An impedance biosensor based on immunoaffinity and its preparation method and application

Abstract

The present invention provides an impedance biosensor based on immunoaffinity and its preparation method and application. The preparation method includes: surface modification using mercaptopropionic acid or chitosan based on a wireless passive sensor, or surface modification using nitrocellulose membrane based on a three-electrode sensor, and then binding a probe that can capture tumor protein markers on the surface to prepare an impedance biosensor based on immunoaffinity. The present invention provides for the first time such a preparation method of an impedance biosensor based on immunoaffinity and its application in tumor protein marker detection. Compared with the preparation method of traditional biosensors, the method of the present invention can ensure the high density, integrity and number of effective binding sites of biological molecules, realize the detection of trace proteins of tumor proteins, and the detection limit reaches the pg level. At the same time, it can also help solve the problem of accurate calibration of protein markers, and has the advantages of low detection cost and short detection cycle.

Description

Technical Field

[0001] The present invention relates to the field of biosensors, and more specifically to an impedance biosensor based on immunoaffinity and a preparation method and application thereof. Background Art

[0002] Due to many reasons, such as changes in people's lifestyles and the increase in the aging population, cancer has become a major chronic disease that seriously threatens people's health. The 5-year survival rate of patients with advanced cancer is low, and early diagnosis is of great significance in reducing the mortality rate of cancer. Therefore, early diagnosis and early treatment are regarded as the most important cancer prevention and control measures in my country. Tumor protein markers such as alpha-fetoprotein (AFP), transferrin (TRF), and carcinoembryonic antigen (CEA) are typical markers for early cancer screening.

[0003] The content of tumor protein markers in normal human tissues is low, and the sensitivity and specificity of the detection method are required to be high. At present, the application of proteomics to the study of tumor markers has become one of the hot spots in recent years. It has the advantages of high throughput, miniaturization, and automation, but it also has the disadvantages of high detection cost and high technical personnel operation requirements. Liquid biopsy is a method for diagnosing and detecting tumors. It can screen and detect tumor markers at an early stage, overcome the temporal and spatial heterogeneity of tumors, and has the advantages of non-invasive, easy repeated sampling, simple operation, and real-time monitoring. However, it also has the disadvantages of high price and inconsistent detection standards. Molecular biological methods for detecting tumor markers include immunoblotting, liquid chromatography-mass spectrometry, etc., which have the advantages of high throughput, strong specificity, and high sensitivity, but also have the disadvantages of high price and long detection cycle. Protein chips can be used to quantitatively detect specific types of proteins, which are of great significance to the occurrence of diseases, the screening of tumor markers, and the study of disease development mechanisms, and have become an important tool for biological research. The basic principle is to use protein molecules as ligands (probe proteins), and to fix them in order on the surface of solid phase carriers (gold surface, filter membrane, glass slide, etc.) through non-covalent adsorption, covalent binding, and affinity capture with streptavidin to form a microarray. The protein to be detected is directly labeled with fluorescent dyes (Cy3, Cy5, FITC, etc.), or the secondary antibody of the protein is labeled with fluorescent dyes. After binding with the protein on the chip, the excited fluorescence signal is detected by laser scanning and CCD photography technology, and analyzed by computer software system. However, since the light intensity of fluorescence is not high, the linear situation is sometimes not very ideal. For low-abundance protein samples, the sensitivity of fluorescence detection methods is not high. Therefore, there is an urgent need to develop a simple, low-cost, label-free, and rapid monitoring platform to detect cancer biomarkers for clinical diagnosis and screening. Summary of the invention

[0004] The purpose of the present invention is to provide an impedance biosensor based on immunoaffinity and its preparation method and application, so as to solve the problems of high price, long detection cycle and low sensitivity in the existing cancer biomarker detection technology.

[0005] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0006] According to a first aspect of the present invention, a method for preparing an impedance biosensor based on immunoaffinity is provided, the preparation method comprising: surface modification using mercaptopropionic acid or chitosan based on a wireless passive sensor, or surface modification using a nitrocellulose membrane based on a three-electrode sensor, and then combining a probe that can capture a tumor protein marker on the surface to prepare an impedance biosensor based on immunoaffinity.

[0007] According to a preferred embodiment of the present invention, the preparation method comprises the following steps: A1: the wireless passive sensor is soaked in anhydrous ethanol and blown dry with nitrogen; A2: 40-60 mM mercaptopropionic acid and 8-12 mM 6-mercapto-1-hexanol are mixed in a volume ratio of 1:1, and the wireless passive sensor treated in step A1 is placed in it, and incubated at room temperature for 12-20 hours; A3: 0.05-0.15 MEDC and thiosuccinimide of the same concentration are mixed in a volume ratio of 1:1 and then dropped on the chip, incubated at room temperature in the dark for 1-3 hours, gently rinsed with PBS, and blown dry with nitrogen.

[0008] According to another preferred embodiment of the present invention, the preparation method comprises the following steps: B1: placing the wireless passive sensor in a beaker filled with 100% ethanol solution, shaking it on a shaker for 10 to 20 minutes, then placing it in deionized water for ultrasonic treatment for 1 to 3 minutes, blowing it dry with nitrogen, and then placing it in an oxygen plasma cleaning machine for sample surface activation treatment; B2: dissolving chitosan in 2% acetic acid solution, stirring the chitosan magnetically to completely dissolve the chitosan, and the solution becomes clear and transparent to obtain a 1wt.% chitosan solution, weighing 1.8 to 2.2 mg of single-walled carbon nanotubes in a 1.5 mL plastic centrifuge tube, and adding 1 mL The 1% chitosan solution was ultrasonicated for 20-40 min to obtain a chitosan solution containing 1.8-2.2 mg / mL single-walled carbon nanotubes, and the mixed solution was obtained by ultrasonicating for 20-40 min; B3: 10-30 uL of the chitosan solution was dropped on the wireless passive sensor, heated and dried on a hot plate for 2-30 min, and placed in a ventilated place overnight; B4: 10-30 uL of the antibody was dropped on the wireless passive sensor prepared in step B3, placed in a 37°C incubator for 2 h, and completely dried.

[0009] According to another preferred embodiment of the present invention, the preparation method comprises the following steps: C1: dissolving a 2.5cm*2cm nitrocellulose membrane using 5mL of ethanol solution, shaking it on a shaker for 20-25min, centrifuging it at 6000rpm for 30min, removing the supernatant, and concentrating the volume to 60uL; C2: taking 10-30uL of the above-mentioned nitrocellulose membrane ethanol solution and dropping it on a three-electrode sensor, and drying it at room temperature; C3: taking 10-30uL of the antibody and dropping it on the three-electrode sensor obtained in step C2, and drying it.

[0010] According to a second aspect of the present invention, there is provided an immunoaffinity-based impedance biosensor prepared according to the preparation method described above.

[0011] According to the third aspect of the present invention, there is provided an application of an immunoaffinity-based impedance biosensor for detecting tumor protein markers, the application is not used for diagnosis and treatment of diseases, and comprises the following steps: 1) providing an immunoaffinity-based impedance biosensor as described above; 2) dropping tumor protein marker samples with gradient concentrations and the sample to be tested on the electrodes of the impedance biosensor respectively, and after the same incubation time, when the tumor protein marker is captured by the capture probe, the capacitance between the electrodes changes, thereby changing the frequency characteristics of the entire chip, and the detection of the tumor protein marker in the sample to be tested can be achieved by reading the change signal through a back-end vector network analyzer or an electrochemical workstation.

[0012] The tumor protein markers include: alpha-fetoprotein, transferrin, carcinoembryonic antigen and the like.

[0013] According to the present invention, the above method can also be used to modify lectins, thereby realizing the monitoring of specific glycosylation changes in biological samples.

[0014] The purpose of the present invention is to achieve the monitoring of relevant tumor protein markers under the condition of introducing less interference in the antigen-antibody binding environment. The bioelectric sensor prepared by modifying the wireless passive sensor with mercaptopropionic acid or chitosan and modifying the three-electrode sensor with nitrocellulose membrane has the advantages of no need for labeling, easy miniaturization and real-time online monitoring. The method based on the mixed thiol probe can increase the modification efficiency of the capture probe through the modification method of the mixed probe, thereby improving the detection sensitivity; the non-covalent modification method based on chitosan / carbon nanotubes, chitosan has good biocompatibility, which is conducive to the activity maintenance of antibodies or aptamers; the non-covalent modification method based on nitrocellulose membrane, nitrocellulose is a commonly used protein transfer medium, and has a very high affinity for antibody proteins. These three methods are all versatile and are suitable for the construction and preparation of a variety of impedance biosensors based on metal electrodes. According to the present invention, such a method for detecting tumor markers does not require fluorescent labeling of antibodies, does not rely on fluorescence intensity to judge the binding efficiency of antigens and antibodies, and the reflection coefficient changes regularly when the antigens and antibodies bind.

[0015] However, the prior art has never disclosed a technical solution for detecting tumor markers by combining thiol probes or chitosan with wireless passive sensors, nor has it disclosed a technical solution for detecting tumor markers by modifying nitrocellulose membranes on three-electrode sensors, and no relevant technical inspiration has been given.

[0016] In summary, the present invention provides for the first time such an impedance biosensor based on immunoaffinity and its application in the detection of tumor protein markers. Compared with the preparation method of traditional biosensors, the method of the present invention can ensure the high density, integrity and number of effective binding sites of biological molecules, realize the detection of trace proteins of tumor proteins, and the detection limit reaches the pg level. At the same time, it can also help solve the problem of accurate calibration of tumor markers, and has the advantages of low detection cost and short detection cycle. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 It is a preparation process flow chart (A) and a detection principle diagram (B) of an impedance biosensor prepared based on a wireless passive sensor according to the present invention;

[0018] Figure 2 The results of the impedance biosensor prepared by surface modification of the wireless passive sensor using mercaptopropionic acid are shown below; (A) the response curve of the AFP wireless passive biosensor to protein standards of different concentrations, and (B) the comparison of the response values ​​of different wireless passive sensors to AFP.

[0019] Figure 3The results of the impedance biosensor prepared by surface modification of chitosan based on wireless passive sensors; (A) the response curve of TRF wireless passive biosensor to protein standards of different concentrations; (B) the comparison of TRF response values ​​of different wireless passive sensors;

[0020] Figure 4 It is the detection result of the impedance biosensor prepared by surface modification of nitrocellulose membrane based on the three-electrode sensor, and shows the response curve of the impedance biosensor to CEA protein standards of different concentrations. DETAILED DESCRIPTION

[0021] The present invention is further described below in conjunction with specific examples. It should be understood that the following examples are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0022] Example 1 Preparation of impedance biosensor based on wireless passive sensor using mercaptopropionic acid for surface modification

[0023] In this embodiment, a modification method based on a thiol mixed probe (mercaptopropionic acid and 6-mercapto-1-hexanol) is provided. The method based on the mixed thiol can increase steric hindrance, improve the binding efficiency of the target molecule, and help improve the detection sensitivity. The specific steps are as follows:

[0024] 1) Cleaning of wireless passive sensors: Soak the wireless passive sensors in anhydrous ethanol for 5 minutes and blow dry with nitrogen;

[0025] 2) Modification of carboxyl groups: 50 mM mercaptopropionic acid and 10 mM 6-mercapto-1-hexanol were mixed in a volume ratio of 1:1, placed in the wireless passive sensor treated in step 1), and incubated at room temperature for 16 h;

[0026] 3) Activation of carboxyl groups: 0.1 M EDC and 0.1 M sulfosuccinimide (Sulfo-NHS) were mixed in a volume ratio of 1:1 and dropped onto the chip. The mixture was incubated at room temperature for 2 h in the dark, gently rinsed with PBS, and dried with nitrogen gas.

[0027] 4) Antigen-antibody binding: AFP protein standards with concentrations of 1 μg / ml, 100 ng / ml, 10 ng / ml, 1 ng / ml, and 100 pg / ml (each concentration volume was 60 μL) were dropped on different wireless passive chips, and after incubation for 10 min at each concentration, the reflection coefficient was measured using a vector network analyzer. PBS was used as the control group.

[0028] The experimental results showed that after adding 100 pg / mL, 1 ng / mL, 10 ng / mL, 100 ng / mL and 1 μg / mL AFP samples into the reaction chamber, the amplitude gradually decreased with the increase of the added sample concentration after the same incubation time.

[0029] Example 2 Preparation of impedance biosensor based on wireless passive sensor using chitosan and carbon nanotubes for surface modification

[0030] In this embodiment, a modification method based on chitosan and carbon nanotubes is provided. This is a non-covalent modification method. Chitosan has good biocompatibility, is conducive to maintaining the activity of antibodies or aptamers, and has the potential for repeated use. The modification method is simple and rapid, and can be used to detect tumor-related proteins in biological samples such as tissues, cells, and serum. The specific steps are as follows:

[0031] 1) Cleaning of wireless passive sensors: Place the wireless passive sensor in a 100% ethanol solution in a beaker, shake it on a shaker for 15 minutes, then place it in deionized water for 2 minutes of ultrasonic treatment, and blow it dry with nitrogen; then place it in an oxygen plasma cleaning machine for sample surface activation treatment;

[0032] 2) Preparation of chitosan solution: Dissolve 0.1g chitosan in 10mL 2% acetic acid solution, stir magnetically for 1h to completely dissolve the chitosan, and the solution becomes clear and transparent, thus obtaining a 1% (wt%) chitosan solution; weigh 2mg single-walled carbon nanotubes in a 1.5mL plastic centrifuge tube, add 1mL 1% chitosan solution and ultrasonicate for 30min to obtain a chitosan solution containing 2mg / mL single-walled carbon nanotubes. Ultrasonicate for 30min to obtain a mixed solution;

[0033] 3) Drying of chitosan: Take 20uL of chitosan solution and drop it on the wireless passive sensor treated in step 1), heat and dry it on a hot plate for 30min, and place it in a ventilated place overnight;

[0034] 4) Antibody drying: Take 20uL of antibody and drop it on the wireless passive sensor prepared in step 3), place it in a 37°C incubator for 2h to completely dry;

[0035] 5) TRF protein standards with concentrations of 1 μg / ml, 100 ng / ml, 10 ng / ml, 1 ng / ml, and 100 pg / ml (each concentration volume is 60 μL) were dropped onto the different wireless passive chips prepared in step 4), incubated for 10 min at each concentration, and the reflection coefficient was measured using a vector network analyzer, with PBS as a control;

[0036] The experimental results showed that after adding 100 pg / mL, 1 ng / mL, 10 ng / mL, 100 ng / mL and 1 μg / mL SCC protein samples into the reaction chamber, the amplitude gradually decreased with the increase of the added sample concentration after the same incubation time.

[0037] Example 3 Antibody modification method based on three-electrode sensor

[0038] In this embodiment, an antibody modification method based on a three-electrode sensor is provided. Nitrocellulose membrane is a capture reagent commonly used to fix proteins to detect samples. The nitrocellulose membrane is dissolved by an organic reagent and then physically adsorbed and combined with the three-electrode sensor, thereby achieving the detection of multiple tumor-related protein markers while maintaining the activity of antibodies and antigens.

[0039] 1) Dissolution of organic reagents for nitrocellulose membrane: Dissolve nitrocellulose membrane of size 2.5 cm*2 cm in 5 mL of ethanol solution, shake on a shaker for 15 min, centrifuge at 6000 rpm for 30 min, remove the supernatant, and concentrate the volume to 60 uL;

[0040] 2) Take 20uL of the above nitrocellulose membrane alcohol solution and drop it on the three-electrode sensor and dry it at room temperature for 10 minutes;

[0041] 3) Antibody drying: After diluting the CEA antibody 2 times, take 20uL of the antibody solution and drop it on the three electrodes, and dry it in a 37°C constant temperature incubator for 1h (or at room temperature for 2h);

[0042] 4) CEA protein standards with concentrations of 1 μg / ml, 100 ng / ml, 10 ng / ml, 1 ng / ml, 100 pg / ml, 10 pg / ml, and 1 pg / ml (each concentration volume is 60 μL) were dropped on different three-electrode sensors, and the incubation time of each concentration of CEA protein standard and antibody was 10 min. After the incubation, the electrochemical workstation was used for measurement, and PBS was used as a control;

[0043] The experimental results show that when 100pg / mL, 1ng / mL, 10ng / mL, 100ng / mL and 1μg / mL CEA protein samples were dropped on the three electrodes, after the same incubation time, the resistance gradually decreased with the increase of the added sample concentration.

[0044] The above is only a preferred embodiment of the present invention, and is not intended to limit the scope of the present invention. The above embodiments of the present invention can also be modified in various ways. All simple, equivalent changes and modifications made according to the claims and the contents of the specification of the present invention fall within the scope of protection of the claims of the present invention. The contents not described in detail in the present invention are all conventional technical contents.

Claims

1. A method for preparing an immunoaffinity-based impedance biosensor, characterized in that: The preparation method comprises: using mercaptopropionic acid or chitosan to perform surface modification based on a wireless passive sensor, or using a nitrocellulose membrane to perform surface modification based on a three-electrode sensor, and then combining a probe capable of capturing a tumor protein marker on the surface, thereby preparing an impedance biosensor based on immunoaffinity; The preparation method comprises the following steps: A1: The wireless passive sensor is soaked in anhydrous ethanol and dried with nitrogen; A2: Mix 40-60 mM mercaptopropionic acid and 8-12 mM 6-mercapto-1-hexanol in a volume ratio of 1:1, put into the wireless passive sensor treated in step A1, and incubate at room temperature for 12-20 h; A3: 0.05~0.15M EDC and thiosuccinimide of the same concentration were mixed in a volume ratio of 1:1 and dropped onto the chip. Incubate at room temperature for 1~3 hours in the dark, rinse gently with PBS, and blow dry with nitrogen gas. The preparation method comprises the following steps: B1: Place the wireless passive sensor in a beaker filled with 100% ethanol solution, shake it on a shaker for 10-20 minutes, then place it in deionized water for ultrasonic treatment for 1-3 minutes, blow it dry with nitrogen, and then place it in an oxygen plasma cleaner for sample surface activation treatment; B2: Dissolve chitosan in 2% acetic acid solution, stir magnetically to completely dissolve chitosan, and make the solution clear and transparent to obtain a 1 wt.% chitosan solution. Weigh 1.8-2.2 mg of single-walled carbon nanotubes in a 1.5 mL plastic centrifuge tube, add 1 mL of 1% chitosan solution, and sonicate for 20-40 min to obtain a chitosan solution containing 1.8-2.2 mg / mL single-walled carbon nanotubes. Sonicate for 20-40 min to obtain a mixed solution. B3: Take 10~30uL of chitosan solution and drop it on the wireless passive sensor, heat and dry it on a hot plate for 2~30min, and place it in a ventilated place overnight; B4: Take 10~30uL of antibody and drop it on the wireless passive sensor prepared in step B3, place it in a 37℃ incubator for 2h, and dry it completely; or The preparation method comprises the following steps: C1: Dissolve 2.5cm*2cm nitrocellulose membrane in 5mL ethanol solution, shake on a shaker for 20-25min, centrifuge at 6000rpm for 30min, remove the supernatant, and concentrate the volume to 60uL; C2: Take 10~30uL of the above nitrocellulose membrane ethanol solution and drop it on the three-electrode sensor and dry it at room temperature; C3: Take 10~30uL of antibody and drop it on the three-electrode sensor obtained in step C2, dry it, and you are done.

2. An immunoaffinity-based impedance biosensor prepared according to the preparation method of claim 1.

3. An application of an immunoaffinity-based impedance biosensor in the detection of tumor protein markers, the application not being used for the diagnosis and treatment of diseases, characterized in that: The following steps are involved: 1) providing an immunoaffinity-based impedance biosensor according to claim 2; 2) Tumor protein marker samples with gradient concentrations and the sample to be tested are dropped onto the electrodes of the impedance biosensor respectively. After the same incubation time, when the tumor protein marker is captured by the capture probe, the capacitance between the electrodes changes, thereby changing the frequency characteristics of the entire chip. The change signal can be read by a back-end vector network analyzer or an electrochemical workstation to detect the tumor protein marker in the sample to be tested.

4. The use according to claim 3, characterized in that: The tumor protein markers include: alpha-fetoprotein, transferrin, and carcinoembryonic antigen.

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