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CN114397455B - A microfluidic chip-based magnetic nanoparticle detection system without spin exchange relaxation - Google Patents

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

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CN114397455B
CN114397455B CN202111523515.6A CN202111523515A CN114397455B CN 114397455 B CN114397455 B CN 114397455B CN 202111523515 A CN202111523515 A CN 202111523515A CN 114397455 B CN114397455 B CN 114397455B
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CN114397455A (en
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栗大超
鲍博
王日东
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Tianjin University
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Abstract

The invention provides a micro-fluidic chip magnetic nanoparticle detection system based on spin-free exchange relaxation, which utilizes a micro-fluidic chip device to fix a biological macromolecule to be detected on a chip substrate, and uses magnetic nanoparticles to mark the biological macromolecule to be detected, wherein the biological macromolecule to be detected comprises tumor-related antigens, tumor-related glycolipids, glycoprotein and hormone peptide, the adopted magnetic nanoparticle size is 10nm-10 mu m, the modification fixing process of the biological macromolecule can be controlled by a rapid stay technology, the magnetic nanoparticles in the micro-fluidic chip are uniformly magnetized by a magnet placement device, then the micro-fluidic chip containing molecules to be detected is arranged on a linear scanning sample stage, and the surface space magnetic field distribution of the micro-fluidic chip is obtained by utilizing a scanning magnetic imaging mode. Fitting the spatial magnetic field distribution according to the dipole magnetic field distribution, and inverting to obtain the quantity and the spatial information of the magnetic particles; the detection system has the advantages of strong specificity, high sensitivity, and quick, visual and quantifiable reading result.

Description

Microfluidic chip magnetic nanoparticle detection system based on spin-free exchange relaxation
Technical Field
The invention relates to a micro-fluidic chip magnetic signal detection system, in particular to a micro-fluidic chip magnetic nanoparticle detection system based on spin-free exchange relaxation.
Background
Early diagnosis of disease relies on molecular specific cell detection techniques. Molecular-specific cellular diagnostics are typically accomplished with the aid of optical and magnetic labels. The current well-established molecular specificity assay methods in medicine are fluorescence and chemiluminescence assays using luminescent substances as labels, which have the disadvantages of high cost of the fluorophores used and stability affected by the light source and the detection environment. The magnetic nanoparticles used for magnetic labeling have better biocompatibility, stability and lower toxicity than optical labeling, and can be applied under opaque conditions, including in vivo environments. For the application of magnetic labels in molecular specificity detection, how to improve sensitivity, spatial resolution and detection efficiency is a hotspot problem of current academic research.
At present, two types of weak magnetic sensors can realize ultra-high sensitivity and high spatial resolution scanning magnetic imaging, namely a superconducting quantum weak magnetic sensor and a weak magnetic sensor based on atomic spin. In recent years, with the progress of laser technology, a plurality of weak magnetic sensors based on atomic spin and based on the interaction of laser, wherein the weak magnetic sensor based on spin-free relaxation can reach the magnetic field sensitivity of the fT/HZ 1/2 level equivalent to that of the superconducting quantum weak magnetic sensor, and the magnetic signal detection of single magnetic nano particles can be realized. Compared with a superconducting quantum weak magnetic sensor, the weak magnetic sensor based on spin-free exchange relaxation does not need low-temperature working conditions, so that the expensive cost of liquid helium is saved, and the running cost of the weak magnetic sensor is greatly reduced. And the size of the weak magnetic sensor based on no spin-exchange relaxation is much smaller than that of the superconducting quantum weak magnetic sensor, and can provide higher spatial resolution.
The micro-fluidic chip technology is a micro-reaction operation technology developed in recent years, and the detection method prepares a micro-reaction area through a micro-pipeline chip processed by a certain material, and has the most outstanding advantages of very small used sample quantity and reagent quantity, high throughput and simultaneous detection of multiple samples. The microfluidic technology is used for binding and separating magnetic nano particles, so that the detection step is simplified, in-situ measurement can be realized, and the detection time is greatly shortened. The combination of the arrayed design of the microfluidic chip and the scanning atomic magnetometer enables the high-throughput in-situ screening of biological macromolecules (antibodies-antigens, receptor-ligands, DNA, proteins).
Disclosure of Invention
The invention aims to solve the technical problem of providing a micro-fluidic chip magnetic nanoparticle detection system based on spin-free exchange relaxation, which can realize the specific detection of micro-biological macromolecules in a sample.
In order to solve the technical problems, the technical scheme of the invention is as follows:
a microfluidic chip magnetic nanoparticle detection system based on spin-free relaxation comprises 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,
The microfluidic system consists of a peristaltic pump, a microfluidic chip, a waste liquid pond and a magnet placement device, wherein the microfluidic chip consists 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 in a surface plasma treatment mode, fluid pipelines for guiding liquid to flow in and out are encapsulated and communicated on fluid channel inlets and outlets at two ends of the middle PDMS channel layer, the peristaltic pump and the waste liquid pond are respectively communicated with two ends of the fluid pipelines through flexible pipes, a gold film is plated on the surface of the lower glass sheet, a-COOH functional group is modified on the gold film and can react with amino groups on monoclonal antibodies to bind biological macromolecules on the surface of the chip, the magnet placement device is divided into an upper interlayer, a middle interlayer and a lower interlayer, a rectangular neodymium iron boron powerful magnet is respectively placed in the upper interlayer and the lower interlayer, and the microfluidic chip is placed in the middle layer;
the weak magnetic sensor is arranged in the magnetic shielding device and right above the track;
The linear scanner is arranged at one side of the magnetic shielding device and used for delivering the micro-fluidic chip, the micro-fluidic chip is arranged on a quartz rod of the linear scanner, and the quartz rod moves along the track;
the LabVIEW controller is respectively connected with the micro-fluidic chip, the linear scanner and the weak magnetic sensor in a line way, and simultaneously controls the micro-fluidic chip, the linear scanner and the weak magnetic sensor by utilizing LabVIEW software, so that in-situ magnetic marking and in-situ measurement of magnetic signals of an object to be measured are realized, and the accuracy and the working efficiency of magnetic nanoparticle concentration measurement are improved.
Preferably, in the microfluidic chip magnetic nanoparticle detection system based on spin-free relaxation, the microfluidic chip is encapsulated and reinforced by epoxy resin glue.
Preferably, in the microfluidic chip magnetic nanoparticle detection system based on spin-free exchange relaxation, the fluid pipeline is a polytetrafluoroethylene fluid pipeline.
Preferably, in the microfluidic chip magnetic nanoparticle detection system based on spin-free relaxation, the weak magnetic sensor is a weak magnetic sensor based on spin-free relaxation for detecting magnetic signals of magnetic nanoparticles.
According to the microfluidic chip magnetic nanoparticle detection system based on spin-free exchange relaxation, the microfluidic chip device is utilized to fix the biomacromolecule to be detected on the chip substrate, the biomacromolecule to be detected comprises tumor-related antigens, tumor-related glycolipids, glycoprotein and hormone peptide, the adopted magnetic nanoparticle size is 10nm-10 mu m, and the modification and fixation process of the biomacromolecule can be controlled by a rapid residence technology.
According to the microfluidic chip magnetic nanoparticle detection system based on spin-free exchange relaxation, the magnetic nanoparticles in the microfluidic chip are uniformly magnetized through the magnet placement device, then the microfluidic chip containing molecules to be detected is mounted on a linear scanning sample table, and the spatial magnetic field distribution of the surface of the microfluidic chip is obtained by using a scanning magnetic imaging mode. Fitting the spatial magnetic field distribution according to the dipole magnetic field distribution, and inverting to obtain the quantity and the spatial information of the magnetic particles.
The beneficial effects are that:
the microfluidic chip magnetic nanoparticle detection system based on spin-free exchange relaxation has the following advantages:
1. And delivering the microfluidic chip to move along a scanning channel through a precise linear scanner, scanning a detection area of the microfluidic chip based on a weak magnetic sensor without spin-exchange relaxation to obtain spatial magnetic field distribution, fitting the spatial magnetic field distribution according to dipole magnetic field distribution, and inverting to obtain the quantity and the spatial information of the magnetic particles.
2. The microfluidic chip has the advantages of small sample size, strong specificity, high sensitivity, low price, quantifiable, quick and visual reading result and the like, and combines efficient fluid driving and control to realize accurate sample injection and efficient and quick distribution of samples to be detected and detection reagents.
3. The magnetic nano particles are modified to the surface of a specific antibody through biochemical means, the micro-flow channel in the micro-flow chip is used for conveying the magnetic nano particles to mark the biological macromolecules to be detected, the flow rate of the fluid in the micro-flow channel is controlled to separate the combined and unbound magnetic nano particles, and the concentration measurement of the biological macromolecules to be detected is directly realized on the chip.
Drawings
FIG. 1 is a schematic diagram of a microfluidic chip magnetic nanoparticle detection system based on spin-free exchange relaxation in accordance with the present invention;
FIG. 2 is a schematic diagram of a magnetized microfluidic chip device according to the present invention;
Fig. 3 is a schematic diagram of a microfluidic chip magnetic nanoparticle detection system based on spin-free exchange relaxation according to the present invention.
In the figure, a 1-micro-fluidic chip 2-magnet placement device 1-1-upper glass sheet
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
The following describes a micro-fluidic chip magnetic nanoparticle detection system based on spin-free exchange relaxation and an implementation method thereof in detail by referring to the embodiments and the accompanying drawings.
The weak magnetic sensor based on spin-free relaxation in the following apparatus was QZFM weak magnetic sensor from QUSPIN. The magnetic shielding device uses five-layer shielding barrels made of high-permeability alloy materials. The signal reading device uses a multifunctional data acquisition card of Beijing Altai technology company.
Example 1
A microfluidic chip magnetic nanoparticle detection system based on spin-free exchange relaxation has the following structure:
As shown in fig. 1 and 2, a linear scanner 3 is installed at the entrance of a magnetic shielding device 6, a quartz rod 4 with the diameter of 5mm is placed at the front end of the linear scanner, a microfluidic chip 1 is arranged on the quartz rod, the linear scanner transmits the microfluidic chip to the position right below a weak magnetic sensor 5 based on spin-free relaxation in the magnetic shielding device through the quartz rod, and in the transmitting process, the microfluidic chip passes through a magnet placing device 2 arranged between the linear scanner and the magnetic shielding device and penetrates through a middle layer of the magnet placing device.
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 in a surface plasma treatment mode, polytetrafluoroethylene fluid pipelines for guiding liquid to flow in and out are packaged and communicated on fluid channel inlets and outlets at two ends of the middle PDMS channel layer, two ends of the fluid pipeline are respectively communicated with a peristaltic pump 1-4 and a waste liquid tank 1-5 through soft pipes, an object to be detected is injected into the fluid channels through the peristaltic pump, the object to be detected finally enters the waste liquid tank and is recovered after detection is completed, a gold film is plated on the surface of the lower glass sheet, a-COOH functional group is modified on the gold film and can react with amino groups on a monoclonal antibody, and biological macromolecules are bound on the surface of the chip.
The scanning speed of the linear scanner is 0.01-20 mm/s, and the scanning length is 0-500 mm. The magnetic nano particles fixed on the lower glass sheet 1-3 in the micro-fluidic chip are magnetized to generate a magnetic field, the magnetic field is detected by a weak magnetic sensor based on spin-free exchange relaxation in a magnetic shielding device and then converted into a voltage signal, and a signal reading circuit connected with the weak magnetic sensor and a LabVIEW controller reads out the accurate voltage signal.
The LabVIEW controller is a workstation which is constructed based on LabVIEW software and is used for controlling a microfluidic system, a signal reading circuit and a weak magnetic sensor based on spin-free relaxation. The method is used for realizing automatic sample injection of a microfluidic system, and state adjustment and signal reading of a weak magnetic sensor based on spin-free exchange relaxation. The experimental time is greatly shortened, the environmental interference to the system is reduced, and the accuracy of experimental results is further improved.
In order to uniformly magnetize the magnetic nano particles fixed in the micro-fluidic chip, so that a weak magnetic sensor based on spin-free relaxation can detect a larger magnetic signal, a magnet placement device 2 is specially designed, the magnet placement device is divided into an upper interlayer, a middle interlayer and a lower interlayer, a rectangular NdFeB powerful magnet is respectively placed in parallel in the upper interlayer and the lower interlayer, a uniform strong magnetic field is formed in the middle interlayer of the magnet placement device, and magnetization occurs when the micro-fluidic chip moves along with a quartz rod and passes through a middle interlayer.
The application method of the microfluidic chip magnetic nanoparticle detection system based on spin-free relaxation is further described based on immunomagnetic detection study of AFP concentration in a sample solution (solution containing macromolecules to be detected), and FIG. 3 is a detection schematic diagram of the system.
Firstly, washing a microfluidic chip with a-COOH functional group modified on the surface of a flow channel by using deionized water and absolute ethyl alcohol in sequence, and then introducing PBS (PBS is phosphate buffer solution) buffer solution into the microfluidic chip to finish the cleaning of the surface of the flow channel.
Secondly, activating the surface of a flow channel of the microfluidic chip, namely uniformly mixing EDC (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride) and NHS (N-hydroxysuccinimide) according to a volume ratio of 1:1, slowly introducing the mixture into the flow channel of the microfluidic chip, standing for a preset period (for example, 15 min), flushing the mixture by using PBS buffer solution, and then sending the microfluidic chip into a magnetic shielding device for scanning magnetic imaging to obtain the spatial magnetic field distribution (used for background reference) of the surface of the microfluidic chip. In the invention, EDC is used as a coupling agent in combination with NHS, and antibody molecules with amino, carboxyl or sulfhydryl groups can be connected to the surface of a sensor through the coupling action of a biological coupling agent. The binding anti-AFP monoclonal antibody in this example contains protein molecules with multiple amino groups, so EDC and NHS are selected to be attached to the surface of the microfluidic chip.
Thirdly, slowly introducing an anti-AFP monoclonal antibody diluent into a flow channel of the microfluidic chip, standing for a preset period of time (for example, 15 min) to form stable covalent bond connection, and then introducing PBS buffer solution for flushing to remove the residual antibody on the surface of the microfluidic chip;
And fourthly, slowly introducing an ethanolamine-hydrochloric acid solution into a flow channel of the microfluidic chip, standing for a preset period of time (for example, 15 min) to seal redundant active sites on the surface of the microfluidic chip, and then introducing PBS buffer solution for flushing. In the invention, the ethanolamine-hydrochloric acid solution can seal redundant active sites to form a thin non-reactive layer to prevent non-specific binding of AFP and the sites;
Fifthly, slowly introducing AFP solutions with the concentrations of 25, 50, 100 and 200ng/mL into a flow channel of a microfluidic chip, standing for a preset period of time (for example, 10 min), and waiting for the combination of an antigen and an antibody;
And sixthly, slowly introducing a polyclonal antibody solution with one end modified with magnetic nano particles into a flow channel of the microfluidic chip, standing for a preset period of time (for example, 20 min), and then introducing PBS buffer solution to wash out the polyclonal antibody which is not specifically bound on the surface of the microfluidic chip. And magnetizing the microfluidic chip for 10min by using a magnet magnetizing device, then sending the microfluidic chip into a magnetic shielding device for scanning magnetic imaging to obtain the spatial magnetic field distribution on the surface of the microfluidic chip, fitting the spatial magnetic field distribution according to the dipole magnetic field distribution, and inverting to obtain the quantity and the spatial information of the magnetic particles. In the sample scanning process, when the specifically adsorbed magnetic nano particles are closest to the weak magnetic sensor, the measured magnetic signals reach the strongest points, namely 52pT, 98pT, 213pT and 430pT respectively, and the concentration of AFP molecules in the corresponding solution is measured to be 25, 50, 100 and 200ng/mL according to the magnetic signal intensity.
The foregoing is merely a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, it is possible to make several modifications and adaptations of the structure of the present invention without departing from the principle of the present invention, and those modifications and adaptations of the structure based on the method of the present invention are intended to be within the scope of the present invention.

Claims (4)

1. A microfluidic chip magnetic nanoparticle detection system based on spin-free relaxation is characterized by comprising 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,
The micro-fluidic system consists of a peristaltic pump, a micro-fluidic chip, a waste liquid pond and a magnet placement device, wherein the micro-fluidic chip consists 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 in a surface plasma treatment mode, fluid pipelines for guiding liquid to flow in and out are encapsulated and communicated on fluid channel inlets and outlets at two ends of the middle PDMS channel layer, and two ends of the fluid pipelines are respectively communicated with the peristaltic pump and the waste liquid pond through flexible pipes;
the weak magnetic sensor is arranged in the magnetic shielding device and right above the track;
the linear scanner is arranged at one side of the magnetic shielding device and used for delivering the micro-fluidic chip, the micro-fluidic chip is arranged on a quartz rod of the linear scanner, and the quartz rod moves along the track;
The LabVIEW controller is respectively connected with the micro-fluidic chip, the linear scanner and the weak magnetic sensor in a line way, and simultaneously controls the micro-fluidic chip, the linear scanner and the weak magnetic sensor by using LabVIEW software to realize in-situ magnetic marking and in-situ measurement of magnetic signals of an object to be detected.
2. The microfluidic chip magnetic nanoparticle detection system based on spin-free relaxation according to claim 1, wherein the microfluidic chip is reinforced by an epoxy resin glue package.
3. The microfluidic chip magnetic nanoparticle detection system based on spin-free relaxation according to claim 1, wherein the fluid pipeline is a polytetrafluoroethylene fluid pipeline.
4. The microfluidic chip magnetic nanoparticle detection system based on spin-free relaxation according to claim 1, wherein the weak magnetic sensor is a weak magnetic sensor based on spin-free relaxation for detecting magnetic signals of magnetic nanoparticles.
CN202111523515.6A 2021-12-14 2021-12-14 A microfluidic chip-based magnetic nanoparticle detection system without spin exchange relaxation Active CN114397455B (en)

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