Magnetic control coupling chemiluminescence detection method based on magnetic drive cluster immune probe

Abstract

The invention provides a magnetically-controlled coupled chemiluminescence detection method based on a magnetically-driven cluster immunoprobe, which comprises the following steps: adding a magnetic donor sphere, an acceptor sphere, a buffer solution and a sample to be detected into a sample pool, uniformly mixing, attracting the magnetic donor sphere by using a permanent magnet, and gathering the magnetic donor sphere to the bottom of the sample pool to form a heterogeneous cluster, putting the sample pool into a sample groove in the center of the three-dimensional Helmholtz coil; applying a rotating magnetic field with a pitch angle of 0 degree to the three-dimensional Helmholtz coil, incubating, applying a rotating magnetic field with a pitch angle of 0-4 degrees and a rightward direction angle, translating the heterogeneous cluster, and closing the magnetic field; irradiating the heterogeneous cluster by using 680nm laser, and collecting an afterglow signal in the sample cell by using a photomultiplier to obtain light-activated chemiluminescence intensity; and obtaining the concentration of the to-be-detected antigen according to the standard curve of the to-be-detected antigen. According to the technical scheme, the incubation efficiency is improved, and the problems that in the prior art, the incubation efficiency is low, and non-specific adsorption is high are solved.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical detection, and in particular to a magnetically controlled coupled chemiluminescence detection method based on magnetically driven cluster immune probes. Background Art

[0002] Magnetic nanoparticles are widely used in the biomedical field, involving various in vivo imaging, drug delivery, and in vitro detection. Especially in in vitro immunoassay, many achievements have been made. After being modified with antibodies, magnetic nanoparticles become magnetic immune probes. The probes specifically immunologically bind to the analyte, and thus the analyte can be separated from the biological matrix by a magnetic field. Magnetic immune probes have been successfully used for the detection of various antigens, antibodies, receptors, enzymes, tumor markers, inflammatory markers, viruses, bacteria, toxins, and tumor cells.

[0003] The photoactivated chemiluminescence immunoassay method has broad development prospects in biomedical detection. This method involves two types of microspheres - donor spheres and acceptor spheres. The donor spheres generate singlet oxygen under laser irradiation, and the acceptor spheres react with the singlet oxygen to emit light. The donor and acceptor spheres are loaded with antibodies against the analyte. The analyte binds to the donor and acceptor spheres to form a sandwich, bringing the two microspheres closer within the diffusion range of singlet oxygen, causing the acceptor spheres to emit light. The luminescence intensity is proportional to the content of the analyte. The emission wavelength of the probe is shorter than the excitation wavelength, so this method can eliminate the interference of background fluorescence. Since the unbound donor and acceptor spheres are separated by a distance exceeding the diffusion distance of singlet oxygen, they cannot emit light, and there is no need to wash the unbound probes. Therefore, the photoactivated chemiluminescence immunoassay method is a homogeneous and wash-free detection method, with simple operation and time-saving detection.

[0004] Existing magnetic immune probes focus on magnetic separation and the interaction between the analyte and a single probe, without taking advantage of the controllable and manipulable magnetic motion, especially the advantage of controllable active magnetic cluster motion. In addition, the integration level of the instrument is low and the operation is cumbersome. Moreover, the existing photoactivated chemiluminescence immunoassay method still has interference caused by non-specific adsorption, with low incubation efficiency and long detection time. Summary of the Invention

[0005] In view of the above technical problems, the present invention discloses a magnetically controlled coupled chemiluminescence detection method based on magnetically driven cluster immune probes, which combines magnetic immune probes with the photoactivated chemiluminescence immunoassay method and uses the controllable active cluster motion of magnetic nanomotors to solve the problems of low incubation efficiency and strong non-specific adsorption in the photoactivated chemiluminescence immunoassay method.

[0006] For this purpose, the technical solution adopted by the present invention is as follows:

[0007] A magnetically controlled coupled chemiluminescence detection method based on magnetically driven cluster immune probes, comprising:

[0008] Step S1, add magnetic donor spheres, acceptor spheres, buffer solution, and the sample to be tested into the sample cell and mix them evenly. The surface of the magnetic donor spheres is loaded with a photosensitizer, and the acceptor spheres are polystyrene chemiluminescent nanospheres. Use a permanent magnet to attract and gather the magnetic donor spheres to the bottom of the sample cell to form a heterogeneous cluster, and then place the sample cell into the sample slot at the center of the three-dimensional Helmholtz coil. Among them, the three-dimensional Helmholtz coil functions as a magnetic stirrer. The surface of the magnetic donor spheres is coupled with a capture antibody. The surface of the acceptor spheres is coupled with a detection antibody, and the acceptor spheres contain a chemiluminescent agent, such as the thioxene derivative PCU.

[0009] Step S2, first apply a rotating magnetic field with a pitch angle of 0° to the three-dimensional Helmholtz coil to make the magnetic donor spheres form a stable vortex cluster, and perform incubation for a time within 10 minutes. Then apply a rotating magnetic field with a pitch angle of 0 - 4° and a direction angle to the right to translate the heterogeneous cluster and separate the heterogeneous cluster from the substances in the sample solution that have not undergone immunological binding with the donor spheres, and turn off the magnetic field.

[0010] Step S3, irradiate the separated heterogeneous cluster in the sample cell with a 680 nm laser, and turn off the laser. Then open the incident window of the photomultiplier tube to collect the afterglow signal of the heterogeneous cluster in the sample cell to obtain the photoactivated chemiluminescence intensity of the sample.

[0011] Step S4, use the standard solution of the antigen to be tested to measure the relationship curve between the concentration and the photoactivated chemiluminescence intensity. According to the photoactivated chemiluminescence intensity of the sample measured in Step S3, the concentration of the antigen to be measured in the sample can be calculated. Among them, the antigen to be tested is any antigen in the sample to be tested.

[0012] Adopting this technical solution, a stable vortex cluster is formed by the magnetic donor spheres using the magnetic field. When the vortex cluster rotates, due to the active movement of the magnetic donor spheres, the collision frequency between the magnetic donor spheres and the acceptor spheres increases, improving the incubation efficiency and solving the problems of low incubation efficiency and strong non-specific adsorption in the photoactivated chemiluminescence immunoassay.

[0013] As a further improvement of the present invention, in Step S1, the sample cell is a microchannel chip, and the microchannel chip includes a first chamber and a second chamber, and the first chamber and the second chamber are connected by a connecting channel. Add the magnetic donor spheres, acceptor spheres, buffer solution, and the sample to be tested into the first chamber, and add a buffer solution with the same total volume as the solution in the first chamber to the second chamber. Among them, the second chamber is used to accommodate the separated and transferred heterogeneous cluster.

[0014] As a further improvement of the present invention, in Step S2, the heterogeneous cluster is translated into the second chamber using the rotating magnetic field. Adopting this technical solution, the separated heterogeneous cluster can be transferred to the second chamber, which is convenient for the next detection and improves the detection accuracy.

[0015] As a further improvement of the present invention, in step S3, the magnetic field strength of the rotating magnetic field with a pitch angle of 0° is not greater than 16 mT, and the frequency is not higher than 30 Hz.

[0016] As a further improvement of the present invention, the magnetic donor sphere is a magnetic photosensitive nanosphere, and the magnetic donor sphere is loaded with a photosensitizer Ce6.

[0017] The present invention also discloses a detection device adopted by the magnetically controlled coupled chemiluminescence detection method based on a magnetically driven cluster immune probe as described above, that is, a magnetically controlled coupled chemiluminescence detection device based on a magnetically driven cluster immune probe, which includes a magnetic control module and a photoactivated chemiluminescence detection module. The magnetic control module includes a three-dimensional Helmholtz coil, and a sample slot for placing a sample cell, which can be withdrawn, is provided at the center of the three-dimensional Helmholtz coil; the photoactivated chemiluminescence detection module includes an excitation light source and a photomultiplier tube. The excitation light source is located obliquely above the sample slot for irradiating the sample cell, and the incident window of the photomultiplier tube faces the sample slot. As a further improvement of the present invention, a filter is provided in the incident direction of the photomultiplier tube.

[0018] As a further improvement of the present invention, the sample cell is made of a transparent material.

[0019] As a further improvement of the present invention, the support platform below the sample slot is made of a transparent material.

[0020] As a further improvement of the present invention, a camera is provided below the three-dimensional Helmholtz coil, and the camera faces the sample slot for photographing the situation inside the sample cell.

[0021] As a further improvement of the present invention, the camera is connected to a height adjustment mechanism.

[0022] As a further improvement of the present invention, the height adjustment mechanism includes a support column and an adjustment fixing member, and the camera is connected to the support column through the adjustment fixing member. By moving the adjustment fixing member up and down on the support column and fixing it, the height position of the camera can be adjusted. With this technical solution, the situation inside the sample cell can be monitored, and relevant process parameters can be set and adjusted, such as the irradiation time of the laser, the time interval between stopping irradiation and opening the incident window, the afterglow acquisition time, and so on.

[0023] As a further improvement of the present invention, a supplementary light source for supplementing light to the camera is provided above the three-dimensional Helmholtz coil.

[0024] As a further improvement of the present invention, the excitation light source is a 100 mW 680 nm laser.

[0025] As a further improvement of the present invention, the incident window of the photomultiplier tube is connected to a motor, and the opening and closing of the incident window are controlled by the motor. With this technical solution, the operation is more convenient.

[0026] The magnetically controlled coupled chemiluminescence detection device based on magnetically driven cluster immune probes further includes a control module, and the control module is electrically connected to the three-dimensional Helmholtz coil, the excitation light source, and the photomultiplier tube.

[0027] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0028] Adopting the technical solution of the present invention, the magnetic immune probe is combined with the photoactivated chemiluminescence immunoassay method. By using a rotating magnetic field to manipulate the active and controllable movement of magnetic donor spheres to form a vortex cluster, the incubation is accelerated and in-situ separation is achieved. This solves the problems of strong interference caused by non-specific adsorption, low incubation efficiency, strong non-specific adsorption, and long detection time in the photoactivated chemiluminescence immunoassay method, and the operation is simple. Description of the Drawings

[0029] Figure 1 It is a schematic flow chart of a magnetically controlled coupled chemiluminescence detection method based on magnetically driven cluster immune probes according to an embodiment of the present invention.

[0030] Figure 2 It is a schematic diagram of a rotating magnetic field according to an embodiment of the present invention.

[0031] Figure 3 It is a schematic diagram of the rotation and translation of a heterogeneous cluster in a magnetic field according to an embodiment of the present invention; where (a) is rotation and (b) is translation.

[0032] Figure 4 It is a schematic diagram of a heterogeneous cluster in a rotating magnetic field according to an embodiment of the present invention; (a) is a schematic diagram of a three-dimensional Helmholtz coil, (b) is a schematic diagram of the state of the heterogeneous cluster in the magnetic field, and (c) is a schematic diagram of the binding of an antibody to an antigen.

[0033] Figure 5 It is a schematic structural diagram of a magnetically controlled coupled chemiluminescence detection device based on magnetically driven cluster immune probes according to an embodiment of the present invention.

[0034] Figure 6 It is a schematic diagram of the placement of a sample cell according to an embodiment of the present invention.

[0035] The reference numerals include:

[0036] 1 - frame, 2 - three-dimensional Helmholtz coil, 3 - sample groove, 4 - sample cell, 5 - excitation light source, 6 - filter, 7 - photomultiplier tube, 8 - motor, 9 - camera, 10 - support column, 11 - adjustment and fixing member, 12 - supplementary light source, 13 - touch screen, 14 - control module. Detailed implementation manners

[0037] The following further elaborates on the preferred embodiments of the present invention in detail.

[0038] Embodiment 1

[0039] A magnetically controlled coupled chemiluminescence detection method based on magnetically driven cluster immune probes, as Figure 1 shown, includes:

[0040] I. Magnetic field manipulation of heterogeneous clusters;

[0041] Step S1, rotation of heterogeneous clusters, specifically including:

[0042] Magnetic photosensitive nanospheres, namely magnetic donor spheres (MDB), polystyrene chemiluminescent nanospheres, namely acceptor spheres (AB), buffer solution, and a sample to be measured are added to a sample cell and mixed evenly. A permanent magnet is used to attract and gather the magnetic donor spheres to the bottom of the sample cell to form a heterogeneous cluster, and then the sample cell is placed in a sample slot at the center of a three-dimensional Helmholtz coil. Among them, capture antibodies are coupled to the surface of the magnetic donor spheres; detection antibodies are coupled to the surface of the acceptor spheres, and chemiluminescent agents, such as thioxanthene derivative PCU, are contained in the acceptor spheres.

[0043] Furthermore, the sample cell is a microchannel chip made by 3D printing. The microchannel chip includes a first chamber and a second chamber, and the first chamber and the second chamber are connected through a connecting channel. The magnetic donor spheres, acceptor spheres, buffer solution, and the sample to be measured are added to the first chamber, and buffer solution with the same total volume as the solution in the first chamber is added to the second chamber. The microchannel chip is made of a transparent material.

[0044] A rotating magnetic field is applied, as Figure 2 shown. Let the pitch angle (θ) be 0°, and the magnetic field intensity (0 - 16 mT) and frequency (0 - 30 Hz) are adjusted to make the magnetic donor spheres form a stable vortex cluster for incubation, as Figure 3 (a) shown. The vortex cluster morphology is maintained for incubation for a specified time (within 10 min). The situation in the sample cell during this process is as Figure 4 shown.

[0045] In this technical solution, a three-dimensional Helmholtz coil can generate a uniform magnetic field in any direction in the sample cell at its center to control the incubation and separation of heterogeneous clusters. When the magnetic field frequency is low, the magnetic donor spheres form short rod-shaped clusters; when the magnetic field frequency is high, the magnetic donor spheres form unstable vortex clusters; when the magnetic field frequency is moderate, the magnetic donor spheres form stable vortex clusters; the greater the magnetic field strength, the narrower the frequency range for forming stable vortex clusters; the range depends on the magnetism of the magnetic donor spheres, the mass of the clusters, and the immune binding inside the clusters. When the vortex clusters rotate, due to the active movement of the magnetic donor spheres, the collision frequency between the magnetic donor spheres and the receptor spheres increases, improving the incubation efficiency.

[0046] Step S2, the translational motion of the heterogeneous clusters

[0047] As Figure 3 (b) shows, apply a rotating magnetic field with a pitch angle (as Figure 2 shown) to the above-mentioned heterogeneous clusters, so that they perform translational motion while rotating, and set the direction angle to control the direction of the translational motion. The greater the magnetic field strength, the greater the pitch angle, and the faster the translational motion speed of the clusters. An excessive pitch angle (θ≥5°) will cause the clusters to deform or even disintegrate. Specifically, in this embodiment, a rotating magnetic field with a pitch angle of 0-4° and a direction angle to the right is applied. By using the controllable translational motion of the heterogeneous clusters, the clusters are separated from the substances in the sample solution that have not undergone immune binding with the donor spheres and translated to the right chamber. During this process, the magnetic field parameters are adjusted to ensure that the cluster morphology remains stable without deformation and that the clusters do not collide with the sample cell wall.

[0048] Then, turn off the magnetron system, and irradiate the heterogeneous clusters in the sample cell with a 680 nm laser for a specified time (within 1 s). After the irradiation is completed, turn off the laser; then open the incident window of the photomultiplier tube to collect the afterglow signal of the heterogeneous clusters in the sample cell, and thus obtain the photoinduced chemiluminescence intensity of the sample.

[0049] In this step, the photosensitizer Ce6 loaded on the magnetic donor spheres generates singlet oxygen under the irradiation of a 680 nm laser. The singlet oxygen has a short lifetime and a diffusion distance within 200 nm in aqueous solution. Therefore, only when the donor and receptor spheres are bound together due to immune action can the singlet oxygen diffuse to the receptor spheres. The receptor spheres react with the singlet oxygen and emit chemiluminescence at 615 nm. This chemiluminescence has a long lifetime, so there is still afterglow after the laser irradiation stops. After the excitation stops, open the incident window of the photomultiplier tube to allow the afterglow photons emitted by the heterogeneous clusters in the sample cell to enter the photomultiplier tube through the filter. The filter can filter out interfering light such as scattered light. By collecting the afterglow signal and integrating, the photoinduced chemiluminescence signal intensity can be obtained and output to the screen for display.

[0050] Step S4: Measure the standard curve (i.e., the relationship curve between concentration and photochemiluminescence intensity) using the standard solution of the antigen to be measured. According to the photochemiluminescence intensity of the sample measured in Step S3, the concentration of the antigen to be measured in the sample can be calculated. The irradiation time of the laser, the time interval between stopping irradiation and opening the incident window, and the afterglow acquisition time can be set by the operator before detection. Among them, the antigen to be measured is any antigen contained in the sample to be measured.

[0051] Example 2

[0052] As Figure 5 and Figure 6 shown, a magnetically controlled coupled chemiluminescence detection device based on a magnetically driven cluster immune probe, that is, the device used in the method of the above Example 1, includes a frame 1, and a magnetic control module, a photochemiluminescence detection module, a control module 14, and a display module are provided on the frame 1.

[0053] The magnetic control module includes a three-dimensional Helmholtz coil 2, and a sample slot 3 for placing a sample cell 4 that can be detached is provided at the center of the three-dimensional Helmholtz coil 2; the photochemiluminescence detection module includes an excitation light source 5 and a photomultiplier tube 7. The excitation light source 5 is located obliquely above the sample slot 3 for irradiating the sample cell 4, and the incident window of the photomultiplier tube 7 faces the sample slot 3. A filter 6 is provided in the incident direction of the photomultiplier tube 7. The control module 14 is electrically connected to the three-dimensional Helmholtz coil 2, the excitation light source 5, and the photomultiplier tube 7.

[0054] The display module includes a touch screen 13, and the operator can control the operation of the instrument on the screen. The screen is also used to display the real-time motion image of the heterogeneous cluster and the data result of the photochemiluminescence detection. The control module 14 is responsible for transmitting the instructions of the touch screen 13 to each component of the instrument, and transmitting the output signals of each component of the instrument to the screen for display.

[0055] The support platform below the sample slot 3 is made of a transparent material. A camera 9 is provided below the three-dimensional Helmholtz coil 2, and the camera 9 faces the sample slot 3 for photographing the situation inside the sample cell 4. The camera 9 is connected to a height adjustment mechanism. A supplementary light source 12 for supplementing light to the camera 9 is provided above the three-dimensional Helmholtz coil 2. It is a white light lamp, which is turned on during magnetic control operation and turned off during photochemiluminescence detection.

[0056] Specifically, the height adjustment mechanism includes a support column 10 and an adjustment fixing member 11. The camera 9 is connected to the support column 10 through the adjustment fixing member 11. By moving the adjustment fixing member 11 up and down on the support column 10 and fixing it, the height position of the camera 9 can be adjusted. The camera 9 can be used to monitor the situation inside the sample cell 4 and set and adjust relevant process parameters, such as the irradiation time of the laser, the time interval between stopping irradiation and opening the incident window, the afterglow acquisition time, and so on.

[0057] Further, the excitation light source 5 is a 680 nm laser with a power of 100 mW.

[0058] Further, the incident window of the photomultiplier tube 7 is connected to the motor 8, and the opening and closing of the incident window are controlled by the motor 8, making the operation more convenient.

[0059] The technical solution of this embodiment integrates the magnetron system, the detection system, the light source, the control system, and the data processing system into a single device, namely, a magnetically controlled coupled chemiluminescence detection system based on magnetically driven cluster immune probes, which solves the problem of cumbersome operation. By using this device, the active and controllable movement of magnetic donor spheres is manipulated by a rotating magnetic field to form a vortex cluster, accelerating the incubation and achieving in-situ separation, thus solving the problems of strong interference caused by non-specific adsorption, low incubation efficiency, and long detection time in the photochemiluminescence immunoassay.

[0060] The above content is a further detailed description of the present invention in combination with specific preferred embodiments. It cannot be determined that the specific implementation of the present invention is only limited to these descriptions. For those of ordinary skill in the technical field to which the present invention belongs, without departing from the concept of the present invention, several simple deductions or substitutions can still be made, and all should be regarded as belonging to the protection scope of the present invention.

Claims

1. A magnetically controlled coupled chemiluminescence detection method based on magnetically driven cluster immune probes, characterized in that: include: Step S1, adding magnetic donor balls, acceptor balls, buffer, and samples to be tested into a sample pool and mixing them evenly, wherein the surface of the magnetic donor balls is loaded with a photosensitizer, and the acceptor balls are polystyrene chemiluminescent nanospheres; using a permanent magnet to attract and gather the magnetic donor balls to the bottom of the sample pool to form a heterogeneous cluster, and then placing the sample pool into a sample slot at the center of the three-dimensional Helmholtz coil; Step S2, first applying a rotating magnetic field with a pitch angle of 0° to the three-dimensional Helmholtz coil to make the magnetic donor spheres form a stable vortex cluster, and incubating for less than 10 minutes; then applying a rotating magnetic field with a pitch angle of 0-4° and a rightward direction angle to translate the heterogeneous clusters, separating the heterogeneous clusters from substances in the sample solution that are not immunologically bound to the donor spheres, and turning off the magnetic field; Step S3, irradiating the separated heterogeneous clusters in the sample pool with a 680nm laser, turning off the laser; then opening the incident window of the photomultiplier tube, collecting the afterglow signal of the heterogeneous clusters in the sample pool, and obtaining the photoinduced chemiluminescence intensity of the sample; Step S4, using the standard solution of the antigen to be tested to measure the relationship curve between the concentration and the photochemiluminescence intensity, the concentration of the antigen to be tested in the sample can be calculated according to the photochemiluminescence intensity of the sample measured in step S3.

2. The magnetically controlled coupled chemiluminescence detection method based on magnetically driven cluster immunoprobe according to claim 1, characterized in that: In step S1, the sample pool is a microchannel chip, and the microchannel chip includes a first chamber and a second chamber, and the first chamber and the second chamber are connected by a connecting channel; the magnetic donor ball, the acceptor ball, the buffer solution, and the sample to be tested are added to the first chamber, and the buffer solution equal to the total volume of the solution in the first chamber is added to the second chamber; In step S2, the heterogeneous cluster is translated into the second chamber using a rotating magnetic field.

3. The magnetically controlled coupled chemiluminescence detection method based on magnetically driven cluster immunoprobe according to claim 1, characterized in that: In step S3, the magnetic field intensity of the rotating magnetic field with a pitch angle of 0° is not greater than 16 mT, and the frequency is not higher than 30 Hz.

4. The magnetically controlled coupled chemiluminescence detection method based on magnetically driven cluster immunoprobe according to claim 1, characterized in that: The magnetic donor sphere is a magnetic photosensitive nanosphere, and the magnetic donor sphere is loaded with photosensitizer Ce6.

5. The detection device used in the magnetically controlled coupled chemiluminescence detection method based on magnetically driven cluster immunoprobe according to any one of claims 1 to 4, characterized in that: It includes a magnetoelastic module and a photochemiluminescence detection module. The magnetoelastic module includes a three-dimensional Helmholtz coil. A removable sample slot for placing a sample pool is provided at the center of the three-dimensional Helmholtz coil. The photoinduced chemiluminescence detection module includes an excitation light source and a photomultiplier tube. The excitation light source is located obliquely above the sample tank and is used to irradiate the sample pool. The incident window of the photomultiplier tube faces the sample tank, and a filter is provided in the incident direction of the photomultiplier tube.

6. The magnetically controlled coupled chemiluminescence detection device based on magnetically driven cluster immunoprobe according to claim 5, characterized in that: A camera is provided below the three-dimensional Helmholtz coil, and faces the sample tank, and is used to photograph the situation in the sample tank.

7. The magnetically controlled coupled chemiluminescence detection device based on magnetically driven cluster immunoprobe according to claim 6, characterized in that: The camera is connected to the height adjustment mechanism.

8. The magnetically controlled coupled chemiluminescence detection device based on magnetically driven cluster immunoprobe according to claim 7, characterized in that: The height adjustment mechanism includes a support column and an adjustment fixing member, and the camera is connected to the support column via the adjustment fixing member.

9. The magnetically controlled coupled chemiluminescence detection device based on magnetically driven cluster immunoprobe according to claim 5, characterized in that: The excitation light source is a 100mW 680nm laser; the incident window of the photomultiplier tube is connected to a motor, and the opening and closing of the incident window is controlled by the motor.

10. The magnetically controlled coupled chemiluminescence detection device based on magnetically driven cluster immunoprobe according to any one of claims 5 to 9, characterized in that: It also includes a control module, which is electrically connected to the three-dimensional Helmholtz coil, the excitation light source, and the photomultiplier tube.

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