阅读说明：本技术 基于太赫兹衰减全反射模式的早期癌症检测方法 (Early cancer detection method based on terahertz attenuated total reflection mode ) 是由 刘钱 府伟灵 刘璐 张明焜 于 2019-09-09 设计创作，主要内容包括：本发明公开了基于太赫兹衰减全反射模式的早期癌症检测方法,建立全反射面-样品池底面-细胞层-缓冲液层的多界面与倏逝波相互作用的物理模型,由于全反射面和样品池底面为同一材质,所以全反射发生在样品池底面-细胞层界面。本发明基于细胞的太赫兹特征指纹谱的检测将会比基于细胞形态的检测具有更高的灵敏度和准确度。(The invention discloses an early cancer detection method based on a terahertz attenuated total reflection mode, which is used for establishing a physical model of interaction between a total reflection surface, a sample pool bottom surface, a cell layer and a buffer liquid layer and an evanescent wave. The cell-based terahertz characteristic fingerprint spectrum detection method based on the terahertz characteristic fingerprint spectrum has higher sensitivity and accuracy than the cell morphology-based detection.)

1. The early cancer detection method based on the terahertz attenuated total reflection mode is characterized in that a physical model of interaction between a multi-interface of a total reflection surface, a sample pool bottom surface, a cell layer and a buffer liquid layer and an evanescent wave is established, because the total reflection surface and the sample pool bottom surface are made of the same material, total reflection occurs at the sample pool bottom surface-cell layer interface, and a critical angle theta of internal reflection of the contact surface can be obtained according to the following formula_cThe relationship is as follows:

in the above formula n_Sam、n_Sirefractive index of sample and silicon prism, penetration depth d of evanescent wave in cell layer-buffer layer_pThe relationship with wavelength λ is as follows:

The above formula characterizes the penetration of the evanescent wave through the cell and buffer layers with exponential decay, d_pIs the characteristic penetration depth at which the electric field intensity of the terahertz wave decays to 1/e at the surface, λ is the incident wave wavelength, and θ is the incident angle.

2. The early cancer detection method based on terahertz attenuated total reflection mode as claimed in claim 1, wherein the clinical sample is placed in the sample area of the sample cell, the sample cell is loaded on the total reflection surface, the sample cell is rotated to make the evanescent wave fully act on the reference area of the sample cell, the output electric field amplitude ERef is the reference signal, the clinical sample is placed in the sample area of the sample cell, the sample cell is loaded on the total reflection surface, the sample cell is rotated to make the evanescent wave fully act on the reference area of the sample cell, the sample cell is rotated again to make the evanescent wave fully act on the sample area of the sample cell, the terahertz emergent signal is obviously changed, and the output electric field amplitude is changed at the momentIs the sample signal. If the amplitude of the electric field of the incident terahertz wave is E_InThen according to the law of reflection, can obtain

In the formula r_RefThe reflection coefficient when no sample is injected, in this case the reflection coefficient of the reference zone;Is a sample areathe reflection coefficient of (2). According to Fresnel reflection law, the reflection coefficient of the interface of the total reflection surface and the cell layer under P polarizationIs composed of

In the formula of_SiIs the complex dielectric constant of the bottom surface of the sample cell,The complex dielectric constant of the material close to the total reflection surface. Reflection coefficient of cell layer-buffer interface under P polarizationIs composed of

in the formulaIs the complex dielectric constant of the buffer. Then the simultaneous formulas (4) and (5) can be obtainedIs composed of

Wherein d is the thickness of the cell layer, and the terahertz complex dielectric characteristic spectrum of the cell layer can be obtained by combining the formulas (3) and (6).

Technical Field

The invention belongs to the technical field of early cancer detection, and particularly relates to an early cancer detection method based on a terahertz attenuated total reflection mode.

background

Cancer is the first cause of death of urban and rural residents in China, and every year, 429 ten thousand of new cancer cases in China account for 20 percent of new cases in the world, and 281 ten thousand of cases of death. Cancer prevention and treatment have become an important public health problem in China. A large number of researches show that the treatment effect on early cancer is obviously superior to that of middle and late cancers. If the cancer can be accurately diagnosed at the early stage, the cancer can be cured radically, and the onset period of the cancer can be effectively controlled or prolonged, so that the life quality of patients is improved, and the life span is prolonged. Therefore, in many countries including our country, early diagnosis and treatment of cancer is the most important means for preventing and treating cancer.

Accurate detection of early stage carcinogenesis is a prerequisite for early stage treatment of cancer. Currently, methods for detecting cancer can be classified into imaging methods, immunohistochemical methods, puncture pathology methods, and the like. The imaging method is to form different images according to different reflection intensities of tissues with different densities by using an electron computed tomography or a nuclear magnetic resonance technology, and judge the cancerated tissues by contrasting normal tissues. The imaging method is mainly used for detecting the tumor tissue level and is suitable for detecting the middle and late stages of cancer. Immunohistochemistry is a novel pathological detection method for detecting tumor markers based on immunohistochemistry, molecular biology, flow cytometry and the like, and is suitable for early detection of cancers. However, this method has problems that sensitivity and specificity of tumor markers are not good, false positives and false negatives easily occur, because many tumor markers are produced not only when canceration occurs but also expressed to different degrees in the case of normal and benign tumors. The "gold standard" for accurate cancer detection requires microscopic observation of cancer cells, and the puncture pathology is the direct observation of tissue sections of suspicious sites or exfoliated cells using an optical microscope, depending on the morphology of the cells, such as: the method is the most reliable method for confirming the cancer and is suitable for early and medium stage detection of the tumor. However, such pathology detection methods based on cell morphology observation are affected by many factors including sample processing procedure, optical microscope resolution, and subjective experience of doctors, resulting in poor accuracy, such as: the misdiagnosis rate of breast cancer in the United states can reach 43 percent at most. It can be seen that the current detection of early cancer has the problems of poor sensitivity and specificity. Therefore, we must develop a new method to realize accurate detection of early cancer cells, so as to achieve the purpose of early accurate diagnosis of cancer.

In recent years, with the development of terahertz biomedicine, terahertz biological detection technology has a great potential in the field of cancer diagnosis. Terahertz waves are electromagnetic waves having a frequency range of 0.1-10THz, and have a wavelength (0.03-3mm) between the microwave and far infrared (as shown in fig. 1). Because the energy level of the terahertz wave and the low-frequency vibration of the biological molecules (such as the vibration of a biological molecule framework, the rotation and the twisting of residues in protein and the like) has a large boundary and is very sensitive to the weak interaction between the biological molecules (such as hydrogen bonds, van der waals force and the like) and the water content in a living body, the qualitative and quantitative analysis of a biological sample can be realized through the characteristic spectrum (namely complex dielectric spectrum and characteristic absorption spectrum) of the living body in the terahertz frequency band. Different biomolecules, different conformations of the same biomolecule, different cells and intracellular chemical compositions, different physiological stages of the same cell and different water contents of different bacteria have different responses in the terahertz waveband; meanwhile, the terahertz photon energy is low, almost no ionization damage is caused to organisms, and the terahertz photon energy is very safe. Therefore, terahertz diagnosis has been recognized as a next-generation extremely competitive biomedical technology. At present, the diagnosis of cancer by using terahertz technology is reported internationally: on the aspect of tissue detection, the terahertz detection result of the cancerous tissue is very consistent with the pathological detection, such as brain glioma [13] and gastric cancer tissue [14 ]; in the aspect of cell detection, different cancer cells have obvious terahertz dielectric difference [10 ]; on the aspect of molecular detection, the combination action of a tumor marker and a specific ligand thereof can be sensitively detected by terahertz characteristic spectrum, and the detection sensitivity reaches 1 pmol/mu L. The chemical composition and proportion of substances contained in cancerous cells change earlier than normal cells in terms of changes in cell morphology observed by light microscopy, and these changes in cells can sensitively affect the physicochemical properties of the cells.

The terahertz technology is applied to early detection of cancers, and the terahertz biomedical detection is tried to be put into clinical application from a laboratory by utilizing an attenuated total reflection detection mode which is independently researched and developed, belongs to the advanced technology of clinical medical treatment, and has certain practical significance for enhancing the technical competitiveness of digital diagnosis and treatment equipment in China and promoting the development of the medical instrument industry. The existing cancer cell detection mainly depends on a marking technology, the sample preparation is complex, and the cell damage is strong. Due to the unique spectrum advantage, the terahertz can detect information of intracellular biomolecules and chemical components which cannot be analyzed by other electromagnetic wave bands and the existing living cell detection technology, can identify early cancer cells through the change of chemical components and proportion of substances contained in the cells, has detection sensitivity superior to that of an optical microscope, and is expected to become an indispensable brand new means for the existing early cancer detection.

at present, the terahertz technology has prominent obvious advantages in three layers of tissue-cell-molecule for cancer detection, and especially can discover early-stage cancerous cells at a cell layer earlier than a traditional optical microscope, so that the terahertz technology is an early-stage cancer detection technology with great potential. The traditional terahertz biological detection generally adopts a transmission mode, and because the active biological sample has abundant water content, the water content in the sample and the thickness of the sample can have great influence on the terahertz detection result, people usually adopt a proper liquid sample cell to load the biological sample so as to control the thickness of the sample. Therefore, the transmission detection has several problems: (1) the pretreatment process of the sample is complex and fine, the requirements on experimental conditions and operation technology are high, and the direct application of the terahertz diagnosis technology in clinical environment is limited. (2) The detection result is influenced by the thickness error of the sample, and the detection precision is reduced. (3) The transmission detection brings interference of standing wave resonance in the detection result, and increases the post-processing difficulty of data. Therefore, developing a suitable detection mode to rapidly and accurately detect the terahertz spectrum of the biological sample is a problem that the terahertz technology needs to be broken through in practical clinical application.

Disclosure of Invention

In order to solve the problems, the invention provides an early cancer detection method based on a terahertz attenuated total reflection mode, provides a revolutionary technical means for early cancer detection, provides a new technology and a new method for qualitative and quantitative detection for detection research of cancer cells and tumor markers, and the constructed terahertz spectrum fingerprint library aiming at the cancer cells classified by different histologies is expected to become a new index for cancer detection, can effectively improve the diagnosis and monitoring capability of tumor diseases, and has important clinical significance for early diagnosis and prevention of cancers.

In order to achieve the purpose, the technical scheme of the invention is as follows:

An early cancer detection method based on a terahertz attenuated total reflection mode is characterized in that a physical model of interaction between multiple interfaces of a total reflection surface, a sample pool bottom surface, a cell layer and a buffer liquid layer and an evanescent wave is established, total reflection occurs at the sample pool bottom surface-cell layer interface due to the fact that the total reflection surface and the sample pool bottom surface are made of the same material, and a critical angle theta of internal reflection of a contact surface can be obtained according to the following formula_cThe relationship is as follows:

In the above formula n_Sam、n_SiRefractive index of sample and silicon prism, penetration depth d of evanescent wave in cell layer-buffer layer_pThe relationship with wavelength λ is as follows:

The above formula characterizes the penetration of the evanescent wave through the cell and buffer layers with exponential decay, d_pIs the characteristic penetration depth at which the electric field intensity of the terahertz wave decays to 1/e at the surface, λ is the incident wave wavelength, and θ is the incident angle.

Further, placing the clinical sample in the sample area of the sample cell, loading the sample cell on the total reflection surface, rotating the sample cell to make the evanescent wave completely act on the reference area of the sample cell, the output electric field amplitude EREf is the reference signal, placing the clinical sample in the sample area of the sample cell, loading the sample cell on the total reflection surface, rotating the sample cell to make the evanescent wave completely act on the reference area of the sample cell, rotating the sample cell again to make the evanescent wave completely act on the sample area of the sample cell, the terahertz emergent signal can be obviously changed, the output electric field amplitude at this momentis the sample signal. If the amplitude of the electric field of the incident terahertz wave is E_InThen according to the law of reflection, can obtain

In the formula r_RefThe reflection coefficient when no sample is injected, in this case the reflection coefficient of the reference zone;is the reflectance of the sample area. According to Fresnel reflection law, the reflection coefficient of the interface of the total reflection surface and the cell layer under P polarizationis composed of

In the formula of_SiIs the complex dielectric constant of the bottom surface of the sample cell,The complex dielectric constant of the material close to the total reflection surface. Reflection coefficient of cell layer-buffer interface under P polarizationIs composed of

In the formulaIs the complex dielectric constant of the buffer. Then the simultaneous formulas (4) and (5) can be obtainedIs composed of

Wherein d is the thickness of the cell layer, and the terahertz complex dielectric characteristic spectrum of the cell layer can be obtained by combining the formulas (3) and (6).

compared with the prior art, the invention has the beneficial effects that: the change in the biological molecules and chemical components contained in cancerous cells relative to normal cells is earlier than the change in cell morphology that can be observed with light microscopy. Intermolecular interaction forces (hydrogen bonds, van der waals forces and the like) and intramolecular forces (molecular skeleton vibration, dipole rotation and the like) of intracellular biological molecules and chemical molecules are just positioned in the terahertz frequency band. When the cells cancerate, the changes of intracellular water content, biological molecules and chemical substances can be sensitively reflected on the terahertz characteristic spectrum. Therefore, the detection of the terahertz characteristic fingerprint spectrum based on the cell has higher sensitivity and accuracy than the detection based on the cell morphology. According to the physical optical principle of attenuated total reflection, the acting distance between the evanescent wave and the sample is irrelevant to the thickness of the sample, the technology effectively avoids the error caused by the thickness in the detection of the active biological sample, and improves the detection precision. In addition, evanescent waves firstly enter the living cell layer, the action depth of the evanescent waves is matched with the cell thickness, and the emergent terahertz signals mainly reflect the terahertz physicochemical property of the cell layer, so that the detection sensitivity and specificity are improved. Finally, the technology simplifies the pretreatment process of the sample, can meet the requirements of clinical detection, and realizes a new technology for real-time and rapid detection of cancer without pretreatment and reagent.

Drawings

Fig. 1 is an integrated diagram of an attenuated total reflection module and a terahertz time-domain spectroscopy system.

FIG. 2 is a tumor marker detection map of terahertz spectroscopy and microfluidics.

FIG. 3 is a DNA terahertz spectrum detection diagram based on an array micro-channel and a metamaterial biosensing chip.

FIG. 4 is a conformational diagram of alanine polypeptides of different chain lengths in aqueous solution.

Fig. 5 is a diagram showing the position of a terahertz wave in the electromagnetic spectrum and molecular spectrum information reflected by each electromagnetic band.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.