Capillary optical fiber optical funnel capable of being used for generating single cell flow

Abstract

The invention provides a capillary optical fiber optical funnel capable of generating single cell flow. The device is characterized by comprising a capillary optical fiber and a light source. Experiment sample liquids of different types of cells (particles) are injected into the microfluidic channel 1 of the optical funnel, light emitted by the light source 2 can form the optical funnel structure 3 at the emergent end through the annular core of the capillary optical fiber, the different cells (particles) in the experiment sample liquids are constrained by the optical funnel, and the motion trails are focused on the central axis. The discrete flowing cell (particle) flow forms a regular linear cell (particle) flow, and during the period, the cell (particle) flow can be monitored and observed in real time through an imaging system. The micro-fluidic chip can be used as a functional module in the micro-fluidic chip, is used for cell sorting, circulating tumor cell detection and the like, and can realize detection and analysis of various medical biological components in different forms by combining with the structural design of a micro-channel in the chip.

Description

Technical Field

[0001] The present invention relates to a capillary fiber optic funnel capable of generating single-cell flow. The funnel can be used as a functional module in a microfluidic chip for applications such as cell sorting and circulating tumor cell detection. By combining the structural design of the chip's internal microchannels, it can enable detection and analysis of various medical biological components in different forms. Background Art

[0002] Circulating tumor cells (CTCs) usually enter the blood in the early stages of cancer. Therefore, the development of new CTCs detection technologies is of great significance for the early detection and screening of cancer.

[0003] Metastasis is the leading cause of cancer-related death and can occur in patients with aggressive cancers at an early stage of tumor development. The first step in metastatic spread is the invasion of cancer cells into the blood circulation, through which the cells can spread to other parts of the body. Even in patients with early-stage small tumors, early metastases can spread to distant sites. Circulating tumor cells (CTCs) in the blood may be the first indicator of the early steps of cancer metastasis. Metastasis begins with the local invasion of primary tumor cells into their surrounding microenvironment and the subsequent migration across the endothelial barrier and into the blood or lymphatic system. Once in the blood circulation, CTCs are carried by the blood to other parts of the body, where they can extravasate, proliferate and form metastatic lesions.

[0004] As we all know, cancer is one of the leading causes of death worldwide, and cancer is usually diagnosed in the late stages. Late-stage cancer diagnosis severely limits the choice of treatment options and usually leads to poor prognosis. Therefore, early detection of cancer is not only important for optimizing early treatment options and improving cure rates, but also crucial for improving patient prognosis. Currently, many strategic planning projects for early cancer detection have been launched around the world, including the European Commission-led Europe's Beating Cancer Plan (launched in 2020), the UK Research and Innovation Agency's Accelerating Detection of Disease Challenge (2019-2025), and the Cancer Moonshot led by the National Institutes of Health / National Cancer Institute of the United States.

[0005] The spread of cancer cells occurs early but is usually discovered late. Currently, the gold standard for cancer diagnosis is histopathology, which usually involves invasive biopsy of solid tumor tissue. Compared with the tissue biopsy technology commonly used in clinical practice, liquid biopsy, as a means of cancer detection, has many significant advantages, such as early detection, non-invasiveness, high accuracy, simple sampling, and low price. Liquid biopsy has shown great potential in the early detection, dynamic monitoring and targeted treatment of various cancers, including lung cancer, breast cancer, cervical cancer, prostate cancer, etc., especially the separation of CTCs in the blood, the development of mass spectrometry analysis and single-cell multiplex fluorescence detection technology, which has made early detection and early treatment of cancer possible.

[0006] Growing evidence suggests that metastasis is an early event in patients with aggressive cancers, occurring even before the primary lesion is clinically detectable. Metastasis is typically formed by cancer cells that spread through the bloodstream to distant, non-malignant tissues. As early as 2013, circulating tumor cells (CTCs) were defined as an early cancer detection method, providing patients with a new way to accelerate diagnosis and initiation of treatment while avoiding overdiagnosis and treatment of slow-growing, milder tumors.

[0007] Physical separation and enrichment of CTCs is based on differences in size, density, deformability, and electrical properties between CTCs and blood cells. CTCs are typically larger. Microfluidics-based cell sorting methods use a combination of intrinsic (e.g., fluid dynamics) and extrinsic (e.g., magnetic, electric, acoustic, and optical) techniques to separate cells. Target cells are then selected from a heterogeneous sample based on their distinct physical and biological properties. Because CTCs are larger than other blood cells and exhibit greater geometric variability, mechanical microstructured cell diversion offers the advantages of simplicity and low cost, but has the disadvantage of being susceptible to cell damage. Therefore, in microfluidic chips, focusing the sample fluid minimizes contact with the microchannel walls, reducing the potential for sample contamination and the risk of clogging within the microchannel. For microfluidic chips with cell (particle) screening capabilities, pre-focusing to form a single-file stream of cells (particles) is an essential and important step. Hydraulic focusing is a commonly used method for fluid focusing.

[0008] The article published by Cheng Jingmeng et al. in 2017 is "Simulation of single-cell flow formation based on hydrodynamic focusing in microfluidics" (Cheng Jingmeng, Zhang Sixiang, Li Xinran, etc. Simulation of single-cell flow formation based on hydrodynamic focusing in microfluidics [J]. Micro-Nano Electronic Technology, 2017, 54(03): 168-172.), and the article published by Yogesh M. Patel et al. in 2020 is "An inexpensive microfluidic device for three-dimensional hydrodynamic focusing in imaging flow cytometry" (Patel YM, Jain S, Singh AK, et al. An inexpensive microfluidic device for three-dimensional hydrodynamic focusing in imaging flow cytometry[J]. Biomicrofluidics, 2020, 14(6): 064110.), Song Feifei et al. published an article in 2020 entitled "Microfluidic chip design and flow field analysis based on countercurrent sheath liquid" (Song Feifei, Ma Yuting, Wu Yunliang, et al. Microfluidic chip design and flow field analysis based on countercurrent sheath liquid[J]. Laboratory Research and Exploration, 2020, 39(04): 29-32.) A set of hydraulic focusing models with countercurrent sheath liquid were established using the two-phase flow module, and the countercurrent sheath liquid hydraulic focusing model reflected the focusing law of the sample liquid.

[0009] Inertial microfluidics has attracted widespread attention in recent years due to its good application prospects in cell filtration, sorting and blood cell counting. As a passive manipulation technology, it can manipulate cells and particles in microchannels without an external field and has the advantage of extremely high throughput. In this phenomenon, cells and particles migrate along streamlines and sequentially at equilibrium positions near the channel wall. This phenomenon is due to the action of inertial forces, but in microfluidics with low Reynolds numbers, this inertial force is often ignored. In a straight channel, inertial offset is generally considered to be caused by the balance force of the lift (shear-induced lift) caused by the curvature of the velocity profile and the interaction force (wall-induced lift) between the particle and the channel wall. In a square channel, the inertial migration of cells or particles causes them to focus on the four equilibrium position centers on the channel surface (Jian Zhou and Ian Papautsky, Fundamentals of inertial focusing in microchannels, Lab Chip, 2013, 13, 1121-1132.).

[0010] To address the shortcomings of prior technologies, the present invention designs a photodynamic manipulation device capable of single-cell focusing using a capillary optical fiber. This device utilizes a conical shell-shaped light field distribution at the end of the tapered capillary optical fiber to focus cells flowing through the conical shell-shaped light field into a single-cell stream. We figuratively call this device an "optical funnel." Summary of the Invention

[0011] The purpose of the present invention is to provide a capillary fiber optic funnel that can generate single-cell flow. As a functional module in a microfluidic chip, it is used for applications such as cell sorting and detection of circulating tumor cells. By combining the structural design of the microchannels inside the chip, it can realize different forms of detection and analysis of various medical biological components.

[0012] The object of the present invention is achieved like this:

[0013] This capillary fiber optical funnel that can generate single cell flow is based on the capillary fiber structure. The capillary fiber is composed of a ring core and a hollow air hole. This type of fiber has a hollow air hole running through the central axis and a ring core surrounding the hole. Figure 1 As shown. A small hole channel is processed on the side of the capillary fiber by femtosecond laser micromachining, and discrete cells (particles) are injected into the air hole of the hollow capillary fiber through the hole. One end of the capillary fiber is collapsed by heating to close the air hole, and a single-mode optical fiber is welded to the collapsed point for injecting a 980nm control light source. The other end of the capillary fiber is processed into a cone by grinding the optical fiber end. After the 980nm light source is transmitted to the cone reflection surface through the waveguide on the inner wall of the optical fiber, a funnel-shaped light field is formed, which is called an "optical funnel". When cells (particles) pass through the "optical funnel" area, due to the effect of light radiation pressure, discrete cells (particles) will form a three-dimensional single cell (particle) flow after passing through the funnel area, as shown Figure 2 shown.

[0014] When cells (particles) flow through the capillary fiber and enter the outgoing light field with an annular cone distribution, the cells (particles) are pushed by the light radiation pressure. The stronger the light field distribution, the greater the light radiation pressure. Therefore, the cells will accelerate along the area with stronger light field distribution until they reach the central axis of the annular light field focus. The discrete flowing cells (particles) can be focused to form a single cell (particle) flow, which is like the function of a "funnel". Therefore, it is figuratively called an "optical funnel". Figure 3 The comparison diagram of capillary optical fiber before and after polishing is shown in the figure. Figure 3 (c) is a three-dimensional stereogram of the numerical simulation calculation of the light field focusing of the cone angle of the capillary optical fiber cone.

[0015] The "optical funnel" effect of the annular cone of light formed by the capillary fiber enables the transport of living single cells (particles) into other microchannels within the microfluidic chip. Furthermore, the focusing ability and transport speed of the optical funnel can be controlled by adjusting the injected light power according to the movement speed of the cells (particles).

[0016] The light field at the end of the capillary fiber cone acts as a focusing "optical funnel" for the flowing cells (particles) therein. 980nm laser light is injected from the ring fiber core and converges after passing through the fiber end cone. The force on the cells (particles) in the "optical funnel" can be considered in two areas: the inner wall and the outer wall. The force analysis is as follows: Figure 4 As shown, Figure 4 (a) is a force analysis diagram of the inner wall of the "funnel". Assuming that the flow velocity distribution of the liquid in the optical fiber is U0(R), when the cell (particle) reaches the inner wall of the funnel, it will be subject to two forces. One is the viscous resistance F 流 , and the other is the light radiation pressure F 光 , the direction of light radiation pressure is outward, F 流 and F 光 The combined force changes the direction of movement of the cell (particle), and the cell (particle) moves toward the outer wall of the "funnel". When the cell (particle) moves to the outer wall of the "funnel", the force analysis is as follows: Figure 4 As shown in (b), the light radiation pressure F 光 , the direction is inward, at this time F 流 and F 光 The direction of the resultant force is inward. Therefore, cells (particles) will move along the wall of the "funnel" to the funnel mouth, forming a single cell (particle) flow.

[0017] The constraint equations describing the motion of cells (particles) are:

[0018] F 光 =ma 光 (1)

[0019] F 流 =σ*(v cell -U0(R)) (2)

[0020] F 合 =F 光 +F 流 (3)

[0021] Compared to prior art, the present invention offers significant advantages: by utilizing a capillary optical fiber structure, the fiber tip is ground into a cone with an appropriate taper angle. This allows the development of a novel "optical funnel" device that can focus cells (particles) in a cell (particle) liquid into a single cell (particle) stream. Furthermore, the present invention can be used as a functional module within a microfluidic chip for applications such as cell sorting and circulating tumor cell detection. Combined with the chip's internal microchannel design, it can perform diverse medical and biological component detection and analysis, adding a novel functional component to the microfluidic chip. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 It is a schematic diagram of the capillary optical fiber structure.

[0023] Figure 2 Schematic diagram of the principle implementation of a capillary fiber optical funnel capable of generating single-cell flow.

[0024] Figure 3 Comparison of capillary optical fiber before and after polishing: (a) is the theoretical calculation result when the capillary optical fiber is not polished, (b) is the theoretical calculation result when the capillary optical fiber end is polished at an angle of 32°, and (c) is a three-dimensional stereogram of the numerical simulation calculation of light field focusing.

[0025] Figure 4 This is a force analysis diagram showing that the light field at the end of the capillary optical fiber cone has a focusing "optical funnel" function for the flow cell therein.

[0026] Figure 5 Schematic diagram of a microfluidic cell detection chip with a capillary fiber optic funnel function that can generate single-cell flow. DETAILED DESCRIPTION

[0027] Below is Figure 5 The present invention is specifically described by taking the microfluidic cell detection chip having the function of a capillary fiber optic funnel capable of generating single-cell flow as an example.

[0028] In the designed CTCs microfluidic chip, before analyzing and identifying cells, all cells must first be arranged in a column, that is, all cells must be focused to form a single-cell stream before the next operation can be performed in the chip. Since the microfluidic channels in the microfluidic chip are on the order of hundreds of microns, while the cells are only a few microns to a dozen microns in size, the present invention allows cell fluid to flow through the hollow air holes of a capillary optical fiber with an annular optical waveguide layer. This design can enable cells in the microfluidics within the capillary optical fiber to converge and form a new type of optical manipulation device for single-cell flow, namely an "optical funnel." Among them, a through hole is machined on the side of the hollow capillary optical fiber using femtosecond laser micromachining, and discrete cells are injected into the air hole of the hollow capillary optical fiber through the through hole. One end of the inner wall waveguide hollow capillary optical fiber is heated and collapsed to eliminate the air hole, and a single-mode optical fiber is welded to the collapsed point for injecting a 980nm manipulation light source. The other end of the hollow capillary fiber is machined into a cone by grinding the fiber end. The 980nm light source is transmitted through the waveguide on the inner wall of the fiber to the cone, where it is reflected to form a funnel shape, which we call an "optical funnel." When cells pass through the "optical funnel" area, due to the effect of light radiation pressure, discrete cells will form a three-dimensional single-cell flow after passing through the funnel area.

[0029] The present invention proposes a capillary fiber optic funnel that can generate single-cell flow. The specific preparation process is as follows:

[0030] The method for transmitting light through a ring core in a hollow capillary fiber involves collapsing the air space using a fused cone method, while simultaneously pumping out the air. After the cone collapses, it is cut open with a cleaver and welded to a single-mode fiber, completing the coupling of light from the single-mode fiber to the ring core. To minimize insertion loss, different cone waist angles can be tried to find the one that minimizes loss.

[0031] Capillary fiber side drilling process: strip the coating off the light and place it in a fixture, import the pattern to be removed into the processing system, and after processing, use hydrofluoric acid to wash away the residual material to complete the side drilling of the fiber end.

[0032] Experimental process of focusing effect of cells of different sizes: Rhodamine solution is added to the experimental cell fluid, and a 532nm laser is passed as an indicator light to evaluate the funnel focusing situation; a high-power 980nm laser is passed as the light to form the "funnel" effect. By mixing it with cells of different diameters, the cell focusing effect can be observed under a microscope, and the cell movement trajectory can be recorded with the help of high-speed camera acquisition.

[0033] Then, the capillary fiber optical funnel is combined with the microfluidic chip, and the corresponding chip channel structure and packaging part are formulated according to the functional structure of the chip. The microfluidic chip channel is formed by bonding a quartz substrate and a PDMS cover sheet. The microfluidic channel is made on the quartz substrate by femtosecond laser micromachining. The cell fluid inflow outlet is realized by punching holes on the PDMS with a puncher. Among them, the capillary fiber-based optical funnel for realizing single cell flow is embedded in the microfluidic chip (1) area, and the micropores processed on the side of the fiber are placed upward, opposite to the cell fluid inlet. The cell fluid flows into the air hole of the capillary fiber through the injection port; after passing through different functional modules, the cells will be diverted (2), and the corresponding waste cells 2 and cells 3 will be discharged. The CTC cells (3) after analysis flow out from the outlet and are collected for subsequent use.

Claims

1. The present invention provides a capillary fiber optic funnel capable of generating single-cell flow. Its characteristics are: It consists of a capillary optical fiber and a light source. Experimental sample liquids of different types of cells (particles) are injected into the microfluidic channel 1 of the optical funnel. The light energy emitted by the light source 2 passes through the annular core of the capillary optical fiber to form an optical funnel structure 3 at the output end. The different cells (particles) in the experimental sample liquid are bound by the optical funnel, and the movement trajectory is focused on the central axis. The discrete flow of cell (particle) streams forms a regular straight cell (particle) stream, during which the cell (particle) stream can be monitored and observed in real time by an imaging system. The present invention can be used as a functional module in a microfluidic chip for applications such as cell sorting and detection of circulating tumor cells. By combining the structural design of the microchannel inside the chip, different forms of detection and analysis of various medical biological components can be realized.

2. The capillary fiber optic funnel capable of generating single cell flow according to claim 1, characterized in that: The capillary optical fiber structure is composed of an annular fiber core and a hollow air hole. This type of optical fiber has a hollow air hole running through the central axis and an annular fiber core surrounding the hole.

3. The capillary fiber optic funnel capable of generating single cell flow according to claim 1, wherein: The side of the capillary optical fiber can be micro-processed and punched to increase the cell (particle) inlet, so that one or more cells (particles) can be injected into the hollow hole of the capillary optical fiber at the same time.

4. The capillary fiber optic funnel capable of generating single cell flow according to claim 1, wherein: The optical funnel can be further combined with a traditional microfluidic chip to connect the cell (particle) inlet with the microfluidic material channel, serving as a functional module in the traditional microfluidic chip.

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