Hollow optical fiber optical tweezers

Abstract

The invention provides a pair of novel hollow optical fiber optical tweezers. The device is characterized by comprising a hollow optical fiber and a light source. Experiment sample liquids of different types of cells (particles) are injected into the microfluidic channel 1 of the hollow optical fiber optical tweezers, light emitted by the light source 2 can form an optical tweezers effect at the emergent end through a centrosymmetric fiber core of the hollow optical fiber and capture the cells (particles), and the different cells (particles) in the experiment sample liquids are controlled in the capture area 3 through constraint of light field force. The micro-fluidic chip can be used as a functional module in the micro-fluidic chip, has the functions of capturing, shunting and detecting cells (particles) and the like, is additionally provided with a novel optical fiber photodynamic control device by combining with the structural design of a micro-channel in the chip, and is used for realizing detection and analysis of various medical biological components in different forms; and a new technical basis is provided for early screening of cancers.

Description

Technical Field

[0001] The present invention relates to a novel hollow fiber optic tweezers, which can be used as a functional module in a microfluidic chip for applications such as cell capture, diversion, and detection. By combining the structural design of the microchannels inside the chip, a new fiber optic photodynamic manipulation device is added to the chip to realize different forms of detection and analysis of multiple medical biological components, providing a new technical foundation for early cancer screening. 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 methods for CTCs are based on differences between CTCs and blood cells in size, density, deformability, and electrical properties. CTCs are typically larger. Microfluidics-based cell sorting methods use a combination of "intrinsic" (such as fluid dynamics) and "extrinsic" (such as magnetic fields, electric fields, acoustics, and optical forces) 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 their geometric shape is highly variable, mechanical microstructure cell diversion offers the advantages of simple operation and low cost, but has the disadvantage of being prone to cell damage. Therefore, in microfluidic chips, focusing the sample fluid minimizes contact with the microchannel walls, reducing the possibility of sample contamination and the risk of clogging within the microchannel. For microfluidic chips with cell (particle) screening capabilities, the ability to capture and manipulate single cells (particles) is an essential and important function.

[0008] The invention patent with patent number CN201510295509.8 proposes a tunable liquid microsphere laser. In this patent, two fiber optic tweezers are required to capture the microspheres at the same time, and the signal light is collected by using the fiber output at one end and the fiber reception at the other end; the invention patent with patent number CN201510267391.8 proposes a droplet whispering gallery mode laser and its manufacturing method. In this patent, the input light is coupled into the ring core by hot-melt taper of single-mode fiber and ring core fiber. The droplet also needs to contact the micro-nano fiber to transmit the signal light out; the invention patent with patent number 201810169543.4 proposes a living single-cell multifunctional spectrometer based on coaxial dual-waveguide fiber. The cell micro-light hand mentioned in this patent is similar to the principle used in this patent, which is to capture cells (particles) with a centrally symmetrical fiber core, but the device structures are different. The present invention uses a hollow fiber structure and also adds a variety of new functions such as fiber optic tweezers capture, diversion and detection.

[0009] In response to the shortcomings of the prior art, the present invention adopts a novel hollow fiber optical tweezers composed of a circular fiber core. The optical tweezers, with the help of the conical shell-shaped light field distribution at the end of the conical hollow fiber, have the functions of capturing, diverting and detecting cells (particles) flowing through the conical shell-shaped light field. Summary of the Invention

[0010] The purpose of the present invention is to provide a novel hollow fiber optic tweezers that can be used as a functional module in a microfluidic chip for applications such as cell capture, diversion and detection. By combining the structural design of the microchannels inside the chip, a new fiber optic photodynamic control device is added to the chip to realize different forms of detection and analysis of various medical biological components, providing a new technical basis for early cancer screening.

[0011] The object of the present invention is achieved like this:

[0012] This novel hollow fiber optical tweezers is based on the hollow fiber structure. The hollow fiber is composed of a circular fiber core and a hollow air hole. The fiber has a hollow air hole running through the central axis and a circular fiber core. Figure 1As shown. A small hole channel is processed on the side of the capillary fiber by femtosecond laser micromachining, and the mixed cells (particles) are injected into the air hole of the hollow fiber through the hole. One end of the hollow fiber is collapsed by heating to close the air hole, and is welded to the collapsed point with a single-mode fiber for injecting a 980nm control light source. The other end of the hollow fiber is processed into a cone by grinding the fiber end. The light field formed after the 980nm light source is transmitted to the cone reflection surface through the waveguide on the inner wall of the optical fiber can be directly used to capture cells (particles) flowing out of the optical fiber, so it is called a hollow fiber tweezers. When cells (particles) pass through the capture area of ​​the hollow fiber tweezers, the discrete cells (particles) will be stabilized in the capture area due to the action of the light trap force. Figure 2 The outer dotted line part is the optical radiation area, and the middle dotted line part is the optical tweezers capture area.

[0013] In order to stably capture different types of cells (particles) in a microfluidic environment, it is necessary not only to analyze the force on the captured cells (particles), but also to verify the "power-resistance" balance condition of the cells. Figure 3 When a spherical particle moves slowly in a static unbounded viscous fluid, the resistance it encounters is the Stokes resistance, which is expressed as

[0014] F D =6πμaV p (1)

[0015] Where μ is the viscosity coefficient of the liquid, a is the particle radius, V p is the particle speed. When the fluid has a velocity V, the viscous resistance expression is

[0016] F D =6πμa(VV p ) (2)

[0017] Consider a single ray of power P, whose incident power per unit time is n1P / c, incident on the dielectric sphere at an angle θ. The resultant force through the center of the sphere can finally be decomposed into two components in the z and y directions: F z and F y Quantity

[0018]

[0019]

[0020] where θ and r are the angle of incidence and the angle of reflection, respectively.

[0021] The force analysis diagram of the interaction between the hollow optical tweezers formed at the end of the hollow cone fiber and the cells (particles) flowing through the hollow fiber is shown in the figure. Figure 4In order to capture cells, the grinding cone angle needs to be between 16° and 21°. Figure 2 The outer dotted line is the optical radiation field, and the middle dotted line is the optical tweezers capture area. The optical tweezers capture area can be divided into two areas, with the focus point as the center, the left side as the cell acceleration area, and the right side as the deceleration area. In the cell acceleration area, the cell performs uniform acceleration motion, and the force on the cell can be expressed as:

[0022] f 阻 =σ*v (5)

[0023] f 加 =ma 加 = Light trapping force in region A (6)

[0024] In the cell deceleration zone, the cell performs uniform deceleration motion. The force on the cell in the deceleration zone can be expressed by the following equation:

[0025] f 阻 =σ*v (7) f 减 =ma 减 = Optical trapping force in region B (8)

[0026] The force diagram of a single cell in a flow field is shown in the figure below. Figure 4 (a) and 4 (b). The off-axis light a and light b pass through the cell symmetrically without loss. The cell is located to the left of the focal point. The force diagram is shown in Figure 4 (a) The force F exerted by light a and light b on the cell a and F b Equal, F a and F b The component F in the vertical direction ay and F by Equal in magnitude and opposite in direction; in the direction of beam propagation, the cell is subject to viscous resistance F D and the force F exerted by rays a and b on the cell a and F b The component 2F in the direction of propagation z , F D and 2F z In the same direction, the cells move toward the point where the beams converge.

[0027] When the cell is located to the right of the focal point, such as Figure 4 (b) F a The horizontal component F ay With F b The horizontal component F by Phase equilibrium, F a With F b The components in the direction of beam propagation are equal, in the same direction, and the magnitude is 2F z , and Fz In the opposite direction, the cells will be subject to the viscous resistance F of the fluid. D .

[0028] When the cell is in position 4(b), that is, the center of the cell is on the principal optical axis, the fluid viscous resistance and the optical trap force in the z direction are equal in magnitude and opposite in direction, and the single cell reaches dynamic equilibrium, that is,

[0029] 2F y +F D =0 (9)

[0030] Then we can get the dynamic equilibrium equation about flow velocity,

[0031]

[0032] Based on the micro-flow rate balance condition, the finite element analysis method is used to determine the effective radius of the optical potential well. The calculation results are as follows: Figure 5 shown. Figure 5 (a) is the lateral optical trap force curve of cells (particles) with different diameters. It can be seen from the figure that the larger the particle size, the larger the effective radius of the optical potential well. When the cell (particle) diameter is 16um, the effective radius of the optical potential well is about 10um. Figure 5 (b) is the axial optical trap force curve of cells (particles) with different diameters. It can be seen from the figure that the larger the particle size, the larger the effective radius of the optical potential well. When the cell diameter is 16um, the effective radius of the optical potential well is about 20um.

[0033] Based on the above theoretical analysis results, the experimental results are further verified. Figure 6 As shown, Figure 6 (a) is a diagram showing the experimental processing of microholes on the side of an inner-wall waveguide-type hollow capillary optical fiber using a femtosecond laser micromachining system; Figure 6 (b) is a schematic diagram of the optical fiber end after polishing, with a cone angle of 19°; Figure 6 (c) is the numerical calculation result of the fiber end containing a frustum; Figure 6 (d) shows the test results of the polished fiber under rhodamine after the application of a 532nm laser. The experimental results are in close agreement with the theoretical results. The delivery of the cell (particle) stream into the optical trapping potential well at the end of the hollow tapered fiber ensures stable cell (particle) capture. Furthermore, the focusing capability and delivery speed of the hollow fiber optical tweezers can be controlled by adjusting the injected optical power, depending on the cell (particle) velocity. This allows for specific applications such as cell (particle) capture, diversion, and detection to be tailored. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 It is a schematic diagram of the hollow optical fiber structure.

[0035] Figure 2 This is a schematic diagram of the principle implementation of hollow fiber optical tweezers.

[0036] Figure 3 It is a schematic diagram of the fluid resistance of particles and their relative motion speed.

[0037] Figure 4 Schematic diagram of force analysis of the interaction between the hollow optical tweezers formed at the end of the hollow cone fiber and the cells (particles) flowing through the hollow fiber; (a) is the cell located to the left of the focal point; (b) is the cell located to the right of the focal point.

[0038] Figure 5 This is the preliminary simulation calculation result of determining the effective radius of the optical potential well using the finite element analysis method; (a) is the lateral optical trap force curve of cells with different diameters; (b) is the axial optical trap force curve of cells with different diameters.

[0039] Figure 6 Experimental photos: (a) is an experimental diagram of using a femtosecond laser micromachining system to machine microholes on the side of an inner-wall waveguide-type hollow capillary optical fiber; (b) is a schematic diagram of the polished optical fiber end, with a cone angle of 19°; (d) is the numerical calculation result of the optical fiber end containing a frustum; (d) is the test result of the polished optical fiber under rhodamine after passing a 532nm laser.

[0040] Figure 7 This is a single-cell analysis method using hollow fiber tweezers: a schematic diagram of the working principles of the CTC cell capture and fluorescence spectrum analyzer functional areas. DETAILED DESCRIPTION

[0041] Below is Figure 1 The present invention is specifically described by taking the microfluidic cell detection chip of the hollow fiber optical tweezers with an annular core as an example.

[0042] In the designed CTCs microfluidic chip, before single cell analysis and identification can be achieved, it is first necessary to be able to stably capture each cell in the fluid. That is, all cells must flow out through the air holes of the hollow optical fiber and be stably captured in the focal area before the next operation can be performed in the chip. Because the microfluidic channels in the microfluidic chip are on the micrometer scale, and the cells are also within the range of a few microns to more than ten microns, the present invention allows cell fluid to flow through the air holes of the hollow optical fiber. This design can capture, detect and analyze cells in the microfluidics within the hollow optical fiber one by one. This new optical manipulation device is the hollow optical fiber optical tweezers proposed in this invention.

[0043] In this process, a through-hole is machined on the side of the hollow fiber using femtosecond laser micromachining, and discrete cells are injected into the air hole of the hollow fiber through the through-hole. One end of the inner wall waveguide hollow fiber is collapsed by heating to eliminate the air hole, and a single-mode fiber is welded to the collapsed point to inject a 980nm capture 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 inner wall waveguide of the fiber to the cone, and after reflection, a light radiation zone is formed. A capture zone is formed near the focus. When the cells flow out of the fiber to the fiber end area, the light trapping force can stably capture the single cells.

[0044] The present invention proposes a novel hollow fiber optical tweezers, and the specific preparation process is as follows:

[0045] To achieve light transmission through a hollow fiber ring core, the air cavity is collapsed using a fused cone method, while simultaneously pumping out the air. After the collapse, the cone is cut open with a cleaver, and finally, a single-mode fiber is welded to it, 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.

[0046] Hollow 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.

[0047] The experimental process of capturing 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 focusing of the funnel; a high-power 980nm laser is passed to form a light trap force field. By mixing in cells of different diameters, the cell capture effect can be observed under a microscope. With the adjustment of the 980nm laser, the light intensity required to capture cells of different sizes can be recorded.

[0048] Select multiple wavelengths of laser light, including 980nm cell capture light, 345nm, 490nm, 511nm, 515nm, 530nm, 596nm and 639nm fluorescence excitation light sources, which can be injected into the ring core of the optical fiber at the same time through a multi-wavelength coupler, and a conical focusing light field is formed through the tapered optical fiber end. The wavelength of multiple fluorescence excitation light sources can be selected according to needs, such as Figure 7As shown. A fluorescent light source excites the fluorescence of CTC cells, and the excited fluorescence is collected into a spectroscopic device through a large numerical aperture optical fiber, thereby extracting various fluorescence signals. Fluorescence spectroscopy technology plays an important role in biomedicine and disease diagnosis. The hollow fiber optical tweezers proposed in the present invention have a diameter of less than or equal to several hundred microns and can directly receive reflected light and fluorescence signals emitted from the captured cells for analysis of single cells. Because intercellular heterogeneity is common in microorganisms and tumor cells, the analysis of the fluorescence spectrum of living single cells can be achieved by combining optical tweezers. The hollow annular waveguide optical fiber tapered end has an illumination light field with high spatial resolution, which is more convenient for single cell capture and fluorescence spectrum excitation.

[0049] Then, the ring tube fiber tweezers were combined with the microfluidic chip, and the corresponding in-chip channel structure and packaging part were formulated according to the functional structure of the chip. The microfluidic chip channel was formed by bonding a quartz substrate and a PDMS cover sheet. The microfluidic channel was made on the quartz substrate by femtosecond laser micromachining, and the cell fluid inflow outlet was realized by punching holes in the PDMS with a puncher.

Claims

1. The present invention provides a novel hollow fiber optical tweezers. Its characteristics are: It consists of a hollow optical fiber and a light source. Experimental sample liquids of different cell (particle) types are injected into the microfluidic channel 1 of the hollow optical fiber optical tweezers. The light energy emitted by the light source 2 forms an optical tweezer effect at the output end through the annular core of the hollow optical fiber and captures the cells (particles). Different cells (particles) in the experimental sample liquid are bound by the optical field force and controlled within the capture zone 3. During this period, the cells (particles) 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 functions such as cell (particle) capture, diversion, and detection. By combining the structural design of the microchannel inside the chip, a new optical fiber photodynamic control device is added to the chip to realize different forms of detection and analysis of multiple medical biological components, providing a new technical foundation for early cancer screening.

2. A novel hollow fiber optical tweezers according to claim 1, characterized in that: The hollow optical fiber is composed of a circular fiber core and a hollow air hole. The optical fiber has a hollow air hole running through the central axis and the circular fiber core.

3. The novel hollow fiber optical tweezers according to claim 1, characterized in that: The side of the hollow optical fiber can be punched with a micro-machining process to increase the cell (particle) entrance, so that one or more cells (particles) can be injected into the air holes of the hollow optical fiber at the same time.

4. The novel hollow fiber optical tweezers according to claim 1, characterized in that: The hollow fiber tweezers 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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