Leu-Pro-Gly-Gly-Leu-His-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 A method for preparing a giant magnetoresistance (GMR) biosensor for detecting β-secretase

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

[0069] (2) First, the glass piece plated with Au film is immersed in the functional polypeptide-containing phosphate buffered saline solution obtained in step (1), and incubated at room temperature for 20 min, then high-concentration sodium chloride (NaCl) solution is gradually added to the mixed solution, so that the concentration of NaCl in the mixed solution reaches 300 mmol / L, and the functional polypeptide is fixed on the Au film of the glass piece by incubating at 4°C for 24 hours, finally, 0.1 mmol / L hexanethiol and 1% bovine serum albumin are used for blocking;

[0070] (3) First, 0.02 mg / mL Ni 2+ Chelated magnetic beads are uniformly dispersed in phosphate buffered saline solution (20 mmol / L PBS, pH = 7.4, containing 500 mmol / L NaCl), then the glass piece obtained in step (2) is immersed in the solution, and the magnetic bead coupling reaction is carried out by oscillation incubation, then the glass piece is taken out, washed with phosphate buffered saline solution for three times, and dried with N2, so that the magnetic beads are fixed on the Au film of the glass piece through the functional polypeptide;

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

[0072] Figure 1 The flow chart for preparing the giant magnetoresistance (GMR) biosensor for detecting β-secretase in Example 1; from the figure, it can be seen that the functional polypeptide is fixed on the region plated with Au film on the glass piece by using the action between thiol (-SH) and Au, and the magnetic beads are linked to the functional polypeptide by using the chelation between Ni 2+ and His tag on the polypeptide, so that the magnetic labeling of the polypeptide probe is realized.

[0073] Figure 2 and Figure 3 are respectively the scanning electron microscope (SEM) and X-ray energy spectrum analysis (EDS) of the Ni 2+ Chelated nanometer magnetic beads used in Example 1. From the figures, it can be seen that the Ni Figure 2It can be seen that the magnetic beads have uniform size, and the average size is about 200 nm. Figure 3 It can be seen that the magnetic beads contain Si, O, Fe and Ni elements, which indicates that the Ni 2+ The 6His functional polypeptide is provided with good chelation sites on the surface of the magnetic beads.

[0074] Figure 4 The surface Ni 2 The microscope image of the chelated magnetic beads dispersion, from Figure 4 It can be seen that the Ni 2+ The chelated magnetic beads can be uniformly fixed on the Au film on the surface of the GMR sensor detection area.

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

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

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

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

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

[0080] Example 4 Preparation method 4 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 example 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 the concentration of the Ni2+ The concentration of the chelated magnetic beads was 0.01 mg / mL, and the reaction time of the functional polypeptide with the magnetic beads was 40 min.

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

[0083] This example relates to the preparation steps of a giant magnetoresistance (GMR) biosensor for detecting β-secretase as in Example 1, except that the functional polypeptide used has the amino acid sequence: Cys-Asp-Asp-Asp-Glu-Val-Asn-Leu-Asp-Ala-Phe-Asp-Asp-Asp-His-His-His-His-His-His, and the concentration of the functional polypeptide was 5 μmol / L, and the magnetic beads were Co 2+ The concentration of the chelated magnetic beads was 0.01 mg / mL, and the reaction time of the functional polypeptide with the magnetic beads was 40 min. Co 2+ Transmission electron microscopy characterization of the chelated magnetic beads is shown in Figure 5 The magnetic beads have a uniform particle size, and the average particle size is about 1 μm. Figure 6 Co 2+ X-ray energy dispersive spectroscopy (EDS) analysis of the chelated magnetic beads shows that the magnetic beads contain Si, O, Fe and Co elements, which indicates that Co 2+ exists on the surface of the magnetic beads, so Co 2+ The chelated magnetic beads are linked to the 6His-labeled functional polypeptide through chelation.

[0084] Example 6 Application of the giant magnetoresistance (GMR) biosensor for detecting β-secretase

[0085] The test system for detecting β-secretase using the GMR biosensor prepared in Example 1 includes a Hohmertz coil, a current source and a nanovoltmeter, and the specific functions are as follows: the Hohmertz coil is used to provide an external bias magnetic field, and the field strength is controlled by changing the current output of the direct current source. 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 of the sensor caused by different concentrations of magnetic beads, the influence of the analyte on the GMR sensor is analyzed.

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

[0087] (1) A bias magnetic field of 50e is applied by the Hohmertz coil to magnetize the magnetic beads that have been 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 drop 20 μL of acetate buffer solution of the sample to be tested onto the Au membrane. 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 to carry out the enzymatic digestion reaction. Then, rinse the glass slide three times with the buffer solution to remove the magnetic beads that have been removed after the enzymatic digestion reaction. Dry it with N2. Finally, place the glass slide on the surface of the GMR sensor and fix it so that the Au membrane area covers the detection area of ​​the GMR sensor. The sample is ready for testing.

[0089] (3) Apply an external bias magnetic field of 5 Oe through a Helmholtz coil to magnetize the remaining magnetic beads on the Au membrane of the biosensor detection area obtained in step (2), and measure the output voltage of the GMR sensor at this time, denoted 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 The content of β-secretase was obtained based on the linear regression equation between the difference in output voltage of the GMR sensor and the concentration of β-secretase.

[0091] Figure 7 This is a schematic diagram illustrating the principle of the GMR biosensor for detecting β-secretase according to the present invention. As shown in the figure, when the β-secretase to be tested is not added to the sensor surface, a large number of magnetic beads are fixed on its surface. When an external bias magnetic field of a certain intensity is applied to magnetize the magnetic beads, it causes a significant change in the resistance of the GMR sensor, thus outputting a high voltage value. When the target β-secretase is added to the surface of the GMR sensor, under certain conditions, it can specifically hydrolyze and cleave the magnetically labeled peptide probe, and the magnetic beads will be released from the surface of the GMR sensor. After rinsing with a buffer solution, the magnetic beads are removed from the sensor surface, and the voltage value output by the sensor will decrease. The change in voltage value detected by the sensor is negatively correlated with the change in the concentration of the β-secretase to be tested, thereby enabling quantitative analysis of the β-secretase to be tested.

[0092] Figure 8 This graph shows the changes in the output voltage of the detection system after different concentrations of β-secretase react with the peptide probes on the surface of the GMR sensor. Figure 9 This is a linear graph of the logarithmic value of β-secretase concentration versus the sensor response voltage.

[0093] from Figure 8 As can be seen, 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, indicating that the sensor has a good response to changes in the concentration of β-secretase.

[0094] fromFigure 9 The data show a good linear relationship between the concentration change of β-secretase and the output voltage of the detection system. The linear range for detecting β-secretase activity is 0.5 U / mL–80.0 U / mL, with a detection limit of 0.1 U / mL. The linear equation is y = 0.06425x + 0.06175, and the correlation coefficient R0 is [value missing]. 2 =0.99616.

[0095] Example 7 demonstrates the anti-interference capability of the giant magnetoresistive (GMR) biosensor in detecting β-secretase.

[0096] This embodiment aims to verify the anti-interference capability of the GMR biosensor prepared in Example 1 for detecting the magnetism of β-secretase. The detection steps are the same as in Example 6, except that the target analyte β-secretase in the detection system is replaced sequentially with interfering substances that may be present in the actual sample, namely glucose oxidase (GOX), trypsin, matrix metalloproteinase-2 (MMP-2), bovine serum albumin (BSA), and human immunoglobulin (IgG). The changes in the output voltage of the detection system are then analyzed, and the results are as follows. Figure 10 As shown.

[0097] from Figure 10 It can be seen that when there are anti-interference substances in the analyte that may coexist with β-secretase, only β-secretase can significantly cause a change in the output voltage of the detection system, while other interfering substances do not show significant changes. This indicates that the GMR biosensor described in this invention has strong anti-interference capabilities and is suitable for the detection of β-secretase in complex environments.

[0098] In summary, the application provides a giant magnetoresistance biosensor for detecting protease, comprising a giant magnetoresistance sensor, a glass sheet plated with a gold film, a functional polypeptide and metal ion chelated magnetic beads; the glass sheet plated with a gold film is fixed on the giant magnetoresistance sensor, one end of the peptide chain of the functional polypeptide is connected with the glass sheet plated with a gold film through the sulfhydryl on the cysteine residue, and the other end is connected with the metal ion chelated magnetic beads through a His tag; the polypeptide probe of the biosensor is magnetically labeled, and compared with traditional fluorescent labeling, it has higher stability, is not affected by light bleaching, and has no strong environmental noise; the giant magnetoresistance biosensor has high sensitivity, strong anti-interference ability, fast response speed and stable performance, the linear range for detecting the activity of beta-secretase is 0.5 U / mL-80.0 U / mL, and the detection limit is 0.1 U / mL. Compared with the existing method for detecting protease, the giant magnetoresistance biosensor of the application is used for detecting protease, does not need to rely on large instruments, can quickly and accurately quantitatively analyze the activity of protease, is easy to integrate, and can develop 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 application is not used for the recognition of a to-be-detected biological molecule by taking DNA or an antigen and an antibody as a biological recognition element of a sensor, but is used for the recognition of a to-be-detected biological molecule by taking a polypeptide easy to design and synthesize as a biological recognition element of a sensor, the sequence of the polypeptide probe can be changed to design the GMR biosensor for monitoring the activity of different types of protease, the sensing principle is designed ingeniously, and has a certain universality.

[0099] Finally, it should be noted that: the above only for the preferred embodiments of the application, and does not limit the application, although the application has been described in detail with reference to the foregoing embodiments, for those skilled in the art, it still can modify the technical solutions recorded in the foregoing embodiments, or make equivalent replacement to part of the technical features, any modification, equivalent replacement, improvement, etc. within the spirit and principles of the application, should be included in the protection scope of the application.

Claims

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

2. The giant magnetoresistive biosensor as described in claim 1, characterized in that, The recognition peptide of the protease includes any one of the recognition peptides of β-secretase, trypsin, matrix metalloproteinase-2, matrix metalloproteinase-7, and matrix metalloproteinase-9.

3. The giant magnetoresistive biosensor as described in claim 1, characterized in that, The metal ions in the metal ion chelating magnetic beads are Co. 2+ or Ni 2+ .

4. The method for preparing the giant magnetoresistive biosensor according to any one of claims 1-3, characterized in that, Includes the following steps: (1) Design and synthesize functional peptides, then dissolve them in phosphate buffer solution for later use; (2) Immerse the glass slide coated with gold film into the phosphate buffer solution containing functional peptides obtained in step (1) to react, fix the functional peptides on the glass slide coated with gold film, and block them with hexamethylenetetramine and bovine serum albumin. (3) The glass slide with gold film obtained in step (2) is immersed in a phosphate buffer solution containing metal ion chelating magnetic beads for coupling reaction. The metal ion chelating magnetic beads are linked to the functional polypeptide by utilizing the chelation between the His tag and the metal ions to obtain a glass slide with magnetic beads fixed. (4) Place the glass slide obtained in step (3) on the surface of the giant magnetoresistive sensor and fix it so that the gold film area covers the detection area of ​​the giant magnetoresistive sensor, thus obtaining a giant magnetoresistive biosensor for detecting protease.

5. The preparation method according to claim 4, characterized in that, The phosphate buffer solution described 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 peptide 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 chelating magnetic beads in step (3) is 0.005 mg / mL ~ 0.05 mg / mL, the average particle size is 200 nm - 1µm, and the reaction time with the peptide is 20-60 min.

7. The method of using the giant magnetoresistive biosensor as described in any one of claims 1 to 3 for detecting proteases for non-disease diagnosis and treatment purposes, characterized in that, Includes the following steps: (1) An external bias magnetic field of a certain strength is applied through a Helmholtz coil to magnetize the magnetic beads fixed on the gold film in the detection area of ​​the giant magnetoresistive biosensor. The output voltage of the giant magnetoresistive biosensor is measured and denoted as . V 0 ; (2) Take out the glass slide of the detection area of ​​the giant magnetoresistive biosensor, add the buffer solution containing the protease to be tested to the gold film on the glass slide, incubate, react, then rinse the glass slide with the buffer solution to remove the magnetic beads that have been removed after the enzyme digestion reaction, blow dry with N2, place the glass slide on the surface of the giant magnetoresistive biosensor and fix it so that the gold film area covers the detection area of ​​the giant magnetoresistive biosensor, and then test. (3) Apply an external bias magnetic field of a certain strength through a Helmholtz coil to magnetize the remaining magnetic beads on the detection area of ​​the giant magnetoresistive biosensor obtained in step (2), and measure the output voltage of the giant magnetoresistive sensor at this time, denoted as V. X ; (4) Calculate the output voltage obtained in step (1). V 0 The output voltage V obtained in step (3) X The difference ΔV=V 0 -V X The content of protease was obtained by using the linear regression equation between the difference in output voltage of the giant magnetoresistive biosensor and the protease concentration.

8. The application of the giant magnetoresistive biosensor according to any one of claims 1 to 3 or the giant magnetoresistive 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 for purposes other than disease diagnosis and treatment; (2) Screening of protease inhibitors for purposes other than disease diagnosis and treatment; (3) Quantitative comparison or efficacy evaluation of protease inhibitors for purposes other than disease diagnosis and treatment.

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

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