A microfluidic chip-based magnetic nanoparticle detection system without spin exchange relaxation

Abstract

The present invention provides a microfluidic chip magnetic nanoparticle detection system based on spin exchange relaxation-free. The biomacromolecules to be detected are fixed on the chip substrate by a microfluidic chip device and marked with magnetic nanoparticles. The biomacromolecules to be detected include tumor-associated antigens, tumor-associated glycolipids and glycoproteins, and hormone peptides. The size of the magnetic nanoparticles used is 10nm-10μm. The modification and fixation process of the biomacromolecules can be controlled by a fast dwelling technology. The magnetic nanoparticles in the microfluidic chip are uniformly magnetized by a magnet placement device, and then the microfluidic chip containing the molecules to be detected is mounted on a linear scanning sample stage, and the spatial magnetic field distribution on the surface of the microfluidic chip is obtained by scanning magnetic imaging. The spatial magnetic field distribution is fitted according to the dipole magnetic field distribution, and the number and spatial information of the magnetic particles are obtained by inversion. The detection system has strong specificity and high sensitivity, and the reading results are fast, intuitive, and quantifiable.

Description

Technical Field

[0001] The invention relates to a microfluidic chip magnetic signal detection system, in particular to a microfluidic chip magnetic nanoparticle detection system based on spin-free exchange relaxation. Background Art

[0002] Early diagnosis of diseases relies on molecular-specific cell detection technology. Molecular-specific cell diagnosis is usually accomplished with the aid of optical and magnetic markers. Currently, the more mature molecular-specific analysis methods in medicine are fluorescence analysis and chemiluminescence analysis using luminescent substances as markers. The disadvantages of this method are the high cost of the fluorescent groups used, and the stability is affected by the light source and the detection environment. Compared with optical markers, the magnetic nanoparticles used in magnetic markers have better biocompatibility, stability, and lower toxicity, and can be used under opaque conditions, including in vivo environments. For the application of magnetic markers in molecular-specific detection, how to improve sensitivity, spatial resolution, and detection efficiency is a hot topic in academic research.

[0003] There are two main types of weak magnetic sensors that can achieve ultra-high sensitivity and high spatial resolution scanning magnetic imaging: superconducting quantum weak magnetic sensors and weak magnetic sensors based on atomic spins. In recent years, with the advancement of laser technology, a variety of weak magnetic sensors based on atomic spins based on the interaction between atoms and lasers have emerged. Among them, weak magnetic sensors based on spin-free exchange relaxation can already reach fT / HZ comparable to superconducting quantum weak magnetic sensors. 1 / 2 The magnetic field sensitivity of the level can realize the magnetic signal detection of a single magnetic nanoparticle. Compared with the superconducting quantum weak magnetic sensor, the weak magnetic sensor based on spin exchange relaxation does not require low temperature working conditions, saving the expensive cost of liquid helium, and its operating cost will be greatly reduced. Moreover, the size of the weak magnetic sensor based on spin exchange relaxation is much smaller than that of the superconducting quantum weak magnetic sensor, which can provide higher spatial resolution.

[0004] Microfluidic chip technology is a micro-reaction operation technology developed in recent years. This detection method uses a micro-channel chip made of a certain material to prepare a micro-reaction area. The most prominent advantage is that the amount of sample and reagent used is very small, and high-throughput and simultaneous detection of multiple samples can be achieved. Microfluidic technology is used for the binding and separation of magnetic nanoparticles to simplify the detection steps, realize in-situ measurement, and greatly reduce the detection time. The combination of the array design of the microfluidic chip and the scanning atomic magnetometer makes it possible to screen biological macromolecules (antibodies-antigens, receptor-ligands, DNA, proteins) in high-throughput in situ. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a microfluidic chip magnetic nanoparticle detection system based on spin exchange relaxation-free, wherein the detection system can realize the specific detection of trace biomacromolecules in a sample.

[0006] In order to solve the above technical problems, the technical solution of the present invention is:

[0007] A microfluidic chip magnetic nanoparticle detection system based on spin exchange relaxation-free includes a microfluidic system, a linear scanner, a magnetic shielding device for shielding external magnetic field interference, a weak magnetic sensor and a LabVIEW controller, wherein:

[0008] The microfluidic system is composed of a peristaltic pump, a microfluidic chip, a waste liquid pool and a magnet placement device. The microfluidic chip is composed of an upper glass sheet, a middle PDMS channel layer and a lower glass sheet. The middle PDMS channel layer is bonded between the upper glass sheet and the lower glass sheet by surface plasma treatment. The inlet and outlet of the fluid channel at both ends of the middle PDMS channel layer are encapsulated and connected with a fluid pipeline for guiding the inflow and outflow of liquid. The two ends of the fluid pipeline are respectively connected to the peristaltic pump and the waste liquid pool through a soft catheter; the surface of the lower glass sheet is plated with a gold film, and the gold film is modified with a -COOH functional group, which can react with the amino group on the monoclonal antibody to bind the biomacromolecule to the chip surface; the magnet placement device is divided into three compartments, upper, middle and lower, and a rectangular NdFeB strong magnet is placed in each of the upper and lower compartments, and the microfluidic chip is placed in the middle compartment;

[0009] A track is provided in the middle of the magnetic shielding device, and the weak magnetic sensor is placed in the magnetic shielding device and directly above the track;

[0010] The linear scanner is placed on one side of the magnetic shielding device and is used to deliver the microfluidic chip. The microfluidic chip is placed on a quartz rod of the linear scanner, and the quartz rod moves along the track. The magnet placement device is placed between the linear scanner and the magnetic shielding device, and the quartz rod can pass through the middle partition of the magnet placement device. The linear scanner can deliver the microfluidic chip containing the sample to be tested to the position directly below the weak magnetic sensor in the magnetic shielding device in the horizontal direction.

[0011] The LabVIEW controller is connected to the microfluidic chip, the linear scanner and the weak magnetic sensor circuit respectively; the LabVIEW controller uses LabVIEW software to simultaneously control the microfluidic chip, the linear scanner and the weak magnetic sensor, thereby realizing in-situ magnetic labeling of the object to be measured and in-situ measurement of the magnetic signal, which is beneficial to improving the accuracy and work efficiency of the concentration measurement of magnetic nanoparticles.

[0012] Preferably, in the above-mentioned microfluidic chip magnetic nanoparticle detection system based on no spin exchange relaxation, the microfluidic chip is encapsulated and reinforced by epoxy resin glue.

[0013] Preferably, in the above-mentioned microfluidic chip magnetic nanoparticle detection system based on no spin exchange relaxation, the fluid pipeline is a polytetrafluoroethylene fluid pipeline.

[0014] Preferably, in the above-mentioned microfluidic chip magnetic nanoparticle detection system based on no spin exchange relaxation, the weak magnetic sensor is a weak magnetic sensor based on no spin exchange relaxation for detecting magnetic signals of magnetic nanoparticles.

[0015] The above-mentioned microfluidic chip magnetic nanoparticle detection system based on spin exchange relaxation-free uses a microfluidic chip device to fix the biological macromolecules to be detected on the chip substrate and label them with magnetic nanoparticles. The biological macromolecules to be detected include tumor-associated antigens, tumor-associated glycolipids and glycoproteins, and hormone peptides. The size of the magnetic nanoparticles used is 10nm-10μm, and the modification and fixation process of the biological macromolecules can be controlled by rapid dwell technology.

[0016] The above-mentioned microfluidic chip magnetic nanoparticle detection system based on spin exchange relaxation-free uniformly magnetizes the magnetic nanoparticles in the microfluidic chip through a magnet placement device, and then mounts the microfluidic chip containing the molecules to be tested on a linear scanning sample stage, and uses scanning magnetic imaging to obtain the spatial magnetic field distribution on the surface of the microfluidic chip. The spatial magnetic field distribution is fitted according to the dipole magnetic field distribution, and the number and spatial information of the magnetic particles are inverted.

[0017] Beneficial effects:

[0018] The microfluidic chip magnetic nanoparticle detection system based on spin exchange relaxation-free method of the present invention has the following advantages:

[0019] 1. The microfluidic chip is delivered to move along the scanning channel through a precise linear scanner. The weak magnetic sensor without spin exchange relaxation is used to scan the detection area of ​​the microfluidic chip to obtain the spatial magnetic field distribution. The spatial magnetic field distribution is fitted according to the dipole magnetic field distribution, and the number and spatial information of the magnetic particles are inverted.

[0020] 2. Microfluidic chips have the advantages of small sample volume, strong specificity, high sensitivity, low price, quantifiable, fast and intuitive reading results, etc. Combined with efficient fluid drive and control, they can achieve accurate injection and efficient and rapid distribution of samples and detection reagents.

[0021] 3. Magnetic nanoparticles are modified onto the surface of specific antibodies through biochemical means, and the magnetic nanoparticles are transported through the microchannels in the microfluidic chip to label the biomacromolecules to be tested. The flow rate of the fluid in the microchannel is controlled to separate the bound and unbound magnetic nanoparticles, and the concentration of the biomacromolecules to be tested is measured directly on the chip. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 It is a structural schematic diagram of a microfluidic chip magnetic nanoparticle detection system based on spin exchange relaxation-free according to the present invention;

[0023] Figure 2 It is a schematic diagram of the structure of the magnetized microfluidic chip device of the present invention;

[0024] Figure 3 It is a detection principle diagram of the microfluidic chip magnetic nanoparticle detection system based on no spin exchange relaxation of the present invention.

[0025] In the figure, 1- microfluidic chip 2- magnet placement device 1-1- upper glass sheet

[0026] 1-2-middle PDMS channel layer 1-3-lower glass sheet 1-4-peristaltic pump 1-5-waste liquid pool 3-linear scanner 4-quartz rod 5-weak magnetic sensor 6-magnetic shielding device DETAILED DESCRIPTION

[0027] The following is a detailed description of a microfluidic chip magnetic nanoparticle detection system based on no spin exchange relaxation and its implementation method of the present invention in conjunction with the embodiments and drawings.

[0028] The weak magnetic sensor based on spin exchange relaxation in the following instrument is the QZFM weak magnetic sensor of QUSPIN. The magnetic shielding device is a five-layer shielding tube made of high magnetic permeability alloy material. The signal readout device is a multi-function data acquisition card of Beijing Altai Technology Co., Ltd.

[0029] Example 1

[0030] A microfluidic chip magnetic nanoparticle detection system based on spin exchange relaxation-free has the following structure:

[0031] like Figure 1 , Figure 2 As shown, first, a linear scanner 3 is installed at the entrance of the magnetic shielding device 6, a quartz rod 4 with a diameter of 5 mm is placed at the front end of the linear scanner, and a microfluidic chip 1 is placed on the quartz rod. The linear scanner transfers the microfluidic chip to the magnetic shielding device directly below the weak magnetic sensor 5 based on spin exchange relaxation without through the quartz rod. During the transfer process, the microfluidic chip passes through the magnet placement device 2 arranged between the linear scanner and the magnetic shielding device, and passes through the middle partition of the magnet placement device.

[0032] The microfluidic chip consists of an upper glass sheet 1-1, a middle PDMS channel layer 1-2 and a lower glass sheet 1-3, wherein the middle PDMS channel layer is bonded between the upper glass sheet and the lower glass sheet by surface plasma treatment, and the inlet and outlet of the fluid channel at both ends of the middle PDMS channel layer are encapsulated and connected with polytetrafluoroethylene fluid pipelines for guiding the inflow and outflow of liquid, and the two ends of the fluid pipeline are respectively connected to a peristaltic pump 1-4 and a waste liquid pool 1-5 through a soft catheter, and the object to be tested is injected into the fluid channel by the peristaltic pump. After the detection is completed, the object to be tested is finally recovered in the waste liquid pool; the surface of the lower glass sheet is plated with a gold film, and the -COOH functional group is modified on the gold film, which can react with the amino group on the monoclonal antibody to bind the biomacromolecule to the chip surface.

[0033] The scanning speed of the linear scanner is 0.01 to 20 mm / s, and the scanning length is 0 to 500 mm. The magnetic nanoparticles fixed on the lower glass sheet 1-3 in the microfluidic chip generate a magnetic field after magnetization, which is detected by the weak magnetic sensor based on spin exchange relaxation in the magnetic shielding device and converted into a voltage signal. The precise voltage signal is read out by a signal readout circuit connecting the weak magnetic sensor and the LabVIEW controller.

[0034] The LabVIEW controller is a workstation built based on LabVIEW software to control the microfluidic system, signal readout circuit and weak magnetic sensor based on spin exchange relaxation, respectively. It can realize automatic sampling of the microfluidic system, state adjustment and signal reading of the weak magnetic sensor based on spin exchange relaxation, greatly shorten the experimental time, reduce environmental interference to the system, and further improve the accuracy of the experimental results.

[0035] In order to uniformly magnetize the magnetic nanoparticles fixed in the microfluidic chip so that the weak magnetic sensor based on spin exchange relaxation can detect a larger magnetic signal, a magnet placement device 2 is specially designed. The magnet placement device is divided into three layers: upper, middle and lower. A rectangular NdFeB strong magnet is placed in parallel in each of the upper and lower layers, so that the middle layer of the magnet placement device forms a uniform strong magnetic field. When the microfluidic chip moves with the quartz rod through the middle layer, it is magnetized.

[0036] Based on the immunomagnetic detection study of AFP concentration in the sample solution (solution containing the macromolecule to be detected), the application method of the above-mentioned microfluidic chip magnetic nanoparticle detection system based on spin exchange relaxation is further explained below. Figure 3 This is the detection principle diagram of the system.

[0037] In the first step, the microfluidic chip whose channel surface is modified with -COOH functional groups is rinsed with deionized water and anhydrous ethanol in turn, and then PBS (PBS is phosphate buffered saline) buffer is introduced into the microfluidic chip to complete the cleaning of the channel surface.

[0038] The second step is to activate the surface of the flow channel of the microfluidic chip: EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) and NHS (N-hydroxysuccinimide) are mixed in a volume ratio of 1:1, and then slowly introduced into the flow channel of the microfluidic chip. After standing for a preset time (for example, 15 minutes), it is rinsed with PBS buffer, and then the microfluidic chip is sent into a magnetic shielding device for scanning magnetic imaging to obtain the spatial magnetic field distribution on the surface of the microfluidic chip (for background reference). In the present invention, EDC is used as a coupling agent in combination with NHS, and antibody molecules with amino, carboxyl or thiol groups can be connected to the sensor surface through the coupling action of the biocoupling agent. The bound anti-AFP monoclonal antibody in this embodiment contains protein molecules with multiple amino groups. Therefore, EDC and NHS are selected to connect them to the surface of the microfluidic chip.

[0039] The third step is to slowly pass the anti-AFP monoclonal antibody dilution into the flow channel of the microfluidic chip, let it stand for a preset time (e.g., 15 minutes) to form a stable covalent bond, and then pass it into PBS buffer for washing to remove the residual antibodies on the surface of the microfluidic chip;

[0040] In the fourth step, the ethanolamine-hydrochloric acid solution is slowly introduced into the flow channel of the microfluidic chip, and allowed to stand for a preset time (e.g., 15 minutes) to block the excess active sites on the surface of the microfluidic chip, and then the PBS buffer is introduced for washing. It should be noted that in the present invention, the ethanolamine-hydrochloric acid solution can block the excess active sites, forming a thin non-reactive layer to prevent non-specific binding of AFP to the sites;

[0041] Step 5: slowly pass AFP solutions with concentrations of 25, 50, 100, and 200 ng / mL into the flow channel of the microfluidic chip, and let them stand for a preset time (e.g., 10 minutes) to wait for the combination of antigen and antibody;

[0042] In the sixth step, a polyclonal antibody solution modified with magnetic nanoparticles at one end is slowly passed into the flow channel of the microfluidic chip, and allowed to stand for a preset time (e.g., 20 minutes), and then PBS buffer is passed to wash away the polyclonal antibodies that are not specifically bound to the surface of the microfluidic chip. The microfluidic chip is magnetized for 10 minutes using a magnet magnetization device and then sent to a magnetic shielding device for scanning magnetic imaging to obtain the spatial magnetic field distribution on the surface of the microfluidic chip. The spatial magnetic field distribution is fitted according to the dipole magnetic field distribution, and the number and spatial information of the magnetic particles are inverted. During the sample scanning process, when the specifically adsorbed magnetic nanoparticles are closest to the weak magnetic sensor, the measured magnetic signal reaches the strongest point, which is 52pT, 98pT, 213pT, and 430pT, respectively. According to the magnetic signal intensity, the concentration of AFP molecules in the corresponding solution is measured to be 25, 50, 100, and 200ng / mL.

[0043] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. The construction steps of the structure of the present invention are not in any particular order. Improvements and modifications such as structural transformation carried out by technicians in this technical field based on the method of the present invention or on the basis of the method are deemed to be within the scope of protection of the present invention.

Claims

1. A microfluidic chip magnetic nanoparticle detection system based on spin exchange relaxation free, characterized in that: It includes a microfluidic system, a linear scanner, a magnetic shielding device for shielding external magnetic field interference, a weak magnetic sensor and a LabVIEW controller, among which: The microfluidic system is composed of a peristaltic pump, a microfluidic chip, a waste liquid pool and a magnet placement device. The microfluidic chip is composed of an upper glass sheet, a middle PDMS channel layer and a lower glass sheet. The middle PDMS channel layer is bonded between the upper glass sheet and the lower glass sheet by surface plasma treatment. The inlet and outlet of the fluid channel at both ends of the middle PDMS channel layer are encapsulated and connected with a fluid pipeline for guiding the inflow and outflow of liquid. The two ends of the fluid pipeline are respectively connected to the peristaltic pump and the waste liquid pool through a soft catheter; the surface of the lower glass sheet is plated with a gold film, and the gold film is modified with a -COOH functional group; the magnet placement device is divided into three compartments, namely, upper, middle and lower. A rectangular NdFeB strong magnet is placed in each of the upper and lower compartments, and the microfluidic chip is placed in the middle compartment; A track is provided in the middle of the magnetic shielding device, and the weak magnetic sensor is placed in the magnetic shielding device and directly above the track; The linear scanner is placed on one side of the magnetic shielding device and is used to deliver the microfluidic chip. The microfluidic chip is placed on a quartz rod of the linear scanner, and the quartz rod moves along the track. The magnet placement device is placed between the linear scanner and the magnetic shielding device, and the quartz rod passes through the middle partition of the magnet placement device. The linear scanner delivers the microfluidic chip containing the sample to be tested to the position directly below the weak magnetic sensor in the magnetic shielding device in the horizontal direction. The LabVIEW controller is connected to the microfluidic chip, the linear scanner and the weak magnetic sensor circuit respectively; the LabVIEW controller uses LabVIEW software to simultaneously control the microfluidic chip, the linear scanner and the weak magnetic sensor, thereby realizing in-situ magnetic labeling of the object to be measured and in-situ measurement of the magnetic signal.

2. The microfluidic chip magnetic nanoparticle detection system based on spin exchange relaxation-free according to claim 1, characterized in that: The microfluidic chip is encapsulated and reinforced by epoxy resin glue.

3. The microfluidic chip magnetic nanoparticle detection system based on spin exchange relaxation-free according to claim 1, characterized in that: The fluid pipeline is a polytetrafluoroethylene fluid pipeline.

4. The microfluidic chip magnetic nanoparticle detection system based on spin exchange relaxation-free according to claim 1, characterized in that: The weak magnetic sensor is a weak magnetic sensor based on spin exchange relaxation and used for detecting magnetic signals of magnetic nanoparticles.

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