CN111610155A - A terahertz device for the capture and detection of circulating tumor cells - Google Patents

Abstract

The present disclosure discloses a terahertz device for capturing and detecting circulating tumor cells, comprising: a silicon dioxide substrate, a graphene film coated on the silicon dioxide substrate; the graphene film is etched with A plurality of annular nano-gap, the plurality of annular nano-gap are arranged in an array to form a periodic annular nano-gap array; the annular area of each annular nano-gap is a capture and detection area, which is used for the detection of circulating tumors. Cells are captured and used for detection of captured circulating tumor cells. The present disclosure utilizes graphene coaxial holes to form terahertz plasmonic tweezers to achieve near-field capture of tumor cells, and at the same time combines the tunable optical properties of graphene to meet the requirements of tumor cells of different sizes and refractive indices for capture performance , to achieve specific capture of circulating tumor cells.

Description

A terahertz device for the capture and detection of circulating tumor cells

technical field

The present disclosure relates to a terahertz device, in particular to a terahertz device for capturing and detecting circulating tumor cells and a preparation method thereof.

Background technique

As a global public health problem, cancer greatly endangers human life and health. It is worth noting that a large number of cancer patients have already developed distant tumor metastasis at the time of initial diagnosis, thus missing the best treatment period. Therefore, early diagnosis of cancer is of great significance for prolonging the life of patients. At present, the clinical detection of cancer mainly includes imaging and pathological detection. However, the hysteresis of imaging information makes early detection and diagnosis impossible for imaging detection; pathology-based tissue biopsy technology has the advantages of accurate diagnosis and so on. However, it has limitations such as large trauma, difficult sampling, limited sampling window period, and possible increased risk of metastasis, and has limited effects on early diagnosis, metastasis, and prognosis evaluation of cancer.

Liquid biopsy is an emerging non-invasive detection technology. It monitors malignant tumors in vivo by collecting patient's body fluids (including blood, saliva, sweat and secretions, etc.) At present, the most studied blood markers include: microRNA, circulating tumor DNA and circulating tumor cells. Among them, circulating tumor cells are cells that fall off from the primary tumor spontaneously or during treatment and enter the peripheral blood, and enter other tissues and organs along with the blood circulation, and grow into new tumor tissue. The presence of circulating tumor cells in the blood indicates the presence of tumors in the body and may have metastasized, which is often a major factor in high mortality in cancer patients. Therefore, circulating tumor cells have become an important marker for monitoring the dynamic development of tumors in real time, and their number and type can be effectively used for early diagnosis, treatment and prognosis evaluation of cancer. However, the content of circulating tumor cells in blood is extremely low, accounting for only one millionth of all blood cells, which requires the detection technology used can efficiently and accurately isolate and detect very few target cells from a large number of cells.

The detection technology based on terahertz wave plasmon resonance sensor is expected to solve the detection problem of circulating tumor cells. The terahertz plasmon resonance sensor is very sensitive to the change of the refractive index of the surrounding environment. The refractive index is an inherent property of the medium, and different types of media have Different refractive indices, so any medium attached to the plasmonic resonance sensor structure corresponds to different resonance frequencies in terms of transmission characteristics, so that the detection of different media can be realized. At present, terahertz plasmon resonance sensors have been widely used in the sensing and detection of various cells, biomolecules and viruses. Terahertz waves refer to electromagnetic waves with a frequency range of 0.1 to 10THz. Since the photon energy of terahertz waves is much smaller than the energy of X-rays, it will not cause harmful ionization to biological macromolecules, biological cells and tissues, which is a perfect fit for circulating tumor cell liquid biopsy. requirements. In addition, the rotation and vibration frequencies of biological macromolecules such as lipids, nucleic acids, proteins, and carbohydrates are just in the THz band, which can effectively generate resonance absorption under the excitation of terahertz wave energy to provide a specific feature identification fingerprint spectrum. Therefore, terahertz wave It has great application potential in the fields of non-destructive, label-free sensing and cancer detection.

At present, the detection technology of circulating tumor cells often adopts traditional tumor cell separation and enrichment technology based on physical properties. This technology mainly separates cancer cells according to the difference in size between cancer cells and normal blood cells. White blood cells (8-10 μm) and red blood cells (<7 μm). This method has the advantages of simple operation and better maintenance of cell integrity and activity, but it has disadvantages such as poor specificity and easy loss of tumor cells beyond a certain size, making it difficult to achieve efficient and specific separation and enrichment of circulating tumor cells. In addition, terahertz plasmonic resonance sensors composed of noble metals as plasmonic materials have problems such as limited sensitivity and non-tunability, and it is difficult to meet the requirements of high-sensitivity detection of different types of microcirculating tumor cells.

SUMMARY OF THE INVENTION

In view of the deficiencies in the prior art, the purpose of the present disclosure is to provide a terahertz device for capturing and detecting circulating tumor cells, using graphene coaxial holes to form terahertz plasmon tweezers to achieve near-field to tumor cells The capture, while combining the tunable optical properties of graphene to meet the capture performance requirements of tumor cells of different sizes and different refractive indices, realizes the specific capture of circulating tumor cells. In addition, the remarkable plasmonic enhancement effect of graphene in the terahertz wave band, the hydrophobic properties of multilayer graphene, and the tunability of graphene are used to improve the interaction between electromagnetic waves and substances and achieve high efficacies to different types of circulating tumor cells. Sensitivity detection.

To achieve the above object, the present disclosure provides the following technical solutions:

A terahertz device for capturing and detecting circulating tumor cells, comprising: a silicon dioxide substrate and a graphene film coated on the silicon dioxide substrate;

A plurality of annular nano-gap is etched on the graphene film, and the plurality of annular nano-gap is arranged in an array to form a periodic annular nano-gap array;

The annular region of each annular nanogap is a capture and detection region for capturing circulating tumor cells and for detecting the captured circulating tumor cells.

Preferably, the periodic annular nanogap array obtains a near-field trapping force by localizing terahertz waves in the trapping region.

Preferably, the detection of the captured circulating tumor cells is performed by detecting the shift amount of the resonance frequency of the terahertz device.

Preferably, the period of the periodic annular nanogap array is 2-20 μm.

Preferably, the inner diameter of each annular nanogap is 1-10 μm.

Preferably, the gap width of each annular nanogap is 1-200 μm.

The present disclosure also provides a method for preparing a terahertz wave-based sensor, comprising the following steps:

S100: Graphene films are formed on silica substrates by chemical vapor deposition;

S200 : forming a plurality of annular nano-gap on the graphene film by focused ion beam etching.

Preferably, the plurality of annular nano-gap is arranged in an array to form a periodic annular nano-gap array.

Preferably, the period of the periodic annular nanogap array is 2-20 μm.

Preferably, the inner diameter of each annular nanogap is 1-10 μm.

Compared with the prior art, the beneficial effects brought by the present disclosure are:

1. A new circulating tumor cell separation and capture technology is proposed, a circulating tumor cell capture technology based on terahertz plasmon tweezers, which achieves specific non-destructive capture of circulating tumor cells by combining the tunable optical properties of graphene. , compared with the traditional circulating tumor cell separation technology, it has the advantages of simple operation and high specificity;

2. A new circulating tumor cell enrichment technology, based on multi-layer graphene, is proposed. Using the hydrophobic properties of the surface of multi-layer graphene, the concentration of circulating tumor cells in the blood can be indirectly increased. To improve the enrichment effect and detection sensitivity of circulating tumor cells;

3. A new terahertz plasmon resonance sensor, a graphene-based terahertz plasmon resonance sensor, is proposed. Compared with the traditional plasmon resonance sensor composed of noble metals, it has the advantages of simple structure, tunability and high sensitivity. High-sensitivity detection of different types of microcirculating tumor cells can be achieved.

Description of drawings

FIG. 1 is a schematic structural diagram of a terahertz device for capturing and detecting circulating tumor cells according to an embodiment of the present disclosure;

FIG. 2 is a top view of a terahertz device for capturing and detecting circulating tumor cells according to an embodiment of the present disclosure;

3 is a schematic diagram of a single graphene coaxial hole in a terahertz device for capturing and detecting circulating tumor cells according to an embodiment of the present disclosure;

Figure 4(a) and Figure 4(b) are schematic diagrams of the shapes of blood droplets on the surfaces of the graphene film and the precious metal film, respectively;

FIG. 5 is a schematic diagram of circulating tumor cells being trapped at the nanogap according to an embodiment of the present disclosure;

FIG. 6 is a schematic diagram of a reflectivity curve of a terahertz wave-based sensor provided by another embodiment of the present disclosure.

Detailed ways

Specific embodiments of the present disclosure will be described in detail below with reference to FIGS. 1 to 6 . While specific embodiments of the present disclosure are shown in the drawings, it should be understood that the present disclosure may be embodied in various forms and should not be limited by the embodiments set forth herein. Rather, these embodiments are provided so that the present disclosure will be more thoroughly understood, and will fully convey the scope of the present disclosure to those skilled in the art.

It should be noted that certain terms are used in the description and claims to refer to specific components. It should be understood by those skilled in the art that the same component may be referred to by different nouns. The present specification and claims do not take the difference in terms as a way to distinguish components, but take the difference in function of the components as a criterion for distinguishing. As referred to throughout the specification and claims, "comprising" or "including" is an open-ended term and should be interpreted as "including but not limited to". Subsequent descriptions in the specification are preferred embodiments for implementing the present invention, however, the descriptions are for the purpose of general principles of the specification and are not intended to limit the scope of the present invention. The scope of protection of the present disclosure should be defined by the appended claims.

To facilitate the understanding of the embodiments of the present disclosure, the following will take specific embodiments as examples for further explanation and description in conjunction with the accompanying drawings, and each accompanying drawing does not constitute a limitation to the embodiments of the present disclosure.

In one embodiment, as shown in FIG. 1 , a terahertz device for capturing and detecting circulating tumor cells includes: a silicon dioxide substrate, a graphene film coated on the silicon dioxide substrate, and the A plurality of annular nano-gap is etched on the graphene film, and the plurality of annular nano-gap is arranged in an array to form a periodic annular nano-gap array; the annular area of each annular nano-gap is used for capturing and detecting Region for the capture of circulating tumor cells and for detection of the captured circulating tumor cells.

In this embodiment, since the noble metal has fixed free electrons, once the sensing device composed of the noble metal is fabricated, its working performance is difficult to change, and cannot meet the requirements for capturing and detecting different types of circulating tumor cells. In contrast, the carrier density of graphene can be regulated by the gate voltage, which provides the possibility for the fabrication of tunable sensing devices. In addition, compared with noble metals, graphene also has the advantages of high carrier concentration, high mobility, and low optical loss, which can significantly improve the field localization of electromagnetic waves, thereby enhancing the interaction between terahertz waves and the object to be tested. , which provides conditions for the realization of high-sensitivity detection. Therefore, compared with the problems of non-tunability and limited sensitivity of the sensor composed of precious metals used in the prior art, this embodiment uses a graphene-based terahertz device for the analysis and detection of circulating tumor cells, which can obtain better detection results. Exemplarily, Figure 4(a) and Figure 4(b) are schematic diagrams of the shapes of the same volume of blood dropped on the graphene film (hydrophobic interface) and the surface of the noble metal film (hydrophilic interface), respectively, as shown in Figure 4 ( As shown in a) to Fig. 4(b), the blood droplets form contact angles θ ₁ and θ ₂ on the surface of the multilayer graphene and the surface of the noble metal film, respectively, and θ ₁ >θ ₂ . The larger contact angle _θ1 indicates that the blood droplet has a smaller contact area and higher height on the graphene surface. When the concentration of circulating tumor cells in the measured blood is constant, a smaller contact area will indirectly increase the spatial density of circulating tumor cells per unit area, thereby increasing the probability of being measured. Therefore, compared with the traditional sensor composed of metal thin films, due to the hydrophobicity of the surface of the sensor composed of multilayer graphene, the concentration of circulating tumor cells in the blood per unit area can be indirectly increased, thereby enhancing the enrichment effect of circulating tumor cells and improving the its detection sensitivity.

In another embodiment, the periodic annular nanogap array achieves near-field trapping force by localizing terahertz waves within the trapping region.

In this embodiment, under the radiation of the terahertz electromagnetic wave, the free electrons in the graphene film collectively oscillate and gather at the boundary of the annular nanogap, which makes the terahertz electromagnetic wave strongly localized near the nanogap, And a local hot spot and a huge electromagnetic field enhancement are generated at the nanogap. Since the electromagnetic field intensity decreases rapidly with the distance from the center of the local hot spot, a near-field trapping force exerted on the cell is generated and a three-dimensional trapping potential is formed on the graphene surface. When the size of the three-dimensional trapping potential well is large enough to overcome the irregular Brownian motion of the cells, the cells will move to the local hot spot center (ie the lowest potential energy point) and be bound there under the impetus of the trapping force. Since the magnitude of the near-field capture force is proportional to the strength of the electromagnetic field, the size and refractive index of the captured cells, and the size and refractive index parameters of circulating tumor cells are higher than other cells in the blood (eg, red blood cells, white blood cells). Therefore, when the graphene plasmon structure is determined, it is only necessary to adjust the Fermi level to control the graphene plasmon resonance intensity to realize the regulation of the capture ability of the device, and then obtain enough to capture circulating tumor cells but not enough to capture other cells. The capture ability finally achieves the specific non-destructive capture of circulating tumor cells. Compared with the traditional separation and enrichment technology based on the size difference of circulating tumor cells, this example is based on the difference of two key physical properties of circulating tumor cell size and refractive index, so it has higher specificity.

In another embodiment, the detection of the captured circulating tumor cells is performed by detecting the shift of the resonance frequency of the terahertz device.

In this example, due to the localization and enhancement of the electromagnetic field of the terahertz wave, the terahertz wave is very sensitive to the dielectric properties of the graphene film surface. When the circulating tumor cells are stably captured in the capture area, the PBS buffer is used to rinse the blood The presence of circulating tumor cells will perturb the dielectric properties of the graphene film surface, resulting in a shift in the resonance frequency of the terahertz device, detected by a terahertz time-domain spectroscopy system. The resonant frequency of the reflection spectrum/transmission spectrum of the device and its shift amount can realize the detection of the biological information carried by the circulating tumor cells. Exemplarily, as shown in Fig. 5, when the period of the graphene annular nanogap array is P=3μm, the inner diameter of the gap is D=2μm, the gap width is w=20nm, and the graphene Fermi level is 0.6eV, when the terahertz wave is Under the effect of the radiation, the circulating tumor cells in the blood will be captured in the nano-gap, and the red blood cells and white blood cells in the blood are washed with PBS buffer at this time, and finally the captured circulating tumor cells are left. Assuming that there are no circulating tumor cells, the refractive index of the blood to be tested is n=1.5, and the resonant frequency f ₀ of the device is 5.0THz. When the blood to be tested contains circulating tumor cells, the refractive index n=2.0, that is The rate disturbance Δn is 0.5, as shown in Fig. 6, and the resonant frequency shift Δf of the terahertz device is 0.9 THz at this time. Due to the different absorption frequency bands of terahertz waves by different biological cells, information such as the number and type of circulating tumor cells can be obtained by comparing and analyzing the resonant frequency of the terahertz device and its movement amount, so as to realize the diagnosis of cancer patients. The normalized sensitivity S' _f of the terahertz device used for the capture and detection of circulating tumor cells can be expressed as S' _f =Δf/(Δn·f ₀ ), and the normalized sensitivity of the terahertz device described in this example It can reach 0.36THz/RIU, while the normalized sensitivity of traditional plasmonic resonance sensors is only 0.01-0.1THz/RIU. In addition, according to the requirements of different types of circulating tumor cells for the capture parameters and the different resonance frequencies of intracellular biomolecules in the terahertz frequency range, the Fermi level of the graphene film is regulated by the external field voltage regulator to realize the capture ability and The resonance frequency is adjusted to meet the capture performance requirements of different circulating tumor cells and the resonance frequency of molecules in tumor cells, and finally realize the specific capture and high-sensitivity detection of different types of circulating tumor cells.

In another embodiment, the period of the periodic annular nanogap array is 2-20 μm.

In another embodiment, the inner diameter of the annular nanogap is 1-10 μm.

In this embodiment, the feature sizes of devices based on metamaterial structures are all sub-wavelength scales (ie, 0.1 to 1 times the wavelength), and the wavelength range of terahertz waves is 30 μm-3000 μm, so the inner diameter of the annular nanogap is selected as 1 -10μm.

In another embodiment, the gap width of the annular nanogap is 1-200 nm.

In this embodiment, in order to achieve localization and enhancement of terahertz waves, the width of the nanogap is selected to be 1-200 nm.

What needs to be understood is that the width of the nanogap is set to the nanometer level to achieve localization and enhancement of the terahertz wave, thereby exciting the resonance of the device.

It also needs to be understood that when the circulating tumor cells are stably trapped in the trapping region, as the nanogap width w decreases, the field localization and enhancement effect of the electromagnetic wave becomes stronger, making the electromagnetic wave change more on the interface dielectric properties. In order to be sensitive, the detection sensitivity of the sensing device can be improved by reducing the nanogap width w.

In another embodiment, the present disclosure also provides a method for preparing a terahertz wave-based sensor, comprising the following steps:

S100: Graphene films are formed on silica substrates by chemical vapor deposition;

S200 : forming a plurality of annular nano-gap on the graphene film by focused ion beam etching.

The basic principles of the present application have been described above in conjunction with specific embodiments. However, it should be pointed out that the advantages, advantages, effects, etc. mentioned in the present application are only examples rather than limitations, and these advantages, advantages, effects, etc., are not considered to be Required for each embodiment of this application. In addition, the specific details disclosed above are only for the purpose of example and easy understanding, rather than limiting, and the above-mentioned details do not limit the application to be implemented by using the above-mentioned specific details.

The foregoing description has been presented for the purposes of illustration and description. Furthermore, this description is not intended to limit the embodiments of the application to the forms disclosed herein. Although a number of example aspects and embodiments have been discussed above, those skilled in the art will recognize certain variations, modifications, changes, additions and sub-combinations thereof.

Claims (10)

1. A terahertz device for capturing and detecting circulating tumor cells, comprising: a silicon dioxide substrate, a graphene film coated on the silicon dioxide substrate; A plurality of annular nano-gap is etched on the graphene film, and the plurality of annular nano-gap is arranged in an array to form a periodic annular nano-gap array; The annular region of each annular nanogap is a capture and detection region for capturing circulating tumor cells and for detecting the captured circulating tumor cells. 2 . The terahertz device according to claim 1 , wherein, preferably, the periodic annular nanogap array obtains a near-field trapping force by localizing terahertz waves in the trapping region. 3 . 3. The terahertz device according to claim 1, wherein the detection of the captured circulating tumor cells is performed by detecting the amount of shift of the resonance frequency of the terahertz device. 4. The terahertz device according to claim 1, wherein the period of the periodic annular nanogap array is 2-20 μm. 5. The terahertz device of claim 1, wherein the inner diameter of each annular nanogap is 1-10 μm. 6. The terahertz device of claim 1, wherein the gap width of each annular nanogap is 1-200 nm. 7. A method for preparing the arbitrary described terahertz device of claim 1-6, comprising the steps: S100: Graphene films are formed on silica substrates by chemical vapor deposition; S200 : forming a plurality of annular nano-gap on the graphene film by focused ion beam etching. 8 . The method of claim 7 , wherein the plurality of annular nanogaps are arranged in an array to form a periodic annular nanogap array. 9 . 9. The method of claim 7, wherein the periodic annular nanogap array has a period of 2-20 [mu]m. 10. The method of claim 7, wherein each annular nanogap has an inner diameter of 1-10 [mu]m.

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