Micro-nuclear magnetic resonance probe and method for detecting nucleic acid and antibody by nuclear magnetic resonance

Abstract

The present application relates to a kind of micro nuclear magnetic resonance probe and nucleic acid, antibody nuclear magnetic resonance detection method.Micro nuclear magnetic resonance probe includes the parallel arrangement of first plane shimming coil, nuclear magnetic resonance micro coil chip, digital microfluidic chip and second plane shimming coil in sequence, digital microfluidic chip is single pole plate form, the microstrip of medium substrate on nuclear magnetic resonance micro coil chip is annular microstrip coil, nuclear magnetic resonance micro coil chip and digital microfluidic chip are spaced apart.There is beneficial effect: through digital microfluidic chip control droplet, with simple structure, easy integration advantage;Annular microstrip coil can generate a uniform B1 field that matches the shape of droplet, can improve the filling factor of trace sample droplet;Nucleic acid, antibody modified magnetic particle probe can enhance nuclear magnetic resonance detection sensitivity, by quantitative analysis the change amount of relaxation time and the concentration of target detection substance, nucleic acid, antibody quantitative detection can be realized.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of micro nuclear magnetic resonance detection, and particularly relates to a micro nuclear magnetic resonance probe and a nucleic acid and antibody nuclear magnetic resonance detection method. BACKGROUND

[0002] Digital microfluidic chip utilizes the electrowetting phenomenon on the medium to realize the control of liquid drops. An array of driving electrodes is made on a substrate material, and the digital microfluidic chip sequentially changes the wettability of different regions of the medium layer by changing the state of the driving electrodes in a programmatic manner, thereby controlling the free movement of the liquid drops.

[0003] Chinese patent document CN112090456A discloses a planar double microstrip microcoil probe, which comprises a first gradient coil, a dielectric substrate, a microfluidic chip, a grounding plate and a second gradient coil. The dielectric substrate has parallel double microstrip lines, the parallel double microstrip lines are placed parallel to the direction of the static magnetic field B0, and the fluid channel is placed between the parallel microstrip lines. When the magnetic resonance signal is excited and received, the radio frequency current passes through the microstrip line to generate a uniform radio frequency field B1 perpendicular to the plane of the coil. The microfluidic chip is composed of a sandwich structure of an upper Teflon sheet, a lower Teflon sheet and a layer of hole-shaped PDMS film. The fluid channel of the detection area of the microfluidic chip is parallel to the double microstrip lines.

[0004] The existing technology has the following disadvantages: first, the microfluidic channel of the microfluidic chip is complex to process, and requires additional mechanical components such as micropumps and microvalves, which are difficult to integrate; second, although the microcoil reduces the sample volume of the detection area, the pump and the pipeline related area for making the sample flow still have a large useless volume, which increases the actual sample usage. At the same time, continuous fluid control also increases the sample usage and is easy to cause cross contamination between liquids.

[0005] The virus nucleic acid detection reagent in the prior art includes a new coronavirus nucleic acid detection reagent, which is mostly based on the real-time fluorescent quantitative PCR (qPCR) technology. The reaction process usually consists of 20-40 PCR cycles, each cycle consisting of three steps of high-temperature denaturation, low-temperature renaturation (annealing) and appropriate temperature extension. The temperature rising and falling process takes time, and it takes several hours to get accurate results. To shorten the time, the number of cycles can be reduced, which will affect the sensitivity and cause "false negative" results. In order to accelerate the virus diagnosis, LAMP nucleic acid amplification technology can be used to avoid frequent temperature rising and falling process, and the time required for nucleic acid amplification is shortened from 1 hour to 15 minutes. The method for detecting the LAMP amplification product is usually to observe the magnesium pyrophosphate precipitation of the reaction solution, Mg 2+Color change methods such as indicator calcein and hydroxynaphthol blue. All these detection methods are based on the DNA mass produced by LAMP reaction, regardless of whether the product sequence is specific or non-specific. Non-specific amplification, primer dimer formation or contamination can easily lead to false positives.

[0006] Currently, virus antibody detection, including new coronavirus antibody detection, mostly uses immunochromatography, which is fast and convenient for detection, but cannot be accurately quantified. While using traditional enzyme-linked immunoassay to detect antibodies, the quantification is more accurate, but the requirement for the operator is high, the cost is high and the steps are complicated and time-consuming. SUMMARY

[0007] The technical problem to be solved by the present application is to provide a micro nuclear magnetic resonance probe and a nucleic acid and antibody nuclear magnetic resonance detection method, and to improve the integration of the probe.

[0008] The technical solution adopted by the present application to solve the technical problem is: a micro nuclear magnetic resonance probe, comprising a first planar field coil, a nuclear magnetic resonance micro coil chip, a digital microfluidic chip and a second planar field coil arranged in parallel in sequence, the digital microfluidic chip is in the form of a single pole plate, the microstrip on the dielectric substrate of the nuclear magnetic resonance micro coil chip is a loop microstrip coil, the loop microstrip coil is a loop formed by two symmetrical half-loop microstrips, the upper and lower junctions of the half-loop microstrip are the current input end and the current output end of the loop microstrip coil, for making the electric field directions transmitted in the two half-loop microstrips the same, the nuclear magnetic resonance micro coil chip and the digital microfluidic chip are spaced apart to form a sandwich structure, and the sandwich cavity of the sandwich structure is a detection sandwich cavity for nuclear magnetic resonance detection.

[0009] Further limitation, the nuclear magnetic resonance micro coil chip comprises a dielectric substrate, a loop microstrip coil, a first front conductive strip, a second front conductive strip and a back conductive strip, the first front conductive strip, the second front conductive strip and the loop microstrip coil are located on the front surface of the dielectric substrate, the back conductive strip is located on the back surface of the dielectric substrate, the inner end of the first front conductive strip and the second front conductive strip are connected to the current input end and the current output end of the loop microstrip coil respectively, the first front conductive strip and the second front conductive strip are on a straight line, the extension direction of the back conductive strip is the same as the extension direction of the first front conductive strip or the second front conductive strip, the back conductive strip passes through the projection area of the loop microstrip coil on the back surface of the dielectric substrate, one end of the back conductive strip is connected to the outer end of the first front conductive strip, the other end of the back conductive strip and the outer end of the second front conductive strip are the current input end and the current output end of the nuclear magnetic resonance micro coil chip, for connecting the coil tuning matching circuit.

[0010] Further limitation, the surface of the nuclear magnetic resonance micro coil chip and the digital microfluidic chip facing the detection sandwich cavity has a hydrophobic layer.

[0011] Further limited, the detection clamp cavity is filled with fluorine oil.

[0012] Further limited, the first planar field coil, the nuclear magnetic resonance micro coil chip, the digital microfluidic chip and the second planar field coil are fixedly installed through the mounting seat, and the mounting seat is located at the lower end of the first planar field coil, the nuclear magnetic resonance micro coil chip, the digital microfluidic chip and the second planar field coil.

[0013] Further limited, the nuclear magnetic resonance micro coil chip and the first planar field coil on the same side of the nuclear magnetic resonance micro coil chip have at least one liquid injection hole communicated with the detection clamp cavity and at least one liquid discharge hole communicated with the detection clamp cavity, and the bottom of the digital microfluidic chip has a heater and a temperature sensor.

[0014] Further limited, the driving electrode array region on the digital microfluidic chip has a nucleic acid amplification region, a probe reaction region and a nuclear magnetic resonance detection region, the bottom of the nucleic acid amplification region, the probe reaction region and the nuclear magnetic resonance detection region has an independently controlled heater and a temperature sensor, and the nuclear magnetic resonance detection region corresponds to the annular microstrip coil region of the nuclear magnetic resonance micro coil chip.

[0015] Further limited, the driving electrode array region on the digital microfluidic chip has a detection function region, the bottom of the detection function region has a heater and a temperature sensor, and the detection function region corresponds to the annular microstrip coil region of the nuclear magnetic resonance micro coil chip.

[0016] A nucleic acid nuclear magnetic resonance detection method, which adopts the above-mentioned micro nuclear magnetic resonance probe to perform nucleic acid nuclear magnetic resonance detection, first loads a nucleic acid detection sample into the detection clamp cavity of the micro nuclear magnetic resonance probe for RT-LAMP amplification; then mixes and reacts the amplified droplets with a nucleic acid probe modified magnetic particle probe, the magnetic particle probe captures target nucleic acid, causing the magnetic particle probe to aggregate; then measures the nuclear magnetic resonance of the mixed reaction droplets through the annular microstrip coil of the micro nuclear magnetic resonance probe, analyzes the relaxation time signal change, and realizes rapid nucleic acid detection.

[0017] An antibody nuclear magnetic resonance detection method, which adopts the above-mentioned micro nuclear magnetic resonance probe to perform antibody nuclear magnetic resonance detection, first loads an antibody detection sample into the detection clamp cavity of the micro nuclear magnetic resonance probe and mixes and reacts with an antibody probe modified magnetic particle probe, the magnetic particle probe captures target antibody, causing the magnetic particle probe to aggregate; then measures the nuclear magnetic resonance of the mixed reaction droplets through the annular microstrip coil of the micro nuclear magnetic resonance probe, analyzes the relaxation time signal change, and realizes rapid antibody detection.

[0018] The application has the advantages that: the liquid drops are controlled by the digital microfluidic chip, the required detection sample volume is small, the liquid drops can be accurately controlled, the analysis speed is fast, the pump and the valve are not required to be used in cooperation, the structure is simple, the integration is easy, and the requirements of the sample on-chip nuclear magnetic resonance detection and analysis are met;

[0019] The annular microstrip coil of the nuclear magnetic resonance micro coil chip can generate a uniform B1 field matched with the shape of the liquid drop controlled by the digital microfluidic chip, the filling factor of the trace sample liquid drop can be improved, the signal sensitivity can be greatly improved, and the nuclear magnetic signal of the microliter level liquid drop sample can be obtained;

[0020] The nuclear magnetic resonance micro coil chip and the digital microfluidic chip are arranged in a sandwich structure, the annular microstrip coil is tightly attached to the detection sample liquid drop, the uniform field space is small, the detection sample liquid drop space needs to be subjected to the uniform field, the uniform field effect is remarkable, and therefore a grounding plate is not required to be separately arranged to ensure the uniform field effect, the design is beneficial to simplifying the structure and providing the integration degree;

[0021] The nucleic acid and antibody nuclear magnetic resonance detection method using the nuclear magnetic resonance probe of the application is a non-contact detection means, does not depend on optical or electrochemical signals, but is based on the atomic nucleus information of the sample, and is not easy to generate non-specific interference signals like optical or electrochemical methods, so that the complex and time-consuming sample pretreatment steps can be omitted, and the detection time is effectively shortened;

[0022] The nucleic acid and antibody modified magnetic particle probe can enhance the nuclear magnetic resonance detection sensitivity, the quantitative detection of the nucleic acid and the antibody can be realized by quantitatively analyzing the relationship between the change amount of the relaxation time and the concentration of the target detection object. BRIEF DESCRIPTION OF DRAWINGS

[0023] The application will be further described below in combination with the drawings and examples;

[0024] Figure 1 is a structure principle diagram of the micro nuclear magnetic resonance probe of example 1 of the application;

[0025] Figure 2 is a front structure schematic diagram of the nuclear magnetic resonance micro coil chip of the application;

[0026] Figure 3 is a back structure schematic diagram of the nuclear magnetic resonance micro coil chip of the application;

[0027] Figure 4 is a structure schematic diagram of the micro nuclear magnetic resonance probe of example 1 of the application;

[0028] Figure 5 is an exploded view of the micro nuclear magnetic resonance probe of example 1 of the application;

[0029] Figure 6 Circuit diagram of the coil tuning matching circuit of the present application;

[0030] Figure 7 NMR spectrum a of Example 1 of the present application;

[0031] Figure 8 NMR spectrum b of Example 1 of the present application;

[0032] Figure 9 NMR spectrum c of Example 1 of the present application;

[0033] Figure 10 Layout diagram of the driving electrode array of the digital microfluidic chip of Example 2 of the present application;

[0034] Figure 11 Process schematic of modifying nucleic acid probes on magnetic polymer nanoparticles in Example 2 of the present application;

[0035] Figure 12 Principle schematic of the nucleic acid NMR detection method based on magnetic particle probes in Example 2 of the present application;

[0036] Figure 13 Layout diagram of the driving electrode array of the digital microfluidic chip of Example 3 of the present application;

[0037] Figure 14 Process schematic of modifying antibody probes on magnetic polymer nanoparticles in Example 3 of the present application;

[0038] Figure 15 Principle schematic of the antibody NMR detection method based on magnetic particle probes in Example 3 of the present application;

[0039] In the figure, 1. first planar shim coil, 2. NMR microcoil chip, 2-1. dielectric substrate, 2-2. ring-shaped microstrip coil, 2-3. first front conductive strip, 2-4. second front conductive strip, 2-5. back conductive strip, 2-6. copper-plated through hole, 3. digital microfluidic chip, 3-1. driving electrode array, 4. second planar shim coil, 5. mounting seat, 6. heater, 7. temperature sensor, 8. droplet, 9. liquid injection hole, 10. liquid discharge hole, 11. hydrophobic layer, 12. nucleic acid amplification region, 13. probe reaction region, 14. NMR detection region, 15. magnetic particle probe, 15-1. DNA1 probe, 15-2. DNA2 probe, 15-3. polystyrene shell, 15-4. magnetic europium core, 15-5. Anti-IgM antibody, 15-6. protein antigen, 16. target nucleic acid, 17. detection functional region, 18. target antibody. DETAILED DESCRIPTION

[0040] Example 1, as Figures 1-6 As shown, a miniature nuclear magnetic resonance (NMR) probe comprises a first planar shim coil 1, a NMR microcoil chip 2, a digital microfluidic chip 3, and a second planar shim coil 4, arranged in parallel. The digital microfluidic chip 3 is a monopole plate. The microstrip on the dielectric substrate 2-1 of the NMR microcoil chip 2 is a ring-shaped microstrip coil 2-2, which is formed by two symmetrical semi-circular microstrip lines. The upper and lower junctions of the semi-circular microstrip lines serve as the current input and output terminals of the ring-shaped microstrip coil 2-2, ensuring that the electric field transmitted in the two semi-circular microstrip lines has the same direction. The NMR microcoil chip 2 and the digital microfluidic chip 3 are spaced apart to form a sandwich structure. The sandwich cavity is a detection cavity for NMR detection. The digital microfluidic chip 3 has a drive electrode array 3-1 for driving droplets 8 in the detection cavity. The first planar shim coil 1 and the second planar shim coil 4 are the first and second gradient coils disclosed in Chinese Patent Document CN112090456A.

[0041] Through simulation with the electromagnetic simulation software CST, it can be found that a uniform B1 field in the shape of a droplet is generated in the area directly above the center of the two semi-annular microstrip lines, which matches the shape of the droplet 8 controlled by the digital microfluidic chip 3. This uniform field area is the detection area that can be used for nuclear magnetic resonance detection.

[0042] like Figure 1 、 2 As shown in Figure 3, the nuclear magnetic resonance microcoil chip 2 includes a dielectric substrate 2-1, a ring-shaped microstrip coil 2-2, a first front conductive strip 2-3, a second front conductive strip 2-4 and a back conductive strip 2-5. The first front conductive strip 2-3, the second front conductive strip 2-4 and the ring-shaped microstrip coil 2-2 are located on the front side of the dielectric substrate 2-1, and the back conductive strip 2-5 is located on the back side of the dielectric substrate 2-1. The inner ends of the first front conductive strip 2-3 and the second front conductive strip 2-4 are connected to the current input end and the current output end of the ring-shaped microstrip coil 2-2 respectively. The first front conductive strip 2-3, the second front conductive strip 2-4 and the ring-shaped microstrip coil 2-2 are located on the front side of the dielectric substrate 2-1. -3. The second front conductive strip 2-4 is in a straight line. The extension direction of the back conductive strip 2-5 is the same as that of the first front conductive strip 2-3 or the second front conductive strip 2-4. The back conductive strip 2-5 passes through the projection area of ​​the annular microstrip coil 2-2 on the back of the dielectric substrate 2-1. The upper end of the back conductive strip 2-5 is connected to the outer end of the first front conductive strip 2-3. The lower end of the back conductive strip 2-5 and the outer end of the second front conductive strip 2-4 are the current input and current output ends of the nuclear magnetic resonance microcoil chip 2, which are used to connect to the coil tuning and matching circuit. Figure 1 The first front conductive strip 2-3, the second front conductive strip 2-4 and the back conductive strip 2-5 are not drawn.

[0043] Preferably, the first front conductive strip 2-3 and the second front conductive strip 2-4 have a width of 20 mm, the inner end of the first front conductive strip 2-3 and the second front conductive strip 2-4 is narrowed to connect the annular microstrip coil 2-2, the narrowed region is triangular, the angle A of the two sides is 45 degrees, the width of the annular microstrip coil 2-2 is 800 um, the radius of the center is 1 mm, the thickness of the dielectric substrate 2-1 is 3 mm, which can make the structure of the annular microstrip coil 2-2 region in the circuit inductance much larger than capacitance, the material of the dielectric substrate 2-1 is F4B substrate or Rogers 5880, the dielectric constant is 2.2, and the loss tangent value is small. The nuclear magnetic resonance microcoil chip 2 adopts a PCB processing technology, and the annular microstrip coil 2-2, the first front conductive strip 2-3, the second front conductive strip 2-4 and the back conductive strip 2-5 are printed and prepared on the dielectric substrate 2-1, the annular microstrip coil 2-2, the first front conductive strip 2-3, the second front conductive strip 2-4 and the back conductive strip 2-5 are 35 um copper foil, and the upper end of the back conductive strip 2-5 is connected with the upper end of the first front conductive strip 2-3 through the copper-plated through hole 2-6.

[0044] As shown in Figure 6 The coil tuning matching circuit includes impedance matching capacitors C1 and tuning capacitors C2 and C3. C2 and C3 are connected to the lower end of the back conductive strip 2-5 and the second front conductive strip 2-4, respectively. This voltage division design makes the voltage at both ends of the annular microstrip coil 2-2 V / 2 and -V / 2, respectively, which reduces the voltage required to be borne by the tuning capacitor C2, and at the same time makes the center of the annular microstrip coil 2-2 in a virtual ground state.

[0045] The surface of the nuclear magnetic resonance microcoil chip 2 and the digital microfluidic chip 3 facing the detection clamping cavity has a hydrophobic layer 11. The hydrophobic layer 11 is Teflon.

[0046] The detection clamping cavity is filled with fluorine oil, specifically FC-40 fluorine oil or FC-43 fluorine oil. Filling the detection clamping cavity with fluorine oil can reduce the magnetic field inhomogeneity and instability caused by the digital microfluidic chip 3.

[0047] The nuclear magnetic resonance microcoil chip 2 and the first planar shim coil 1 located on the same side of the nuclear magnetic resonance microcoil chip 2 have at least one liquid injection hole 9 and at least one liquid discharge hole 10 communicating with the detection clamping cavity. In the accompanying drawings of the first embodiment, the number of the liquid injection hole 9 and the liquid discharge hole 10 is one.

[0048] The first planar shim coil 1, the nuclear magnetic resonance micro coil chip 2, the digital microfluidic chip 3 and the second planar shim coil 4 are fixed by the mounting seat 5, and the mounting seat 5 is located at the lower end of the first planar shim coil 1, the nuclear magnetic resonance micro coil chip 2, the digital microfluidic chip 3 and the second planar shim coil 4.

[0049] The specific process of the method for performing nuclear magnetic resonance detection by using the micro nuclear magnetic resonance probe of the embodiment 1 is as follows by taking a glucose solution as an example:

[0050] The heavy water solution containing 0.1M glucose is loaded into the detection cavity through the liquid injection hole 9 to perform nuclear magnetic resonance detection, and as shown in FIG. 5, no peak is observed in the obtained spectrum. Next, the digital microfluidic chip 3 drives the glucose sample droplet to the center of the annular microstrip coil 2-2, and as shown in FIG. 6, a clear and resolved glucose spectrum is observed in the obtained spectrum. Finally, the glucose sample droplet is removed from the annular microstrip coil 2-2, and a blank spectrum is obtained again, as shown in FIG. 7, and a weak signal is emitted by the sample remaining at the annular microstrip coil 2-2. Figure 7 Figure 8 Figure 9

[0051] The embodiment 2 is basically the same as the embodiment 1, and the difference lies in that the reaction temperature has specific requirements in the process of processing the biological sample, and in order to realize high-performance nuclear magnetic resonance detection, specific temperatures need to be provided in each functional area of the digital microfluidic chip 3. For example, the suitable temperature for the specific binding between the antigen and the antibody is 37℃, the best reaction temperature needs to be maintained at 65℃ in the RT-LAMP amplification process of the nucleic acid, the probe reaction needs to be set at 37℃, and the working temperature of the static magnetic field magnet required in the nuclear magnetic resonance detection process needs to be set at 25℃. Therefore, the heater 6 and the temperature sensor 7 need to be provided at the bottom of the digital microfluidic chip 3.

[0052] As shown in FIG. 8, the micro nuclear magnetic resonance probe of the embodiment 2 is specifically a micro nuclear magnetic resonance probe for nucleic acid nuclear magnetic resonance detection, the driving electrode array 3-1 area on the digital microfluidic chip 3 has a nucleic acid amplification area 12, a probe reaction area 13 and a nuclear magnetic resonance detection area 14, the bottom of the nucleic acid amplification area 12, the probe reaction area 13 and the nuclear magnetic resonance detection area 14 all have independently controlled heaters 6 and temperature sensors 7, and the nuclear magnetic resonance detection area 14 corresponds to the annular microstrip coil 2-2 area of the nuclear magnetic resonance micro coil chip 2. Figure 10 The specific process of the method for performing nucleic acid nuclear magnetic resonance detection by using the micro nuclear magnetic resonance probe is as follows:

[0053] (1) Prepare a nucleic acid probe modified magnetic particle probe 15.

[0054]

[0055] ​​​​The specific process of step 1 is:

[0056] (1.1) preparing surface carboxylated magnetic polymer nanoparticles;

[0057] (1.2) Modifying nucleic acid probes on magnetic polymer nanoparticles.

[0058] like Figure 11 As shown, 1 mL of carboxylated magnetic polymer nanoparticles was dissolved in 2 mL of MES buffer (50 mM, pH 6.0), 8 mg of EDC and 12 mg of NHS were added, and the mixture was sonicated for 3 minutes and shaken at 37 degrees for 15 minutes. The carboxyl groups were activated in a weakly acidic environment, and a DNA1 probe 15-1 having a complementary sequence to the target nucleic acid 16 was added (without washing), and the mixture was shaken at 37 degrees for 1 hour. A blocking solution was added to block the reaction site, and the mixture was shaken at 37 degrees for another hour to obtain a magnetic particle probe 15 modified with the DNA1 probe 15-1. The magnetic particle probe 15 modified with the DNA1 probe 15-1 was separated, washed, and then dispersed in a neutral MES buffer.

[0059] The same steps are followed to obtain the magnetic particle probe 15 modified with the DNA2 probe 15-2 having a complementary sequence to the target nucleic acid 16.

[0060] The magnetic polymer nanoparticles are specifically nanoparticles with a polystyrene shell 15-3 wrapping a magnetic europium core 15-4. The polystyrene shell 15-3 wrapping the magnetic europium core 15-4 can effectively improve the stability of the magnetic particle solution under an external magnetic field. Only when the detection target is present, the probe-modified magnetic particle probe 15 will cause aggregation changes of the magnetic particle probe 15 through specific coupling with the target, resulting in a significant decrease in the T1 / T2 of the water proton system, thereby obtaining qualitative and quantitative information of biological molecules.

[0061] (2) Nucleic acid nuclear magnetic resonance detection is performed using the micro-nuclear magnetic resonance probe of Example 2 and the magnetic particle probe 15 modified with the nucleic acid probe of step 1.

[0062] The entire RT-LAMP nucleic acid amplification and detection process is completed in the detection chamber of the micro-nuclear magnetic resonance probe. The reaction droplets controlled by the digital microfluidic chip 3 include RT-LAMP reagent droplets, nucleic acid detection sample droplets and magnetic particle probe droplets. The magnetic particle probe droplets contain equal amounts of magnetic particle probes 15 modified with DNA1 probes 15-1 and magnetic particle probes 15 modified with DNA2 probes 15-2.

[0063] Firstly, the nucleic acid detection sample is loaded into the detection cavity of the micro nuclear magnetic resonance probe for RT-LAMP amplification; then the amplified droplet 8 is mixed with the nucleic acid probe modified magnetic particle probe 15, the magnetic particle probe 15 captures the target nucleic acid 16, causing the magnetic particle probe 15 to aggregate; then the mixed reaction droplet 8 is measured by the annular microstrip coil 2-2 of the micro nuclear magnetic resonance probe, and the relaxation time signal change is analyzed to realize rapid nucleic acid detection.

[0064] The specific process is as follows: the digital microfluidic chip 3 controls the nucleic acid detection sample droplet and the RT-LAMP reagent droplet to move to the nucleic acid amplification area 12 for mixing and nucleic acid amplification, the nucleic acid amplification adopts the RT-LAMP amplification method and is carried out at 65℃ constant temperature, which eliminates the sample cycle temperature rising and falling time, and simultaneously carries out RNA reverse transcription and nucleic acid amplification; after the nucleic acid amplification is completed, the digital microfluidic chip 3 controls the amplified droplet 8 in the nucleic acid amplification area 12 and the magnetic particle probe droplet to move to the probe reaction area 13 for mixing and reaction, the nucleic acid fragment of the target nucleic acid 16 causes the DNA1 probe 15-1 modified magnetic particle probe 15 and the DNA2 probe 15-2 modified magnetic particle probe 15 to aggregate, as shown in Figure 12 ; the digital microfluidic chip 3 controls the reaction completed droplet 8 to move to the nuclear magnetic resonance detection area 14 for nuclear magnetic resonance detection, analyzes the relaxation time signal change, and realizes rapid nucleic acid detection.

[0065] Example 3 is basically the same as Example 1, the difference is that the driving electrode array area on the digital microfluidic chip 3 has a detection function area 17, the bottom of the detection function area 17 has a heater 6 and a temperature sensor 7, and the detection function area 17 corresponds to the annular microstrip coil 2-2 area of the nuclear magnetic resonance microcoil chip 2.

[0066] The specific process of the method for antibody nuclear magnetic resonance detection using the micro nuclear magnetic resonance probe is as follows:

[0067] (1) Preparation of antibody probe modified magnetic particle probe 15.

[0068] As shown in Figure 14 , the specific process of step 1 is as follows:

[0069] (1.1) Preparation of carboxylated magnetic polymer nanoparticles with surface carboxylation;

[0070] (1.2) Modification of antibody probe on magnetic polymer nanoparticles.

[0071] (2) Antibody nuclear magnetic resonance detection using the micro nuclear magnetic resonance probe of this embodiment 3 and the antibody probe modified magnetic particle probe 15 of step 1.

[0072] The whole detection process is completed in the detection cavity of the micro-NMR probe. The reaction droplets controlled by the digital microfluidic chip 3 include antibody detection sample droplets and magnetic particle probe droplets.

[0073] First, the antibody detection sample is loaded into the detection cavity of the micro-NMR probe. Then, the antibody detection sample is mixed with the antibody probe modified magnetic particle probe 15, the magnetic particle probe 15 captures the target antibody 18, causing the magnetic particle probe 15 to aggregate. Then, the mixed reaction droplet 8 is measured by the annular microstrip coil 2-2 of the micro-NMR probe, and the relaxation time signal change is analyzed to realize rapid antibody detection.

[0074] Taking the detection of IgM as an example, the specific process is as follows: the digital microfluidic chip 3 controls the antibody detection sample droplet and the magnetic particle probe droplet to move to the detection function area 17 for mixing and reaction. When the target antibody 18 exists in the solution, such as IgM, the target antibody 18 IgM will interact with the Anti-IgM antibody 15-5 and the protein antigen 15-6 on the surface of the magnetic particle probe 15, causing the originally freely dispersed magnetic particle probe 15 in the solution to aggregate. Figure 15 As shown in FIG. 6, the aggregation of the magnetic particle probe 15 will cause the magnetic field in the local area around it to increase, while the magnetic field in other local areas will decrease, causing the magnetic field around the particles to fluctuate. This fluctuation of the magnetic field will accelerate the process of molecular spin relaxation in the vicinity, which is manifested as a decrease in the relaxation time T2 value of the solution. The degree of aggregation is related to the concentration of the target antibody 18 IgM. By quantitatively analyzing the relationship between the change in T2 value and the concentration of the target antibody 18, quantitative detection of antibodies can be realized.

[0075] After the reaction is completed, NMR detection is performed to analyze the relaxation time signal change and realize rapid nucleic acid detection.

Claims

1. A micro nuclear magnetic resonance probe characterized by: The application relates to a nuclear magnetic resonance (NMR) microcoil chip and a digital microfluidic chip, and belongs to the technical field of NMR. The NMR microcoil chip (2) comprises a dielectric substrate (2-1), a ring-shaped microstrip coil (2-2), a first front conductive strip (2-3), a second front conductive strip (2-4) and a back conductive strip (2-5), the first front conductive strip (2-3), the second front conductive strip (2-4) and the ring-shaped microstrip coil (2-2) are located on the front of the dielectric substrate (2-1), the back conductive strip (2-5) is located on the back of the dielectric substrate (2-1), the inner end portions of the first front conductive strip (2-3) and the second front conductive strip (2-4) are connected to the current input end and the current output end of the ring-shaped microstrip coil (2-2) respectively, the first front conductive strip (2-3) and the second front conductive strip (2-4) are on a straight line, the extension direction of the back conductive strip (2-5) is the same as the extension direction of the first front conductive strip (2-3) or the second front conductive strip (2-4), the back conductive strip (2-5) passes through the projection area of the ring-shaped microstrip coil (2-2) on the back of the dielectric substrate (2-1), one end of the back conductive strip (2-5) is connected to the outer end portion of the first front conductive strip (2-3), the other end of the back conductive strip (2-5) and the outer end portion of the second front conductive strip (2-4) are the current input end and the current output end of the NMR microcoil chip (2) and are used for connecting a coil tuning matching circuit.

2. The micro-NMR probe of claim 1, wherein: The surface of the NMR microcoil chip (2) and the digital microfluidic chip (3) facing the detection cavity is provided with a hydrophobic layer (11).

3. The micro-NMR probe of claim 1, wherein: The detection cavity is filled with fluorine oil.

4. The micro-NMR probe of claim 1, wherein: The first planar shim coil (1), the NMR microcoil chip (2), the digital microfluidic chip (3) and the second planar shim coil (4) are fixedly installed through a mounting seat (5), and the mounting seat (5) is located at the lower end portions of the first planar shim coil (1), the NMR microcoil chip (2), the digital microfluidic chip (3) and the second planar shim coil (4).

5. The micro-NMR probe of claim 1, wherein: The NMR microcoil chip (2) and the first planar shim coil (1) located on the same side of the NMR microcoil chip (2) are provided with at least one liquid injection hole (9) and at least one liquid discharge hole (10) which communicate with the detection cavity, and the bottom of the digital microfluidic chip (3) is provided with a heater (6) and a temperature sensor (7).

6. The micro-NMR probe of claim 1, wherein: The driving electrode array (3-1) region on the digital microfluidic chip (3) has a nucleic acid amplification region (12), a probe reaction region (13) and a nuclear magnetic resonance detection region (14), the bottom of the nucleic acid amplification region (12), the probe reaction region (13) and the nuclear magnetic resonance detection region (14) has an independently controlled heater (6) and a temperature sensor (7), and the nuclear magnetic resonance detection region (14) corresponds to the annular microstrip coil (2-2) region of the nuclear magnetic resonance microcoil chip (2).

7. The micro-NMR probe of claim 1, wherein: The driving electrode array (3-1) region on the digital microfluidic chip (3) has a detection function region (17), the bottom of the detection function region (17) has a heater (6) and a temperature sensor (7), and the detection function region (17) corresponds to the annular microstrip coil (2-2) region of the nuclear magnetic resonance microcoil chip (2).

8. A method of detecting a nucleic acid by nuclear magnetic resonance, characterized by: The micro nuclear magnetic resonance probe of claim 1 is used for nucleic acid nuclear magnetic resonance detection, first, the nucleic acid detection sample is loaded into the detection clamping cavity of the micro nuclear magnetic resonance probe for RT-LAMP amplification, then the amplified droplet (8) is mixed with the nucleic acid probe modified magnetic particle probe (15) for reaction, the magnetic particle probe (15) captures the target nucleic acid (16), causing the magnetic particle probe (15) to aggregate, then the mixed reaction droplet (8) is measured by the annular microstrip coil (2-2) of the micro nuclear magnetic resonance probe, the relaxation time signal change is analyzed, and rapid nucleic acid detection is realized.

9. An antibody NMR detection method, characterized by: The micro nuclear magnetic resonance probe of claim 1 is used for antibody nuclear magnetic resonance detection, first, the antibody detection sample is loaded into the detection clamping cavity of the micro nuclear magnetic resonance probe and mixed with the antibody probe modified magnetic particle probe (15) for reaction, the magnetic particle probe (15) captures the target antibody (18), causing the magnetic particle probe (15) to aggregate, then the mixed reaction droplet (8) is measured by the annular microstrip coil (2-2) of the micro nuclear magnetic resonance probe, the relaxation time signal change is analyzed, and rapid antibody detection is realized.

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