Magnetic levitation synergized non-immobilized electrochemical immunosensor and preparation method and application thereof

Abstract

The application belongs to the technical field of sensors and the field of electrochemical analysis, and relates to a magnetic floating and non-immobilized electrochemical immunosensor, a preparation method and application thereof. The electrochemical immunosensor comprises a floating probe, a magnetic signal reporting probe and a magnetic electrode. The floating probe is formed by connecting hollow silica microspheres and a capture antibody 1. The magnetic signal reporting probe is composed of a magnetic mesoporous silica, a signal molecule and a capture antibody 2. The target biomarker is specifically combined with the capture antibody 1 and the capture antibody 2 to form a sandwich immunocomplex. The sandwich immunocomplex and the remaining free floating probe are separated and removed by buoyancy. The remaining free magnetic signal reporting probe is collected by magnetic separation. The magnetic electrode is matched with the remaining free magnetic signal reporting probe collected by magnetic separation. The electrochemical immunosensor provided by the application has the advantages of simple assembly process, simple operation, high sensitivity, good specificity, low cost and the like, and can realize quantitative detection of early biomarkers.

Description

Technical Field

[0001] This invention belongs to the fields of sensor technology and electrochemical analysis, and relates to a magnetically levitated non-immobilized electrochemical immunosensor, its preparation method, and its application. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] According to the inventors' research, current methods for detecting biomarkers such as alpha-fetoprotein (AFP) using electrochemical biosensors require immobilizing biorecognition elements onto the sensor surface. This necessitates precise control of immobilization conditions to effectively adjust the assembly density of the biorecognition elements, ensuring good biosensing performance. Furthermore, biorecognition events at the solution-electrode interface can easily affect biorecognition efficiency and signal amplification. Therefore, immobilization-based electrochemical biosensing technology suffers from problems such as cumbersome modification processes, high operational difficulty, and poor reproducibility and stability. Summary of the Invention

[0004] To address the shortcomings of existing technologies, the present invention aims to provide a magnetically levitated, non-immobilized electrochemical immunosensor, its preparation method, and its application. The electrochemical immunosensor provided by the present invention has advantages such as simple assembly process, easy operation, high sensitivity, good specificity, and low cost, and can realize the quantitative detection of early biomarkers.

[0005] To achieve the above objectives, the technical solution of the present invention is as follows:

[0006] On the one hand, a magnetically levitated, non-immobilized electrochemical immunosensor includes a flotation probe, a magnetic signal reporting probe, and a magnetic electrode;

[0007] The flotation probe is formed by connecting hollow silica microspheres and capture antibody 1, wherein the capture antibody 1 can specifically bind to the target biomarker;

[0008] The magnetic signal reporter probe consists of magnetic mesoporous silica, a signal molecule, and capture antibody 2. The magnetic mesoporous silica is loaded with the signal molecule and is connected to the capture antibody 2. The capture antibody 2 can specifically bind to the target biomarker.

[0009] The target biomarker in the solution binds specifically to capture antibody 1 and capture antibody 2, causing the flotation probe and the magnetic signal reporter probe to bind into a sandwich immune complex. The sandwich immune complex and the remaining free flotation probe are removed by buoyancy separation, while the remaining free magnetic signal reporter probe is collected by magnetic separation. The magnetic electrode works in conjunction with the magnetic separation to collect the remaining free magnetic signal reporter probe.

[0010] On the other hand, a method for preparing the above-mentioned magnetic levitation-coordinated non-immobilized electrochemical immunosensor includes a flotation probe preparation process and a magnetic signal reporting probe preparation process.

[0011] The preparation process of the flotation probe is as follows: hollow silica microspheres, capture antibody 1 and glutaraldehyde are cross-linked to connect the hollow silica microspheres and capture antibody 1.

[0012] The preparation process of the magnetic signal reporter probe is as follows: magnetic mesoporous silica and signal molecules are dispersed in a solvent so that the signal molecules are loaded in the mesopores of the magnetic mesoporous silica, thus obtaining magnetic mesoporous silica loaded with signal molecules; the magnetic mesoporous silica loaded with signal molecules is electrostatically adsorbed with PEI, and then capture antibody 2 is added for incubation to link the magnetic mesoporous silica loaded with signal molecules with capture antibody 2.

[0013] Thirdly, the application of the above-mentioned magnetic levitation-coordinated non-immobilized electrochemical immunosensor in the preparation of reagents, components or systems for detecting tumor markers, wherein the tumor marker is a target biomarker.

[0014] Fourthly, a liver cancer detection kit includes the aforementioned magnetically levitated non-immobilized electrochemical immunosensor and buffer solution, wherein the target biomarker for liver cancer detection is alpha-fetoprotein.

[0015] The beneficial effects of this invention are as follows:

[0016] (1) This invention uses flotation microspheres (hollow silica microspheres) as the target recognition element carrier to prepare a buoyancy-mediated high-efficiency capture and separation probe. The flotation microsphere separation carrier can achieve target recognition without instruments or external forces. Magnetic nanoparticles (magnetic mesoporous silica) are used as the electrical signal reporting molecular carrier to design a magnetically controlled electrode signal output mode that does not require fixing. The signal reporting of the magnetic signal probe on the magnetic electrode can avoid the complex fixing and modification of the electrode.

[0017] (2) The non-interface quasi-homogeneous biorecognition system in the solution phase of the present invention avoids the cumbersome steps of electrode interface modification and can maximize the spatial freedom of biomolecules, which is conducive to achieving efficient molecular recognition events.

[0018] (3) The dual synergy of magnetic levitation separation in this invention simplifies operation and shortens analysis time, and is expected to realize the concept of minimalist detection without the need for external force, and is expected to be used in the field of Point-of-Care detection.

[0019] (4) The magnetic levitation-coordinated non-immobilized electrochemical immunosensor provided by the present invention has versatility and flexibility. It can be easily extended to the quantitative detection of other biomarkers by changing the recognition element, providing a reference for the establishment of a universal and highly sensitive testing method for complex life systems. Attached Figure Description

[0020] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0021] Figure 1 This is a schematic diagram of the magnetic levitation-assisted non-immobilized electrochemical immunosensor in an embodiment of the present invention.

[0022] Figure 2 The diagram shows the zeta (ζ) potential during the probe preparation process in this embodiment of the invention. A represents the ζ-potential of different modified microspheres, and B represents the ζ-potential of different modified MMS.

[0023] Figure 3 The following are the (A) DPV response and (B) electrochemical impedance spectroscopy of the electrode under different conditions in the embodiments of the present invention: (a) bare mGCE, (b) MMS / mGCE, (c) MMS(MB)-Ab2 / mGCE, and (d) and (e) are the detection of the solution phase reaction system in the presence of blank and target analyte AFP, respectively.

[0024] Figure 4 (A) shows the electrochemical immunosensor in this embodiment of the invention reacting with different concentrations of the target analyte AFP (from a to h, 0.01, 0.1, 1, 10, 100, 1000, 1 × 10⁻⁶). 4 and 1×10 5 (a) DPV response of pg / mL; (b) DPV peak current versus target AFP concentration (in the range of 0.1 to 1 × 10⁻⁶ pg / mL); 4 Linear calibration curves between logarithms within the range of pg / mL, with error bars representing the standard deviation of three parallel experiments;

[0025] Figure 5 This is a specific detection histogram of the electrochemical immunosensor in this embodiment of the invention, showing AFP, Mixer, HSA, CEA, PSA, HIgG, and BSA. ΔI represents the difference in DPV response with and without the target (or interfering agent), and the error bars represent the standard deviation of three parallel experiments.

[0026] Figure 6 The figure shown is a result of verifying the feasibility of the electrochemical immunosensor in clinical diagnosis in this embodiment of the invention. A is a histogram of AFP detection in clinical serum samples. Clinical samples were collected from 6 liver cancer patients (diagnosed as hepatocellular carcinoma (1-3), cholangiocarcinoma (4,5) and liver metastasis (6)). The dashed line represents the theoretical current change of clinical reference AFP concentration. B is the DPV response corresponding to the same color AFP detection in A. The error bars represent the standard deviation of three parallel experiments. Detailed Implementation

[0027] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0028] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of exemplary embodiments according to the invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0029] Given that current immobilized electrochemical biosensing technologies suffer from problems such as cumbersome modification processes, high operational difficulty, and poor reproducibility and stability, this invention proposes a magnetic levitation-assisted non-immobilized electrochemical immunosensor, its preparation method, and its application.

[0030] A typical embodiment of the present invention provides a magnetically levitated non-immobilized electrochemical immunosensor, comprising a flotation probe, a magnetic signal reporting probe, and a magnetic electrode;

[0031] The flotation probe is formed by connecting hollow silica microspheres and capture antibody 1, wherein the capture antibody 1 can specifically bind to the target biomarker;

[0032] The magnetic signal reporter probe consists of magnetic mesoporous silica, a signal molecule, and capture antibody 2. The magnetic mesoporous silica is loaded with the signal molecule and is connected to the capture antibody 2. The capture antibody 2 can specifically bind to the target biomarker.

[0033] The target biomarker in the solution binds specifically to capture antibody 1 and capture antibody 2, causing the flotation probe and the magnetic signal reporter probe to bind into a sandwich immune complex. The sandwich immune complex and the remaining free flotation probe are removed by buoyancy separation, while the remaining free magnetic signal reporter probe is collected by magnetic separation. The magnetic electrode works in conjunction with the magnetic separation to collect the remaining free magnetic signal reporter probe.

[0034] In some embodiments, hollow silica microspheres and capture antibody 1 are formed by a cross-linking reaction with glutaraldehyde.

[0035] In some embodiments, the signaling molecule is methylene blue.

[0036] In some embodiments, the magnetic mesoporous silica is nanoparticles.

[0037] In some embodiments, the magnetic electrode is a magnetic glassy carbon electrode.

[0038] Another embodiment of the present invention provides a method for preparing the above-mentioned magnetic levitation-coordinated non-immobilized electrochemical immunosensor, including a flotation probe preparation process and a magnetic signal reporting probe preparation process;

[0039] The preparation process of the flotation probe is as follows: hollow silica microspheres, capture antibody 1 and glutaraldehyde are cross-linked to connect the hollow silica microspheres and capture antibody 1.

[0040] The preparation process of the magnetic signal reporter probe is as follows: magnetic mesoporous silica and signal molecules are dispersed in a solvent so that the signal molecules are loaded in the mesopores of the magnetic mesoporous silica, thus obtaining magnetic mesoporous silica loaded with signal molecules; the magnetic mesoporous silica loaded with signal molecules is electrostatically adsorbed with PEI, and then capture antibody 2 is added for incubation to link the magnetic mesoporous silica loaded with signal molecules with capture antibody 2.

[0041] In some embodiments, hollow silica microspheres are activated using a piranha solution. The activated hollow silica microspheres are then coupled with 3-aminopropyltriethoxysilane (APTES) to obtain amino-functionalized hollow silica microspheres. The amino-functionalized hollow silica microspheres, glutaraldehyde, and capture antibody 1 are then subjected to a cross-linking reaction. The cross-linking reaction is carried out at 3–5°C for 20–30 h. Specifically, the piranha solution is obtained by mixing H₂SO₄ (≥96%) and H₂O₂ (30%) at a volume ratio of 2.5–3.5:1.

[0042] In some embodiments, bovine serum albumin (BSA) is added after the cross-linking reaction to block the remaining active sites, thereby improving detection sensitivity.

[0043] In one or more embodiments, HSB was first activated overnight with freshly prepared piranha solution, then aminosilanized overnight with 5% APTES (v / v) solution diluted with methanol, and washed extensively with methanol and DMF to obtain aminofunctionalized HSB. Then, 1 mL of 5% glutaraldehyde was added to 25 μL of the aminofunctionalized HSB solution and shaken for 6 h. After washing several times with PBS, the mixture was dispersed in 1 mL of PBS. Subsequently, 25 μL of Ab1 containing 1 mg / mL AFP was added, and the mixture was incubated at 4 °C with shaking for 24 h to form an HSB-Ab1 conjugate. Then, 3% BSA was added to block the remaining active sites. Finally, the product was separated by buoyancy, washed with PBS, resuspended in 1 mL of PBS buffer, and stored at 4 °C for later use.

[0044] In some embodiments, after incubation with capture antibody 2, bovine serum albumin (BSA) is added to block the remaining active sites, thereby improving detection sensitivity.

[0045] In some or more embodiments, 2 mg of MMS nanocomposite material was dispersed in 1 mL of PBS, and then 100 μL of 1 mM MB was added. The mixture was gently shaken overnight in the dark. With the aid of magnetic separation, the MB-loaded MMS was separated, and washed with PBS buffer to remove unbound MB. Next, 1 mL of 0.2 mg / mL PEI was added, and the mixture was gently shaken in the dark for 12 hours to achieve adsorption of positively charged PEI molecules onto negatively charged MMS (MB). The resulting product was magnetically separated, washed, and redispersed in 1 mL of PBS buffer. 20 μL of 0.1 mg / mL Ab2 was added, and the mixture was incubated at 4 °C with shaking for 24 h. Then, 3% BSA was added to block the remaining active sites. Finally, the resulting signal reporter probe MMS (MB)-Ab2 was magnetically separated, washed, and redispersed in 1 mL of PBS, and stored at 4 °C for later use.

[0046] A third embodiment of the present invention provides an application of the above-mentioned magnetic levitation-coordinated non-immobilized electrochemical immunosensor in the preparation of reagents, components or systems for detecting tumor markers, wherein the tumor marker is a target biomarker.

[0047] In some embodiments, the tumor markers are alpha-fetoprotein, carcinoembryonic antigen, prostate-specific antigen, etc.

[0048] A fourth embodiment of the present invention provides a liver cancer detection kit, comprising the above-mentioned magnetic levitation-coordinated non-immobilized electrochemical immunosensor and a buffer solution, wherein the target biomarker for liver cancer detection is alpha-fetoprotein.

[0049] In some embodiments, the buffer solution is a phosphate buffer solution.

[0050] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0051] Example

[0052] HSB (IM16K) hollow silica microspheres (average particle size 20 μm, density 0.46 g / cm³) were activated using a freshly prepared piranha solution (H₂SO₄ (98%):H₂O₂ (30%) = 3:1). -3 The product (purchased from 3M, USA) was incubated overnight, then aminosilanized overnight with 5% APTES (v / v) solution diluted in methanol, and washed extensively with methanol and DMF to obtain amino-functionalized HSB. Then, 1 mL of 5% glutaraldehyde was added to 25 μL of the amino-functionalized HSB solution (5 mg / mL), and the mixture was shaken for 6 h. After washing several times with PBS, the mixture was dispersed in 1 mL of PBS. Subsequently, 25 μL of 1 mg / mL AFP Ab1 (Anti-AFP McAb (coating), purchased from Shanghai Lingchao Biotechnology Co., Ltd.) was added, and the mixture was incubated at 4 °C with shaking for 24 h to form the HSB-Ab1 conjugate. Then, 3% BSA was added to block the remaining active sites. Finally, the product was separated by buoyancy, washed with PBS, and resuspended in 1 mL of PBS buffer. It was stored at 4 °C for later use.

[0053] Preparation of magnetic signal reporting probes:

[0054] 800 mg of the prepared Fe3O4 NPs were ultrasonically dispersed in 72 mL of ultrapure water. Then, 1.36 g of CTAB and 8 g of TEA were added to the solution, and the mixture was mechanically stirred at 85 °C for 1 h. Next, 11.16 mL of TEOS was rapidly added dropwise to the reaction mixture, and stirring continued for 2 h to obtain a brown colloidal suspension. The prepared brown colloidal nanoparticles were collected using a magnet, washed several times with ethanol, and vacuum dried at 60 °C for 24 h. Finally, the dried colloidal nanoparticles were calcined at 550 °C for 5 h to remove the residual surfactant CTAB within the mesopores, yielding the MMS nanocomposite material.

[0055] 2 mg of MMS nanocomposite was dispersed in 1 mL of PBS, and then 100 μL of 1 mM MB was added. The mixture was gently shaken overnight in the dark. With the aid of magnetic separation, the MB-loaded MMS was separated, and washed with PBS buffer to remove unbound MB. Next, 1 mL of 0.2 mg / mL PEI (polyethyleneimine) was added, and the mixture was gently shaken in the dark for 12 hours to achieve adsorption of positively charged PEI molecules onto negatively charged MMS (MB). The resulting product was magnetically separated, washed, and redispersed in 1 mL of PBS buffer. 20 μL of 0.1 mg / mL Ab2 (Anti-AFP McAb (labeling), purchased from Shanghai Lingchao Biotechnology Co., Ltd.) was added, and the mixture was incubated at 4 °C with shaking for 24 h. Then, 3% BSA was added to block the remaining active sites. Finally, the obtained signal reporter probe MMS (MB)-Ab2 was magnetically separated, washed, and redispersed in 1 mL of PBS, and stored at 4 °C for later use.

[0056] Electrochemical Measurement: First, 10 μL of HSB-Ab1 multifunctional flotation probe was mixed with different concentrations of AFP target protein and incubated at room temperature (RT) for 50 min with shaking to achieve target protein recognition. Then, 20 μL of MMS(MB)-Ab2 magnetic signal reporter probe was added, and the mixture was incubated on a shaker at RT for 40 min. Subsequently, with the help of buoyancy, the HSB-Ab1 / AFP / MMS(MB)-Ab2 conjugate floated to the surface of the liquid, and the residual MMS(MB)-Ab2 probe in the solution under the microspheres was magnetically collected and dropped onto a magnetic glassy carbon electrode (mGCE). The electrochemical response was recorded using differential pulse voltammetry (DPV) with a scan potential of 0.1 V to -0.6 V, a pulse amplitude of 50 mV, a pulse width of 50 ms, a pulse period of 0.2 s, and a potential increment of 4 mV (with Ag / AgCl as the reference electrode).

[0057] The principle is as follows Figure 1 As shown, a multifunctional flotation probe with good recognition and separation capabilities was prepared by cross-linking HSB with the primary antibody (Ab1) using glutaraldehyde. Simultaneously, a multifunctional magnetic reporter probe with signal transduction, amplification, and output functions was prepared by loading magnetic mesoporous silica (MMS) nanocomposite materials with the electrochemical signal molecule methylene blue (MB) for labeling the signal antibody (Ab2). The presence of the target protein AFP promoted the formation of a sandwich immune complex, and the buoyancy enabled the separation of bound and unbound signal reporter probes. The unbound probes were firmly and effectively magnetically adsorbed onto the surface of the magnetic electrode. The electrochemical signal peak of MB at -0.28V can reflect the concentration of the target antigen AFP, enabling precise electrochemical measurement.

[0058] To verify the successful preparation of the probe, the zeta(ζ) potential behavior was investigated. Figure 2 The successful assembly process of the multifunctional flotation probe and magnetic signal reporting probe at each step was verified. Figure 2 As shown in Figure A, the zeta potential of the activated hydroxyl microspheres is -7.30 mV, which originates from the abundant hydroxyl groups on the microsphere surface. After aminosilanization with APTES, the prepared microspheres are aminated and positively charged (4.62 mV). Subsequently, the zeta potential reverses again to negative (-18.2 mV), indicating that the negatively charged primary antibody (Ab1) is linked to the aminated microspheres under glutaraldehyde crosslinking. The comparative results show that the multifunctional flotation probe was successfully prepared; for the magnetic signal reporting probe, the zeta potential of the prepared MMS is -29.13 mV (…). Figure 2 (B) Subsequent loading of MB slightly reduced the negative charge to -22.90 mV. Conversely, further modification of MMS with a high-density positively charged PEI resulted in a zeta potential of +11.60 mV, indicating successful modification of MMS with branched PEI molecules. Notably, when the signal antibody (Ab2) underwent further modification, its zeta potential was -16.67 mV, suggesting that the positively charged MMS(MB)-PEI facilitates the electrostatic adsorption of the negatively charged Ab2. These results demonstrate that a magnetic signal reporter probe was ultimately obtained by modifying MMS with MB and Ab2.

[0059] To verify the feasibility of this method, the electrochemical immunosensor was fabricated through ( Figure 3 The DPV and electrochemical impedance spectroscopy (EIS) were used to characterize the signal. First, the DPV response under different conditions was investigated. Figure 3 As shown in Figure A, no electrochemical response was observed for bare mGCE (curve a) and MMS / mGCE (curve b).

[0060] When MMS(MB)-Ab2 is magnetically adsorbed onto the electrode, a distinct DPV peak for MB is observed (curve c), indicating successful preparation of the magnetic signal reporter probe. As expected, when the quasi-homogeneous immunoreaction system without target AFP is adsorbed onto the electrode (curve d), the DPV response is almost identical to that of MMS(MB)-Ab2 adsorbed only onto the electrode, indicating minimal non-specific adsorption of the reporter probe onto the HSB-Ab1 multifunctional flotation probe. In contrast, the presence of the target protein AFP leads to a significant decrease in DPV (curve e), suggesting that successful immunorecognition captures a certain proportion of MMS(MB)-Ab2, resulting in a reduction in the number of residual reporter probes. These results fully demonstrate the feasibility of the designed AFP-immobilized electrochemical immunosensing strategy.

[0061] Electrochemical impedance spectroscopy (EIS) has proven to be a powerful tool for studying interfacial states and has been further used to verify the implementation of hypothetical strategies. Figure 3As shown in B, the bare mGCE exhibits a very small semicircle (curve a, Ω), indicating that [Fe(CN)6] 3- / 4- The electron transfer process is very fast. EIS increases with the magnetic adsorption of MMS, due to the non-conductive nature of MMS increasing the electron transport resistance (curve b, Ω). After magnetic adsorption of MMS(MB)-Ab2, the charge transfer resistance (R)... et The R value significantly increased (curve c, Ω), which can be attributed to the synergistic effect of MMS and the protein insulating layer forming on mGCE. Furthermore, when mGCE was incubated in a homogeneous immunoreaction system without target AFP, there was no significant change compared to the above-mentioned treatment curve c (curve d, Ω), indicating no significant nonspecific adsorption between the multifunctional flotation probe and the magnetic signal reporter probe. In contrast, the presence of the target protein AFP in the immunoreaction system led to R... et The decrease was even greater (curve e, Ω), because immune recognition led to a reduction in the adsorption of MMS(MB)-Ab2. These results confirm the feasibility and effectiveness of the designed strategy.

[0062] Under optimal experimental conditions, this example evaluated the analytical performance of a non-immobilized electrochemical immunosensor in response to different concentrations of AFP. Figure 4 As shown in Figure A, the DPV signal gradually decreased as the concentration of the target analyte AFP increased from 10 fg / mL to 100 ng / mL (curves a to h). A good linear relationship was observed between the current and the logarithm of the AFP concentration in the range of 100 fg / mL to 10 ng / mL. Figure 4 B) The detection limit was calculated to be 14.52 fg / mL. The sensor designed in this embodiment exhibits a wide linear range and a relatively low detection limit, which can be attributed to the excellent design of the buoyancy-magnetic mediated homogeneous biosensor, providing an efficient and simple analytical method for highly sensitive and accurate quantification of tumor protein biomarkers.

[0063] To investigate the specificity of the biosensor designed in this embodiment, several possible interfering proteins, including human serum albumin (HSA), carcinoembryonic antigen (CEA), prostate-specific antigen (PSA), HIgG, and bovine serum albumin (BSA), were used as interfering substances to systematically evaluate the specificity of the immune sensor. Figure 5 As shown, only the presence of the target protein AFP resulted in a significant current change, while the presence of interfering proteins caused negligible current changes. Furthermore, as expected, the current response of the mixed analytical solution containing both the target AFP and all interfering proteins was not significantly different from that containing only the target AFP. These results demonstrate that the proposed immunosensor exhibits good specificity for AFP detection and can accurately distinguish between the target protein and interfering proteins.

[0064] To evaluate the feasibility of the sensing strategy designed in this embodiment for clinical diagnosis, AFP was measured in human serum samples collected from six liver cancer patients (diagnosed as hepatocellular carcinoma (1-3), cholangiocarcinoma (4-5), and liver metastases (6), respectively). Because AFP levels were above the upper limit of the linear response range, the samples were diluted 1000-fold with PBS (0.01M, pH 7.4) before measurement. Figure 6 As shown, samples 1-3 exhibited higher electrochemical responses, while samples 4-6 showed lower responses, indicating that AFP expression levels were higher in patients 1-3 than in 4-6. The AFP concentrations obtained by this method were consistent with those measured by the clinical reference ELISA method, with acceptable relative error and RSD. This suggests that this method can be used for accurate diagnosis of AFP in clinical samples. Interestingly, the significant difference in AFP levels between samples 1-3 and 4-6 indicates that hepatocellular carcinoma typically shows higher AFP levels, while cholangiocarcinoma and liver metastases show lower AFP levels, providing a useful reference for understanding histological type and clinical stage. Therefore, these results demonstrate the satisfactory practicality of measuring AFP in complex biological samples such as human serum.

[0065] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A magnetically levitated, non-immobilized electrochemical immunosensor, characterized in that, Includes flotation probes, magnetic signal reporting probes, and magnetic electrodes; The flotation probe is formed by connecting hollow silica microspheres and capture antibody 1, wherein the capture antibody 1 can specifically bind to the target biomarker; The magnetic signal reporter probe consists of magnetic mesoporous silica, a signal molecule, and capture antibody 2. The magnetic mesoporous silica is loaded with the signal molecule and is connected to the capture antibody 2. The capture antibody 2 can specifically bind to the target biomarker. The signal molecule is methylene blue; The target biomarker in solution binds specifically to capture antibody 1 and capture antibody 2, causing the flotation probe and the magnetic signal reporter probe to bind into a sandwich immune complex. The sandwich immune complex and the remaining free flotation probe are removed by buoyancy separation, thus separating the bound and free signal reporter probe. At the same time, the remaining free magnetic signal reporter probe is collected by magnetic separation. The magnetic electrode works in conjunction with the magnetic separation to collect the remaining free magnetic signal reporter probe. The electrochemical signal peak of methylene blue in the free magnetic signal reporter probe reflects the concentration of the target biomarker.

2. The magnetically levitated, non-immobilized electrochemical immunosensor as described in claim 1, characterized in that, Hollow silica microspheres and capture antibody 1 are formed by cross-linking with glutaraldehyde.

3. A method for preparing a magnetically levitated, non-immobilized electrochemical immunosensor as described in claim 1 or 2, characterized in that, This includes the preparation process of flotation probes and the preparation process of magnetic signal reporting probes; The preparation process of the flotation probe is as follows: hollow silica microspheres, capture antibody 1 and glutaraldehyde are cross-linked to connect the hollow silica microspheres and capture antibody 1. The preparation process of the magnetic signal reporter probe is as follows: magnetic mesoporous silica and signal molecules are dispersed in a solvent so that the signal molecules are loaded in the mesopores of the magnetic mesoporous silica, thus obtaining magnetic mesoporous silica loaded with signal molecules; the magnetic mesoporous silica loaded with signal molecules is electrostatically adsorbed with PEI, and then capture antibody 2 is added for incubation to link the magnetic mesoporous silica loaded with signal molecules with capture antibody 2.

4. The method for preparing the magnetically levitated, non-immobilized electrochemical immunosensor as described in claim 3, characterized in that, Hollow silica microspheres were activated using a piranha solution. After activation, the hollow silica microspheres were coupled with 3-aminopropyltriethoxysilane to obtain amino-functionalized hollow silica microspheres. The amino-functionalized hollow silica microspheres, glutaraldehyde, and capture antibody 1 were then cross-linked.

5. The method for preparing the magnetically levitated, non-immobilized electrochemical immunosensor as described in claim 3, characterized in that, Bovine serum albumin was added after the cross-linking reaction to block the remaining active sites.

6. The method for preparing the magnetically levitated, non-immobilized electrochemical immunosensor as described in claim 3, characterized in that, After incubation with capture antibody 2, bovine serum albumin was added to block the remaining active sites.

7. The application of the magnetically levitated, non-immobilized electrochemical immunosensor according to claim 1 or 2 in the preparation of reagents, components, or systems for detecting tumor markers, wherein the tumor marker is a target biomarker.

8. The application of the magnetically levitated, non-immobilized electrochemical immunosensor as described in claim 7 in the preparation of reagents, components, or systems for detecting tumor markers, characterized in that, The tumor markers are alpha-fetoprotein, carcinoembryonic antigen, and prostate-specific antigen.

9. A liver cancer detection kit, characterized in that, The invention includes the magnetically levitated non-immobilized electrochemical immunosensor and buffer solution as described in claim 1 or 2, with alpha-fetoprotein as the target biomarker for liver cancer detection.

10. The liver cancer detection kit as described in claim 9, characterized in that, The buffer solution is a phosphate buffer solution.

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