阅读说明：本技术 一种基于磁性纳米颗粒的微流控式即时免疫检测方法 (Microfluidic immediate immunodetection method based on magnetic nanoparticles ) 是由 何赛灵 支正良 于 2021-09-07 设计创作，主要内容包括：本发明公开了一种基于磁性纳米颗粒(MNP)的微流控式即时免疫检测方法。该方法通过采用MNP或MNP@Au作为抗体载体直接捕获复杂样品环境中的目标物,仅需要施加磁场即可实现目标物的分离,并采用配套的固体试剂快速溶解配方,缩短测试时间,延长免疫试剂的保质期。为提高检测的灵敏度和精准度,采用时间分辨荧光检测方法降低背景信号,MNP表面的金层可以对拉曼标记物进行表面增强拉曼散射增强,并且将微流控芯片与共聚焦光谱显微成像系统、差分检测结合。为降低检测成本,将比色反应内置于微流控芯片中,然后与手机APP结合研发显色微流控芯片检测仪。为提高诊断的准确性,提出了一个微流控芯片陈列结构方案,实现对多种疾病标志物的同时检测。(The invention discloses a microfluidic immediate immunodetection method based on Magnetic Nanoparticles (MNP). According to the method, MNP or MNP @ Au is used as an antibody carrier to directly capture the target object in a complex sample environment, the separation of the target object can be realized only by applying a magnetic field, and a matched solid reagent rapid dissolving formula is adopted, so that the testing time is shortened, and the quality guarantee period of the immunoreagent is prolonged. In order to improve the sensitivity and the accuracy of detection, a time-resolved fluorescence detection method is adopted to reduce background signals, a gold layer on the surface of the MNP can perform surface-enhanced Raman scattering enhancement on a Raman marker, and the microfluidic chip is combined with a confocal spectrum microscopic imaging system and differential detection. In order to reduce the detection cost, the colorimetric reaction is arranged in the microfluidic chip, and then the colorimetric reaction is combined with a mobile phone APP to research and develop the chromogenic microfluidic chip detector. In order to improve the accuracy of diagnosis, a scheme of a microfluidic chip array structure is provided, and the simultaneous detection of multiple disease markers is realized.)

1. A microfluidic immediate immunodetection method based on magnetic nanoparticles comprises the following core technical components: 1) freeze-drying the magnetic nanoparticle or gold-coated magnetic nanoparticle-antibody coupling compound and another fluorescence or enzyme or Raman labeled antibody coupling compound to prepare a dry reagent, and packaging the dry reagent into a microcavity with a detection window at one end; 2) after the sample solution to be detected is added into the sample adding port at the other end, the filtered sample solution reaches the microcavity at the detection end to quickly dissolve the dry reagent and release the immune reagent; 3) by an external magnetic field, the magnetic coupler combined with the target object is directionally moved to one end and enters a capture area of the complex, so that the target object is separated by a microfluidic technology; 4) designing a hydrophobic liquid stop valve at the downstream of the capture area, so that liquid cannot pass through the liquid stop valve; 5) and measuring the luminescence or fluorescence or Raman intensity before and after the separation of the target object by adopting a conventional immune luminescence or fluorescence or time-resolved fluorescence detection method or a Raman detection method, and representing the concentration of the target object by the difference value.

2. The method as claimed in claim 1, wherein the magnetic nanoparticles are used as a carrier to directly capture the target in the complex sample environment, and the separation of the target is realized by applying a magnetic field, so as to simplify the experimental operation steps;

the gold layer on the Surface of the magnetic nanoparticles (Au nanoparticles or Au nanorods can be selected) can be used as an Enhanced substrate, and SERS (Surface-Enhanced Raman Scattering) enhancement can also be performed on the Raman marker, so that the detection sensitivity is improved.

3. The method of claim 1, wherein the immunoreagent is stored in a dry condition to prolong the useful life of the reagent, and wherein the reagent is rapidly dissolved after the test sample is added to the test sample to reduce the test time.

4. The method of claim 1, wherein the concentration of the target is characterized using a differential method, and wherein the contribution of the excess fluorescent label or raman antibody conjugate to fluorescence or raman detection is eliminated, thereby improving the accuracy of the detection system.

5. The method of claim 1, wherein the time-resolved fluorescence detection method is an effective means for improving the sensitivity of fluorescence analysis, the influence of stray light of excitation light becomes more serious when ultra trace analysis is performed, and the sensitivity of fluorescence detection can be greatly improved by measuring the fluorescence intensity after the excitation light is turned off by using a long-life fluorescence marker.

6. The method of claim 1, wherein the fluorescence detection is time-resolved fluorescence detection using an ultraviolet pulse train laser as the excitation source of europium (Eu), the pulse duration of the laser being on the order of microseconds to ensure that the excitation source does not contribute to Eu fluorescence collection as background noise light after a certain delay time.

7. The method as claimed in claim 1, wherein in the raman detection, in order to further increase the detection rate of the trace target, the microfluidic chip is combined with the confocal raman spectroscopy imaging system to detect the signal in the detection window of the microfluidic chip, so as to increase the signal-to-noise ratio and reduce the probability of detecting false negative and false positive.

8. The method of claim 1, wherein the integration of nano-column filtration, magnetic nanoparticles, immunoreagent and reaction chamber is integrated by precise microfabrication technology, and basic operation units of sample preparation, immunoreaction, separation, detection, etc. are integrated into a microfluidic chip device to realize a further detection mode of 'sample in and result out';

conventional immunofluorescence excitation may use inexpensive Light Emitting Diodes (LEDs).

9. The method as claimed in claim 1, wherein, to further reduce the detection cost, colorimetric reaction based on enzyme-labeled antibody material is built in the microfluidic chip, the enzyme reacts with the chromogenic substrate pre-encapsulated in the capture region to generate color change, and the negative and positive are qualitatively judged by naked eyes according to the color change degree, or combined with the mobile phone APP, so that qualitative or semi-quantitative detection of the disease marker can be realized without additional detection tools.

10. The method as claimed in claim 1, wherein based on the method, a plurality of joint detection chips for different disease biomarkers are developed, and instant immune joint detection (such as five joint detection or six joint detection) products are developed to realize joint detection of a plurality of disease markers.

Technical Field

The invention belongs to the field of optical sensing, and relates to a microfluidic immediate immunodetection method based on magnetic nanoparticles.

Background

Microfluidic chips, also known as Lab-on-a-chips (Lab-on-a-chips) or microfluidic Lab-on-chips (cfcs), are the main platform for the realization of microfluidic technologies. The lab-on-a-chip has the ability to scale down the basic functions of biological, chemical and other laboratories to a few square centimeters chip, and can complete all the steps of sample pretreatment, separation, dilution, mixing, chemical reaction, detection and the like. The biggest challenge in achieving an immune trace analysis for some biomarkers in a complex sample environment is the separation of target analytes from antibody complexes. The magnetic nano-particles fixed with the antibody have the inherent property of attracting with a magnet, and can be used for directly capturing, separating and pre-concentrating a target substance in a complex environment sample according to the property and provide a new development opportunity for an immunoassay technology. Compared with the traditional lateral flow immunoassay method based on colloidal gold, the method combining the magnetic nanoparticles and the microfluidic chip has the advantages of overwhelming sensitivity, precision, reproducibility and the like.

Conventional analytical detection often characterizes the concentration of the target analyte by measuring the fluorescence intensity of the nanoparticles. The method ignores the influence of the optical properties of the particles on the signal, for example, in the presence of nano-magnetic particles, the stronger absorption of certain wavelengths of light can lead to the reduction of the signal. In the case of trace analytes, the signal is often undetectable due to the limitations of the instrument itself, so that a lower detection limit cannot be reached.

The time-resolved fluorescence analysis is a super-sensitivity detection technology which is characterized by rare earth ion labeled antigen or antibody, nucleic acid probe, cell and the like, and overcomes the defects of instability of an enzyme label, primary luminescence of chemiluminescence, easy environmental interference, indirect labeling of electrochemiluminescence and the like. The kit reduces non-specific signals to a negligible degree, achieves a high signal-to-noise ratio, greatly exceeds the sensitivity which can be achieved by enzyme labeling or radioactive isotopes, and has the advantages of simple preparation of the label, no influence on the biological activity and structure of the labeled substance, long storage time, no radioactive pollution, good detection repeatability, capability of realizing multiple labeling and multiple tests, short operation flow, wide range of standard curves, no interference of natural fluorescence of samples, very wide application range and the like, thereby becoming a new milestone for development of the label after radioimmunoassay.

Compared with the traditional optical fiber and solid laser, the gas microsecond pulse laser can directly excite the high-coherence light beam with the wavelength of 337 nm without complex nonlinear frequency conversion means (frequency doubling and frequency combination), and can output 337 nm laser with the power of 100 milliwatts under continuous work. The continuous laser working mode can be converted into a pulse working mode through external TTL signal modulation. Under the pulse working mode, the laser can output pulse laser with the duration of microsecond and the pulse repetition frequency of continuously adjustable between 100 Hz and 1 kHz, the RMS index of the power stability of the laser is less than 3 percent, and the high stability of a time fluorescence resolution system can be ensured.

The research and development of fluorescent microfluidic instant immunoassay products are very popular at home and abroad. The domestic company has Shenzhen Wei Point, Nanjing lan Yu, Huameixing Wei, Zhongxin Keju, Shijiazhuang Hebai, Zhejiang Pushakang, Chengdu Weikang and the like; however, the detection of the new coronavirus antigen by only 2 overseas research and development manufacturers (LumiraDx and Quidel) can reach more than 97 percent at present; the sensitivity and accuracy of the virus antigen real-time detection products developed in China are obviously insufficient, and the virus antigen real-time detection products rarely meet the strict detection requirements of the FDA, so that technical innovation and research are very necessary to be developed in the field.

Raman scattering, which originates from changes in the relative positions between atoms within a molecule or crystal, i.e. vibrations and rotations, is an important analytical tool for studying the structure of molecules and solid materials. Raman spectrum as a molecular spectrum technology can realize non-contact and nondestructive detection and is widely applied to the field of biological detection. The combination of the microscopic technology and the Raman spectrometer is a revolutionary breakthrough of the Raman experimental technology, the microscopic Raman system focuses laser beams on a sample by using a microscopic objective lens and collects Raman scattered light by using the same objective lens, so that the sample amount required by testing is reduced on one hand, and the laser power required by measurement is reduced on the other hand, thereby greatly widening the application range of the Raman spectrum technology. More importantly, the spatial resolution of Raman measurement is improved to submicron and micron dimensions by the microscopic technology, so that a novel experimental technical method, namely Raman spectrum imaging, is introduced for the Raman spectrum technology. This advantageously allows the extension of conventional single-point analysis to simultaneous comparative analysis of samples over a range of spaces.

Tumors are the result of multiple clones of a single variant cell, and their development is a multistep, polygenic process of carcinogenesis. The biological characteristics of tumor cells have complexity and polymorphism, and are expressed by the difference of pathological types of different tumors after canceration, the constant thinking of tumor cells of the same case type, the difference of the genotype and the phenotype of the tumor cells, and the like. Cells with different characteristics are present in a tumor, and may differ in growth rate, surface receptors, immunological properties, invasiveness, metastasis, and toxicity to drugs. Thus, a single tumor may contain one or more tumor markers, while different tumors or different tissue types of the same tumor may have either a common tumor marker or different tumor markers. In order to improve the positive rate of tumor marker detection, some tumor markers with higher specificity are selected for combined detection, so that the application value of the tumor markers can be improved.

Disclosure of Invention

The invention provides a microfluidic immediate immunodetection method based on magnetic nanoparticles, which comprises the following steps of 1) solving the problem that trace target analytes in a complex sample environment are separated under the action of a magnetic field and the magnetic nanoparticles in a microfluidic system; 2) the gold-plated layer on the surface of the magnetic nano-particle is used as an antibody fixing and enhancing substrate to perform SERS enhancement on the Raman marker, so that the aim of improving the detection sensitivity is fulfilled; 3) the interference of the particles to signals is eliminated by using a difference method, so that the detection limit of immunofluorescence and Raman analysis is improved; 4) the dry formula containing the components such as sucrose and the like and capable of being dissolved rapidly is used for preparing the biological reagent, so that the quality guarantee period of the biological reagent is prolonged, and the testing time is shortened; 4) the purpose of improving the sensitivity is achieved by adopting time-resolved fluorescence labeling; 5) the confocal Raman spectrum imaging system is utilized to improve the detection signal-to-noise ratio and further improve the detection sensitivity of the trace analyte; 6) a colorimetric reaction is arranged in a micro-fluidic chip, and then the colorimetric reaction is combined with a mobile phone APP to develop a new generation of a community or household chromogenic micro-fluidic chip detector; 7) the instant immune joint inspection mode is utilized to realize joint inspection of multiple markers of tumors or cardiovascular or other diseases.

The invention has the advantages of

The invention discloses a magnetic nanoparticle-based microfluidic immediate immunodetection method, which integrates basic operation units such as sample preparation, reaction, separation, detection and the like on a chip to serve as a new generation of ultrasensitive rapid microfluidic technology, realizes further detection of 'sample in and result out', and solves the problems of complex operation, time consumption, insufficient sensitivity and precision and the like of immunodetection.

1) By integrating magnetic particle separation, micro-fluidic and differential detection into a detection system, the invention realizes the purpose of obtaining accurate detection results within 10 minutes by one-step sample adding with simple operation, and is very suitable for field real-time detection scene application.

2) The microfluidic system based on the magnetic particles and the matched solid reagent are quickly formulated, so that the testing time is shortened, the quality guarantee period of the immunoreagent is prolonged, and the detection sensitivity and precision are greatly improved.

3) The SERS detection technology of the time-resolved fluorescence label and the gold-coated magnetic nanoparticles is adopted to realize the aim of further improving the detection sensitivity.

4) The concentration of the target object is represented by using a differential method, the influence of the absorbance of the nanoparticles is eliminated, and the accuracy of a detection system is improved.

5) The micro-fluidic chip is combined with a confocal Raman spectrum imaging system, so that the detection rate of the trace target object is improved, and the probability of false negative and false positive is reduced.

6) Through placing the colorimetric reaction in the micro-fluidic chip in, then combine together with cell-phone camera detection (obtain quantization value) and APP, under the condition that does not need detecting instrument, just can realize the quantitative determination to the target object.

7) Six (but not limited to six) chips aiming at different types of disease biomarkers are arranged on the disc, and synchronous joint detection of 6 markers is realized.