Sorting microfluidic system, chip and preparation method thereof

Abstract

The invention discloses a sorting microfluidic system which sequentially comprises a pretreatment unit, a sample introduction unit, a sorting unit and an outlet unit. The pretreatment unit comprises a centrifugal rotary filter membrane to filter out large-size CTC clusters; the sorting unit comprises a first micro-column array, the first micro-column array at least comprises a first-stage array and a second-stage array connected with the first-stage array in sequence, and the first micro-column array is used for separating CTCs with different sizes; the outlet unit includes a plurality of outlets for particles of different sizes. The sample introduction unit comprises a buffer solution inlet and a preliminary screening sample inlet, the number of gaps of the first micro-column array is configured to be different according to a first preset proportion, the centrifugal rotation moving membrane adopts a silicon nitride filtering membrane, and the microfluidic module is used for extracting circulating tumor cells CTCs.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological detection, and particularly relates to a sorting microfluidic system, a chip and a preparation method thereof. Background Art

[0002] Circulating tumor cells (CTCs), as the key mediators of tumor metastasis, originate from the shedding of the primary tumor and invade the peripheral blood circulation system. They eventually induce the formation of distant organ metastases through migration and colonization. Although a large number of tumor-derived cells can be detected in peripheral blood, only a few CTC subsets with stem cell phenotypes have the potential for metastatic activity. Such special cell populations usually exist in the circulatory system in the form of free single cells or microaggregates. The molecular characteristic information they carry can not only trace the heterogeneity of the primary tumor, but also play a core regulatory role in the tumor invasion and metastasis cascade reaction, making the detection of CTCs of great significance.

[0003] In CTC detection methods, based on the molecular typing and functional analysis of CTCs, a variety of efficient capture and enrichment methods have been developed in recent years. For example, multi-omics analysis at the single-cell level can reveal novel biomarkers during tumor progression and provide multi-dimensional diagnosis and treatment basis for clinical practice. The characteristics of this method include:

[0004] 1) Realize early warning of tumor micrometastasis by dynamically monitoring the changes in the number and molecular characteristics of CTCs;

[0005] 2) Evaluate the sensitivity of treatment regimens and track the efficacy response in real time;

[0006] 3) Construct a prognostic prediction model and optimize individualized treatment strategies;

[0007] 4) Screen drug action targets and guide precision drug use regimens.

[0008] Compared with traditional tissue biopsy, this non-invasive detection technology can break through the spatio-temporal sampling limitations and realize the dynamic monitoring of tumor biological behavior, providing important translational medicine evidence for clinical efficacy evaluation, recurrence monitoring, adjuvant decision-making and the research and development of new anti-cancer drugs.

[0009] In the evolution of circulating tumor cell (CTC) detection technology, microfluidic systems have become core tools with their unique physical control capabilities. Through the coordinated design of microscale flow channels and fluid dynamics, a multidimensional cell manipulation interface has been constructed: on the one hand, the submillimeter structural characteristics of the chip are highly compatible with the biophysical properties of cells (such as size and deformation ability), and can achieve precise capture under low shear conditions; on the other hand, its dynamic flow field control module can integrate multiple functions such as cell sorting, in situ characterization, and activity maintenance, forming a closed-loop experimental system from sample processing to information analysis. This miniaturized platform not only breaks through the dependence of traditional technologies on cell surface markers through the optimization of fluid-cell interaction mechanisms, but also can simultaneously obtain multiple biological information such as mechanical properties and metabolic status at single-cell resolution, providing a multidimensional analytical perspective for tumor metastasis research.

[0010] In 2007, Nagrath's research team published a groundbreaking research result in the journal Nature [Nagrath S, Sequist LV, Maheswaran S, et al. Isolation of rare circulating tumour cells in cancer patients by microchip technology [J]. Nature, 2007, 450 (7173): 1235-9.], which disclosed a cell capture platform based on microcolumn arrays, whose capture efficiency and sensitivity were significantly better than the CellSearch system approved by the FDA in 2004. This breakthrough has promoted the rapid development of microfluidic technology in the field of CTC capture, and subsequently a variety of innovative microchip technologies have been introduced.

[0011] The current mainstream sorting mechanisms can be divided into two major technical routes: biorecognition-oriented and physical feature-dependent. The former constructs a specific molecular capture interface through surface functionalization modification. Although it can achieve accurate recognition at the molecular level, it is limited by the dynamic expression characteristics of antigen epitopes and label-induced cell stress responses. It faces challenges such as sensitivity drift and loss of functional information in clinical transformation. The latter designs a label-free sorting strategy based on the inherent physical parameters of cells (size, density, dielectric properties, etc.). Its advantage is that while maintaining the original state of the cells, it realizes subpopulation feature analysis through a multi-level screening architecture, and realizes living cell functional analysis while maintaining a high recovery rate, showing greater clinical application potential.

[0012] As an important method for circulating tumor cell (CTC) isolation, the core principle of microfluidic microfiltration technology is to set specific filtration structures in microfluidic channels, such as microcolumn arrays, porous membranes, or tapered channels. These structures utilize the significant size difference between CTCs and blood cells (CTCs typically have a diameter of 10 - 20 μm, while blood cells have a diameter of about 8 - 12 μm). When a blood sample flows through the filtration structure, smaller blood cells can pass through smoothly, while CTCs are effectively intercepted. Currently, the capture of CTCs by this technology is mainly achieved through reverse flow, that is, by changing the fluid direction to recover the intercepted cells. Mature commercial devices on the market include Parasortix (Angle Corporation), CellSieve (Creaty MicroTech), and ScreenCell (ScreenCell), although these devices can achieve a capture efficiency of up to 90%, they still face technical bottlenecks such as limited processing throughput and easy clogging of the filter membrane.

[0013] In the development process of photoactuated manipulation technology, cell manipulation methods based on the principle of photon momentum transfer have shown unique advantages. Early studies confirmed that the potential well formed by a focused laser beam can achieve non-contact manipulation of micron-sized biological particles, and this physical mechanism has opened up a new path for precise cell sorting. The research team of K.C. Kim first reported the research results of cell sorting using optical tweezers in the journal Analytical Chemistry in 2008. Subsequently, commercial optical tweezer systems such as Elliot E3500 and Tweez200si were successively launched on the market, and these systems can achieve precise cell separation and multi-point capture operations. However, such technologies face multiple technical bottlenecks in practical applications: 1) The local temperature gradient caused by the photothermal effect may damage cells; 2) Precise waveguide components require sub-micron positioning accuracy, resulting in the system's stability being vulnerable to environmental disturbances; 3) The high-frequency maintenance requirements of optical components significantly increase the usage cost; 4) The complex parameter optimization process limits the universal application in clinical scenarios.

[0014] In microfluidic systems, cell sorting strategies based on hydrodynamic principles mainly cover three core mechanisms:

[0015] 1) Deterministic lateral displacement effect (DLD), which induces the differentiation of particle migration trajectories through a periodic microcolumn array;

[0016] 2) Inertial focusing effect, which realizes spatial positioning by the synergistic effect of Dean flow and inertial lift force;

[0017] 3) Vortex flow field regulation, inducing the generation of local secondary flow by means of the geometric characteristics of microstructures. These methods can achieve label-free sorting according to the biophysical properties such as cell size and stiffness by precisely regulating flow field parameters (such as Reynolds number, flow velocity gradient) and microchannel configurations, providing a technical basis for the functional analysis of living cells.

[0018] Among them, the research team led by Warkiani developed an innovative three-layer spiral microfluidic chip, combined with a pretreatment step for red blood cell (RBC) lysis, achieving a cell line recovery rate of 85% at a flow rate of 3 mL / h. In the analysis of clinical tumor samples, this sorting platform demonstrated outstanding detection efficiency, maintaining a full positive detection rate even in blood samples with a circulating tumor cell proportion as low as 0.1%-10%. By improving the red blood cell clearance process, its processing capacity per unit time can be optimized to 12 mL / h. It is worth noting that the automated device developed based on this principle has passed the EU CE certification (for in vitro diagnostic use), obtained the US FDA access permission, and the Class I medical device certification of the China National Medical Products Administration, becoming one of the few liquid biopsy systems globally that have completed multi-region regulatory approvals. However, there is still significant room for improvement in the existing technology system: 1) The recovery rate and purity of target cells always hover below the 90% critical value; 2) When non-target cells (such as specific subtypes of white blood cells) have similar physical characteristics to CTCs, the existing sorting strategies cannot effectively construct a differential screening field, resulting in an increased mixing degree of the final product. These key technical obstacles restrict its in-depth application in the context of precision medicine and urgently require technological iteration through interdisciplinary means such as the innovation of microfluidic chip structures and the development of multi-parameter sorting algorithms.

[0019] In the evolution of cell precise sorting technology, although the magnetic response manipulation system lags behind the development of light / electric manipulation technology, its unique technical advantages are driving this field into a new research dimension. As the core functional carrier, superparamagnetic microspheres have evolved into advanced forms such as iron-based composite structures and gadolinium-doped nanoparticles through materials engineering innovation. These specially designed micro-nano materials show important application values in tumor liquid biopsy. Its technical characteristics are mainly reflected in:

[0020] 1) Constructing a biocompatible colloidal dispersion system with adjustable magnetic moment;

[0021] 2) Endowing it with the selective recognition ability for specific cell subsets through surface topology optimization and ligand-directed coupling. Especially in the scenario of circulating tumor cell capture, the functionalized magnetic beads can establish a three-dimensional magnetic capture interface in the physiological fluid environment and achieve high-purity enrichment through the synergistic effect of magnetic field gradient and biological recognition.

[0022] Current research trends indicate that the control of the particle size of magnetic-responsive microspheres and the optimization of magnetic properties are breaking through the resolution limits of traditional magnetic separation technologies. By applying an external magnetic field, researchers can precisely control the movement trajectories of cells. However, in the CTC sorting strategy based on immune recognition, the current technical system still faces multiple technical challenges:

[0023] First, there are matching limitations between the kinetic parameters (such as the binding rate constant and dissociation equilibrium time) of the antigen-antibody binding process and the hydrodynamic parameters of the microfluidic system, which restricts the design of high-throughput sorting channels;

[0024] Second, limited by the compatibility between the surface topology of magnetic beads and the curvature of the cell membrane, the biological interface contact efficiency is difficult to break through the theoretical limit value;

[0025] Third, the phenotypic heterogeneity transformation that occurs during the circulation of tumor cells (such as the dynamic drift of the expression profile of EMT-related molecular markers) will lead to a decrease in the accessibility of molecular targets, and then cause a decline in the specific capture efficiency. These limiting factors at the mechanism level jointly affect the clinical applicability of the sorting system, prompting researchers to turn to the development of multi-modal sorting strategies (such as the combination of mechanical properties and molecular markers) and new bionic recognition interfaces (DNA aptamer-modified magnetic beads, topology-matching microstructures) to break through the existing technical bottlenecks. Summary of the Invention

[0026] One embodiment of the present disclosure is a multi-array non-destructive composite sorting microfluidic system for the extraction of high-purity circulating tumor cells. According to the flow order of the sample liquid, the microfluidic system sequentially includes a pretreatment unit, a sample introduction unit, a sorting unit, and an outlet unit.

[0027] The pretreatment unit includes rotary moving membrane filtration to filter out CTC clusters or CTCs in the mesenchymal phenotype; the sorting unit includes a microcolumn array, which in turn at least includes a first-stage array and a second-stage array connected to the first-stage array in sequence, for separating and obtaining CTC cells of different sizes; the outlet unit includes multiple outlets for cells of different sizes.

[0028] The sample introduction unit includes a buffer inlet and a primary screening sample inlet, and the number of gaps in the microcolumn array is configured for different inlets according to a first preset ratio.

[0029] Preferably, the rotary moving membrane uses a silicon nitride filtration membrane.

[0030] Preferably, the microcolumns of the microcolumn array are modified with multiple tumor cell markers.

[0031] The outlet unit includes a secondary micro-column array, and the number of gaps in the secondary micro-column array is configured for different outlets according to a second preset ratio. The micro-column array realizes the sorting function through the principles of hydrodynamics and chemical affinity. Description of the Drawings

[0032] By reading the following detailed description with reference to the accompanying drawings, the above and other objects, features, and advantages of the exemplary embodiments of the present invention will become readily understood. In the drawings, several embodiments of the present invention are shown by way of example and not limitation, wherein:

[0033] Figure 1 Schematic diagram of the rotary moving filtration device in the sorting microfluidic system according to one embodiment of the present invention.

[0034] Figure 2 Schematic diagram of the secondary micro-meter column flexible array sorting chip according to one embodiment of the present invention.

[0035] 1 - Silicon nitride rotary filtration membrane,

[0036] 2 - Filtration membrane rotary cylinder,

[0037] 3 - Sleeve,

[0038] 4 - Connecting chassis. Detailed Embodiments

[0039] In view of the problems faced by the existing microfluidic technology in sorting CTCs, the present disclosure proposes a microfluidic chip integrating microfiltration and multiple flexible micro-meter column sorting technologies. The chip has the characteristics of high-purity and high-throughput cell recovery. Through innovative integrated design, the manufacturing process is simplified, and at the same time, efficient high-viability cell sorting and recovery are achieved.

[0040] According to one or more embodiments, a microfluidic system for sorting CTCs aims to achieve the extraction of high-purity circulating tumor cells (CTCs). The microfluidic system of the embodiments of the present disclosure improves the sorting efficiency through multiple array non-destructive composite sorting. The microfluidic system includes the following modules or units:

[0041] Pretreatment unit: including a low-speed centrifugal rotary moving membrane filtration module for improving the sorting efficiency and throughput and reducing the risk of blockage.

[0042] Sample introduction unit: This unit includes a buffer inlet and a primary screening sample inlet for introducing samples and buffers.

[0043] Micro-meter flexible array composite sorting unit: An optimized micro-column array is adopted, and the traditional circular micro-column structure is fitted by a Bessel curve to improve the capture efficiency and purity.

[0044] Outlet unit: It includes three graded outlets, which are optimally configured for large-sized particles, medium-sized particles, and small-sized particles.

[0045] Imaging and observation unit: It is used to combine high-resolution microfluorescence imaging technology to observe the dynamic behavior of CTCs in real time.

[0046] Temperature control unit: It is used to adjust the temperature of the chip to facilitate the elution of CTCs from the flexible microcolumns.

[0047] The sample is first pretreated through a low-speed rotating centrifugal moving membrane filtration module to sort out large-sized CTC cell clusters. The pretreated sample and buffer enter the micron flexible array composite sorting unit through the inlet unit and are finely sorted through an optimized microcolumn array. During the sorting process, dual capture of CTCs is carried out using Zigzag hydrodynamics and micron columns modified with multiple tumor cell markers. Cells of about 5 μm finally flow out from the bottom outlet 3, and cells of about 10 μm finally flow out from outlet 2 located in the middle position. The sorted CTCs are observed in real time by high-resolution microfluorescence imaging technology in the imaging capture cavity. The sorted sample leaves the chip through three graded outlets to achieve efficient collection of particles of different sizes. The temperature control unit controls the elution of CTCs by adjusting the temperature of the chip. In the microfluidic chip of the embodiment of the present disclosure, fluid channels connect each unit to ensure the flow of the sample and buffer. The microcolumn array is located in the fluid channel, and the capture and sorting of CTCs are achieved through precise geometric design and surface modification. The imaging area covers the entire micron column flexible array sorting area for observing and analyzing the sorted CTCs. The graded outlets are located at the end of the chip for collecting and classifying particles of different sizes.

[0048] The improvements of the multi-array non-destructive composite sorting microfluidic system and chip for high-purity circulating tumor cell extraction in the embodiments of the present disclosure compared with the existing sorting methods include:

[0049] 1. Aiming at the problems of low throughput and easy blockage in microfluidic technology, based on the basic theory of circulating tumor cell (CTCs) sorting, an innovative solution is proposed: the rotary moving membrane filtration and micron flexible array composite sorting method. This method combines the advantages of membrane filtration, physical and chemical affinity sorting microfluidic chips, significantly improving the sorting efficiency and throughput, while reducing the risk of blockage of micron column sorting and the resulting changes in liquid hydrodynamics.

[0050] 2. In view of the problem that magnetic force cell sorting has low capture efficiency and cannot achieve high-efficiency and high-purity capture due to problems such as insufficient antibody-antigen binding time, ineffective control, and limited magnetic bead adsorption area, the present disclosure uses Bessel curves to fit the traditional circular microcolumn structure, abstracts the physical sorting model into a mathematical model, and constructs a parametric model by changing three parameters: the flexible microcolumn gap, the flexible microcolumn size, and the microcolumn array period. With the goal of reducing the first flow width, the flow velocity distribution in the microcolumn gap is re-optimized, and a micron array microcolumn morphology with excellent fluid regulation ability is obtained.

[0051] In view of the separation problem caused by the size overlap (about 10-15μm) between circulating tumor cells (CTCs) and white blood cells, the present disclosure proposes a solution based on a size grading strategy. Since there is a contradiction between efficiency and purity when a conventional deterministic lateral displacement (DLD) chip uses a single critical diameter - using a smaller critical diameter (~10μm) can improve the CTC capture rate but significantly increase the risk of white blood cell contamination, while a larger critical diameter (~15μm) can reduce white blood cell interference but lead to missed detection of medium-sized CTCs, the present disclosure innovatively designs a two-critical-diameter 8μm (microcolumn diameter 40μm, microcolumn gap 30μm) and critical diameter 12μm (microcolumn diameter 40μm, microcolumn gap 25μm) hierarchical array DLD chip system. The microfluidic chip of the present disclosure, through a three-stage size grading strategy: small size (<8μm), medium size (8-13μm), and large size (>13μm), focuses on capturing large-sized CTCs that are significantly different from white blood cells, while retaining medium-sized CTCs that are easily confused, taking into account both the integrity and specificity of sorting. The experimental system uses A549 lung cancer cells as a CTC model, combines computational fluid dynamics (ALE method) simulation and hydrogel microsphere verification to construct a complete performance evaluation system. The results show that the sorting purity of the chip for 12μm / 20μm standard particles reaches 98±0.3% and 97.74±0.5% respectively, and the recovery rate is stable at 93±0.5%; the size resolution of the two-stage critical diameters reaches 85% and 89%; in the whole blood environment test, combined with chemical affinity sorting, the system throughput is increased to 2mL / min, the A549 cell recovery efficiency reaches 96.70±2.10%, and the white blood cell removal rate is ~99.99%. It is particularly worth noting that through the combination with the chemical affinity method, the efficient exclusion of white blood cells is successfully achieved, verifying the significant advantages of this composite sorting strategy in solving the problem of CTC heterogeneity. The selection of flexible hydrogel microcolumns and the post-treatment recovery method significantly reduce CTC cell damage and enable controllable downstream analysis and testing. The two-stage array microfluidic magnetic chip designed based on this morphology exhibits excellent sorting efficiency and sorting performance with high cell viability.

[0052] 3. Aiming at the problems of false negative of EpCAM in the sorting of CTCs based on the affinity mechanism and low purity in separating white blood cells and CTCs with similar sizes by microfluidic hydrodynamic technology, the present disclosure modifies the sorting micro-columns with a variety of tumor cell markers, effectively avoiding the false negative problem easily caused by single EpCAM sorting, and improving the detection efficiency and accuracy of different subsets of CTCs; at the same time, it also effectively realizes the sorting of white blood cells with sizes close to those of CTCs.

[0053] 4. Aiming at the problems of low single-cell capture efficiency, difficult elution of CTCs in the chip, and cell damage during the elution process resulting in reduced cell activity, a micron-scale flexible micro-column is designed. After microfluidic sorting is completed, high-resolution microscopic fluorescence imaging technology is combined in the image capture cavity to observe the dynamic behavior of CTCs in real time. By adjusting the temperature of the chip, CTCs can be easily eluted from the flexible micro-column, facilitating subsequent single-cell analysis or molecular biology detection, providing important data for downstream cancer information analysis, such as the number, morphology, and molecular marker expression of CTCs, which is helpful for early cancer diagnosis, efficacy evaluation, and prognosis monitoring.

[0054] Through the microfluidic chip of the present disclosure, the whole process from sample pretreatment, CTC capture, imaging observation to final sorting is realized.

[0055] According to one or more embodiments, such as Figure 1 and Figure 2 the parts shown constitute a multi-array non-destructive composite sorting microfluidic system for extracting circulating tumor cells with high purity.

[0056] Figure 1This is a schematic diagram of the rotary mobile filtration device in the composite sorting microfluidic system of the present disclosure embodiment. The filter membrane rotating cylinder is located at the center of the device and is made of porous materials such as ceramics or metals, which is used to support the filter membrane and provide structural support. The sleeve is located outside the filter membrane rotating cylinder, which plays a role in protecting and fixing the filter membrane, and may also play a role in thermal insulation or pressure support. The connection chassis is located at the bottom of the device and is used to fix the entire filtration device in place. The connection chassis is usually designed with multiple bolt holes to firmly fix the device. The silicon nitride rotary mobile filter membrane is in a cylindrical shape and is inserted into the filter membrane rotating cylinder, which helps to improve the filtration efficiency and reduce the blockage of the membrane. The working principle of this rotary filtration device is based on centrifugal force and the selective permeability of the filter membrane. When the device rotates, the centrifugal force will retain the circulating tumor cell clusters or large particles larger than the pore size in the blood inside, and push the cell fluid smaller than the pore size in the liquid to the outside of the filter membrane, and the recovery is realized through the external sleeve and the subsequent filtration of the flexible microcolumn chip. In this way, the solid particles are retained inside the filter membrane, while the clean liquid is collected inside the sleeve, thereby realizing the clarification, concentration or separation of the liquid. Further, the use of an ultra-thin silicon nitride rotary mobile filter membrane can further reduce cell damage. Here, the height dimension of the sleeve can be selected as 5 cm, and the height dimension of the connection chassis can be selected as 4 cm. Therefore, in the present disclosure embodiment, the silicon nitride rotary filter membrane system improves the sorting throughput, while the ultra-thin design reduces cell damage.

[0057] In the sorting microfluidic system of the present disclosure, it further includes a microfluidic chip with a specific fluid channel structure inside. The fluid enters the chip from the left whole blood sample inlet, and after being processed by the internal fluid channels, it flows out from the right pre-screened sample outlet. Figure 2 This is a cross-sectional schematic diagram of the microfluidic chip, showing the micro-wire array structure inside the chip. Figure 2 The chip in [description] has different fluid inlets and outlets, namely the pre-screened sample inlet, the buffer inlet, and three grading outlets (Outlet 1, Outlet 2, Outlet 3). The micro-wire array is located in the central area of the chip, and the micro-wire array constitutes a part of the fluid channel, which is used to realize the grading filtration and sorting of CTCs. The arrangement and design of the micro-wires play an important role in controlling the fluid flow, improving the sorting efficiency and reducing the risk of blockage.

[0058] Figure 1 Combined with Figure 2, which shows the design and working principle of the multi-array non-destructive composite sorting microfluidic chip of the present disclosure. The chip realizes the efficient separation of CTCs of different sizes through a hierarchical filtration strategy. The primary filtration uses a silicon nitride filtration membrane with a pore size of 28-30 microns to remove large-sized CTC clusters, while the secondary chip uses 1280 precisely arranged fluid channel gaps. Through optimized hydrodynamic design, it realizes the effective sorting of CTCs of different sizes. The differential cycle design and the optimized configuration of the outlets further improve the sorting efficiency and the rationality of flow distribution.

[0059] According to one or more embodiments, as Figure 2 shown, a multi-array non-destructive composite sorting microfluidic chip with high-purity circulating tumor cell extraction. The chip system mainly consists of a buffer inlet, a primary filtration chip sample inlet, and three hierarchical outlets, and realizes the efficient separation of CTCs of different sizes through a hierarchical filtration strategy. The primary filtration chip, as a pretreatment unit, is mainly used to remove larger-diameter CTC clusters to ensure that the sample entering the microcolumn array as the second stage will not block the filtration channels due to the presence of large particles, resulting in too low CTC sorting efficiency.

[0060] In terms of fluid channel design, the primary rotary filtration system uses a silicon nitride centrifugal rotary filtration membrane with a pore size of 28-30 microns. This pore size design sorts 99.99% of tumor circulating cell clusters. At the same time, the ultra-thin (200 nm thick) silicon nitride membrane can effectively reduce the pressure drop of cells passing through the filter membrane, which helps to separate live CTCs, and finally achieves a cell survival rate higher than 98%. The secondary microcolumn array chip uses 1280 precisely arranged fluid channel gaps, among which the buffer inlet occupies 960 gaps, and the sample inlet is configured with 320 gaps. To ensure hydrodynamic stability, through theoretical calculation and experimental verification, the flow rate ratio of the buffer to the sample solution is optimized to 3:1. This parameter can effectively maintain a stable laminar flow state and avoid the influence of turbulence on the separation effect. Here, the microcolumn array can also be called a microline array.

[0061] To meet the separation requirements of particles of different sizes and ensure the effective contact time between CTCs and the microcolumns, the microcolumn array chip adopts a differential cycle design: the microcolumns with 1280 gaps are divided into two levels. The first-level array is set to 170 cycles. This design fully considers the possible particle collision phenomenon during the migration of medium and large-sized particles to ensure their full migration; the second-level array is optimized to 150 cycles to cope with the possible longitudinal offset phenomenon of small-sized particles during migration. The width of the outlet channel is moderately expanded, providing necessary redundant space for particle movement and effectively reducing the blockage risk.

[0062] In terms of the outlet design, the microcolumn array has three hierarchical outlets, which are optimized and configured for large-sized particles, medium-sized particles, and small-sized particles respectively. The corresponding number of gaps is 320, 480, and 480 respectively, and the resulting flow rate ratio is 2:3:3. This design not only ensures the efficient collection of particles of different sizes but also realizes the reasonable distribution of the flow rate. To improve the process feasibility of chip preparation, circular punching areas with a standardized diameter are provided at the ends of the inlet channel and the outlet channel. This design significantly improves the operational convenience and positioning accuracy in the PDMS chip preparation process, facilitating the batch production of chips.

[0063] According to one or more embodiments, there is a multiplex array non-destructive composite sorting microfluidic chip for high-purity circulating tumor cell extraction. The chip manufacturing and packaging process flow includes:

[0064] (1) Chip design and mask preparation

[0065] The first step in chip manufacturing is to perform a three-dimensional design of the chip structure using professional CAD software (such as AutoCAD, SolidWorks, etc.). When designing, factors such as the functional requirements of the chip, hydrodynamic characteristics, and processing feasibility need to be comprehensively considered. After the design is completed, the design drawing is imported into a high-precision lithography machine, and a quartz plate is used as the base material to fabricate a photolithography mask through laser direct writing or electron beam exposure technology. The line width accuracy of the mask is usually controlled within 1 μm to ensure the accuracy of subsequent processes.

[0066] (2) Lithography and silicon plate preparation

[0067] Place the prepared mask in an ultraviolet exposure machine and expose it using ultraviolet light with a wavelength of 365 nm. During the exposure process, the ultraviolet light irradiates the silicon wafer coated with SU8-2035 negative photoresist through the mask pattern, and the photoresist in the exposed area undergoes a cross-linking reaction. Subsequently, PGMEA developer is used for development, and the photoresist in the unexposed area is dissolved to form the required microstructural pattern. To enhance the surface hydrophobicity, trimethylsiloxane (TMS) is used for vapor deposition treatment on the surface of the silicon plate, and the treatment time is usually controlled within 30 - 60 minutes to complete the preparation of the silicon template.

[0068] (3) PDMS mold preparation

[0069] Precisely weigh the dimethylsiloxane PDMS prepolymer and the curing agent according to a mass ratio of 10:1, and use a planetary stirrer to mix them at a speed of 500 rpm for 5 minutes. Transfer the mixed solution to a vacuum drying oven and degas it for 15 minutes under a vacuum of -0.1 MPa until the mixed solution is completely transparent and bubble-free. Slowly pour the degassed PDMS onto the silicon template, and control the casting thickness in the range of 3-5 mm. The curing process is carried out in a forced-air drying oven at 80 °C, and the curing time is controlled at 45-90 minutes according to the thickness.

[0070] (4) Chip post-treatment

[0071] After curing, in a Class 5 ISO cleanroom, use a special peeling tool to peel the PDMS from the silicon template. Use a surgical blade to cut the PDMS into individual chip units, and keep the blade at a 45° angle to the PDMS surface during cutting to ensure a smooth cut. Use a biopsy needle with a diameter of 0.5-2.0 mm to punch holes at the predetermined positions, and keep the needle perpendicular to the PDMS surface during punching to avoid burrs.

[0072] (5) Surface hydrophilic treatment

[0073] Place the cut and punched PDMS chips in an oxygen plasma cleaner, with a power of 50 W, an oxygen flow rate of 50 sccm, and a treatment time of 30 seconds to enhance surface hydrophilicity and promote hydrogel attachment. Locate the microcolumn growth area according to the design drawing (usually aligned with the PDMS microchannel). Use a micro-injection pump to inject the hydrogel precursor solution (vinyl-functionalized upconversion luminescent NaYF:Er particles crosslinked with acrylate monomers to form a composite gel with both flexibility and high-temperature resistance potential) at a rate of 0.5-1.0 μL / min. Ensure complete filling of the target microcavity by negative pressure suction (pressure -20 kPa, time 5 minutes). Selectively expose with 365 nm ultraviolet light through a mask (light intensity 10 mW / cm 2 , exposure time 30-60 seconds) to crosslink and cure the hydrogel precursor to form a microcolumn structure. The mask pattern needs to match the microstructures of the silicon template to ensure that the microcolumn diameter is 10-200 μm and the height is 50-500 μm. Rinse the uncured residues with phosphate buffer solution (PBS) at a flow rate of 0.2 mL / min for 5 minutes. Age in an incubator at 37 °C for 24 hours to stabilize the swelling performance of the hydrogel. Use a confocal microscope to detect the microcolumn morphology and dimensional tolerance (±5%). Test the fluidity and structural integrity of the microcolumn array through microfluidic fluorescent dye perfusion. In the embodiments of the present disclosure, hydrogel microcolumns are used to achieve flexible capture and sorting of tumor cells, reduce cell damage, and jointly remove white blood cells by chemical affinity, achieving a sorting efficiency of 96.7% and a cell activity higher than 98%.

[0074] (5) Modification of tumor markers

[0075] Immerse the microcolumns in an ethanol solution (1% v / v) containing 3-aminopropyltriethoxysilane (APTES) and react at room temperature for 2 hours to form an aminated surface. Immerse the aminated microcolumns in a glutaraldehyde solution (2.5% v / v) and react at room temperature for 1 hour to form an aldehyde-functionalized surface. Wash three times with PBS buffer (pH 7.4) to remove unreacted glutaraldehyde. Add EpCAM monoclonal antibody solution (10 μg / mL, dissolved in PBS) and incubate overnight at 4 °C so that the antibody forms a Schiff base bond through the amino and aldehyde groups. Reduce the Schiff base bond with PBS containing sodium borohydride (NaBH 4 , 0.1 M) to stabilize the covalent binding (room temperature, 30 minutes). HER2 / CEA / PSMA: Use the same procedure and replace with the corresponding monoclonal antibody. CD44: Use a thiol-gold bonding system. Immerse the modified microcolumns in a PBS blocking solution containing 1% BSA (bovine serum albumin) and 0.05% Tween-20 and shake at room temperature for 2 hours to block unbound active sites and reduce non-specific adsorption. The antibody concentration is controlled at 10–50 μg / mL (too high is likely to cause steric hindrance, and too low results in insufficient binding). The antibody incubation requires >12 hours to ensure sufficient binding.

[0076] The entire process flow needs to be carried out under strictly controlled environmental conditions, with the temperature controlled at 22 ± 1 °C and the relative humidity maintained at 45 ± 5% to ensure the stability and repeatability of the process. Quality inspections are required for each key step, including size measurement, surface morphology observation, and sealing test, etc., to ensure that the performance of the final chip product meets the design requirements.

[0077] Therefore, the beneficial effects of the present disclosure include:

[0078] 1. Adopt a mobile membrane filtration pre-sorting method to improve the sorting efficiency and throughput: Combining the advantages of rotary membrane filtration, physical size, and chemical affinity microfluidic chip sorting can significantly improve the sorting efficiency of circulating tumor cells (CTCs). The low-speed rotary filtration micro-membrane system uses centrifugation to accelerate the filtration time and improve the throughput of the system, which is suitable for processing a large number of samples. At the same time, the design of the initial filtration system of the rotating mobile membrane reduces the clogging problem caused by cell or impurity accumulation in traditional membrane filtration, improving the stability and reliability of the system.

[0079] 2. Optimizing the microcolumn structure by B-spline curve fitting to improve the capture efficiency and purity: By optimizing the microcolumn gap, the size of the flexible microcolumns, and the array period, the flow velocity distribution was significantly improved, thereby enhancing the capture efficiency and purity of cell sorting. The design of the flexible microcolumn array gap optimized the hydrodynamic performance, making the cell sorting process more controllable. Abstracting the physical sorting into a mathematical model provided a theoretical basis for subsequent optimization and improvement, with strong scalability.

[0080] 3. Using a microchip microcolumn modified with multiple tumor cell markers to avoid the false negative problem of EpCAM: By using a microcolumn chip modified with multiple tumor cell markers to sort CTCs, the false negative problem that may be caused by single EpCAM sorting was effectively avoided, the detection rate of different subsets of CTCs was increased, and the interference of non-target cells such as white blood cells was reduced. It is applicable to multiple tumor types, can cover a wider range of CTC subsets, and improves the universality of detection. When separating white blood cells and CTCs with similar sizes, the sorting purity was significantly improved.

[0081] 4. Using a high-temperature-resistant flexible hydrogel microcolumn sorting chip: The selection of the flexible hydrogel reduced the damage to sorted CTC cells and improved the sorting efficiency of CTCs under the condition of appropriately reducing the critical sorting radius. Combining the subsequent step of removing cross-linking groups at low temperature increased the survival rate of CTC cells to over 98%, which is beneficial for downstream analysis and detection experiments of CTCs. Combining with high-resolution microscopy or fluorescence imaging technology, the dynamic behaviors of CTCs (such as movement trajectories, morphological changes, etc.) can be observed in real time, facilitating subsequent single-cell analysis or molecular biology detection, and providing important data for the early diagnosis, efficacy evaluation, and prognosis monitoring of cancer.

[0082] 5. Using a composite sorting method combining rotary microfiltration, a flexible microcolumn array, and chemical affinity for the sorting of CTCs with a recovery rate as high as 96.70 ± 2.10%, a cell purity of 99.99% (white blood cell removal rate), a cell viability higher than 98%, and a processing rate of 1 mL / min.

[0083] The literature related to this disclosure is as follows.

[0084] 1. Smirnov D A, Zweitzig D R, Foulk B W, et al. Global Gene Expression Profiling of Circulating Tumor Cells[J]. Cancer Research, 2005, 65(12): 4993 - 4997.

[0085] 2. Vona G, Sabile A, Louha M, et al. Isolation by size of epithelial tumor cells: a new method for the immunomorphological and molecular characterization of circulating tumor cells[].

[0086] The American journal of pathology, 2000, 156(1): 57 - 63.

[0087] 3. Neoh K H, Hassan A A, Chen A, et al. Rethinking liquid biopsy: Microfluidic assays for mobile tumor cells in human body fluids[J]. Biomaterials, 2018, 150: 112 - 124.

[0088] 4. Tadimety A, Closson A, Li C, et al. Advances in liquid biopsy on - chip for cancer management: Technologies, biomarkers, and clinical analysis[]. Critical Reviews in Clinical Laboratory Sciences, 2018, 55(3): 140 - 162.

[0089] 5. Analytical Chemistry, 2008, 80(7): 2628 - 2630

[0090] 6. Warkiani M E, Khoo B L, Tan D S W, et al. An ultra - high - throughput spiral microfluidic biochip for the enrichment of circulating tumor cells[J]. Analyst, 2014, 139(13): 3245 - 3255.

[0091] 7. Warkiani M E, Guan G, Luan K B, et al. Slanted spiral microfluidics for the ultra-fast, label-free isolation of circulating tumor cells[]. Lab Chip, 2014, 14: 128-137.

[0092] 8. Warkiani M E, Khoo B L, Wu L, et al. Ultra-fast, label-free isolation of circulating tumor cells from blood using spiral microfluidics[JJ. Nature protocols, 2016, 11(1): 134-148

[0093] 9. Lv S-W, Wang J, Xie M, et al. Photoresponsive immunomagnetic nanocarrier for capture and release of rare circulating tumor cells[J]. Chemical Science, 2015, 6(11): 6432-6438.

[0094] It should be noted that although the above content has described the spirit and principle of the present invention with reference to several specific embodiments, it should be understood that the present invention is not limited to the specific embodiments disclosed, and the division of each aspect does not mean that the features in these aspects cannot be combined. This division is only for the convenience of expression. The present invention aims to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims.

Claims

1. A sorting microfluidic system, characterized in that: The microfluidic system includes, in sequence, a pretreatment unit, a sample introduction unit, a sorting unit and an outlet unit. The pretreatment unit includes a centrifugal filter membrane to filter out large-sized CTC clusters; The sorting unit includes a first microcolumn array, which in turn includes at least a first-level array and a second-level array connected to the first-level array, for separating and obtaining CTCs of different sizes; The outlet unit includes a plurality of outlets for particles of different sizes.

2. The sorting microfluidic system according to claim 1, characterized in that: The sample introduction unit includes a buffer inlet and a primary screening sample inlet, and the number of gaps in the first microcolumn array is allocated to different inlets according to a first preset ratio.

3. The sorting microfluidic system according to claim 1, characterized in that: The centrifugal rotating moving membrane adopts a silicon nitride filter membrane.

4. The sorting microfluidic system according to claim 1, characterized in that: The microfluidic system also includes an image capture chamber for observing the dynamic behavior of the sample particles after sorting.

5. The sorting microfluidic system according to claim 1, characterized in that: The microfluidic system is used for extracting circulating tumor cells (CTCs).

6. The sorting microfluidic system according to claim 5, characterized in that: The micropillars of the first micropillar array are modified with a plurality of tumor cell markers.

7. The sorting microfluidic system according to claim 1, characterized in that: The outlet unit includes a second micro-column array, and the number of gaps in the second micro-column array is allocated to different outlets according to a second preset ratio.

8. The sorting microfluidic system according to claim 1, characterized in that: The first micro-column array achieves sorting through fluid mechanics and chemical affinity.

9. A microfluidic chip, characterized in that: It includes a sample introduction unit, a sorting unit and an outlet unit, wherein the sample introduction unit includes a buffer inlet and a primary screening sample inlet. The sorting unit includes a first microcolumn array, which in turn includes at least a first-level array and a second-level array connected to the first-level array, for separating and obtaining CTCs of different sizes; The outlet unit includes a plurality of outlets for particles of different sizes.

10. A method for preparing a microfluidic chip, characterized in that: A method for preparing the microfluidic chip as claimed in claim 9, include, S101, manufacturing a mask for the sorting microfluidic chip according to claim 9; S102, generating a pattern of the sorting microfluidic module on a silicon wafer by a photolithography process according to the mask to make a silicon template, S103, using dimethylsiloxane (PDMS) to form the microfluidic chip array on the silicon template; S104, peeling off and cutting from the silicon template to obtain the microfluidic chip unit; S105, performing cleaning and bonding operations on the microfluidic chip unit.

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