Biomolecule detection method based on micro-cantilever sensing

Abstract

The invention provides a method for detecting biomolecules based on micro-cantilever sensing, and belongs to the technical field of analysis and detection. The method comprises the following steps: providing a micro-fluidic module which comprises a micro-fluidic channel and a temperature controller, arranging a micro-cantilever array in the micro-fluidic channel, uniformly mixing a to-be-detected sample and a magnetic nanoparticle solution through the micro-fluidic channel, and combining the to-be-detected sample and the magnetic nanoparticle solution on the micro-beam surface of the micro-cantilever array; the laser module provides laser and is used for receiving a micro-beam deflection signal of the micro-cantilever beam array; and providing a magnetic field device, and attracting the magnetic nanoparticles by utilizing magnetic force, so that the micro-cantilever deflection deflection of the micro-cantilever array is increased, a deflection signal is enhanced, and the detection of the to-be-detected sample is realized. The micro-beam is driven by magnetic force to generate deformation, and a relatively large micro-beam deformation signal can be generated under extremely low concentration, so that ultra-sensitive detection of trace substances in a micro sample is realized.

Description

Technical Field

[0001] The present invention relates to the technical field of analysis and detection, and in particular to a biomolecule detection method based on micro-cantilever sensing. Background Art

[0002] The principle of traditional microcantilever sensing is that when a biochemical reaction occurs on one side of the microcantilever, the interaction between molecules will cause the stress on the upper and lower surfaces of the microcantilever to change, and the stress difference between the two surfaces drives the microcantilever to bend. The microcantilever sensor device is used to monitor the deflection of the cantilever in real time. However, when the concentration of the target substance is extremely low, the intermolecular force caused by this stress is very small, and there is even no deflection signal. How to improve the sensitivity of microcantilever sensing is a technical problem that researchers urgently need to solve. Summary of the invention

[0003] The purpose of the present invention is to address the above-mentioned deficiencies in the prior art and provide a biomolecule detection method based on microcantilever sensing, which utilizes magnetic force to drive the microbeam to deform and can generate a large microbeam deformation signal even at extremely low concentrations, thereby enabling ultra-sensitive detection of trace substances in trace samples.

[0004] In order to achieve the above object, the present invention adopts the following technical solutions:

[0005] The purpose of the present invention is to provide a biomolecule detection method based on microcantilever sensing, providing a microfluidic module, including a microfluidic channel and a temperature controller, a microcantilever array is arranged in the microfluidic channel, a sample to be tested and a magnetic nanoparticle solution are mixed evenly through the microfluidic channel, and combined on the microbeam surface of the microcantilever array;

[0006] Providing a laser for receiving a micro-beam deflection signal of the micro-cantilever beam array;

[0007] A magnetic field device is provided to attract the magnetic nanoparticles by magnetic force, so that the deflection disturbance of the micro-beams of the micro-cantilever beam array is increased, the deflection signal is enhanced, and the detection of the sample to be tested is realized.

[0008] Furthermore, the biomolecule includes any one of protein, nucleic acid, virus or bacteria.

[0009] Furthermore, the magnetic nanoparticles include Fe3O4 magnetic nanoparticles and graphene oxide coupled magnetic particles.

[0010] Furthermore, the surface of the micro-beams of the micro-cantilever array is modified with probe molecules.

[0011] Furthermore, the magnetic field device is realized by disconnecting and connecting the permanent magnet and the magnetic conductor.

[0012] Furthermore, the relative position of the magnetic field device and the micro-cantilever beam array is adjusted to control the intensity and gradient of the magnetic field applied to the micro-beams, so as to provide a suitable magnetic field environment.

[0013] Furthermore, a temperature control device is also provided in the microfluidic channel.

[0014] Furthermore, each micro-cantilever of the micro-cantilever array consists of a bonded fixed end and a free end.

[0015] Furthermore, the surface of the micro cantilever is plated with a layer of gold film.

[0016] Furthermore, the lasers are mapped one by one to the microbeams of the microcantilever beam array, and each laser irradiates the tip of the mapped microbeam to generate a reflected light spot, which is received by a position sensitive detector to collect a microbeam deflection signal.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] (1) The present invention provides a method for detecting biomolecules based on microcantilever sensing. The method includes providing a microfluidic channel, setting a microcantilever array in the microfluidic channel, mixing the sample to be tested and the magnetic nanoparticle solution evenly through the microfluidic channel, and combining them on the microbeam surface of the microcantilever array; providing a laser for receiving the microbeam deflection signal of the microcantilever array; providing a magnetic field device, using magnetic force to attract the magnetic nanoparticles, so that the microbeam deflection disturbance of the microcantilever array is increased, the deflection signal is enhanced, and the detection of the sample to be tested is achieved. Microfluidics is used to make the trace sample and the magnetic particles combine quickly, the magnetic field device amplifies the microbeam response signal, and the microbeam sensing optical path based on the optical lever principle is combined to detect the cantilever deflection signal. Compared with the traditional method, the detection limit can be reduced by more than five orders of magnitude, and the sensitivity is greatly improved. Compared with the method without enrichment with magnetic particles, the operation is simple, the sample consumption is small, and the detection cycle is greatly shortened, which provides better theoretical value for the practical application of this method.

[0019] (2) For low-concentration target molecules, no microbeam deformation signal can be detected under the traditional surface stress mode. The present invention innovatively uses magnetic force to drive the microbeam to deform, and can generate a large microbeam deformation signal even at extremely low concentrations, thereby achieving ultra-sensitive detection of trace substances in trace samples.

[0020] (3) The detection method provided by the present invention is cheaper than other detection methods, and can simultaneously take into account the sensitive detection of short-term and trace samples, thus shortening the clinical diagnosis time and realizing precision medicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1aA schematic diagram of the structure of a detection device based on micro-cantilever beam sensing provided by the present invention;

[0022] Figure 1b A side view of a microfluidic module used in the embodiment;

[0023] Figure 2 The detection principle diagram based on micro cantilever beam sensing provided by the present invention;

[0024] Figure 3 This is a schematic diagram of the working principle of the magnetic field generating device;

[0025] Figure 4 Schematic diagram of the principle of magnetically induced microbeam deflection;

[0026] Figure 5 This is a graph showing the effect of magnetic particle size on the detection signal;

[0027] Figure 6a-6c It is a trend diagram of the change of the horizontal magnetic force and the vertical magnetic force generated by the magnetic field generating device;

[0028] Figure 7 This is a graph showing the results of using a microcantilever array to detect SOD concentration in embryo culture medium;

[0029] Figure 8 This is a graph showing the results of detecting the HLA-G concentration in embryo culture medium using a microcantilever array;

[0030] Fig. 9 This is a result graph of the cytokine concentration corresponding to VZV detected by the microcantilever array;

[0031] Fig.10 This is a result diagram of the nucleic acid concentration corresponding to VZV detected by the microcantilever array;

[0032] Fig.11 This is a result diagram of the antibody concentration corresponding to VZV detected by the microcantilever array;

[0033] Fig.12a This is a schematic diagram of the principle of using a detection device based on microcantilever sensing to simultaneously detect antibodies, cytokines, and nucleic acids of the fundus disease VZV;

[0034] Figure 12b This is a diagram showing the results of using a microcantilever sensor-based detection device to simultaneously detect antibodies, cytokines, and nucleic acids of the fundus disease VZV;

[0035] Fig.13 Comparison chart of different bacteria to facilitate further amplification of magnetic signals by graphene.

[0036] In the figure: 1. microfluidic module; 11. microfluidic channel; 12. temperature controller; 2. microcantilever array; 3. magnetic field device; 4. laser. DETAILED DESCRIPTION

[0037] In order to make the purpose, technical solution and advantages of the present invention clearer, embodiments of the present invention are described in detail below, and examples of the embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements with the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain the present invention, and cannot be understood as limiting the present invention.

[0038] In traditional microbeam sensing, the sensing is based on the change in the intermolecular force after the probe molecule binds to the target molecule. However, this intermolecular force is often relatively weak, with a single pair of intermolecular forces being only 10 -12 ~10 -17 These factors will lead to very small mechanical effects on the microbeam surface, especially when the concentration of target molecules is very low, the distance between molecules becomes very large, the intermolecular forces will be further reduced, it is difficult to cause significant deformation of the microbeam, and it is difficult to achieve ultra-sensitive (sub-picogram level or even femtogram per milliliter) sensing.

[0039] The present invention found that a Fe3O4 magnetic nanoparticle (MNP) with a diameter of 10nm in a magnetic field is subjected to a force far exceeding the force between a single pair of molecules and the force direction is consistent, theoretically up to 10 -5 pN. This force signal with large value and uniform direction meets the expectation of the new principle of micro-beam force signal generation. Based on this, the present invention establishes a new quantitative mechanical model based on magnetic force and micro-beam deflection signal, using the magnetic force on MNP in the magnetic field instead of the weak intermolecular force to cause the deflection of the beam. This is completely different from the principle that the traditional micro-beam relies on intermolecular force to produce deflection. Based on this new principle, even if the concentration of the target molecule is extremely low, the beam will deflect under the action of a large magnetic force, thus breaking the limitation of the weak intermolecular force on the detection sensitivity, and it is expected to achieve rapid ultra-sensitive detection. When the target molecule exists, the magnetic particles will bind to the beam surface through the target molecule to form a "sandwich" sandwich structure (micro-beam @ target molecule @ MNP), and finally the magnetic particles are driven by the magnetic field force to deform the micro-beam, thereby realizing sensing. Based on this new principle, the present invention has developed a new type of micro-beam array biochemical sensing instrument based on magnetic force.

[0040] refer to Figure 1a, a detection device based on microcantilever sensing provided by the present invention can realize the detection of ultra-low concentration proteins, nucleic acids, bacteria, etc. A microfluidic module 1 is provided, which includes a microfluidic channel 11 and a temperature controller 12. A microcantilever array 2 is arranged in the microfluidic channel. The sample to be tested and the magnetic nanoparticle solution are mixed evenly through the microfluidic channel 2 and combined on the microbeam surface of the microcantilever array 2; a magnetic field device 3 is provided below the microfluidic channel 1, which is located directly below the microcantilever array 2, and uses magnetic force to attract the magnetic nanoparticles, so that the microbeam deflection disturbance of the microcantilever array is increased and the deflection signal is enhanced; a laser 4 set in advance is irradiated to the tip of the microcantilever array 2, and the reflected light spot is received by a position sensitive detector (PSD), and the microbeam deflection signal of the microcantilever array is collected. After data processing, a functional relationship between the microbeam deflection disturbance and the concentration of the sample to be tested is constructed, and the detection of the sample to be tested can be completed;

[0041] It should be noted that the present invention utilizes a microfluidic module to efficiently and stably combine functionalized microbeams with target molecules and magnetic nanoparticles. At the entrance of the microfluidic channel, a magnetic nanoparticle solution and a sample solution are simultaneously introduced through a peristaltic pump and fully combined. The volume of the closed reaction chamber of the microfluidic device does not exceed 300 μL, and the micro sample to be measured (about 10-15 μL) is diluted to 200-300 μL (the volume of the microfluidic chamber) after combining with the magnetic particle solution. The design principle of the microfluidic module: optimize the size of the microfluidic channel to make it miniaturized; design the position of the inlet and outlet ports to promote the discharge of bubbles and the combination of the solution and the microbeam; the peristaltic pump can accurately control a specific flow rate. A microfluidic channel that is small enough and can control the inlet and outlet of the sample can accelerate the biochemical reaction process and make the reaction more complete. The detection device for completing the detection provided by the present invention includes: a closed reaction chamber and a microfluidic channel, a controllable peristaltic pump and a temperature control device, and transparent glass that is convenient for the deformation detection of the microbeam.

[0042] In a specific embodiment of the present invention, the microfluidic module includes a microfluidic channel and a temperature control component, and the dimensions of the fluid channel are: 60 mm long, 20 mm wide, and 10 mm thick. Figure 1bAs shown, the sample inlet is located at the bottom of the right side of the reaction pool, and the sample outlet is located at the upper left side of the reaction pool; the top of the reaction pool is open and sealed with 1mm thick optical glass, the microbeam array is placed at the entrance to accurately fix the microbeam, and a semiconductor temperature controller is installed 2mm below the microfluidic chamber to achieve temperature control; the functionalized microbeam is fixed in the reaction chamber, and the magnetic particle solution modified with probe molecules and the trace sample to be measured are introduced into the sample inlet so that the magnetic particles are coupled with the microbeam through the target to be measured. After full combination, the peristaltic pump drives the liquid to clean the microbeam to reduce the nonspecific adsorption of magnetic particles and improve the accuracy of detection. The capture of the target and the magnetic particles modified with probe molecules in the sample to be measured are both based on the above-mentioned microfluidic module, and the liquid flow rate is controlled by using a peristaltic pump.

[0043] In a specific embodiment of the present invention, the detection process of the sample to be tested is as follows Figure 2 The specific detection process is as follows:

[0044] (1) The designed microcantilever array contains eight microcantilevers, which are functionalized and modified with probe molecules on the surface of the microcantilever. The modified solution (probe molecules) is passed into the capillaries, and the siphon effect of the capillaries will cause the modified liquid to flow spontaneously to the other end of the capillaries. The capillaries are arranged at intervals, and only four microcantilevers are modified in a group of microcantilevers as the experimental group, and the other four unmodified microcantilevers are used as the control group.

[0045] It should be noted that the selection of probe molecules: the core of the microcantilever deflection based on magnetic force is to form a magnetic sandwich structure on the surface of the microcantilever. Then at least two probe molecules are required to specifically bind to the target protein. At present, there are many substances that can be used as probe molecules, and their application scenarios are different. The most common probe molecule is the antibody, which has a strong binding specificity with the antigen. Aptamers are more specific and easier to preserve, and have gradually become the mainstream of probe molecules. In addition, some molecules such as nanoantibodies and PNA have been used in different applications as probe molecules in microcantilever detection. In general magnetic nanoparticle sandwich detection, aptamers are generally the most commonly used as probe molecules from the perspective of cost-effectiveness.

[0046] In the process of modifying the probe molecule, in order to verify the binding of the probe molecule to the microcantilever, the experimental beam was functionally modified during the implementation process, and the control beam was not treated. After binding to the protein and applying the magnetic field, there is a significant difference between the experimental beam and the control beam of the microcantilever. This shows that the aptamer has successfully bound to the microbeam. Another method is to find an aptamer that is complementary to the aptamer probe molecule, and the two DNAs can be well combined. At the same time, when the aptamer is connected to a fluorescent molecule, the modified microcantilever can be characterized by the fluorescent molecule, which can also prove the effective binding of the probe molecule on the microcantilever.

[0047] (2) The modified microcantilever beam (functionalized microcantilever beam) is combined with the target protein and magnetic particles to form a magnetic sandwich structure on the surface of the microcantilever beam. Under the action of the magnetic field, the magnetic particles drive the microcantilever beam to deflect, thereby achieving sensing.

[0048] The magnetic field generating device is realized by the disconnection and connection of the permanent magnet and the magnetic conductor. Its design principle is as follows: Figure 3 As shown: the entire magnetic field generating device is composed of a neodymium magnet that can generate a constant strong magnetic field, a magnetizer made of magnetic material laminates, and non-magnetic brass. When the instrument is in standby mode, the neodymium magnet is rotated to connect the NS pole to the non-magnetic brass. At this time, the magnetic lines of force form a closed loop in the magnetizers on both sides, and the entire device is basically non-magnetic. When the detection needs to generate a magnetic field for magnetic force amplification, the neodymium magnet is rotated to connect the two poles to the magnetizer respectively. At this time, the magnetizer corresponds to the two poles of the neodymium magnet, forming a magnetic field on both sides. At this time, an attractive magnetic force will be generated for the magnetic nanoparticles that are specifically bound to the beam through the target molecule, resulting in a large deformation of the microbeam, realizing ultra-sensitive detection of the target object. At the same time, the magnetic field generating device is loaded on the electric displacement platform, and the relative position of the magnetic field generating device and the microbeam array can be adjusted to control the magnetic field strength and gradient applied to the microbeam, providing a suitable magnetic field environment according to experimental requirements.

[0049] Through biochemical reactions in the microfluidic channel, the target molecules in the sample bind to the magnetic particles and are coupled to the microcantilever beam. The laser is used to irradiate the tip of the microcantilever beam array, and the reflected light spot is received by a position sensitive detector (PSD). After obtaining a stable baseline, the magnetic field device is placed in the reaction pool. Under the action of the magnetic field, the magnetic particles bound to the beam surface are pulled, driving the deflection signal of the microbeam to be recorded in real time.

[0050] like Figure 4 As shown, when the target is present, the magnetic nanoparticles on the beam will be displaced, thereby changing the force on the microbeam to achieve sensing. Under the action of the magnetic field, the magnetic particles bound to the target can be separated from the complex biological sample, thereby achieving the concentration and purification of the target, and then the MNP and the microbeam are connected through the target molecule to form a sandwich structure. This effective method that combines magnetic force and enrichment will further amplify the microbeam detection signal based on magnetic force, and is expected to achieve the detection of single-digit biological hazards (viruses, bacteria).

[0051] The specific steps of the detection method based on micro-cantilever sensing provided by the present invention are as follows:

[0052] (1) Modifying probe molecules on the surface of microcantilever array

[0053] The microcantilever is a structure composed of two materials. It is mainly composed of silicon nitride, and a layer of gold film is plated on the surface of silicon nitride. Gold has good biological affinity. Using a variety of different probe molecules to modify the microcantilever, multiple target molecules can be detected simultaneously.

[0054] Place the array beam in the enzyme well plate, add piranha solution with V(H2SO4):V(H2O2)=3:1 to clean the cantilever beam surface for 10 minutes, wash it three times with deionized water, and blow it dry with nitrogen. Place the array beam in the modification solution, combine the probe molecule solution with the microbeam array for 1.5 hours, take it out and put it in the enzyme well plate, wash it three times with PBST solution, and blow it dry with nitrogen. Since one end of the probe molecule has been modified with thiol, it reacts chemically with the gold surface of the beam to form Au-S bond to the beam surface.

[0055] (2) The sample to be tested and the magnetic particle solution are pumped into the microfluidic channel by a peristaltic pump and enter the reaction chamber containing the modified microcantilever array at a certain flow rate, and the magnetic particles bind to the probe molecules modified on the beam.

[0056] (3) Adjust the laser so that it irradiates the tip of the microcantilever array. The reflected light spot is received by a position sensitive detector (PSD). After obtaining a stable baseline, the magnetic field device is placed in the reaction pool. Under the action of the magnetic field, the magnetic particles bound to the beam surface are pulled, and the deflection signal of the microbeam is recorded in real time. Commercial magnetic nanoparticles (MNPs) can be selected, such as silicon-coated magnetic nanoparticles.

[0057] Example 1

[0058] Explore the effect of magnetic particle size on the detection signal.

[0059] As the magnetic force on the micro-beam increases, the deformation of the micro-beam increases, which will produce a larger mechanical signal. The size of the magnetic particles has a huge impact on the magnetic force. For this reason, the inventors have explored the impact of the size of the magnetic particles on the test results by changing the size of the magnetic particles. Magnetic nanoparticles of 10nm, 30nm, and 100nm were selected for the experiment. As shown in the lower right figure, it is found that as the particle size of the magnetic particles increases, the deflection value of the micro-beam will also increase. However, the larger the size of the magnetic nanoparticles, the better. Through multiple groups of experiments, it was found (such as Figure 5 As shown in the figure, the 10nm magnetic particles make the microbeam deflection signal more stable, while the 100nm experimental results are more discrete. By observing the size of the magnetic particles through TEM, it can also be found that the smaller the magnetic nanoparticles are, the more uniform the particle size is. The stability of the microbeam deflection results is of great significance in engineering practice, so the use of 10nm magnetic particles also has its advantages.

[0060] Example 2

[0061] Explore the impact of the magnetic field generator on the detection signal.

[0062] like Figure 3 As shown in the figure, the magnetic field generating device mainly includes: neodymium magnet, magnetic conductor and brass. When the two poles of the neodymium magnet are connected to the magnetic conductor, a corresponding magnetic field will be generated near the magnetic field excitation module. Here, the influence of the magnetic conductor on the magnetic field is ignored, and only the magnetic field generated by the neodymium magnet is considered. The overall magnetic field distribution is simulated by modeling in COMSOL.

[0063] like Figure 6a-6b As shown in the figure, the results of magnetic field simulation can be obtained. When the magnetic particles are 10 mm away from the top of the neodymium magnet, the magnetic field strength is about 4.6×10 5 A / m, and the magnetic field gradient is about 6.45×10 7 A / m 2 With this point as the center, the curves of the change of magnetic field intensity and gradient are given in the vertical and horizontal directions respectively. At this time, the magnetic force on the magnetic nanoparticles specifically enriched on the beam to be tested can be calculated by the formula:

[0064] F=μ0κVHgradH

[0065] At this time, when the Fe3O4 magnetic nanoparticles with a particle size of 10nm are near the two poles of a normal neodymium magnet, the magnetic force generated is proportional to the magnetic field strength and magnetic field gradient, which is about 8.8×10 -6 pN, which is much larger than the force between a single pair of molecules and has the same force direction, causes the cantilever beam to deform more greatly, thus obtaining higher detection sensitivity.

[0066] In the actual magnetic field loading process, since the distance between the microbeam array and the magnetic field excitation module will vary in different experimental processes, it is necessary to accurately adjust the position of the magnetic field excitation module to control the magnitude of the magnetic force to maintain stability. Figure 6c As shown in the figure, when the magnetic nanoparticles are offset in the vertical and horizontal directions, the simulation results of the magnetic field strength and gradient are substituted into the formula to obtain the curves of the magnetic force changing in different directions. At this time, when the magnetic particles deviate by 0.1 mm in the vertical direction, the deviation of the magnetic force is about ±5%, and when the deviation is 1 mm in the horizontal direction, the magnetic force will be reduced by about 3%. This deviation can be eliminated by controlling the displacement of the magnetic field excitation module to μm accuracy.

[0067] Example 3

[0068] This embodiment provides a method for detecting embryonic metabolite SOD by further amplifying the micro-beam response signal using a magnetic field device. The specific process is as follows:

[0069] Extraction of embryo metabolites: extract 10 μL of metabolite sample, dilute the 10 μL sample 10 times with PBS to obtain 100 μL of mixed solution, centrifuge at room temperature for 3 minutes, and extract the supernatant.

[0070] Microcantilever array modified with probe molecules and detection of leukocyte antigen G (HLA-G): The microcantilever array was placed in an enzyme well plate, and the cantilever surface was cleaned for 10 minutes with a piranha solution of V(H2SO4):V(H2O2)=3:1, and then washed three times with deionized water and dried with nitrogen. The microcantilever array was placed in a differentially modified device, and four capillaries were inserted into the surfaces of beams 1, 3, 5, and 7 to modify the probe molecules. After the probe molecule solution was combined with the microbeam array through the capillaries for 1.5 hours, it was taken out and placed in an enzyme well plate, washed three times with PBST solution, and dried with nitrogen. Beams 2, 4, 6, and 8 without modified probe molecules formed an in situ control with the modified beams. Since one end of the probe molecule has been modified with a thiol group, it reacts chemically with the gold surface of the beam to form an Au-S bond to the beam surface.

[0071] Add blocking reagent 6-mercapto-1-hexanol (MCH) and place at room temperature for 30 minutes, wash with PBST three times, and blow dry with nitrogen. The purpose is to occupy the blank sites on the gold surface that are not bound to the probe molecules to prevent nonspecific adsorption from affecting the deflection detection results.

[0072] The concentrated incubation solution with magnetic particles was added to the array beam modified in the previous step, and the magnetic particles were allowed to bind to the probe molecules modified on the beam for 90 minutes, and then washed once with PBST.

[0073] The beam is placed in the reaction pool, and eight lasers are adjusted to illuminate the tips of the eight array beams. The reflected light spot is received by a position sensitive detector (PSD). After a stable baseline is obtained, the magnetic field device is placed in the reaction pool. Under the action of the magnetic field, the magnetic particles bound to the surface of the beam are pulled, driving the deflection signal of the micro-beam to be recorded in real time.

[0074] According to the above experimental process, the actual samples were tested to detect the deflection of micro-beams caused by the different SOD contents in the embryo metabolites at different culture stages, and the relationship between embryo culture time and micro-beam deflection was established, that is, embryo development-micro-beam deflection. The results are as follows Figure 5 As shown, it was found that the concentration of SOD increased slowly with the growth and development of the embryo.

[0075] The process of aptamer coupling magnetic nanoparticles:

[0076] Main reagents: 15mM MES, pH5.0, purified water, EDC: 10mg / mL, freshly prepared for use

[0077] 10 nm Fe3O4 (1 mg / mL, 2.5 mL) was centrifuged in a 30KD ultrafiltration tube (4500 rpm, 15 min), the supernatant was discarded, and the mixture was washed once with MES and resuspended to 2 mL; 25 μL EDC (10 mg / mL) was added to Fe3O4 by vortexing, and then 3.4 OD (111.71 μg) of aptamer was added after mixing, and MES was added to a volume of 2.5 mL, and the mixture was shaken at 37°C for 20 h after mixing; after the reaction, the reaction solution was taken out, centrifuged in a 30KD ultrafiltration tube (4500 rpm, 15 min), and the filtrate was collected (for detecting the coupling rate of the aptamer); the filtrate was discarded by ultrafiltration, and the magnetic material was washed 3 times with purified water and then fixed to 2.5 mL, thus obtaining magnetic nanoparticles, which were recorded as: 10 nm Fe3O4-aptamer. The magnetic particles coupled with the aptamer were diluted to 10 μg / ml and mixed with a certain concentration of SOD and placed at room temperature for 1.5 h to allow the magnetic particles coupled with the aptamer to fully bind to SOD.

[0078] Example 4

[0079] This embodiment provides a method for detecting the HLA-G concentration in embryo culture medium by further amplifying the micro-beam response signal using a magnetic field device.

[0080] 50 μL of the sample prepared in Example 3 was taken and diluted twice with PBS, and the actual sample was tested. As shown in FIG6 , it was found that the detection signal noise was greater when the sample was tested with a smaller dilution factor. The reason may be that the impurities in the sample were more than those in the sample with a large dilution factor, which affected the optical path. However, it did not have much impact on the experimental results, and the detection limit for HLA-G was lower, with a detection concentration as low as 5 pg / ml.

[0081] Example 5

[0082] This embodiment provides a method for detecting nucleic acid, antibodies and cytokines of herpes zoster virus (VZV) in fundus fluid samples. The specific process is as follows:

[0083] Similar to Example 3, cytokines, nucleic acids, and antibodies of configured concentrations are subjected to micro-beam biochemical detection: cytokines (or nucleic acids or antibodies) and magnetic particles are combined with the surface of the micro-cantilever using a microfluidic module, and after applying a magnetic field, the magnetic particles drive the micro-beam to deflect, thereby achieving sensing.

[0084] like Fig. 9 As shown, different concentrations of cytokines correspond to different deflections of the microbeams, demonstrating the detectability of cytokines corresponding to VZV in the microbeams.

[0085] like Fig.10 As shown, by detecting the nucleic acid corresponding to VZV, the VZV virus concentration can be directly obtained, and the detection data with good detection effect can be obtained.

[0086] like Fig.11 As shown, the detection limit for VZV antibodies was as low as 10 pg / ml.

[0087] Example 6

[0088] This embodiment provides a detection device based on microcantilever sensing to simultaneously detect antibodies, cytokines, and nucleic acids of the fundus disease VZV.

[0089] Among the eight beams in the microcantilever array, four beams were selected for specific modification, three of which were used as experimental groups (corresponding to antibodies, nucleic acids, and cytokines associated with VZV), and one was used as a specific control group, such as Fig.12a The remaining four beams are not modified and are used to make a difference with the experimental beam to reduce the noise during the experiment. The results are shown in Figure 12b As shown, a good detection curve was obtained, and the detection results of VZV-related antibodies, nucleic acids, and cytokines were 100 pg / mL, achieving high-sensitivity detection of VZV.

[0090] Example 7

[0091] This embodiment provides detection of Escherichia coli based on further amplification of magnetic signals by graphene.

[0092] Under the action of the magnetic field, the magnetic particles bound to the target (magnetic particles @ target) can be separated from the complex biological samples, thereby achieving the concentration and purification of the target, and then the MNP and the microbeam are connected through the target molecule to form a sandwich structure. This effective method that combines magnetic force and enrichment will further amplify the microbeam detection signal based on magnetic force, and is expected to achieve the detection of single-digit biological hazards (viruses, bacteria). Fig.13 As shown in the figure, the sensitivity comparison analysis of the detection of E. coli using traditional stress, Fe3O4 magnetic nanoparticles and graphene oxide coupled magnetic particles is shown. It can be seen that the use of graphene oxide coupled magnetic particles can achieve the detection of E. coli, and the detection limit can reach 1cfu / mL. In addition, the present invention also found that it can detect two bacteria, E. coli and Staphylococcus aureus, at the same time, and can also detect proteins in the blood after concentration and purification, which is expected to further improve the detection of disease markers.

[0093] For matters not mentioned above, the prior art applies.

[0094] Although some specific embodiments of the present invention have been described in detail through examples, those skilled in the art should understand that the above examples are for illustration only and are not intended to limit the scope of the present invention. Those skilled in the art to which the present invention belongs may make modifications or supplements to the described specific embodiments or replace them in a similar manner, but they will not deviate from the direction of the present invention or exceed the scope defined by the attached claims. Those skilled in the art should understand that any modifications, equivalent substitutions, improvements, etc. made to the above implementations based on the technical essence of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for detecting biomolecules based on microcantilever sensing, characterized in that: A microfluidic module is provided, comprising a microfluidic channel and a temperature controller, wherein a microcantilever array is arranged in the microfluidic channel, and a sample to be tested and a magnetic nanoparticle solution are mixed evenly through the microfluidic channel and combined on the microbeam surface of the microcantilever array; Providing a laser for receiving a micro-beam deflection signal of the micro-cantilever beam array; A magnetic field device is provided to attract the magnetic nanoparticles by magnetic force, so that the deflection disturbance of the micro-beams of the micro-cantilever beam array is increased, the deflection signal is enhanced, and the detection of the sample to be tested is realized.

2. The method according to claim 1, characterized in that The biomolecules include any one of proteins, nucleic acids, viruses or bacteria.

3. The method according to claim 1, characterized in that The magnetic nanoparticles include Fe3O4 magnetic nanoparticles or graphene oxide coupled magnetic particles.

4. The method according to claim 1, characterized in that The micro-beam surfaces of the micro-cantilever array are modified with probe molecules.

5. The method according to claim 1, characterized in that The magnetic field device is realized by disconnecting and connecting the permanent magnet and the magnetic conductor.

6. The method according to claim 5, characterized in that The relative position of the magnetic field device and the micro-cantilever beam array is adjusted to control the intensity and gradient of the magnetic field applied to the micro-beams, so as to provide a suitable magnetic field environment.

7. The method according to claim 1, characterized in that A temperature control device is also arranged in the microfluidic channel.

8. The method according to claim 1, characterized in that Each micro-cantilever of the micro-cantilever array consists of a bonded fixed end and a free end.

9. The method according to claim 8, characterized in that The surface of the micro cantilever is plated with a layer of gold film.

10. The method according to claim 1, characterized in that The lasers correspond to the micro-beams of the micro-cantilever beam array one by one, and each laser irradiates the tip of the corresponding micro-beam to generate a reflected light spot, which is received by a position sensitive detector to collect a micro-beam deflection signal.

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