WO2024040802A1 - 一种免疫检测方法和系统 - Google Patents

一种免疫检测方法和系统 Download PDF

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WO2024040802A1
WO2024040802A1 PCT/CN2022/138911 CN2022138911W WO2024040802A1 WO 2024040802 A1 WO2024040802 A1 WO 2024040802A1 CN 2022138911 W CN2022138911 W CN 2022138911W WO 2024040802 A1 WO2024040802 A1 WO 2024040802A1
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magneto
optical signal
incident light
optical
mixed solution
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PCT/CN2022/138911
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French (fr)
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刘文中
朱旖雯
王琳
周逸铭
崔鑫超
李兰
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华中科技大学
深圳华中科技大学研究院
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/21—Polarisation-affecting properties
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54326—Magnetic particles
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54346—Nanoparticles

Definitions

  • the invention belongs to the technical field of nanomaterial testing, and more specifically, relates to an immune detection method and system.
  • Immunoassays use a variety of labels to measure antibodies or antigens.
  • the most commonly used methods are enzyme-linked immunosorbent assay, radioimmunoassay, and real-time law polymerase chain reaction.
  • 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 measured that interact with them can be characterized.
  • Magnetic related quantities that are currently widely studied include magnetic relaxation, remanence magnetization, AC magnetic susceptibility, saturation magnetization, and Brownian relaxation time.
  • the use of specific functional groups in the solution can bind the molecules to be measured and the magnetic nanoparticles together, which will change the magnetic information of the magnetic nanoparticles.
  • 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 magneto-optical effect is a new method that can measure the magnetic information of magnetic nanoparticles.
  • the magnetic nanoparticles When a magnetic field is applied to the magnetic fluid, the magnetic nanoparticles will aggregate into chain-like clusters along the direction of the magnetic field, thus showing anisotropic properties.
  • Light propagating in magnetized magnetic fluid will produce a series of magneto-optical effects, resulting in changes in the magneto-optical response signal.
  • the intensity of the magneto-optical response signal is related to the concentration information of the magnetic fluid. Therefore, immune detection can be achieved by detecting the intensity of the magneto-optical effect.
  • magneto-optical immunoassay methods include magnetic fluorescence nanoprobe detection and magnetic particle chemiluminescence immunoassay.
  • magnetic fluorescent nanoprobes unbound fluorescent quantum dots will produce a certain fluorescence intensity and interfere with the detection of conjugates, requiring magnetic field separation.
  • the magnetic particle chemiluminescence immunoassay method requires cleaning after the antigen-antibody reaction is completed to remove substances that are not bound to the immune complex. The washing process will reduce the number of labeled analytes, resulting in an increase in the detection limit.
  • the present invention provides an immune detection method and system, which aims to increase the lower concentration limit of the molecules to be detected and achieve magneto-optical immune detection with higher precision and sensitivity.
  • an immune detection method comprising:
  • step S3 Mix the immunoassay reagent and the solution to be tested so that the incident light is reflected multiple times in the mixed solution excited by the AC magnetic field; the incident light and AC magnetic field are the same as step S1;
  • the magneto-optical signal is the ratio of the second harmonic component and the DC component in the voltage signal.
  • the method further includes passing the incident light through a polarizing plate to form horizontally polarized light.
  • the AC excitation magnetic field is perpendicular to the propagation direction of incident light.
  • the incident light is a laser with a wavelength of 520 nm.
  • an immune detection system including: a laser, a polarizer, a coil, an optical liquid cavity, a photodetector and a computer;
  • Lasers used to generate incident light
  • Polarizer used to change the polarization direction of incident light
  • the optical liquid cavity is used to hold the functional magnetic nanoparticle immunoassay reagent or the mixed solution of the immunoassay reagent and the test solution, so that the incident light can be detected in the functional magnetic nanoparticle immunoassay reagent or the mixed solution under the excitation of the AC magnetic field. Multiple reflections; the structure of the optical liquid cavity is a cubic container with mirrors on the front and rear sides;
  • Photodetector used to convert the emitted light after multiple reflections into a voltage signal
  • a computer is used to extract the magneto-optical signals corresponding to the immunoassay reagent and the mixed solution; when the difference between the magneto-optical signal corresponding to the immunoassay reagent and the magneto-optical signal corresponding to the mixed solution exceeds a preset threshold, it is determined that the solution to be tested contains Target molecule to be measured.
  • the AC excitation magnetic field generated by the coil is perpendicular to the propagation direction of the incident light generated by the laser.
  • the concentration of magnetic nanoparticles that the magneto-optical detection system can detect in a shorter period of time is at least three orders of magnitude smaller.
  • the present invention enables Light is reflected multiple times in the magnetic fluid, the optical path is increased, and the magneto-optical signal intensity can be greatly improved, which can further increase the lower limit of detection concentration of the molecules to be measured and improve the accuracy and sensitivity of immune detection.
  • the present invention uses the ratio of the second harmonic amplitude to the DC component to characterize the magneto-optical signal.
  • Experimental results show that the second harmonic generated by pure water under the excitation of an AC magnetic field is negligible. Therefore, the magneto-optical signal used in this method is The signal has nothing to do with the diamagnetic property of water, so the interference of water on the detection results can be ignored, and the mixed solution has the convenience of no-wash; since the unbound analyte has no effect on the size of the magneto-optical signal, the immunity of the antigen and antibody occurs After the reaction, there is no need to clean the mixed solution and it can be directly detected, which is simpler to operate than the existing magneto-optical immunoassay method.
  • Figure 1 is a diagram of the magneto-optical immunoassay device of the present invention.
  • Figure 2 shows the changes in magneto-optical signals of magnetic nanoparticle solutions with different concentrations as a function of magnetic field
  • Figure 3 shows the change of magneto-optical signal with magnetic field under different reflection times
  • Figure 4 shows the changes in magneto-optical signals of magnetic nanoparticles with different particle sizes as a function of magnetic field.
  • the present invention provides an immune detection method based on an optical liquid cavity.
  • the overall idea is to use light in the optical liquid cavity to Multiple reflections increase the ratio of the second harmonic amplitude of the magneto-optical response signal to the DC component, thereby lowering the lower limit of concentration in immune detection.
  • the method of the present invention specifically includes:
  • a laser with a wavelength of 520 nm is used to generate laser light for detecting liquid concentration.
  • water has a smaller absorption rate of light. It is also possible to comprehensively consider wavelengths where the absorptivity of water is small and the magneto-optical signal is large, thereby reducing the loss caused by absorption when light passes through the magnetic fluid while enhancing the magneto-optical signal.
  • Magnetic nanoparticles under the influence of a magnetic field show anisotropy. After light passes through the magnetic nanoparticle solution, both the absorption coefficient and the scattering coefficient will change, thus causing the transmittance of the incident light to change.
  • the attenuation of light in conventional solutions satisfies the linear absorption law, namely J.H.Lambert's law:
  • I 0 and I are the incident light intensity and the exit light intensity respectively
  • L is the distance that light passes through in the solution
  • ⁇ is the attenuation coefficient
  • H is the excitation magnetic field.
  • the emitted optical signal will contain various harmonics, changing the concentration of the magnetic nanoparticle solution, the intensity of the incident light and other parameters, all The corresponding harmonic amplitude will change.
  • the second harmonic is used in the present invention because among all harmonics, the second harmonic has the largest amplitude, and this harmonic component is positively correlated with the distance of light passing through the magnetic nanoparticle solution under the action of a magnetic field. Using the ratio of the second harmonic to the DC component as the final magneto-optical signal can eliminate the differences in detection results caused by different incident light intensities.
  • the magneto-optical signal obtained by the magneto-optical birefringence effect comes from the phase difference
  • the magneto-optical signal obtained by magneto-optical Faraday comes from the rotation angle ⁇ , through and the sine or cosine of ⁇ to calculate the phase difference and rotation angle.
  • the proportional relationship between ⁇ and the distance traveled by light in the magnetic fluid applies to the range of 0 to ⁇ /2, so the thickness of the liquid container is limited to the order of ⁇ m.
  • the single-pass distance of the optical liquid cavity in the present invention is on the order of mm, so it is not suitable for the traditional magneto-optical birefringence effect and magneto-optical Faraday effect, which is explained through changes in transmittance.
  • the distance the light travels in the magnetic fluid increases with the number of reflections, and the magneto-optical signal is positively correlated with the distance the light travels in the magnetic fluid. , so the magneto-optical signal will be greatly improved.
  • the distance that light travels can also be increased by increasing the thickness of the cuvette, but the volume of the cuvette and the volume of the consumed solution will increase, which will increase the cost and is not conducive to miniaturization and integration.
  • the AC excitation magnetic field is perpendicular to the propagation direction of the incident light, so that a stronger signal can be obtained.
  • the DC component V 0 and the second harmonic amplitude V 2f are obtained, where f represents the excitation frequency of the AC magnetic field.
  • the magneto-optical signals mentioned in the present invention all refer to the ratio of the second harmonic amplitude and the DC component in the photovoltage.
  • step S3 Mix the immunoassay reagent and the antigen (antibody) to be tested. After the immune reaction occurs, an antigen-antibody-magnetic nanoparticle combination is formed, so that the incident light is reflected multiple times in the mixed solution under the excitation of the AC magnetic field; the incident The light and AC magnetic field are the same as step S1;
  • the magneto-optical signal of the immunoidentification reagent is only related to the magnetic nanoparticles, while the magneto-optical signal of the mixed solution contains the total characteristic information of the unbound magnetic nanoparticles and the conjugates that produce an immune reaction with the antigen (antibody) to be tested.
  • the magneto-optical signal is related to the total particle size of magnetic nanoparticles and their combinations. Therefore, when the difference between the magneto-optical signal corresponding to the immunoassay reagent and the magneto-optical signal corresponding to the mixed solution exceeds the preset threshold, it is determined that the test solution contains the target test molecule.
  • the target molecule is contained in the surface solution to be measured.
  • Changing the specific antibodies (antigens) on the surface of functional magnetic nanoparticles can achieve different types of antigen (antibody) detection
  • this magneto-optical immunoassay method based on optical liquid cavity can indeed improve the intensity of magneto-optical signals based on magneto-optical immunoassay and increase the lower limit of detection concentration.

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Abstract

一种免疫检测方法和系统，属于纳米材料测试领域。免疫检测方法包括：使入射光在交流磁场激励下的功能性磁纳米粒子免疫检定试剂中进行多次反射；将多次反射后的出射光转化为电压信号，并提取免疫检定试剂对应的磁光信号；将免疫检定试剂与待测溶液混合，使入射光在交流磁场激励下的混合溶液中进行多次反射；将多次反射后的出射光转化为电压信号，并提取混合溶液对应的磁光信号；当免疫检定试剂对应的磁光信号与混合溶液对应的磁光信号的差异程度超过预设的阈值时，判定待测溶液中含有目标待测分子。方法能提高待测分子的检测浓度下限，提高免疫检测的精度和灵敏度；具有免洗的便利性，较现有磁光免疫检测方法操作简单。

Description

一种免疫检测方法和系统 【技术领域】
本发明属于纳米材料测试技术领域，更具体地，涉及一种免疫检测方法和系统。
【背景技术】
免疫检测使用多种标记来测定抗体或抗原。最常用的方法是酶联免疫吸附测定、放射免疫测定和实时定律聚合酶链反应。最近，磁免疫测定受到了关注。磁纳米粒子由于其独特的磁学性质，可用作免疫检测中的被测物标记。利用磁纳米粒子的磁性，可以对与其相互作用的待测分子的检测特征进行表征。目前被广泛研究的磁相关量有磁弛豫、剩余磁化强度、交流磁化率、饱和磁化强度以及布朗弛豫时间等。在溶液中利用特异功能团可以将待测分子和磁纳米粒子绑定在一起，会改变磁纳米粒子的磁信息。由传感器测量得到的磁纳米粒子磁化响应的变化，与待测分子的浓度相关。
光学测量方法具有非接触性、高灵敏度和高精度等突出的优点。磁光效应是一种能够测量磁纳米粒子磁信息的新方法。对磁流体施加磁场，磁纳米粒子将沿着磁场方向聚集成链状的团簇，从而显示出各向异性的特性。光在被磁化的磁流体中传播，会产生一系列磁光效应，导致磁光响应信号发生变化。磁光响应信号的强度，与磁流体的浓度信息相关，因此通过检测磁光效应强度能够实现免疫检测。
目前普遍使用的磁光免疫检测方法有磁性荧光纳米探针检测、磁微粒化学发光免疫分析法。磁性荧光纳米探针检测中未结合的荧光量子点会产生一定的荧光强度，干扰结合物的检测，需要进行磁场分离。磁微粒化学发光免疫分析法需要在抗原抗体反应结束后，进行清洗，以去除未结合到免疫复合物的物质。洗涤过程会使被标记的待测物减少，导致检测限提高。
【发明内容】
针对现有技术的以上缺陷或改进需求，本发明提供了一种免疫检测方法和系统，其目的在于提高待检测分子的浓度下限，实现更高精度和灵敏度的磁光免疫检测。
为实现上述目的，按照本发明的一个方面，提供了1、一种免疫检测方法，包括：
S1.使入射光在交流磁场激励下的功能性磁纳米粒子免疫检定试剂中进行多次反射；
S2.将多次反射后的出射光转化为电压信号，并提取免疫检定试剂对应的磁光信号；
S3.将免疫检定试剂与待测溶液混合，使入射光在交流磁场激励下的混合溶液中进行多次反射；所述入射光和交流磁场与步骤S1相同；
S4.将多次反射后的出射光转化为电压信号，并提取混合溶液对应的磁光信号；
S5.当免疫检定试剂对应的磁光信号与混合溶液对应的磁光信号的差异程度超过预设的阈值时，判定待测溶液中含有目标待测分子。
进一步地，所述磁光信号为电压信号中二次谐波分量和直流分量的比值。
进一步地，所述方法还包括，将入射光通过偏振片形成水平方向的偏振光。
进一步地，所述交流激励磁场与入射光的传播方向垂直。
进一步地，所述入射光为波长520nm的激光。
按照本发明的另一方面提供了一种免疫检测系统，包括：激光器、偏振片、线圈、光学液体腔、光电探测器和计算机；
激光器，用于产生入射光；
偏振片，用于改变入射光的偏振方向；
线圈，用于产生交流激励磁场；
光学液体腔，用于盛放功能性磁纳米粒子免疫检定试剂或免疫检定试剂与待测溶液的混合溶液，使入射光在交流磁场激励下的功能性磁纳米粒子免疫检定试剂或混合溶液中进行多次反射；所述光学液体腔的结构为前后两面镀有反射镜的立方体容器；
光电探测器，用于将多次反射后的出射光转化为电压信号；
计算机，用于提取免疫检定试剂和混合溶液对应的磁光信号；当免疫检定试剂对应的磁光信号与混合溶液对应的磁光信号的差异程度超过预设的阈值时，判定待测溶液中含有目标待测分子。
进一步地，线圈产生的交流激励磁场与激光器产生的入射光的传播方向垂直。
总体而言，通过本发明所构思的以上技术方案与现有技术相比，能够取得下列有益效果。
(1)与传统检测方案的交流电传感器相比，磁光检测系统在较短时间内能够检测到的磁纳米粒子的浓度至少小三个数量级，而本发明在现有的磁光效应基础上，使光在磁流体中进行多次反射，光程增加，磁光信号强度能够大幅度提升，能够进一步提高待测分子的检测浓度下限，提高免疫检测的精度和灵敏度。
(2)本发明采用二次谐波幅值与直流分量的比值表征磁光信号，实验结果表明，交流磁场激励下的纯水产生的二次谐波可忽略不计，因此本方法采用的磁光信号与水的抗磁性无关，因此可以忽略水对检测结果的干扰性，混合溶液具有免洗的便利性；由于未结合的待测物对磁光信号的大小没有影响，因此在抗原抗体发生免疫反应之后，也无需对混合溶液进行清洗，可直接检测，较现有磁光免疫检测方法操作简单。
【附图说明】
图1是本发明的磁光免疫检测装置图；
图2是不同浓度的磁纳米粒子溶液的磁光信号随磁场的变化；
图3是不同反射次数下磁光信号随磁场的变化；
图4是不同粒径的磁纳米粒子的磁光信号随磁场的变化。
【具体实施方式】
为了使本发明的目的、技术方案及优点更加清楚明白，以下结合附图及实施例，对本发明进行进一步详细说明。应当理解，此处所描述的具体实施例仅仅用以解释本发明，并不用于限定本发明。此外，下面所描述的本发明各个实施方式中所涉及到的技术特征只要彼此之间未构成冲突就可以相互组合。
为了解决先前的基于磁光效应的免疫检测方法效率不高、检测灵敏度低的技术问题，本发明提供了一种基于光学液体腔的免疫检测方法，其整体思路在于：利用光在光学液体腔中多次反射，提高磁光响应信号的二次谐波幅值与直流分量的比值，从而降低免疫检测中的浓度下限。比较免疫检定试剂和混合溶液的信号，若信号差值超过阈值，则可判定待测溶液中含有目标分子。由于水的抗磁性，对磁纳米粒子溶液施加交流激励时，会产生一定的谐波干扰。但实验结果表明，交流磁场激励下的纯水产生的二次谐波可忽略不计，因此本方法采用的磁光信号与水的抗磁性无关，因此可以忽略水对检测结果的干扰性，混合溶液具有免洗的便利性，因此操作较简单；利用光测量技术检测磁信号，能够提高检测的灵敏度，实现快速检测。
本发明方法具体包括：
S1.将待测物抗体(抗原)包被磁纳米粒子形成磁纳米粒子免疫复合物，再配置功能性磁纳米粒子免疫检定试剂。使入射光在交流磁场激励下的功能性磁纳米粒子免疫检定试剂中进行多次反射；
具体地，使用波长为520nm的激光器产生检测液体浓度的激光，在此波长下，水对光的吸收率较小。也可综合考虑水的吸收率较小而磁光信号 较大的波长，从而减小光在磁流体中通过时因吸收造成的损失的同时增强磁光信号。
磁场作用下的磁纳米粒子表现出各向异性，光通过磁纳米粒子溶液后，吸收系数和散射系数均会发生变化，从而使入射光的透射率发生变化。光在常规溶液中的衰减满足线性吸收定律，即朗伯(J.H.Lambert)定律：
I＝I 0exp(-αL)
其中，I 0和I分别为入射光强和出射光强，L为光在溶液中通过的路程，α为衰减系数。根据本发明中经实验得到的磁光信号，对上述吸收定律进行修正，得到出射光强与入射光强之间的关系为：
I＝I 0exp[-α(H)L]
其中，H为激励磁场。根据Taylor公式，有
Figure PCTCN2022138911-appb-000001
由上式可知，当施加的激励磁场为正弦交流场H＝H 0sinωt时，出射的光信号中会包含各次谐波，改变磁纳米粒子溶液的浓度、入射光的光强等参数，均会使相应的谐波幅值发生改变。本发明中采用二次谐波，是因为在各次谐波中，二次谐波幅值最大，且该谐波分量与光通过磁场作用下的磁纳米粒子溶液的路程成正相关。而将二次谐波与直流分量比值作为最终的磁光信号，可消除入射光强不同造成的检测结果的差异。
为了区分本发明中提到的磁场作用下透射率变化产生的磁光效应与传统的磁光双折射效应和磁光法拉第效应，对采用的液体容器长度加以说明。磁光双折射效应获得的磁光信号来源于相位差
Figure PCTCN2022138911-appb-000002
磁光法拉第获得的磁光信号来源于旋转角θ，通过
Figure PCTCN2022138911-appb-000003
和θ的正弦或余弦值来计算相位差和旋转角。
Figure PCTCN2022138911-appb-000004
和θ与光在磁流体中通过的路程的正比关系适用于0到π/2范围内，因此液体容器的厚度被限制为μm量级。而本发明中的光学液体腔单次通过的路程为mm量级，因此不适于传统的磁光双折射效应和磁光法拉第效应，通过 透射率的变化进行了解释。
当水平偏振光在交流磁场激励下磁流体中多次反射后，光在磁流体中经过的距离随着反射次数的增加而增加，而磁光信号随着光在磁流体中经过的距离成正相关，因此磁光信号会大幅度提高。也可以通过增加比色皿厚度的方式来增加光经过的距离，但比色皿的体积和消耗溶液的体积均会增加，成本提高，且不利于小型化和集成化。优选地，交流激励磁场与入射光的传播方向垂直，能获得更强的信号。
S2.将多次反射后的出射光转化为电压信号，并提取免疫检定试剂对应的磁光信号；
通过电压信号进行处理，得到直流分量V 0和二次谐波幅值V 2f，其中，f表示交流磁场的激励频率。用于分析的磁光信号s＝V 2f/V 0，这样可以消除入射光光强不同产生的影响。本发明中提到的磁光信号均指光电压中二次谐波幅值与直流分量的比值。
S3.将免疫检定试剂与待测抗原(抗体)混合，发生免疫反应之后形成抗原-抗体-磁纳米粒子结合物，使入射光在交流磁场激励下的混合溶液中进行多次反射；所述入射光和交流磁场与步骤S1相同；
S4.将多次反射后的出射光转化为电压信号，并提取混合溶液对应的磁光信号；
S5.免疫鉴定试剂的磁光信号仅与磁纳米粒子相关，而混合溶液的磁光信号包含未结合的磁纳米粒子和与待测抗原(抗体)产生免疫反应的结合物的总特征信息。磁光信号与磁纳米粒子及其结合物的总粒径有关。因此，当免疫检定试剂对应的磁光信号与混合溶液对应的磁光信号的差异程度超过预设的阈值时，判定待测溶液中含有目标待测分子
如果功能性磁纳米粒子溶液的磁光信号s 1与混合溶液的磁光信号s 2差值超过阈值，则表面待测溶液中含有目标分子。改变功能性磁纳米粒子表 面的特异性抗体(抗原)，可实现不同种类的抗原(抗体)检测
由于磁光信号只与含有磁纳米粒子的颗粒有关，未结合的待测抗原(抗体)不会对检测结果产生影响，因此本发明提出的免疫检测方法免清洗。实例一：
比较不同浓度磁纳米粒子溶液的磁光信号。如图2所示，为磁纳米粒子溶液浓度分别为100μg/ml、10μg/ml、1μg/ml和蒸馏水时，磁光信号随交流激励磁场幅值的变化曲线。可以看出，当交流激励磁场幅值相同时，磁光信号随着磁纳米粒子溶液浓度的增加而增加。
实例二：
比较不同反射次数的磁光信号。如图3所示，n为光在磁纳米粒子溶液中经过的次数，n与反射次数m的关系为：n＝2m+1。可以看出，当反射次数增加时，磁光信号也会随之增加。
实例三：
比较相同体积分数、不同粒径的磁纳米粒子的磁光信号。如图4所示，为磁纳米粒子粒径分别为30nm、20nm、10nm时，与蒸馏水作对照组的实验结果。可以看出，当粒子粒径增大时，磁光信号也会增加。
因此，这种基于光学液体腔的磁光免疫检测方法确实可以在磁光免疫检测的基础上提升磁光信号的强度，提高了检测的浓度下限。
本领域的技术人员容易理解，以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等，均应包含在本发明的保护范围之内。

Claims (7)

  1. 一种免疫检测方法，其特征在于，包括：
    S1.使入射光在交流磁场激励下的功能性磁纳米粒子免疫检定试剂中进行多次反射；
    S2.将多次反射后的出射光转化为电压信号，并提取免疫检定试剂对应的磁光信号；
    S3.将免疫检定试剂与待测溶液混合，使入射光在交流磁场激励下的混合溶液中进行多次反射；所述入射光和交流磁场与步骤S1相同；
    S4.将多次反射后的出射光转化为电压信号，并提取混合溶液对应的磁光信号；
    S5.当免疫检定试剂对应的磁光信号与混合溶液对应的磁光信号的差异程度超过预设的阈值时，判定待测溶液中含有目标待测分子。
  2. 根据权利要求1所述的一种免疫检测方法，其特征在于，所述磁光信号为电压信号中二次谐波分量和直流分量的比值。
  3. 根据权利要求2所述的一种免疫检测方法，其特征在于，所述方法还包括，将入射光通过偏振片形成水平方向的偏振光。
  4. 根据权利要求1-3任一项所述的一种免疫检测方法，其特征在于，所述交流激励磁场与入射光的传播方向垂直。
  5. 根据权利要求1-4任一项所述的一种免疫检测方法，其特征在于，所述入射光为波长520nm的激光。
  6. 一种免疫检测系统，其特征在于，包括：激光器、偏振片、线圈、光学液体腔、光电探测器和计算机；
    激光器，用于产生入射光；
    偏振片，用于改变入射光的偏振方向；
    线圈，用于产生交流激励磁场；
    光学液体腔，用于盛放功能性磁纳米粒子免疫检定试剂或免疫检定试剂与待测溶液的混合溶液，使入射光在交流磁场激励下的功能性磁纳米粒子免疫检定试剂或混合溶液中进行多次反射；所述光学液体腔的结构为前后两面镀有反射镜的立方体容器；
    光电探测器，用于将多次反射后的出射光转化为电压信号；
    计算机，用于提取免疫检定试剂和混合溶液对应的磁光信号；当免疫检定试剂对应的磁光信号与混合溶液对应的磁光信号的差异程度超过预设的阈值时，判定待测溶液中含有目标待测分子。
  7. 根据权利要求6所述的一种免疫检测系统，其特征在于，线圈产生的交流激励磁场与激光器产生的入射光的传播方向垂直。
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