CN111893023A - Tumor cell capturing device and preparation and application thereof - Google Patents
Tumor cell capturing device and preparation and application thereof Download PDFInfo
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Abstract
The invention discloses a tumor cell capturing device and preparation and application thereof, wherein the capturing device comprises a silicon chip and a microfluid chip assembled with the silicon chip; the silicon chip is provided with a groove structure array and a nanowire array, the silicon chip is provided with a capture surface, the groove structure array is concavely arranged on the capture surface, the nanowire is convexly arranged on the concave surface of the groove structure, and the nanowire is connected with an antibody with specificity for recognizing an antigen on a tumor cell; the microfluidic chip is provided with a connecting surface, and a microfluidic channel is concavely arranged on the connecting surface; the area of the connecting surface provided with the microfluid channel is matched with the area of the capturing surface provided with the groove structure array, so that when the connecting surface covers the capturing surface, the groove structure array is covered by the microfluid channel, and a sealed channel for microfluid to pass through is formed between the silicon chip and the microfluid chip. The technical scheme of the invention solves the technical problem that the traditional microfluid system has low CTC cell capturing precision.
Description
Technical Field
The invention relates to the technical field of biological materials and clinical detection, in particular to a tumor cell capturing device and preparation and application thereof.
Background
With the development of cancer research and cancer treatment methods, the need for the establishment of new methods for monitoring tumor evolution, particularly in tumorigenesis and during treatment, is increasing. Among them, the technology based on liquid biopsy has been developed rapidly with features of real time, less sample demand, and simple instrument and device. Particularly, the method is based on Tumor Cell analysis, such as the capture and analysis of Tumor cells (CTC) in the circulatory system, and shows high compatibility with the existing clinical detection and wide application prospect. Especially, the method has excellent advantages and technical potentials in the following three aspects: one, directly captured circulating tumor cells can be directly targeted to existing clinical assays, particularly based on analysis and detection of pathological tissue samples from patients, such as pathotyping assays for multiple specific cancer markers in pathology. Also for therapeutic targets (e.g. Her2 in breast cancer) FISH staining as well as CISH staining can be performed on the CTCs after capture. The CTC has the advantages that the tumor tissue sampling of a patient is not needed by operation or puncture, and the CTC can be carried out simultaneously with the normal blood sample sampling, so that the condition that some kinds of cancers are not suitable or are very difficult to collect the tumor tissue sample and cannot be detected is avoided, the CTC blood sample is not limited by time and sampling frequency, the illness state of the patient can be reflected more accurately and timely, a treatment scheme most suitable for the cancer development of the patient can be made, and the individualized Medicine (Personal Medicine) is realized. Secondly, with the development of sequencing technology and single cell detection method, gene analysis on single cell level can be realized for a few captured CTCs, so that the oncology evolution characteristics of patients can be determined at molecular level and the targets and targets for accurate positioning therapy in Medicine can be realized (Precision Medicine). Of further interest, unlike the detection of Single gene mutations (Single Mutation) or Free dna (cell Free dna), genetic analysis based on intact CTC cells gives information on the entire Genome (wheel Genome). That is, including individual gene information, the amount of gene expression and Copy Number (Copy Number) and the Variation in Genome Structure (Genome Structure Variation) are also included. Based on this information, it is possible to drill down deeply into patient analysis and treatment options. Thirdly, the practicality, timeliness and detection cost of the CTC detection can bring foreseeable effects and benefits for cancer patients and doctors. The practicality lies in that the CTC technology operation and execution of the traditional blood detection for the fetus derailment are in seamless connection with the conventional operation of the existing pathological analysis. The timeliness is that the collection and analysis of the sample are not limited by time and space, and the monitoring and the tracking of the treatment effect of the tumor patient are realized to the maximum extent. The detection cost is low, and especially the detection and analysis of the pathological layer on the technical platform described in the patent are similar to the cost of the traditional blood analysis, so that the burden of patients and family members is reduced.
Despite these advantages, and many years of development and breakthrough in the CTC technology field, our pursuit for detection efficacy, sensitivity, and detection efficiency (Throughput) and sustainable scalability has never been stopped. The problems that exist at present include: on one hand, in the aspect of detection sensitivity, the existing mature CTC technology needs at least 7.5ml of raw blood to complete detection, and CTC detection is not guaranteed; the false positive cells detected, which are mainly derived from Cytokeratin (Cytokeratin) positive neutrophils in immune cells, mostly resemble the above-mentioned methods based on magnetic nanoparticle capture or reverse immune cell removal, which takes up to 8-16 hours. On the other hand, although many capturing methods developed based on the physical size of cells can reduce the interference of immune cells, the captured cells cannot be guaranteed to belong to CTCs, and the obtained suspected CTCs are not screened by a method verified by tumor biology, and cannot provide clinical pathological information. Nanobiology has shown advantages in capture efficiency and integrated device production as an effective means. It is frequently reported that the strong adsorption capacity of the surface of the nanostructure can well capture cultured cancer cell line cells by utilizing the mechanism and combining with a detection chip of a microfluid technology, but hundreds of millions of immune cells in a blood sample are confronted with immune cells which are not specifically adsorbed on the chip, and great troubles are caused in later pathological immunostaining analysis and subsequent molecular analysis. Furthermore, the technique of separation of CTCs by means of microfluidic technology, through the different fluid properties of the cells in a microfluidic "mixer", does not guarantee any meaningful capture output of CTCs, while reducing the time consumption for capturing and separating CTCs. Thus, CTC detection approaches based on a single capture (or purification) mechanism do not meet the requirements of clinical detection.
Disclosure of Invention
The invention mainly aims to provide a tumor cell capturing device and preparation and application thereof, so as to solve the technical problem that the CTC cell capturing precision of the traditional micro-fluidic system is not high.
In order to achieve the purpose, the invention provides a tumor cell capturing device, which comprises a silicon chip and a microfluid chip assembled with the silicon chip;
the silicon chip is provided with a groove structure array and a nanowire array, the silicon chip is provided with a capture surface, the groove structure array is concavely arranged on the capture surface, the nanowire is convexly arranged on the concave surface of the groove structure, and the nanowire is connected with an antibody with specificity for recognizing an antigen on a tumor cell;
the microfluidic chip is provided with a connecting surface, and a microfluidic channel is concavely arranged on the connecting surface;
the area of the connecting surface provided with the microfluid channel is matched with the area of the capturing surface provided with the groove structure array, so that when the connecting surface covers the capturing surface, the groove structure array is covered by the microfluid channel, and a sealed channel for microfluid to pass through is formed between the silicon chip and the microfluid chip.
Optionally, the groove structure array is a hemispherical groove array.
Optionally, the groove structure array is a hemispherical groove array, and the diameter of the hemispherical groove is 5-45 um; and/or the presence of a gas in the gas,
two adjacent distance between the centre of a circle of hemisphere recess is 3 ~ 20 um.
Optionally, the length of the nanowire is 1-5 um; and/or the presence of a gas in the gas,
the diameter of the nanowire is 10-500 nm.
Optionally, the number of the microfluidic channels is multiple, and the multiple microfluidic channels are sequentially connected end to end and arranged in a snake shape; and/or the presence of a gas in the gas,
the width of the microfluid channel is 0.1-3 mm; and/or the presence of a gas in the gas,
the height of the microfluidic channel is 25-130 um.
Optionally, a plurality of fishbone structures are arranged inside the microfluidic channel part.
Optionally, a plurality of the fishbone structures are arranged at the top of the microfluidic channel at intervals; and/or the presence of a gas in the gas,
the width of the fishbone structure is 5-15 um; and/or the presence of a gas in the gas,
the distance between the fishbone structures is 8-15 um; and/or the presence of a gas in the gas,
the height of fishbone structure is 10 ~ 40 um.
Optionally, the material of the microfluidic chip is one or a combination of plastic, rubber and fiber.
Optionally, the antibody is one or a combination of an EpCAM antibody and/or an EGFR antibody.
The invention also provides a preparation method for preparing the tumor cell capturing device, which comprises the following steps:
s1, preparing a silicon chip:
a. preparing a silicon chip substrate with a groove structure array and nanowires: sequentially cleaning acetone, alcohol and deionized water, and N2Blow drying, O2Etching the silicon wafer by reactive ions for later use; rotationally coating a groove structure micro-mold on a spare silicon wafer, wherein the rotational coating speed is 500-6000 rpm, and the rotational coating time is 10-60 seconds to obtain a template silicon wafer; evaporating a 1-15 nm metal film layer on the template silicon wafer in vacuum, and removing the groove structure micromold to obtain a silicon wafer with an exposed groove structure region; etching the silicon exposed out of the groove area by using inductively coupled plasma etching to obtain a silicon wafer with a groove structure array, wherein the etching depth of the groove structure is 5-15 um, and the etching gas is sixSulfur fluoride, wherein the protective gas is octafluorocyclobutane, the vacuum degree is 10-30 Pa, the power of the inductively coupled plasma is 100-1000W, and the etching speed is 400-700 nm/min; putting the silicon chip with the groove structure array into a mixed solution of 5M ammonium fluoride, 0.01M silver nitrate and 0.5M hydrogen peroxide, carrying out etching reaction for 1-10 minutes to obtain a silicon chip substrate with the groove structure array and the nanowire array, washing with deionized water, and carrying out N-phase oxidation2Drying for later use; before testing, the surface of a standby silicon chip substrate with a groove structure array and a nanowire array is cleaned by PBS buffer solution, and then 1.0-10.0 uM of streptavidin solution is added for incubation for 0.5-2 hours; washing with a PBS (phosphate buffer solution), then co-incubating for 0.5-2 hours in the PBS containing anti-EpCAM antibody and/or anti-EGFR antibody, and washing for later use;
b. modification and biological functionalization of the surface of the silicon chip substrate: washing the silicon chip substrate with the groove structure array and the nanowire array by concentrated sulfuric acid/hydrogen peroxide mixed solution, deionized water and absolute ethyl alcohol in a volume ratio of 45-85: 15-55 in sequence, and carrying out N washing in an ultra-clean environment2Drying for later use; preparing a toluene solution of fresh 3-aminopropyltriethoxysilane with the mass concentration of 0.1-10%, immersing the silicon chip substrate with the groove structure array and the nanowire array in the toluene solution for bonding reaction until the reaction is complete, cleaning with absolute ethyl alcohol, and performing N-phase bonding reaction2Drying to obtain a silicon chip substrate with the surface bonded with 3-aminopropyl triethoxysilane; preparing a fresh N-hydroxysuccinimide-PEG-biotin-based PBS solution with the mass concentration of 10-100 ng/mL, immersing the silicon chip substrate with the surface bonded with the 3-aminopropyltriethoxysilane in the PBS solution for bonding reaction again until the reaction is complete, cleaning with absolute ethyl alcohol, and carrying out N-propyl triethoxysilane-N-propyl-N-ethyl-N-propyl-N2Drying to obtain a silicon chip;
s2, preparing a microfluid chip: designing and printing a mask plate with a microfluid channel structure and a fishbone structure of a microfluid channel region; the method comprises the steps of rotationally coating SU-8 photoresist on a silicon material, drying the photoresist, aligning a mask plate of a microfluidic channel structure and a photoetching area of the microfluidic channel structure corresponding to the photoresist, illuminating and imaging to obtain the microfluidic channel structure, wherein the thickness of the photoresist is 10-150 um; rotationally coating SU-8 photoresist on the silicon material again, wherein the thickness of the photoresist is 50-200 um, drying, aligning a mask plate of a fishbone structure and a photoetching area of a corresponding microfluidic channel on the photoresist, illuminating, and imaging to obtain the fishbone structure constructed in the microfluidic channel area of the microfluidic channel structure; cleaning and drying a photoresist template with a microfluid channel structure, evaporating trichloro (1H, 1H, 2H, 2H perfluorooctyl) silane by using reduced pressure gas, and storing in a dust-free environment; pouring the photoresist template by adopting a Sylgardelelastomer kit, wherein the overall thickness of the microfluidic channel structure is 3-6 mm; cutting the microfluid channel structure into a structure matched with the silicon nanowire chip in size to obtain the required microfluid chip;
s3, assembling of a silicon chip and a microfluid chip: aligning the microfluid channel region on the microfluid chip with the region of the silicon chip etched with the groove structure array, and then clamping the silicon chip and the microfluid chip through a clamp to complete the assembly.
The invention also provides an application of the tumor cell capturing device in tumor cell phenotype analysis and tumor cell gene mutation detection.
The invention also provides application of the tumor cell capturing device in capturing the lung cancer ring tumor cells.
The invention also provides application of the tumor cell capturing device in PD-1 targeted immunotherapy target gene detection.
Compared with the prior art, the invention has the following beneficial effects:
in the technical scheme of the invention, the tumor cell capturing device of the microfluidic channel containing the groove array structure and the nanowire array is formed by constructing the silicon chip and the microfluidic chip and assembling and matching the silicon chip and the microfluidic chip, so that the tumor cells in the blood sample are captured and enriched when the blood sample with the tumor cells passes through the microfluidic channel, and the tumor cells are provided for pathological research and further analysis. Specifically, on the basis of a traditional silicon nanowire bioadsorption mechanism, biotin with a specific recognition function is modified on the surface of the nanowire to specifically recognize and capture tumor cells and reduce the interference of other cells; meanwhile, a micron groove structure array with a function of identifying the size of the tumor cells is etched on the silicon chip, so that the size characteristics of the tumor cells are screened through the groove structure, and the capturing accuracy of the tumor cells is further improved; when the microfluid passes through the microfluid channel, due to the microfluid mixing effect formed by the fish bone structure arranged in the microfluid channel (a turbulent flow phenomenon is formed on the contact surface of the nanowire and the silicon substrate so as to fully prolong the moving time of the blood sample in the microfluid channel), the complete contact between the cells in the blood sample and the nanowire is realized, thus the requirement on the amount of the blood sample is reduced, and the comprehensive detection and the high-efficiency capture of the tumor cells in the blood sample are realized. The invention greatly improves the capture precision and capture capability of the capture device to the tumor cells by capturing the tumor cells in the blood sample at least 4 levels, reduces the requirement on the rich content of the tumor cells in the blood sample, can detect early cancer lesions, and has great clinical medical significance.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the structures shown in the drawings without creative efforts.
FIG. 1 is a schematic view of an assembly structure of a tumor cell capturing device according to an embodiment of the present invention;
FIG. 2 is an exploded view of FIG. 1;
FIG. 3 is a schematic diagram of a process for preparing an array of trench structures on a silicon chip according to an embodiment of the present invention;
FIG. 4 is a flow chart illustrating the process of fabricating a surface modification of a nanowire on a silicon chip according to an embodiment of the present invention;
FIG. 5 is a flow chart illustrating the preparation of bio-functionalization of nanowires on a silicon chip in accordance with an embodiment of the present invention;
FIG. 6 is a flow chart illustrating the preparation of a nanowire-modified antibody assembly on a silicon chip according to an embodiment of the present invention;
FIG. 7 is a schematic diagram of a microfluidic channel structure according to an embodiment of the present invention;
FIG. 8 is a data plot showing the results of tumor cell capture performance of the capture device in accordance with one embodiment of the present invention;
FIG. 9 is a graph of data obtained from the results of the purity of the CTC cells captured and the activity of the CTC cells after capture by different capture devices in one embodiment of the present invention;
FIG. 10 is a fluorescence image of a capture device after capturing CTC cells in accordance with an embodiment of the present invention;
FIG. 11 is an electron micrograph of a CTC cell captured by a capture device according to one embodiment of the present invention;
FIG. 12 is a partial magnified electron micrograph of FIG. 11;
FIG. 13 is a data plot showing the results of a combined antibody assay for capturing CTC cells in accordance with one embodiment of the present invention;
FIG. 14 is a data plot of the results of optimal microfluidic flow rates for a capture device in an embodiment of the invention;
FIG. 15 is a graphical representation of the effect of different microfluidic channel structures on capture efficiency in an embodiment of the invention;
FIG. 16 is a graph showing the effect of different modified substrates on the capture efficiency in one embodiment of the present invention;
FIG. 17 is a graph of electron microscope data of tumor cell capture by a capture device of an embodiment of the present invention during testing of a clinical cancer patient blood sample;
FIG. 18 is a data diagram illustrating the results of a clinical test performed using a capture device in accordance with one embodiment of the present invention.
The implementation, functional features and advantages of the objects of the present invention will be further explained with reference to the accompanying drawings.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
In addition, technical solutions between various embodiments may be combined with each other, but must be realized by a person skilled in the art, and when the technical solutions are contradictory or cannot be realized, such a combination should not be considered to exist, and is not within the protection scope of the present invention.
Referring to fig. 1 to 7, the present invention proposes a tumor cell capturing device, which includes a silicon chip and a microfluidic chip assembled with the silicon chip;
the silicon chip is provided with a groove structure array and a nanowire array, the silicon chip is provided with a capture surface, the groove structure array is concavely arranged on the capture surface, the nanowire is convexly arranged on the concave surface of the groove structure, and the nanowire is connected with an antibody with specificity for recognizing an antigen on a tumor cell;
the microfluidic chip is provided with a connecting surface, and a microfluidic channel is concavely arranged on the connecting surface;
the area of the connecting surface provided with the microfluid channel is matched with the area of the capturing surface provided with the groove structure array, so that when the connecting surface covers the capturing surface, the groove structure array is covered by the microfluid channel, and a sealed channel for microfluid to pass through is formed between the silicon chip and the microfluid chip.
In this embodiment, in order to improve the capturing capability and capturing accuracy of the tumor cell capturing device for the tumor cells, at least 3 levels of capturing checkpoints are designed. Firstly, in the embodiment, a groove structure array is arranged on the silicon chip, the size of the groove structure array is similar to that of the tumor cells, and the tumor cells are captured according to the size of the tumor cells; secondly, the nanowire array is arranged on the concave surface of the groove structure array, and the tumor cells are adsorbed by the biological adsorption capacity of the nanowires; thirdly, in order to further enhance the accurate recognition and capture of the nanowire array to the tumor cells, the nanowire is modified with an antibody combination capable of specifically recognizing the tumor cells, so that the capture accuracy of the tumor cells is improved. In this regard, a microfluidic channel adapted to the region of the groove structure array is disposed on the microfluidic chip, and the microfluidic channel is assembled with the groove structure array region (and the nanowire array disposed on the concave surface of the groove structure) to form a microfluidic channel for the passage of the tumor cell-containing microfluid.
Optionally, to better achieve capture of tumor cells, the groove structure array is a hemispherical groove array. Preferably, the array of groove structures is an array of hemispherical grooves. Specifically, the diameter of hemispherical groove is 5 ~ 45um, adjacent two distance between the centre of a circle of hemispherical groove is 3 ~ 20 um.
Optionally, the length of the nanowire is 1-5 um; and/or the presence of a gas in the gas,
the diameter of the nanowire is 10-500 nm.
Optionally, the number of the microfluidic channels is multiple, and the multiple microfluidic channels are sequentially connected end to end and arranged in a snake shape; and/or the presence of a gas in the gas,
the width of the microfluid channel is 0.1-3 mm; and/or the presence of a gas in the gas,
the height of the microfluidic channel is 25-130 um.
Optionally, in order to improve the flow mixing effect of the microfluid (a turbulent phenomenon is formed on the surface of the nanowire in contact with the silicon substrate to sufficiently increase the moving time of the blood sample in the microfluidic channel), a plurality of fishbone structures are arranged inside the microfluidic channel part.
Optionally, a plurality of the fishbone structures are arranged at the top of the microfluidic channel at intervals; and/or the presence of a gas in the gas,
the width of the fishbone structure is 5-15 um; and/or the presence of a gas in the gas,
the distance between the fishbone structures is 8-15 um; and/or the presence of a gas in the gas,
the height of fishbone structure is 10 ~ 40 um.
Optionally, the material of the microfluidic chip is one or a combination of plastic, rubber and fiber. It should be understood that the material of the microfluidic chip is preferably transparent for easy viewing.
Optionally, the antibody is one or a combination of an EpCAM antibody and/or an EGFR antibody.
In the embodiment of the invention, the tumor cell capturing device of the microfluidic channel containing the groove array structure and the nanowire array is formed by constructing the silicon chip and the microfluidic chip and assembling and matching the silicon chip and the microfluidic chip, so that the tumor cells in the blood sample are captured and enriched when the blood sample with the tumor cells passes through the microfluidic channel, and the tumor cells are used for pathological research and further analysis. Specifically, on the basis of a traditional silicon nanowire bioadsorption mechanism, biotin with a specific recognition function is modified on the surface of the nanowire to specifically recognize and capture tumor cells and reduce the interference of other cells; meanwhile, a micron groove structure array with a function of identifying the size of the tumor cells is etched on the silicon chip, so that the size characteristics of the tumor cells are screened through the groove structure, and the capturing accuracy of the tumor cells is further improved; when the microfluid passes through the microfluid channel, due to the microfluid mixing effect formed by the fish bone structure arranged in the microfluid channel (a turbulent flow phenomenon is formed on the contact surface of the nanowire and the silicon substrate so as to fully prolong the moving time of the blood sample in the microfluid channel), the complete contact between the cells in the blood sample and the nanowire is realized, thus the requirement on the amount of the blood sample is reduced, and the comprehensive detection and the high-efficiency capture of the tumor cells in the blood sample are realized. According to the embodiment of the invention, by capturing the tumor cells in the blood sample at least 4 levels, the capturing precision and the capturing capability of the capturing device on the tumor cells are greatly improved, the requirement on the rich content of the tumor cells in the blood sample is reduced, the early-stage cancer lesion can be detected, and the clinical medicine significance is great. Has great medical significance in the application of tumor cell phenotype analysis and tumor cell gene mutation detection, lung cancer ring tumor cell capture and PD-1 targeted immunotherapy target gene detection.
Specifically, in order to better illustrate the technical effects of the tumor cell capturing device of the present invention, the following example sets and data are provided. It is to be understood that the following group of embodiments is only a few embodiments of the present invention and does not encompass all embodiments.
Example 1
The invention provides a preparation method of a tumor cell capturing device, which comprises the following steps: (in this example, a medical pathological quartz glass slide with a thickness of 0.5 mm is used as a base)
S1, preparing a silicon chip:
a. preparing a silicon chip substrate with a groove structure array and nanowires: referring to fig. 3, acetone, alcohol and deionized water are sequentially cleaned by ultrasonic for 30 minutes, N2Blow-drying the silicon surface and then passing through O2Etching the silicon wafer by reactive ions, further removing organic matters on the surface of the silicon wafer and increasing the hydrophilicity of the surface of the silicon wafer for later use; assembling silicon dioxide microspheres on a spare silicon wafer by adopting a spin coating method, wherein the size of the microspheres is 5-20 um, the spin coating speed is 500-6000 rpm, and the spin coating time is 10-60 seconds, so as to obtain the silicon wafer with the silicon dioxide microspheres; vacuum evaporating a 1-15 nm metal chromium film on the silicon wafer with the silicon dioxide microspheres to form a protective film for dry etching; then removing the silicon dioxide microspheres by using ultrasound to obtain a silicon wafer with exposed circular holes with the diameter of 5-20 um; etching the silicon exposed out of the circular hole area by an Inductively Coupled Plasma (ICP) etching technology to obtain a silicon wafer with a pit (groove structure) with a certain depth, wherein the etching depth of the groove structure (pit) is 5-15 mu m, and the etching gas is sulfur hexafluoride (SF)6) The protective gas is octafluorocyclobutane (C)4F8) The vacuum degree is 10-30 Pa, the power of Inductively Coupled Plasma (ICP) is 800W, and the etching speed is 600 nm/min; and then, further chemically etching the silicon wafer with the circular hole and groove structure array under the condition of not removing the surface metal chromium film to obtain the nanowire structure with larger specific surface area. The conditions of the chemical etching are as follows: putting the silicon chip into 5M ammonium fluoride and 0.01M silver nitrateReacting the mixture with 0.5M hydrogen peroxide for 10 to 15 minutes to perform etching reaction for 1 to 10 minutes to obtain a silicon chip substrate with a round hole groove structure array and a nanowire array, washing with deionized water, and performing N-phase reaction2Drying for later use;
b. modification and biological functionalization of the surface of the silicon chip substrate: referring to fig. 4 to 6, the silicon chip substrate with the circular hole groove structure array and the nanowire array is washed by concentrated sulfuric acid/hydrogen peroxide mixed solution, deionized water and absolute ethyl alcohol in a volume ratio of 70:30 in sequence, and is subjected to N in an ultra-clean environment2Drying for later use; preparing a toluene solution of fresh 3-aminopropyltriethoxysilane with a mass concentration of 1%, immersing the silicon chip substrate with the groove structure array and the nanowire array in the toluene solution for bonding reaction until the reaction is complete, cleaning with absolute ethyl alcohol, and performing N-phase bonding2Drying to obtain a silicon chip substrate with the surface bonded with 3-aminopropyl triethoxysilane; preparing fresh N-hydroxysuccinimide-PEG-biotin-based PBS solution with mass concentration of 50ng/mL, immersing the silicon chip substrate with the surface bonded with the 3-aminopropyltriethoxysilane in the PBS solution for bonding reaction again until the reaction is complete, cleaning with absolute ethyl alcohol, and carrying out N-propyl triethoxysilane-N-propyl-triethoxysilane-based bonding reaction on the silicon chip substrate2Drying to obtain a silicon chip, and placing the silicon chip in a drying environment at 4 ℃ for later use; (before the blood sample test, the chip surface was washed with PBS, and then 200uL of streptavidin solution (5.0uM) was added thereto, and incubated for 1 hour, after the PBS wash, incubated for 1 hour in 200uL of PBS solution containing anti-EpCAM and anti-EGFR antibodies, and used for the capture of CTC cells after the wash)
S2, preparing a microfluid chip: referring to fig. 7, a mask plate having a microfluidic channel structure of a microfluidic channel region and a fishbone structure is designed and printed; spin-coating SU-8 photoresist on the silicon material, wherein the thickness of the photoresist is 70um, drying, aligning a mask plate of the microfluidic channel structure and a corresponding photoetching area of the microfluidic channel structure on the photoresist, illuminating, and imaging to obtain the microfluidic channel structure; rotationally coating SU-8 photoresist on the silicon material again, wherein the thickness of the photoresist is 100um, drying, aligning a mask plate of the fishbone structure and a photoetching area of a corresponding microfluidic channel on the photoresist, illuminating, and imaging to obtain the fishbone structure constructed in the microfluidic channel area of the microfluidic channel structure; cleaning and drying a photoresist template with a microfluid channel structure, evaporating trichloro (1H, 1H, 2H, 2H perfluorooctyl) silane by using reduced pressure gas, and storing in a dust-free environment; pouring the photoresist template by adopting a Sylgardelelastomer kit, wherein the integral thickness of the microfluidic channel structure is 4.5 mm; cutting the microfluid channel structure into a structure matched with the silicon nanowire chip in size to obtain the required microfluid chip; specifically, the width of the microfluidic channel is 1mm and the depth is 100 um. The inside fishbone structure that is equipped with of microfluid channel, the width of fishbone structure is 10um, highly is 30um, and the interval is 10um to when microfluid passes through the microfluid channel, produce the torrent of perpendicular microfluid channel bottom, thereby strengthen the liquid stream and flow through during the contact of cell and silicon nanowire base when microfluid channel, increase the base after the combination catches the antibody and the biological adhesion of target cell.
S3, assembling of a silicon chip and a microfluid chip: referring to fig. 1 and 2, aligning the microfluidic channel region on the microfluidic chip with the region of the silicon chip etched with the array of groove structures, and then clamping the silicon chip and the microfluidic chip by a clamp to complete the assembly.
Specifically, the assembly steps are as follows: preparing 200uL of 5.0uM Streptavidin (Streptavidin, SA) PBS solution, covering the area of the silicon nanowire chip etched with the nanowire, and standing for 45 minutes; washing for three times by using a PBS solution, sucking residual solution on the surface, and assembling the microfluid chip and the silicon nanowire chip by using a clamp; tygon fluid delivery tubing at the inlet and outlet was connected and connected to a 1mL syringe already mounted on the syringe pump. It should be understood that in order to enhance the sealing property between the microfluidic chip and the silicon nanowire chip, the material of the microfluidic channel is one or a combination of polymer such as plastic, rubber and fiber material.
Example 2
1) Preparation of mock samples
The culture abundance is about 95 percentThe non-small cell lung cancer cell line A549 culture dish of (9) prepare cell suspension (accurate counting) with density of 10000/mL, and the solution for sorting is diluted by gradient 200/20uL, 180uL containing 2 x 10 is added5Jurkat cell suspension (cells were both diluted and dispensed with PBS) was used as a 200uL mock sample.
2) Capture performance of tumor cell capture device on tumor cells
The experimental steps are as follows: 500 lung cancer cell lines (A549, HCC827 and H1975) of different sizes were selected, mixed with PBMC extracted from blood, and passed through the chip test group as test samples. Specifically, four kinds of chips with the diameters of the round hole groove structures of 5um, 10um, 15um and 20um are prepared and selected in the experiment for testing. The result data shown in fig. 8 was obtained.
And analyzing results. As can be seen from fig. 8, the circular hole groove structure can distinguish the size of the cells, and can selectively capture tumor cells.
3) Capture device with round hole groove structure array and nanowire array and capture of tumor cells by traditional nanowire array capture device
The experimental steps are as follows: the tumor cells in the mock samples prepared in 1) above were captured in the capturing device of the present invention and the conventional capturing device, respectively, under the same capturing conditions, and the purity of the tumor cells captured on each capturing device, the composition of the non-viable cells, and the activity of the CTC cells after capture were examined, to obtain the result data shown in fig. 9 to 12.
And analyzing results. As can be seen from fig. 9, the capturing device with the groove structure array and the nanowire array has a significantly higher capturing precision for the CTC cells than the conventional nanowire capturing device, and the activity of the CTC cells captured by the capturing device with the groove structure array and the nanowire array is significantly higher than that of the CTC cells captured by the conventional nanowire capturing device. It is shown that the trapping device with the groove structure array and the nanowire array in this embodiment has more excellent properties and advantages. FIG. 10 is a fluorescence image of a capture device with a groove array after capture of CTC cells; FIGS. 11 and 12 are electron micrographs of CT cells captured by a capture device with a groove array; in which fig. 12 is an enlarged view of a portion of fig. 11.
Example 3
1) Combined detection of antibodies for capturing CTC cells
The experimental steps are as follows: and respectively modifying the nanowire by using anti-EpCAM, anti-EGFR antibody and anti-EpCAM and anti-EGFR combined antibody with the same concentration to form a capture device modified with different recognition antibodies. The test object in this example was lung cancer cells of a549 line, and a mock sample was prepared by the preparation method described in 1) in example 2, and then counted by fluorescent staining using the different capturing devices described above. The number of cells caught on the capturing device was measured to obtain the result data as shown in FIG. 10.
And analyzing results. As shown in fig. 10, the combination antibody had better capture efficiency.
2) Testing and selection of optimal flow rates
The experimental steps are as follows:
the mock sample described in 1) of example 2 was selected as the test subject, and the capture efficiency of a549 cells was tested at flow rates of 0.1mL/h, 0.2mL/h, 0.5mL/h, 1.0mL/h, 2.0mL/h and 5.0mL/h, respectively, and the data graph of the results shown in fig. 11 was obtained. The experiment is specifically carried out according to the following operation steps:
a. loading 200uL of simulation sample into a 1mL injector, connecting the system, and injecting the simulation sample into a capturing device according to the flow rate;
b. after the injection, 100uL of 2% PFA (paraformaldehyde) solution was loaded into the syringe and passed through the capturing device at a flow rate of 1.0mL/h to immobilize the captured cells;
c. after the capture device is disassembled, the silicon wafer is taken out, the silicon wafer is washed by PBS solution, then 200uL of prepared mixed solution of 5uM cytokeratin antibody (anti-pan cytokeratin) and 5uM CD45 antibody (anti-CD45) is covered on the area of the silicon nanowire chip etched with the nanowires, the silicon nanowire chip is kept stand for 24 hours at 4 ℃, the silicon nanowire chip is washed, and secondary antibodies are dyed by a standard method, specifically, the CK antibody is marked by Alex488, the CD45 antibody is marked by Alex555, and the dyeing time of the secondary antibodies is 30 minutes; then cleaning, absorbing and removing solution residues on the surface as much as possible, packaging with 80uL of a sealing solution containing a Hoechest nuclear staining reagent, adding a cover glass, performing fluorescence imaging and counting;
d. the cells of CK +/CD45-/DAPI + in the imaging results were counted as captured cancer cells, and the corresponding capture efficiency was calculated by comparing the number of initially added cancer cells with 200.
And analyzing results. Referring to fig. 11, it can be seen that, in a certain microfluidic flow rate range, as the microfluidic flow rate increases, the number of tumor cells captured by the capturing device also increases; specifically, at a microfluidic flow rate of 1.0mL/h, the capture of tumor cells by the capture device reaches a maximum, and the number of captured cells approaches 90 (the initial tumor cell number is 200, i.e., the capture rate approaches 50%); when the flow rate of the microfluid exceeds 1.0mL/h, the capture quantity of the tumor cells by the capture device is gradually reduced along with the increase of the flow rate of the microfluid.
3) Effect of different microfluidic channel structures on Capture efficiency
The experimental steps are as follows: selecting the simulated sample described in 1) in example 2 as a test object, testing the capturing efficiency of the capturing device with the polygonal line microfluidic channel and the capturing device with the straight line microfluidic channel for the CTC cells, respectively, and obtaining the result data as shown in fig. 12. The operation steps are shown in 2), and are not repeated herein.
And analyzing results. As can be seen from fig. 12, the capturing device with the zigzag microfluidic channel has higher capturing efficiency and more advantageous capturing capability for CTC cells.
4) Effect of different modified substrates on Capture efficiency
The experimental steps are as follows: selecting the simulation sample described in 1) of example 2 as a test object, respectively testing the capturing efficiency of the capturing device of the conventional silicon nanowire substrate, the capturing device simultaneously having the combination of the conventional silicon nanowire substrate and the capturing antibody, the capturing device having the array of the circular hole-groove structure, and the capturing device simultaneously having the combination of the array of the circular hole-groove structure and the capturing antibody on the CTC cells, and obtaining the result data shown in fig. 13. The operation steps are shown in 2), and are not repeated herein.
And analyzing results. As can be seen from fig. 13, the capturing device with the combination of the circular hole groove structure array and the capturing antibody has higher capturing efficiency for the CTC cells and more advantageous capturing capability.
5) Interference test
Extraction of leukocytes from healthy blood as interfering cells 2.0 x 106mL, mock samples were prepared and compared for the capture capacity of different types of cancer cells (a549, HCC827 and H1975), demonstrating that interference with the white blood cells in real blood by an equal amount does not affect the efficiency of the assay.
Example 4
1) Capture and phenotypic analysis of CTCs in blood samples of clinical cancer patients:
the method comprises the following steps that 1, 8.5mL of BD Vacutainer Glass ACD Solution A tube is needed for blood collection of clinical samples, so that the phenomenon that EDTA anticoagulation damages antigens on the cell surface in blood, the combination of capture antibodies is affected and the capture efficiency is reduced is avoided; the first tube from which blood was drawn at 2mL and was not used for testing (to detect the appearance of false positive cells due to the shedding of epithelial cells when the needle penetrated the blood).
② taking 4mL of whole blood as an example, the method adopts gradient density centrifugation to primarily purify the blood of a cancer patient. Adding 4mL of PBS solution to dilute the blood sample in an equal volume, mixing uniformly, and slowly adding the diluted blood sample into a 15mL centrifuge tube into which 4mL of gradient density centrifugate (1077) is added; centrifuging at 300g for 40 minutes, removing serum (yellow), and collecting about 2-4 mL of a mononuclear cell layer (PBMC); then, further centrifuging the collected monocyte layer, selecting 400g for 5 minutes, removing supernatant, and washing with 2mL of PBS solution; centrifuging again, removing the supernatant, adding 400uL of a mixed solution of freshly prepared 5uM cytokeratin antibody (anti-pan cytokeratin) and 5uM CD45 antibody (anti-CD45), breaking up cell aggregation, and incubating for 45 minutes; washed and made up to volume in 400uL of PBS.
③ as in embodiment 1, the silicon chip and the microfluidic chip are assembled to form a capturing device, the microfluidic flow rate of the capturing device is 1.0mL/h, the height of the fishbone structure is 30um, and the height of the nanowire is 327 nm. Two parallel runs of 200uL of the mock sample were performed and then stained, resulting in the data shown in figure 14.
2) And (6) analyzing results. Referring to fig. 14, it can be seen that the present example mainly aims at the capture and fluorescent staining of CTC cells contained in the blood of pancreatic cancer patients, and effectively detects and analyzes the CTC phenotype population present in CTCs expressing epithelial metaplasia, and plays an important role in early warning of metastasis. And the method can perform staining detection of various cancer markers on the CTC cells.
Example 5
Post-capture release of CTCs in blood of clinical lung cancer patients and detection of genetic mutations:
1) the experimental steps are as follows: this example tests 2 samples (1 patient with free-form lung cancer and 1 patient with relapsed free-form treatment). The silicon chip and microfluidic chip were then assembled to form a capture device with a microfluidic flow rate of 1.0mL/h, a fishbone structure height of 30um, and a nanowire height of 327nm, as described in example 1. Collecting DNA samples of all cracked CTC cells by an assembled capture device, and then carrying out DNA extraction and amplification by using a WGA4 single cell whole genome amplification kit; amplification and Sanger sequencing were then performed on the lung cancer marker genes EGFR targets L858R and T790M, resulting in the data shown in FIG. 15.
2) And (4) analyzing results: referring to fig. 15, both KRAS subtypes were detected in CTC cells enriched in both testers, but were undetectable in whole blood cells of both testers. Therefore, CTC cells contained in blood of a person with lung diseases can be enriched by the method, and KRAS gene mutation carried by the CTC cells can be rapidly detected. The method provides non-invasive real-time and simple operation for liquid biopsy based on CTC cells, and the result is accurate.
The above description is only an alternative embodiment of the present invention, and not intended to limit the scope of the present invention, and all modifications and equivalents of the present invention, which are made by the contents of the present specification and the accompanying drawings, or directly/indirectly applied to other related technical fields, are included in the scope of the present invention.
Claims (13)
1. A tumor cell capture device, comprising a silicon chip and a microfluidic chip assembled for use with the silicon chip;
the silicon chip is provided with a groove structure array and a nanowire array, the silicon chip is provided with a capture surface, the groove structure array is concavely arranged on the capture surface, the nanowire is convexly arranged on the concave surface of the groove structure, and the nanowire is connected with an antibody with specificity for recognizing an antigen on a tumor cell;
the microfluidic chip is provided with a connecting surface, and a microfluidic channel is concavely arranged on the connecting surface;
the area of the connecting surface provided with the microfluid channel is matched with the area of the capturing surface provided with the groove structure array, so that when the connecting surface covers the capturing surface, the groove structure array is covered by the microfluid channel, and a sealed channel for microfluid to pass through is formed between the silicon chip and the microfluid chip.
2. The tumor cell capture device of claim 1, wherein the array of groove structures is an array of hemispherical grooves.
3. The tumor cell capturing device according to claim 2, wherein the groove structure array is a hemispherical groove array, and the diameter of the hemispherical groove is 5-45 um; and/or the presence of a gas in the gas,
two adjacent distance between the centre of a circle of hemisphere recess is 3 ~ 20 um.
4. The tumor cell capture device of claim 1, wherein the nanowires are 1-5 um in length; and/or the presence of a gas in the gas,
the diameter of the nanowire is 10-500 nm.
5. The tumor cell capturing device according to claim 1, wherein a plurality of the microfluidic channels are provided, and the plurality of the microfluidic channels are connected end to end in sequence and arranged in a serpentine shape; and/or the presence of a gas in the gas,
the width of the microfluid channel is 0.1-3 mm; and/or the presence of a gas in the gas,
the height of the microfluidic channel is 25-130 um.
6. The tumor cell capturing device according to claim 5, wherein a plurality of fishbone structures are provided inside the microfluidic channel section.
7. The tumor cell capturing device according to claim 6, wherein a plurality of the fishbone structures are spaced apart at the top of the microfluidic channel; and/or the presence of a gas in the gas,
the width of the fishbone structure is 5-15 um; and/or the presence of a gas in the gas,
the distance between the fishbone structures is 8-15 um; and/or the presence of a gas in the gas,
the height of fishbone structure is 10 ~ 40 um.
8. The tumor cell capture device of any one of claims 1 to 7, wherein the material of the microfluidic chip is one or a combination of plastic, rubber, and fiber.
9. The tumor cell capture device of any one of claims 1 to 7, wherein the antibody is one or a combination of an EpCAM antibody and/or an EGFR antibody.
10. A method of manufacturing a tumor cell capturing device according to any one of claims 1 to 9, comprising the steps of:
s1, preparing a silicon chip:
a. preparing a silicon chip substrate with a groove structure array and nanowires: sequentially cleaning acetone, alcohol and deionized water, and N2Blow drying, O2Reactive ion etching of silicon wafersUsing; rotationally coating a groove structure micro-mold on a spare silicon wafer, wherein the rotational coating speed is 500-6000 rpm, and the rotational coating time is 10-60 seconds to obtain a template silicon wafer; evaporating a 1-15 nm metal film layer on the template silicon wafer in vacuum, and removing the groove structure micromold to obtain a silicon wafer with an exposed groove structure region; etching the silicon exposed out of the groove area by using inductively coupled plasma to obtain a silicon wafer with a groove structure array, wherein the etching depth of the groove structure is 5-15 um, the etching gas is sulfur hexafluoride, the protective gas is octafluorocyclobutane, the vacuum degree is 10-30 Pa, the power of the inductively coupled plasma is 100-1000W, and the etching speed is 400-700 nm/min; putting the silicon chip with the groove structure array into a mixed solution of 5M ammonium fluoride, 0.01M silver nitrate and 0.5M hydrogen peroxide, carrying out etching reaction for 1-10 minutes to obtain a silicon chip substrate with the groove structure array and the nanowire array, washing with deionized water, and carrying out N-phase oxidation2Drying for later use; before testing, the surface of a standby silicon chip substrate with a groove structure array and a nanowire array is cleaned by PBS buffer solution, and then 1.0-10.0 uM of streptavidin solution is added for incubation for 0.5-2 hours; washing with a PBS (phosphate buffer solution), then co-incubating for 0.5-2 hours in the PBS containing anti-EpCAM antibody and/or anti-EGFR antibody, and washing for later use;
b. modification and biological functionalization of the surface of the silicon chip substrate: washing the silicon chip substrate with the groove structure array and the nanowire array by concentrated sulfuric acid/hydrogen peroxide mixed solution, deionized water and absolute ethyl alcohol in a volume ratio of 45-85: 15-55 in sequence, and carrying out N washing in an ultra-clean environment2Drying for later use; preparing a toluene solution of fresh 3-aminopropyltriethoxysilane with the mass concentration of 0.1-10%, immersing the silicon chip substrate with the groove structure array and the nanowire array in the toluene solution for bonding reaction until the reaction is complete, cleaning with absolute ethyl alcohol, and performing N-phase bonding reaction2Drying to obtain a silicon chip substrate with the surface bonded with 3-aminopropyl triethoxysilane; preparing a fresh N-hydroxysuccinimide-PEG-biotin-based PBS solution with the mass concentration of 10-100 ng/mL, and immersing the silicon chip substrate with the surface bonded with the 3-aminopropyltriethoxysilane in the PBBonding reaction is carried out again in the S solution until the reaction is complete, absolute ethyl alcohol is used for cleaning, and N is carried out2Drying to obtain a silicon chip;
s2, preparing a microfluid chip: designing and printing a mask plate with a microfluid channel structure and a fishbone structure of a microfluid channel region; the method comprises the steps of rotationally coating SU-8 photoresist on a silicon material, drying the photoresist, aligning a mask plate of a microfluidic channel structure and a photoetching area of the microfluidic channel structure corresponding to the photoresist, illuminating and imaging to obtain the microfluidic channel structure, wherein the thickness of the photoresist is 10-150 um; rotationally coating SU-8 photoresist on the silicon material again, wherein the thickness of the photoresist is 50-200 um, drying, aligning a mask plate of a fishbone structure and a photoetching area of a corresponding microfluidic channel on the photoresist, illuminating, and imaging to obtain the fishbone structure constructed in the microfluidic channel area of the microfluidic channel structure; cleaning and drying a photoresist template with a microfluid channel structure, evaporating trichloro (1H, 1H, 2H, 2H perfluorooctyl) silane by using reduced pressure gas, and storing in a dust-free environment; pouring the photoresist template by adopting a Sylgardelelastomer kit, wherein the overall thickness of the microfluidic channel structure is 3-6 mm; cutting the microfluid channel structure into a structure matched with the silicon nanowire chip in size to obtain the required microfluid chip;
s3, assembling of a silicon chip and a microfluid chip: aligning the microfluid channel region on the microfluid chip with the region of the silicon chip etched with the groove structure array, and then clamping the silicon chip and the microfluid chip through a clamp to complete the assembly.
11. Use of a tumor cell capture device according to any one of claims 1 to 9 for phenotypic analysis of tumor cells and detection of genetic mutations in tumor cells.
12. Use of a tumor cell capture device according to any one of claims 1 to 9 for capturing tumor cells in the lung cancer annulus.
13. Use of a tumor cell capture device according to any one of claims 1 to 9 in the detection of a PD-1-targeted immunotherapy target gene.
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| CN112547148A (en) * | 2020-12-09 | 2021-03-26 | 武汉纺织大学 | Micro-fluidic target capture chip based on micro dam array, preparation method and application |
| WO2023109698A1 (en) * | 2021-12-15 | 2023-06-22 | 深圳先进技术研究院 | Microfluidic chip for capturing circulating tumor cell and manufacturing method therefor |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112547148A (en) * | 2020-12-09 | 2021-03-26 | 武汉纺织大学 | Micro-fluidic target capture chip based on micro dam array, preparation method and application |
| WO2023109698A1 (en) * | 2021-12-15 | 2023-06-22 | 深圳先进技术研究院 | Microfluidic chip for capturing circulating tumor cell and manufacturing method therefor |
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