Microcantilever sensing detection method based on magnetic force enhancement

Abstract

A microcantilever sensing detection method based on magnetic force enhancement, the detection method includes fixing a composite coupler to a reaction pool containing a buffer solution; the composite coupler is obtained by combining a microcantilever modified with a first aptamer and a magnetic nanoparticle modified with a second aptamer through the first aptamer and the second aptamer; applying a magnetic field force to the reaction pool, and adding a sample solution to be detected to the reaction pool; wherein the magnetic nanoparticles respond to the magnetic field force so that the second aptamer modified on the magnetic nanoparticles is separated from the first aptamer modified on the microcantilever, and the target molecule in the sample solution to be detected is combined with the first aptamer modified on the microcantilever, and the microcantilever is displaced; the concentration of the target molecule in the sample solution to be detected is obtained according to the displacement generated by the microcantilever. The present invention achieves the purpose of improving the sensitivity of microcantilever immunosensing by replacing the intermolecular force with magnetic force.

Description

Technical Field

[0001] The invention relates to the field of sensing detection, and in particular to a micro-cantilever sensing detection method based on magnetic force enhancement. Background Art

[0002] The principle of immunosensor technology is based on the specific pairing reaction of antigen and antibody, that is, for a certain target molecule (antigen) to be detected, the corresponding probe molecule (antibody) is produced by biological immunity (injecting the antigen into the body of a small animal), the probe molecule is extracted and purified, and the specific binding of antigen and antibody is used to detect the target molecule in the sample. Existing immunosensor technologies such as enzyme-linked immunosorbent assay (ELISA, the principle is to use the specific reaction of antigen and antibody and the catalytic amplification of enzyme label) and immunofluorescence method (IF, the principle is to label antigen or antibody with fluorescent fragments) all require markers to trace the reaction results of antigen and antibody, that is, only antibodies cannot establish corresponding detection methods, and enzyme labels or fluorescent markers or protein complexes are also required. However, the markers of some difficult-to-prepare target molecules are expensive, or almost impossible to prepare or purchase. At the same time, the detection of enzyme-linked immunosorbent assay and protein chip requires cleaning after each step of reaction from the principle operation. The labeling process is cumbersome and time-consuming. It is a post-test and cannot be detected in real time, in situ, and online.

[0003] Microcantilever immunosensing technology based on surface stress detection is a new immuno-biochemical sensing method that has emerged in recent years. Compared with traditional immunosensing methods that require markers, microcantilever immunosensing technology based on immune-specific recognition does not require the use of any enzyme label, fluorescent substance, or radioactivity as a reaction tracer, eliminating the influence of the labeling process, and has high sensitivity (several times higher than ELISA). It can also monitor the reaction process of antigen and antibody in real time and quantitatively by monitoring the deformation of the microcantilever, and obtain more abundant information on immuno-biochemical reactions. After years of development, microcantilever sensing has been used as an emerging technology, and has been compared with traditional methods in bioengineering and environmental pollution monitoring technology, such as RNA transcription factors, enzymes, mercury emissions, and volatile compounds, and is superior to conventional ELISA methods. But even so, the detection sensitivity of microcantilever immunosensor technology is not enough to gain a huge cost advantage over conventional ELISA. That is, if the sensitivity or detection limit of microcantilever immunosensor technology is only several times higher than that of ELISA, and the equipment cost of microcantilever immunosensor technology is relatively high compared with ELISA, it makes microcantilever immunosensor technology difficult to commercialize.

[0004] The upper limit of the sensitivity of the microcantilever is determined by the detection principle. In the traditional model, the antibody-antigen binding causes the microcantilever to deflect based on the interaction between the target molecule and the probe molecule. However, the intermolecular force is often relatively weak, with a single intermolecular force of only 10 -12 -10 -17 pF and the direction of the force is dispersed, which makes it difficult for the detection sensitivity of the microcantilever sensing technology to reach the level of ultra-sensitive detection. This also limits the application scope of the microcantilever technology. For example, it is impossible to achieve ultra-early detection of cancer because it is impossible to detect ultra-early sub-picogram or even femtogram per milliliter (fg / mL) cancer markers. Summary of the invention

[0005] In view of this, one of the main objectives of the present invention is to propose a micro-cantilever sensing detection method based on magnetic force enhancement, in order to at least partially solve at least one of the above-mentioned technical problems.

[0006] In order to achieve the above object, as one aspect of the present invention, a micro-cantilever sensing detection method based on magnetic force enhancement is provided, comprising:

[0007] The composite coupling body is fixed to a reaction pool containing a buffer solution; the composite coupling body is obtained by combining a microcantilever modified with a first aptamer and a magnetic nanoparticle modified with a second aptamer through the first aptamer and the second aptamer;

[0008] Applying a magnetic field force to the reaction pool, and adding a sample solution to be detected into the reaction pool; wherein the magnetic nanoparticles respond to the magnetic field force so that the second aptamer modified on the magnetic nanoparticles is separated from the first aptamer modified on the microcantilever, and the target molecule in the sample solution to be detected is combined with the first aptamer modified on the microcantilever, and the microcantilever is displaced;

[0009] The concentration of the target molecule in the sample solution to be detected is obtained based on the displacement generated by the microcantilever.

[0010] Based on the above technical solution, it can be seen that the micro-cantilever beam sensing detection method based on magnetic force enhancement of the present invention has at least one or part of the following advantages over the prior art:

[0011] 1) The present invention adopts a method of fixing MNPs on the gold surface of a microcantilever, and replaces the intermolecular force with magnetic force to achieve the purpose of improving the sensitivity of microcantilever immunosensing; compared with the existing microcantilever immunosensing method, this magnetic force enhancement method greatly improves the sensitivity of microcantilever detection. For example, in the detection of DA, compared with the traditional method based only on aptamers, the use of magnetic enhancement method reduces the detection limit by five orders of magnitude;

[0012] 2) At the same time, the method of the present invention is not only applicable to small molecules, but also to large molecules, such as CEA protein. At the same time, according to the principle of the method, it is only necessary to select and screen suitable aptamers to complete the detection design of other target molecules, without the need for complex immunization and purification processes like traditional antibody molecules, thus avoiding the shortcomings of long probe molecule preparation cycle, low yield and high price, and greatly reducing the difficulty of designing and executing new target molecule detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 It is a schematic diagram of the principle of the detection method based on magnetic force enhanced micro cantilever beam in an embodiment of the present invention;

[0014] Figure 2 It is a schematic diagram of the micro cantilever beam detection principle in the prior art;

[0015] Figure 3 This is a schematic diagram of the preparation process of functionalized magnetic nanoparticles in an embodiment of the present invention;

[0016] Figure 4 is a transmission electron microscopy image of magnetic nanoparticles (MNP) in an embodiment of the present invention;

[0017] Figure 5 A simulation diagram of the magnetic field of a neodymium magnet in an embodiment of the present invention;

[0018] Figure 6 This is a simulation diagram of the force on MNP in a magnetic field in an embodiment of the present invention;

[0019] Figure 7 It is the hysteresis loop diagram of MNP in the embodiment of the present invention;

[0020] Figure 8 DLS images of the aptamer before and after coupling on the MNP surface in the embodiment of the present invention;

[0021] Fig. 9 UV-Vis images of the aptamer before and after coupling on the MNP surface in the embodiment of the present invention;

[0022] Fig.10 It is a schematic diagram of the process of fixing magnetic particles on the gold surface of a micro-cantilever beam in an embodiment of the present invention;

[0023] Fig.11 This is a SEM image showing the fixation effect of MNP on the gold surface of the microcantilever in the embodiment of the present invention;

[0024] Fig.12 This is a diagram showing the detection results of dopamine using a micro-cantilever beam sensing detection method based on magnetic force enhancement in an embodiment of the present invention;

[0025] Fig.13This is a comparison diagram of the sensitivity of the micro-cantilever beam sensing detection method based on magnetic force enhancement in an embodiment of the present invention;

[0026] Fig.14 This is a diagram showing the specificity detection results of the micro-cantilever beam sensing detection method based on magnetic force enhancement in an embodiment of the present invention;

[0027] Fig.15 This is a diagram showing the detection results of carcinoembryonic antigen (CEA) using the magnetically enhanced microcantilever sensing method in an embodiment of the present invention. DETAILED DESCRIPTION

[0028] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments and with reference to the accompanying drawings.

[0029] In view of the limitation of intermolecular forces on the sensitivity of microcantilever immunosensor detection, the present invention proposes to modify magnetic nanoparticles on the gold-plated surface of the microcantilever, and replace the magnetic particles with target molecules to replace the intermolecular forces to cause the microcantilever to deflect to achieve microcantilever immunosensor detection. The present invention relates to a nano-antibody-modified microcantilever, and the present invention also discloses an immunosensor detection method based on the microcantilever modified with the magnetic nanoparticles, which can be applied to monitoring and detection in the fields of food safety, environmental pollution, biomedicine, scientific research, and production and manufacturing.

[0030] The force on Fe3O4 magnetic nanoparticles (MNP) with a diameter of 10 nm in a magnetic field can be expressed by the formula F 磁 =μ0κVHgradH, where μ0 is the vacuum permeability, κ=M / H, M and V are the saturation magnetization and volume of the MNP, and H and gradH are the magnetic field intensity and gradient. Through simulation calculations in the magnetic field of a neodymium magnet, it is found that a single MNP can be subjected to a force of up to 10 -5 pF and the force direction is consistent, while the interaction between single molecules is (10 -12 -10 -17 ), and the direction of force is dispersed. It can be seen that the force on a single MNP far exceeds the interaction between single molecules and the force direction is uniformly toward the neodymium magnet. Such a force signal with large value and uniform direction meets the expectation of the new principle of microcantilever stress signal generation. The present invention uses the magnetic force on MNP to replace the weak interaction between molecules to cause the deflection of the beam. In this way, even if the concentration of the target molecule is extremely low, the beam will deflect under the action of a considerable magnetic force, thereby breaking the limitation of the interaction between molecules on the detection sensitivity, and is expected to achieve ultra-sensitive detection of microcantilever beams.

[0031] The principle of microcantilever immunosensing technology based on surface stress detection is: the probe (antigen or antibody) molecule is fixed (modified) directly or indirectly to the gold-plated layer on one side of the microcantilever. When the target molecules in the sample liquid to be detected react with the probe molecules on the gold surface of the microcantilever, the surface stress of the microcantilever will change, causing the microcantilever to bend and deform. By detecting this deformation process through optical or electrical methods, real-time information of the immunobiochemical reaction can be obtained, thereby establishing a quantitative relationship between the deflection of the microcantilever and the concentration of the substance to be detected.

[0032] The present invention proposes a universal ultra-sensitive micro-cantilever detection method based on magnetic enhancement in a competitive mode: its principle is as follows Figure 1 As shown, MNP is fixed to the beam through the hybridization of its surface aptamer (nucleic acid aptamer is a functionalized nucleic acid fragment that can bind to a specific target molecule) S2 with the microcantilever surface aptamer S1, and a tensile force is applied to the beam after magnetization ( Figure 1 The target molecule (i.e., the target molecule) will replace the MNP due to its higher affinity with the aptamer S1 on the surface of the microcantilever beam, and the magnetic release caused by the replacement of the MNP will cause a very large upward deflection of the beam ( Figure 1 The upward deflection of the cantilever beam is much greater than the specific binding of the target molecule to the aptamer S1 on the cantilever beam surface ( Figure 2 As shown), the detection sensitivity of the microcantilever is greatly improved.

[0033] The magnetic nanoparticles in the embodiment of the present invention can be first-generation ferrite particles (γ-Fe2O3, Me Fe2O3 (Me = Co, Ni, Mn) and Fe3O4 particles), metal particles (Fe, Co, Ni and their alloy particles), iron nitride particles (FeN, Fe2N, ε-Fe3N, Fe 16 N2, etc.), the particle shapes include but are not limited to spherical, wedge-shaped, linear and flake-shaped, etc.

[0034] At the same time, this method is not only applicable to large molecular target molecules such as protein antigens, but also to small molecular target molecules with smaller molecular weight, simpler structure and more difficult to improve detection sensitivity. The target molecules can be, for example, macromolecules such as CEA (carcinoembryonic antigen), HER2 (human epidermal growth factor receptor-2), AFP (alpha-fetoprotein) and DA (dopamine), ATP (adenosine triphosphate), and clenbuterol hydrochloride.

[0035] Moreover, the aptamers corresponding to the present invention are easy to obtain. It can be said that the magnetically enhanced micro-cantilever sensing platform is not only expected to promote the development of the next generation of super-surface micro-cantilever biochemical sensors, but also to promote the in-depth application of micro-cantilever detection in various fields, especially in medical practices represented by early diagnosis and ultra-early prevention of cancer, and effectively promote the transformation and application of multidisciplinary cross-disciplinary results such as kinetics and analytical chemistry, sensors, nanomaterials, and biomedicine in the medical field.

[0036] Specifically, the present invention discloses a micro-cantilever beam sensing detection method based on magnetic force enhancement, comprising:

[0037] The composite coupling body is fixed to a reaction pool containing a buffer solution; the composite coupling body is obtained by combining a microcantilever modified with a first aptamer and a magnetic nanoparticle modified with a second aptamer through the first aptamer and the second aptamer;

[0038] Applying a magnetic field force to the reaction pool, and adding a sample solution to be detected into the reaction pool; wherein the magnetic nanoparticles respond to the magnetic field force so that the second aptamer modified on the magnetic nanoparticles is separated from the first aptamer modified on the microcantilever, and the target molecule in the sample solution to be detected is combined with the first aptamer modified on the microcantilever, and the microcantilever is displaced;

[0039] The concentration of the target molecule in the sample solution to be detected is obtained based on the displacement generated by the microcantilever.

[0040] In some embodiments of the invention, the first aptamer comprises a DNA or RNA oligonucleotide sequence;

[0041] In some embodiments of the invention, the second aptamer comprises a DNA or RNA oligonucleotide sequence.

[0042] In some embodiments of the present invention, the magnetic nanoparticles include any one or more combinations of first-generation ferrite particles, metal-type particles, and iron nitride particles;

[0043] In some embodiments of the present invention, the first generation ferrite particles include any one or more combinations of γ-Fe2O3, MeFe2O3, and Fe3O4 particles; wherein Me=Co, Ni or Mn;

[0044] In some embodiments of the present invention, the metal type particles include any one or more combinations of Fe, Co, and Ni;

[0045] In some embodiments of the present invention, the iron nitride particles include FeN, Fe2N, ε-Fe3N, Fe 16 Any one or more combinations of N2.

[0046] In some embodiments of the present invention, the shape of the magnetic nanoparticles includes any one of spherical, wedge-shaped, linear, and flake-shaped.

[0047] In some embodiments of the present invention, the particle size of the magnetic nanoparticles is 1nm to 10μm, for example, it can be 1nm, 2nm, 5nm, 10nm, 20nm, 50nm, 80nm, 100nm, 200nm, 500nm, 800nm, 1μm, 2μm, 5μm, 8μm, 10μm.

[0048] In some embodiments of the present invention, the target molecule includes any one of carcinoembryonic antigen, human epidermal growth factor receptor-2, alpha-fetoprotein, dopamine, adenosine triphosphate, and clenbuterol hydrochloride.

[0049] In some embodiments of the present invention, the concentration of magnetic nanoparticles in the composite couple is 5 to 50 μg / mL, for example, 5 μg / mL, 10 μg / mL, 15 μg / mL, 20 μg / mL, 25 μg / mL, 30 μg / mL, 35 μg / mL, 40 μg / mL, 45 μg / mL, 50 μg / mL.

[0050] In some embodiments of the present invention, the concentration of the first aptamer in the composite couple is 2 to 10 μM, for example, 2 μM, 5 μM, 8 μM, or 10 μM.

[0051] In some embodiments of the present invention, the concentration of the target molecule in the sample solution to be detected is 1pM to 1000μM, for example, it can be 1pM, 5pM, 8pM, 10pM, 50pM, 80pM, 100pM, 500pM, 1000pM, 2nM, 5nM, 10nM, 100nM, 500nM, 1μM, 2μM, 5μM, 10μM, 50μM, 100μM, 200μM, 500μM, 1000μM.

[0052] In some embodiments of the present invention, the buffer comprises any one of PBS buffer, Tris-HCl buffer, and MES buffer.

[0053] The technical solution of the present invention is further described below by means of specific embodiments in combination with the accompanying drawings. It should be noted that the following specific embodiments are only used as examples, and the protection scope of the present invention is not limited thereto.

[0054] The chemicals and raw materials used in the following examples are either commercially available or homemade by known preparation methods.

[0055] Example 1

[0056] Preparation of functionalized magnetic nanoparticles S2@MNP

[0057] See also Figure 3 , Figure 3 Schematic diagram of the structure of the preparation process of functionalized magnetic nanoparticles.

[0058] MNPs can be Fe 2+ , Fe 3+ The raw materials are prepared by coprecipitation method, and spherical Fe3O4 MNPs of different particle sizes (10nm, 100nm, 1000nm) can be prepared by controlling the experimental conditions and the ratio of FeCl3·6H2O, FeSO4·7H2O, ethylene glycol (EG) and diethylene glycol (DEG) (but other shapes, such as wedges, other magnetic materials, such as Fe2O3, using the same microcantilever detection method as the design concept of the present invention should also be within the scope of protection). After cleaning and purification, the surface can be further functionalized. The magnetic enhancement method based on the competition mode in this embodiment requires coupling a segment of aptamer on the surface of MNP. MNP has a three-layer core-shell structure, the innermost core is composed of Fe3O4, covered by a hydrophobic oleic acid layer, and the outermost layer is distearoylphosphatidylethanolamine polyethylene glycol carboxyl (DSPE-PEG-COOH). As can be seen from the molecular structure of DSPE-PEG-COOH, it is composed of a hydrophobic tail and a hydrophilic head with a carboxyl group. The tail is combined with oleic acid through a hydrophobic effect, and the head is dissolved in the dispersion through a hydrophilic effect. Thus, a stable three-layer core-shell structure is formed, which not only improves the stability of MNPs in the dispersion, so that it is no longer necessary to prevent MNPs from flocculation by adjusting the pH and isoelectric point, but also brings carboxyl groups to the surface of MNPs, providing a functional group basis for further surface functionalization. First, the carboxyl group (-COOH) is modified on the surface of MNPs through the hydrophobic effect between the hydrophobic tail of DSPE-PEG-COOH and the oleic acid layer on the surface of MNPs. The aptamer modified with an amino group at the 5' end can be customized from a biotechnology company. The carboxyl group on the surface of MNPs is activated under the action of EDC, and the activated group remains stable under the maintenance of NHS and reacts with the amino group on the aptamer to form a peptide bond, thereby preparing MNPs (S2) with surface modified with an aptamer (S2) Figure 3 State C). S2 can complement the aptamer S1 and can be replaced by the target molecule (i.e., the target molecule).

[0059] The specific operation details are as follows: After ultrafiltration three times, MNPs (10 mL) dispersed in 0.015 M MES buffer (pH 5.5) were added with 114 μL EDC (0.2 M, 10 mg / mL) and NHS (0.02 M, 10 mg / mL) respectively, and reacted at room temperature for about 1 hour. After ultrafiltration three times, 0.04 OD / μL aptamer S2 was added to the activated MNP solution, and S2@MNP was formed by esterification of amino and carboxyl groups by gently vortexing or shaking the test tube at room temperature for 20 hours. The mixture was then centrifuged at 10,000 rpm for 30 minutes and rinsed three times with deionized water to remove any unbound aptamer S2. Finally, S2@MNP was dispersed in a solution of 10 mM Tris-HCl (pH 7.4) containing 150 mM sodium chloride and stored in a cool place at 2-8°C.

[0060] Evaluation of the coupling efficiency of S2 on the MNP surface

[0061] The MNPs were prepared and functionalized and then characterized accordingly.

[0062] Figure 4 Transmission electron microscopy image of magnetic nanoparticles (MNP). Figure 5 This is a simulation diagram of the magnetic field of a neodymium magnet. Figure 6 This is the simulation diagram of the force on MNP in a magnetic field. Figure 7 The MNPs were characterized by transmission electron microscopy (TEM) and vibrating sample magnetometer (VSM). Figure 4 As shown in Figure 2, the average particle size d of the synthesized MNPs is 11.3 nm (the designed d is 10 nm). Figure 6 As shown, it shows that the saturation magnetization intensity M of MNP is 40emu / g. Combined with the magnetic field distribution of neodymium magnet ( Figure 5 ) can further calculate the magnetic force on MNP in the magnetic field ( Figure 7 ).

[0063] In order to characterize the coupling efficiency of the aptamer on the MNP surface, dynamic light scattering (DLS) and ultraviolet absorption spectroscopy (UV-Vis) experiments were performed before and after coupling.

[0064] The DLS test results are as follows Figure 8 As shown: the average hydrodynamic diameter of MNP measured before coupling with S2 was 23.54 nm. After coupling, the particle dispersibility (PDI) remained basically unchanged, but the average hydrodynamic diameter increased to 28.07 nm. The increased particle size was equivalent to the length of S2, which was about 4.5 nm, indicating that there was an additional S2 layer on the outer layer of MNP, thus proving that S2 and MNP were successfully coupled.

[0065] UV-Vis results are as follows Fig. 9As shown: the absorption value of the S2 solution at the characteristic wavelength of 260nm before coupling is 2a.u., after adding MNP, coupling reaction is carried out, and then S2@MNP and MNP are washed away to obtain the supernatant, and the absorption value of the supernatant at the characteristic wavelength of 260nm is 0.182au. The significant reduction in the absorption value is because the remaining S2 concentration in the supernatant is very low, which means that most of the S2 is bound to the surface of MNP, so it proves that S2 and MNP are successfully coupled. At the same time, the coupling rate can be calculated according to the formula of coupling rate = (absorption value of 260nm wavelength before coupling - absorption value of 260nm wavelength after coupling) / absorption value of 260nm wavelength before coupling * 100%, which may be due to the large number of active carboxyl groups on the surface of MNP and the high affinity between carboxyl groups and amino groups after activation.

[0066] Immobilization of MNPs on the Gold Surface of Microcantilever

[0067] Fig.10 Flow chart for the fixation of MNPs on the gold surface of microcantilever, as shown in Fig.10 As shown:

[0068] 1) Cleaning: Place an unused microcantilever (purchased from Micromotive, Germany) into an ELISA plate, soak it in Piranha Dip solution (V(H2O2):V(H2SO4)=1:3) for several minutes, rinse with deionized water, and blow dry with nitrogen.

[0069] 2) Fixing S1: Place the cleaned microcantilever into a new plate well, add 200 μL of 2 μM DA aptamer S1, react at room temperature for 3 h, then rinse with deionized water and blow dry with nitrogen to obtain a microcantilever with S1 fixed.

[0070] 3) Blocking: The modified microcantilever array was placed in an ELISA plate, and an alcohol solution of 6-mercapto-1-hexanol (MCH, 1 mM) was added and incubated for 1 hour, then washed with deionized water and dried with nitrogen gas.

[0071] 4) Immobilization of MNPs: Place the sealed microcantilever into a new plate well, add 150 μL of aptamer S2@MNP, incubate at 37°C for 1.5 hours, rinse with deionized water, and blow dry with nitrogen to obtain a microcantilever immobilized with S2@MNPs.

[0072] Evaluation of the binding of MNPs to the gold surface of the microcantilever using SEM

[0073] The modified microcantilever was observed under a scanning electron microscope. The diameter of the MNP particles used was 100 nm. The electron microscope scanning image is shown in Fig.11 As shown, it can be seen that the MNP has been modified onto the gold surface of the microcantilever, and it can be read that the diameter of the modified MNP is indeed 100 nm.

[0074] Detection of dopamine and carcinoembryonic antigen using magnetically enhanced microcantilever sensing

[0075] 1) Cleaning: Anhydrous ethanol, acetone and PBS flow through the microcantilever sensor system respectively, with a flow rate of 0.5 mL / min;

[0076] 2) Fix the microcantilever and apply a magnetic field: The microcantilever modified with MNPs is fixed to the reaction pool of the microcantilever sensing system, and PBS is flowed through the reaction pool. After removing the bubbles in the reaction pool, PBS is flowed at 0.1 mL / min. The magnetic field is applied by a neodymium magnet and the specific position, magnetic field size and gradient are adjusted by an XZ axis translation stage.

[0077] 3) Adjust the optical path: adjust the position of the laser and PSD (photoelectric position sensor) so that the laser light emitted by the laser shines on the tip of the micro cantilever and is received by the PSD.

[0078] 4) Sample measurement: When the microcantilever deflection signal received by the PSD is stable, add 2 mL of dopamine (DA) standard sample diluted with PBS, and record the microcantilever tip displacement information in real time through the PSD.

[0079] like Fig.12 As shown in FIG. 1 , the magnetic enhancement method was used to detect the dopamine standard sample at 100 nM, 10 nM, 1 nM, 0.1 nM, and 0.01 nM, and the displacements were approximately 65 nm, 35 nm, 19 nm, and 8 nm, respectively. Then, only the aptamer was used for detection, and the S-shaped curves of the two were compared, as shown in FIG. Fig.13 As shown, the sensitivity was found to be improved by 5 orders of magnitude.

[0080] like Fig.14 As shown, in order to verify the specificity of the magnetic enhancement method, DA analogs such as AA, Lys, and L-DOPA were detected, and it was found that there was a specific response only when DA was added, thus verifying the specificity of the method.

[0081] like Fig.15 As shown, in order to verify the universality of the method, the magnetic enhancement method was used to detect CEA (carcinoembryonic antigen), and compared with the detection effect using only the aptamer (the detection limit was 50 ng / mL), and a huge improvement in sensitivity was also found, which proved the universality of the method and its broad application prospects in disease diagnosis, environmental monitoring, food safety and other fields.

[0082] Example 2

[0083] The composite coupling body is fixed to a reaction pool containing a buffer solution; the composite coupling body is obtained by combining a microcantilever modified with a first aptamer and a magnetic nanoparticle modified with a second aptamer through the first aptamer and the second aptamer; wherein the first aptamer is DNA, the second aptamer is DNA, the magnetic nanoparticles are Fe3O4 particles; and the buffer solution is PBS buffer solution;

[0084] Apply a magnetic field force to the reaction pool, and add the sample solution to be detected into the reaction pool; wherein the magnetic nanoparticles respond to the magnetic field force so that the second aptamer modified on the magnetic nanoparticles is separated from the first aptamer modified on the microcantilever, and the target molecule in the sample solution to be detected is combined with the first aptamer modified on the microcantilever, and the microcantilever is displaced; wherein the target molecule is a carcinoembryonic antigen molecule with a concentration of 1 μM;

[0085] The concentration of the target molecule in the sample solution to be detected is obtained based on the displacement generated by the microcantilever.

[0086] Embodiments 3 to 10

[0087] The difference between Examples 3 to 10 and Example 2 is that the magnetic nanoparticles in Example 2 are replaced by γ-Fe2O3 particles, MeFe2O3 particles, Fe and Co alloy particles, FeN particles, Fe2N particles, ε-Fe3N particles, Fe 16 N2 particles, the concentration of the target molecule was also measured.

[0088] Examples 11 to 15

[0089] The only difference between Examples 11 to 15 and Example 2 is that the target molecules in Example 2 are replaced with human epidermal growth factor receptor-2, alpha-fetoprotein, dopamine, adenosine triphosphate, and clenbuterol hydrochloride in sequence, and the concentrations of the target molecules are also measured.

[0090] Embodiments 16 to 21

[0091] The only difference between Examples 16 to 21 and Example 2 is that the concentrations of the target molecules in Example 2 are replaced with 1 pM, 100 pM, 1000 pM, 100 μM, 500 μM, and 1000 μM in sequence, and the concentrations of the target molecules are also measured.

[0092] Embodiments 22-23

[0093] The only difference between Examples 22 to 23 and Example 2 is that the buffer in Example 2 is replaced with Tris-HCl buffer and MES buffer in sequence, and the concentration of the target molecule is also measured.

[0094] The specific embodiments described above further illustrate the objectives, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above description is only a specific embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A micro-cantilever sensing detection method based on magnetic force enhancement for non-disease diagnosis and treatment purposes, comprising: immobilizing the composite conjugate into a reaction pool containing a buffer; The composite coupling body is obtained by combining a microcantilever modified with a first aptamer and a magnetic nanoparticle modified with a second aptamer through the first aptamer and the second aptamer; Applying a magnetic field force to the reaction pool, and adding a sample solution to be detected into the reaction pool; wherein the magnetic nanoparticles respond to the magnetic field force so that the second aptamer modified on the magnetic nanoparticles is separated from the first aptamer modified on the microcantilever, and the target molecule in the sample solution to be detected is combined with the first aptamer modified on the microcantilever, and the microcantilever is displaced; The concentration of the target molecule in the sample solution to be detected is obtained based on the displacement generated by the microcantilever.

2. The micro-cantilever sensing detection method according to claim 1, characterized in that: The first aptamer comprises a DNA or RNA oligonucleotide sequence; The second aptamer comprises a DNA or RNA oligonucleotide sequence.

3. The micro-cantilever sensing detection method according to claim 1, characterized in that: The magnetic nanoparticles include any one or more combinations of first-generation ferrite particles, metal particles, and iron nitride particles; Wherein, the first generation ferrite particles include any one or more combinations of γ-Fe2O3, MeFe2O3, and Fe3O4 particles; wherein Me=Co, Ni or Mn; Wherein, the metal type particles include any one or more combinations of Fe, Co, and Ni; Wherein, the iron nitride particles include FeN, Fe2N, ε-Fe3N, Fe 16 Any one or more combinations of N2.

4. The micro-cantilever sensing detection method according to claim 1, characterized in that: The shape of the magnetic nanoparticles includes any one of spherical, wedge-shaped, linear, and flake-shaped.

5. The micro-cantilever sensing detection method according to claim 1, characterized in that: The particle size of the magnetic nanoparticles is 1 nm to 10 μm.

6. The micro-cantilever sensing detection method according to claim 1, characterized in that: The target molecule includes any one of carcinoembryonic antigen, human epidermal growth factor receptor-2, alpha-fetoprotein, dopamine, adenosine triphosphate, and clenbuterol hydrochloride.

7. The micro-cantilever sensing detection method according to claim 1, characterized in that: The concentration of the magnetic nanoparticles in the composite couple is 5 to 50 μg / mL.

8. The micro-cantilever sensing detection method according to claim 1, characterized in that: The concentration of the first aptamer in the composite conjugate is 2 to 10 μM.

9. The micro-cantilever sensing detection method according to claim 1, characterized in that: The concentration of the target molecule in the sample solution to be detected is 1 pM to 1000 μM.

10. The micro-cantilever sensing detection method according to claim 1, characterized in that: The buffer includes any one of PBS buffer, Tris-HCl buffer and MES buffer.

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