CN115326716A - Immunodetection method and system - Google Patents
Immunodetection method and system Download PDFInfo
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- CN115326716A CN115326716A CN202211019708.2A CN202211019708A CN115326716A CN 115326716 A CN115326716 A CN 115326716A CN 202211019708 A CN202211019708 A CN 202211019708A CN 115326716 A CN115326716 A CN 115326716A
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Abstract
The invention discloses an immunodetection method and system, and belongs to the field of nano material testing. The method comprises the following steps: making incident light reflect for many times in the functional magnetic nanoparticle immunoassay reagent under the excitation of the alternating-current magnetic field; converting emergent light after multiple reflections into a voltage signal, and extracting a magneto-optical signal corresponding to the immunoassay reagent; mixing the immunoassay reagent with a solution to be detected, and reflecting incident light in the mixed solution for multiple times under the excitation of an alternating current magnetic field; converting emergent light after multiple reflections into a voltage signal, and extracting a magneto-optical signal corresponding to the mixed solution; and when the difference degree of the magneto-optical signal corresponding to the immunoassay reagent and the magneto-optical signal corresponding to the mixed solution exceeds a preset threshold value, determining that the solution to be detected contains the target molecule to be detected. The invention can improve the lower limit of the detection concentration of the molecules to be detected, and improve the precision and the sensitivity of the immunodetection; the method has the advantages of no washing convenience and simple operation compared with the existing magneto-optical immunodetection method.
Description
Technical Field
The invention belongs to the technical field of nano material testing, and particularly relates to an immunodetection method and system.
Background
Immunoassays use a variety of labels to assay antibodies or antigens. The most commonly used methods are enzyme linked immunosorbent assays, radioimmunoassays and real-time law polymerase chain reaction. Recently, magnetic immunoassays have received attention. Magnetic nanoparticles can be used as analyte labels in immunoassays due to their unique magnetic properties. The detection characteristics of the molecules to be detected interacting with the magnetic nanoparticles can be characterized by utilizing the magnetism of the magnetic nanoparticles. The magnetic correlation quantities that are widely studied at present are magnetic relaxation, residual magnetization, alternating-current magnetic susceptibility, saturation magnetization, brownian relaxation time, and the like. The molecule to be detected and the magnetic nano-particles can be bound together by utilizing the specific functional group in the solution, and the magnetic information of the magnetic nano-particles can be changed. The change in the magnetization response of the magnetic nanoparticles measured by the sensor is related to the concentration of the molecule to be measured.
The optical measurement method has the outstanding advantages of non-contact property, high sensitivity, high precision and the like. The magneto-optical effect is a new method capable of measuring the magnetic information of the magnetic nanoparticles. When a magnetic field is applied to the magnetic fluid, the magnetic nanoparticles are aggregated into chain-like clusters along the direction of the magnetic field, thereby exhibiting anisotropic characteristics. Light propagates in the magnetized magnetic fluid, and a series of magneto-optical effects are generated, so that magneto-optical response signals are changed. The strength of the magneto-optical response signal is related to the concentration information of the magnetic fluid, so that the immunodetection can be realized by detecting the strength of the magneto-optical effect.
The magneto-optical immunity detection method commonly used at present comprises magnetic fluorescence nanometer probe detection and magnetic particle chemiluminescence immunoassay. The unbound fluorescent quantum dots in the magnetic fluorescent nano probe detection can generate certain fluorescent intensity to interfere the detection of the conjugate, and magnetic field separation is needed. The magnetic particle chemiluminescence immunoassay method requires washing after the antigen-antibody reaction is finished to remove substances not bound to the immune complex. The washing process reduces the amount of labeled analyte, resulting in an increased detection limit.
Disclosure of Invention
In view of the above drawbacks or needs for improvement in the prior art, the present invention provides an immunoassay method and system, which aims to increase the lower limit of the concentration of a molecule to be detected and to achieve magneto-optical immunoassay with higher accuracy and sensitivity.
To achieve the above object, according to one aspect of the present invention, there is provided 1 an immunoassay method comprising:
s1, enabling incident light to be reflected for multiple times in a functional magnetic nanoparticle immunoassay reagent under the excitation of an alternating-current magnetic field;
s2, converting emergent light after multiple reflections into a voltage signal, and extracting a magneto-optical signal corresponding to the immunoassay reagent;
s3, mixing the immunoassay reagent with the solution to be detected, and enabling incident light to be reflected for multiple times in the mixed solution under the excitation of an alternating-current magnetic field; the incident light and the alternating current magnetic field are the same as the step S1;
s4, converting emergent light after multiple reflections into a voltage signal, and extracting a magneto-optical signal corresponding to the mixed solution;
and S5, when the difference degree of the magneto-optical signal corresponding to the immunoassay reagent and the magneto-optical signal corresponding to the mixed solution exceeds a preset threshold value, determining that the solution to be detected contains the target molecule to be detected.
Further, the magneto-optical signal is a ratio of a second harmonic component to a direct current component in the voltage signal.
Further, the method includes passing the incident light through a polarizer to form horizontally polarized light.
Further, the alternating-current excitation magnetic field is perpendicular to the propagation direction of the incident light.
Further, the incident light is laser light having a wavelength of 520 nm.
According to another aspect of the present invention there is provided an immunoassay system comprising: the device comprises a laser, a polaroid, a coil, an optical liquid cavity, a photoelectric detector and a computer;
a laser for generating incident light;
a polarizing plate for changing a polarization direction of incident light;
a coil for generating an alternating current excitation magnetic field;
the optical liquid cavity is used for containing a functional magnetic nanoparticle immunoassay reagent or a mixed solution of the immunoassay reagent and a solution to be detected, so that incident light can be reflected for multiple times in the functional magnetic nanoparticle immunoassay reagent or the mixed solution under the excitation of an alternating-current magnetic field; the optical liquid cavity is a cubic container with front and back surfaces plated with reflectors;
the photoelectric detector is used for converting emergent light after multiple reflections into a voltage signal;
the computer is used for extracting magneto-optical signals corresponding to the immunoassay reagent and the mixed solution; and when the difference degree of the magneto-optical signal corresponding to the immunoassay reagent and the magneto-optical signal corresponding to the mixed solution exceeds a preset threshold value, determining that the solution to be detected contains the target molecule to be detected.
Further, the ac excitation magnetic field generated by the coil is perpendicular to the propagation direction of the incident light generated by the laser.
In general, the above technical solutions contemplated by the present invention can achieve the following advantageous effects compared to the prior art.
(1) Compared with the alternating current sensor of the traditional detection scheme, the magneto-optical detection system can detect the concentration of the magnetic nano particles at least three orders of magnitude less in a short time, and on the basis of the existing magneto-optical effect, the magneto-optical detection system enables light to be reflected in magnetic fluid for multiple times, increases the optical path, greatly improves the strength of magneto-optical signals, can further improve the lower limit of the detection concentration of molecules to be detected, and improves the accuracy and the sensitivity of immunodetection.
(2) The invention adopts the ratio of the amplitude of the second harmonic to the direct current component to represent the magneto-optical signal, and the experimental result shows that the second harmonic generated by pure water under the excitation of the alternating current magnetic field can be ignored, so that the magneto-optical signal adopted by the method is irrelevant to the diamagnetism of water, the interference of the water to the detection result can be ignored, and the mixed solution has the convenience of no-washing; since the uncombined object to be detected has no influence on the magnitude of the magneto-optical signal, the mixed solution does not need to be cleaned after the antigen-antibody immunoreaction, the detection can be directly carried out, and the operation is simpler than that of the existing magneto-optical immunodetection method.
Drawings
FIG. 1 is a diagram of a magneto-optical immunoassay device of the present invention;
FIG. 2 is a graph of magneto-optical signal as a function of magnetic field for different concentrations of magnetic nanoparticle solutions;
FIG. 3 is a graph of magneto-optical signal as a function of magnetic field at different reflection times;
FIG. 4 is a magneto-optical signal as a function of magnetic field for magnetic nanoparticles of different particle sizes.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and do not limit the invention. In addition, the technical features involved in the embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.
In order to solve the technical problems of low efficiency and low detection sensitivity of the prior immunoassay method based on the magneto-optical effect, the invention provides an immunoassay method based on an optical liquid cavity, which has the overall thought that: the ratio of the second harmonic amplitude of the magneto-optical response signal to the direct current component is improved by utilizing multiple reflections of light in the optical liquid cavity, so that the lower limit of concentration in the immunoassay is reduced. And comparing the signals of the immunoassay reagent and the mixed solution, and if the difference value of the signals exceeds a threshold value, judging that the solution to be detected contains the target molecules. Due to the diamagnetism of water, certain harmonic interference can be generated when alternating current excitation is applied to the magnetic nanoparticle solution. However, the experimental result shows that the second harmonic generated by pure water under the excitation of the alternating-current magnetic field can be ignored, so that the magneto-optical signal adopted by the method is irrelevant to the diamagnetism of water, the interference of the water on the detection result can be ignored, and the mixed solution has the convenience of no-washing, so that the operation is simpler; the magnetic signal is detected by utilizing the optical measurement technology, so that the detection sensitivity can be improved, and the rapid detection is realized.
The method specifically comprises the following steps:
s1, coating magnetic nanoparticles with an antibody (antigen) of an object to be detected to form a magnetic nanoparticle immune complex, and then preparing a functional magnetic nanoparticle immune assay reagent. Making incident light reflect for many times in the functional magnetic nanoparticle immunoassay reagent under the excitation of the alternating-current magnetic field;
specifically, a laser for detecting the liquid concentration is generated using a laser having a wavelength of 520nm at which the absorption of light by water is small. The wavelength that the absorption rate of water is small and the magneto-optical signal is large can also be comprehensively considered, so that the loss caused by absorption when light passes through the magnetic fluid is reduced, and the magneto-optical signal is enhanced.
The magnetic nanoparticles show anisotropy under the action of a magnetic field, and after light passes through a magnetic nanoparticle solution, both an absorption coefficient and a scattering coefficient can be changed, so that the transmittance of incident light is changed. The attenuation of light in conventional solutions satisfies the linear absorption law, i.e., lambertian (j.h.lambert) law:
I＝I 0 exp(-αL)
wherein, I 0 And I is the incident light intensity and the emergent light intensity respectively, L is the path of light passing through the solution, and alpha is the attenuation coefficient. According to the magneto-optical signal obtained by experiments in the invention, the absorption law is corrected, and the relationship between the emergent light intensity and the incident light intensity is obtained as follows:
I＝I 0 exp[-α(H)L]
wherein H is an excitation magnetic field. According to Taylor's formula, there are
From the above formula, when the applied excitation magnetic field is a sinusoidal ac field H =H 0 sin ω t, the emergent optical signal will contain each harmonic, and changing the concentration of the magnetic nanoparticle solution, the light intensity of the incident light, etc., will all change the corresponding harmonic amplitude. The second harmonic is adopted in the invention because the amplitude of the second harmonic is the largest in each harmonic, and the harmonic component is in positive correlation with the path of light passing through the magnetic nano particle solution under the action of the magnetic field. And the ratio of the second harmonic to the direct current component is used as a final magneto-optical signal, so that the difference of detection results caused by different incident light intensities can be eliminated.
In order to distinguish the magneto-optical effect caused by the change in transmittance under the action of the magnetic field mentioned in the present invention from the conventional magneto-optical birefringence effect and the magneto-optical faraday effect, the length of the liquid container used will be described. Magneto-optical signal obtained by magneto-optical birefringence effect is derived from phase difference
The magneto-optical signal obtained by magneto-optical faraday is derived from the rotation angle theta by
And the sine or cosine value of theta to calculate the phase difference and the rotation angle.
And the proportional relationship of theta to the path of light through the magnetic fluid is applicable in the range 0 to pi/2, so that the thickness of the liquid container is limited to the order of μm. The single-pass path of the optical liquid cavity in the invention is in mm order, so the optical liquid cavity is not suitable for the traditional magneto-optical birefringence effect and the magneto-optical Faraday effect, and is explained by the change of the transmissivity.
When horizontal polarized light is excited by an alternating-current magnetic field and is reflected for multiple times in the magnetic fluid, the distance of the light passing through the magnetic fluid is increased along with the increase of the reflection times, and magneto-optical signals are in positive correlation along with the distance of the light passing through the magnetic fluid, so that the magneto-optical signals can be greatly improved. The distance that light passes can also be increased through the mode that increases the cell thickness, but the volume of cell and the volume of consuming solution all can increase, and the cost is improved, and is unfavorable for miniaturization and integration. Preferably, the alternating excitation magnetic field is perpendicular to the propagation direction of the incident light, enabling a stronger signal to be obtained.
S2, converting the emergent light after multiple reflections into a voltage signal, and extracting a magneto-optical signal corresponding to the immunoassay reagent;
processing the voltage signal to obtain a DC component V 0 And second harmonic amplitude V 2f Where f represents the excitation frequency of the alternating magnetic field. Magneto-optical signal s = V for analysis 2f /V 0 Thus, the influence of the difference in the intensity of the incident light can be eliminated. The magneto-optical signals mentioned in the present invention all refer to the ratio of the second harmonic amplitude to the dc component in the photovoltage.
S3, mixing the immunoassay reagent with an antigen (antibody) to be detected, forming an antigen-antibody-magnetic nanoparticle conjugate after immunoreaction, and enabling incident light to be reflected for multiple times in a mixed solution excited by an alternating-current magnetic field; the incident light and the alternating current magnetic field are the same as the step S1;
s4, converting the emergent light after multiple reflections into a voltage signal, and extracting a magneto-optical signal corresponding to the mixed solution;
s5, the magneto-optical signal of the immunoassay reagent is only related to the magnetic nano particles, and the magneto-optical signal of the mixed solution contains the total characteristic information of the unbound magnetic nano particles and the conjugate which generates immunoreaction with the antigen (antibody) to be detected. The magneto-optical signal is related to the total particle size of the magnetic nanoparticles and combinations thereof. Therefore, when the difference degree of the magneto-optical signal corresponding to the immunoassay reagent and the magneto-optical signal corresponding to the mixed solution exceeds a preset threshold value, the target molecule to be detected in the solution to be detected is judged to be contained
Magneto-optical signal s if functional magnetic nanoparticle solution 1 Magneto-optical signal s with mixed solution 2 If the difference exceeds the threshold value, the surface solution to be detected contains target molecules. The specific antibody (antigen) on the surface of the functional magnetic nano particle is changed, so that the detection of different kinds of antigens (antibodies) can be realized
The magneto-optical signal is only related to the particles containing the magnetic nano particles, and the unbound antigen (antibody) to be detected can not influence the detection result, so the immunoassay method provided by the invention is free from cleaning.
Example one:
and comparing the magneto-optical signals of the magnetic nanoparticle solutions with different concentrations. As shown in FIG. 2, the magneto-optical signal is plotted with the amplitude of the AC excitation magnetic field when the concentration of the magnetic nanoparticle solution is 100. Mu.g/ml, 10. Mu.g/ml, 1. Mu.g/ml and distilled water, respectively. It can be seen that when the amplitude of the ac excitation field is the same, the magneto-optical signal increases with increasing concentration of the magnetic nanoparticle solution.
Example two:
the magneto-optical signals of different reflection times are compared. As shown in fig. 3, n is the number of times light passes through the magnetic nanoparticle solution, and the relationship between n and the number of reflections m is: n =2m +1. It can be seen that as the number of reflections increases, the magneto-optical signal also increases.
Example three:
the magneto-optical signals of magnetic nanoparticles of the same volume fraction and different particle sizes were compared. As shown in FIG. 4, the results of the experiment using distilled water as a control group when the magnetic nanoparticles have particle diameters of 30nm, 20nm and 10nm, respectively. It can be seen that as the particle size increases, the magneto-optical signal also increases.
Therefore, the magneto-optical immunodetection method based on the optical liquid cavity can really improve the strength of magneto-optical signals on the basis of magneto-optical immunodetection, and improve the lower limit of the detection concentration.
It will be understood by those skilled in the art that the foregoing is only an exemplary embodiment of the present invention, and is not intended to limit the invention to the particular forms disclosed, since various modifications, substitutions and improvements within the spirit and scope of the invention are possible and within the scope of the appended claims.
Claims (7)
1. An immunoassay method, comprising:
s1, enabling incident light to be reflected for multiple times in a functional magnetic nanoparticle immunoassay reagent under the excitation of an alternating-current magnetic field;
s2, converting emergent light after multiple reflections into a voltage signal, and extracting a magneto-optical signal corresponding to the immunoassay reagent;
s3, mixing the immunoassay reagent with the solution to be detected, and enabling incident light to be reflected for multiple times in the mixed solution under the excitation of an alternating-current magnetic field; the incident light and the alternating current magnetic field are the same as the step S1;
s4, converting the emergent light after multiple reflections into a voltage signal, and extracting a magneto-optical signal corresponding to the mixed solution;
and S5, when the difference degree of the magneto-optical signal corresponding to the immunoassay reagent and the magneto-optical signal corresponding to the mixed solution exceeds a preset threshold value, determining that the solution to be detected contains the target molecule to be detected.
2. The immunoassay method of claim 1, wherein the magneto-optical signal is a ratio of a second harmonic component to a dc component of the voltage signal.
3. The immunoassay method of claim 2, further comprising passing the incident light through a polarizer to form a horizontally polarized light.
4. An immunoassay method according to any one of claims 1 to 3, wherein the AC excitation magnetic field is perpendicular to the direction of propagation of the incident light.
5. An immunoassay according to any one of claims 1 to 4, wherein said incident light is laser light having a wavelength of 520 nm.
6. An immunoassay system, comprising: the device comprises a laser, a polaroid, a coil, an optical liquid cavity, a photoelectric detector and a computer;
a laser for generating incident light;
a polarizing plate for changing a polarization direction of incident light;
a coil for generating an alternating current excitation magnetic field;
the optical liquid cavity is used for containing a functional magnetic nanoparticle immunoassay reagent or a mixed solution of the immunoassay reagent and a solution to be detected, so that incident light can be reflected for multiple times in the functional magnetic nanoparticle immunoassay reagent or the mixed solution under the excitation of an alternating-current magnetic field; the optical liquid cavity is a cubic container with front and back surfaces plated with reflectors;
the photoelectric detector is used for converting emergent light after multiple reflections into a voltage signal;
the computer is used for extracting magneto-optical signals corresponding to the immunoassay reagent and the mixed solution; and when the difference degree of the magneto-optical signal corresponding to the immunoassay reagent and the magneto-optical signal corresponding to the mixed solution exceeds a preset threshold value, determining that the solution to be detected contains the target molecule to be detected.
7. The immunoassay system of claim 6, wherein the ac excitation magnetic field generated by the coil is orthogonal to the propagation direction of the incident light generated by the laser.
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| CN202211019708.2A CN115326716A (en) | 2022-08-24 | 2022-08-24 | Immunodetection method and system |
| PCT/CN2022/138911 WO2024040802A1 (en) | 2022-08-24 | 2022-12-14 | Immunoasssay method and system |
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| WO2024040802A1 (en) * | 2022-08-24 | 2024-02-29 | 华中科技大学 | Immunoasssay method and system |
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| JPS62118255A (en) * | 1985-11-19 | 1987-05-29 | Toshimitsu Musha | Detection of immunological reaction by using magnetic field |
| US5238811A (en) * | 1988-04-26 | 1993-08-24 | Nippon Telegraph And Telephone Corporation | Laser magnetic immunoassay method and apparatus therefor and superparamagnetic material-labeled body and method for the manufacture of same |
| MY176182A (en) * | 2013-06-28 | 2020-07-24 | Univ Danmarks Tekniske | Biosensor based on measurements of the clustering dynamics of magnetic particles |
| CN106596473A (en) * | 2016-10-27 | 2017-04-26 | 中国科学院电子学研究所 | Optic detector based on electric control magnet enrichment-separation and total internal reflection magnetic imaging |
| EP3724637B1 (en) * | 2017-12-15 | 2021-11-24 | Politecnico di Milano | Opto-magnetic sensor device and molecular recognition system |
| US10161856B1 (en) * | 2018-01-19 | 2018-12-25 | Ping-Chieh Wu | Magneto-optical bio-detection devices having high sensitivity |
| CN115326716A (en) * | 2022-08-24 | 2022-11-11 | 华中科技大学 | Immunodetection method and system |
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