CN113444630A - Automatic control system for filtering and separating circulating tumor cells by using porous filter membrane micro-fluidic chip - Google Patents
Automatic control system for filtering and separating circulating tumor cells by using porous filter membrane micro-fluidic chip Download PDFInfo
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Abstract
The invention discloses an automatic control system for filtering and separating Circulating Tumor Cells (CTCs) by a porous filter membrane micro-fluidic chip. The invention comprises the following steps: (1) developing and constructing a combined system comprising a CTCs porous filter membrane chip filtration separation platform and a micro-flow automatic control method; (2) developing a CTCs filtering and separating experiment, performing theoretical analysis, determining the through hole time of a CTC by combining the experiment and theoretical results, and further determining system control parameters such as injection time and flushing time; (3) and the control system opens and closes the injection pump and the inlet and outlet electromagnetic valves according to the control parameters, so that two operations of filtering and capturing the CTCs and reversely removing the captured CTCs are alternately carried out, and the blood without the CTCs is recovered in real time. The invention combines the technology of filtering and separating CTCs by the porous filter membrane chip with the micro-flow automatic control method, the proposed system is simple and easy to implement, has low cost and strong practicability, and has important significance for eliminating CTCs in a blood circulation system, preventing cancer cells from diffusing and treating cancers.
Description
Technical Field
The invention relates to a method which is closely related to the technologies of biology, medicine, microfluidics and the like. More particularly, the invention relates to a system combining the technology of filtering and separating CTCs by a porous filter membrane chip and a micro-flow automatic control method.
Background
The research on the capture and separation of circulating tumor cells in blood is of great significance to the treatment of cancer. Tumor cells are shed from primary foci and enter the blood circulation system to form Circulating Tumor Cells (CTCs). CTCs migrate with blood circulation to distant organs to form metastases, causing cancer to spread. Tumor metastasis is the leading cause of death in cancer patients, accounting for 90% of tumor-related deaths, and seriously threatening human health. Current common medical approaches to cancer treatment include: surgical treatment, radiotherapy (radiotherapy), chemotherapy (chemotherapy), targeted drug therapy, immunotherapy, and the like. Surgical treatment is the most important treatment means for early and medium-term cancer, and achieves the aim of curing by removing a tumor at a certain part of a patient body. Radiotherapy, chemotherapy, targeted drug therapy and the like have the problems of high cost, discomfort of patients in some cases, frequent relapse after treatment and the like. Therefore, the development of new effective methods for capturing and isolating CTCs from blood is a key to preventing cancer cell spreading and treating cancer.
At present, most of the existing research works are to capture and enrich CTCs from blood to achieve the purpose of detection. The research methods for capturing, separating and enriching the CTCs in the blood mainly comprise the traditional antigen-antibody specific binding method and the microfluidic technology-based method. The most representative method for capturing and separating CTCs based on antigen-antibody specific binding is the CellSearch system. The semi-automatic detection technology combines an immunomagnetic bead enrichment technology and an immunoquartz optical recognition technology, and has the advantage of specifically sorting and enriching specific CTCs. However, there are different subgroups of CTCs, there is no universal marker, and the expression level of some markers of CTCs is in dynamic change, so many highly differentiated cells are not easy to find antigens, and the specificity is limited. And for active CTCs with important clinical significance, false negative of missed detection or detection is easy to cause due to the fact that specific expression of cells is weakened. In addition, the Cellsearch system has high price, high single detection cost, long analysis time and complicated operation, and the defects limit the clinical application of the CTCs based on antigen-antibody reaction. Cell research methods based on microfluidic technology have become the main methods for capturing and separating CTCs. The sorting and enriching method of CTCs in the microfluidic chip is mainly divided into a biochemical method and a physical method. In biochemical methods, microfluidic chips containing microstructures such as cylindrical arrays, fishbone structures, etc. are common CTCs capturing chips. After the microstructure is modified with the ligand or the capture molecule combined with the CTCs surface marker, the CTCs can be captured by adsorption. Since the contact and contact of antigen and antibody on the microfluidic chip directly affect the sorting and enrichment effect, many research teams are dedicated to increasing the contact of antigen and antibody, i.e. increasing the contact of cells with the surface of the structure to which the antibody is bound. Also, the lack of 100% specific antibodies in CTCs and the very limited throughput limit the utility of this approach. The physical method mainly comprises porous membrane filtration and microcolumn array filtration, and the rest of the physical methods also comprise inertial migration separation and enrichment, dielectrophoresis separation and enrichment, acoustic dynamic separation and enrichment, magnetic field force separation and enrichment, deterministic lateral displacement separation and enrichment and the like. The CTCs are sorted and enriched in the micro-fluidic chip according to the difference of physical properties without being limited by the expression of cell surface markers, and the chip has the advantages of easy design of structure, low cost, short time consumption, simple and convenient operation and capability of realizing high-throughput sorting. However, in the filtration method based on the size, deformability, and the like of cells, it is difficult to distinguish CTCs having a size close to that of background blood cells, so that the purity of the captured CTCs is low, and the cells are often continuously retained in the filtration structure, which causes a clogging problem. The inertial migration method can rapidly sort, enrich and maintain high activity of cells by utilizing fluid acting force, but the design and the manufacture are more complex, and the problem of low purity of the captured CTCs is also existed. For dielectrophoresis, magnetic field force and other selection and enrichment methods, complicated devices such as magnetic fields, electric fields and the like are required to be added in design and manufacture, so that the methods become more complicated.
In conclusion, most of the studies are about the enrichment of CTCs from blood for detection purposes, which is of great significance for clinical diagnosis and therapeutic evaluation of CTCs levels in cancer patients. However, in addition to the enrichment assay for CTCs, much work has overlooked the ability to isolate CTCs from blood for cancer therapy. If CTCs in the blood of the patient are separated, the blood without CTCs can be delivered to the patient again, so as to achieve the purposes of gradually eliminating CTCs in the blood circulation system and preventing cancer cells from spreading. Finally, cancer can be treated by surgical treatment, where tumors are removed from the patient's body.
Disclosure of Invention
Under the background, the invention provides an automatic control system for filtering and separating circulating tumor cells by a porous filter membrane microfluidic chip, which helps cancer patients to eliminate CTCs in a blood circulation system, prevent cancer cells from diffusing and treat cancers.
In order to achieve the purpose, the invention provides the following technical scheme:
the invention relates to an automatic control system for filtering and separating circulating tumor cells by a porous filter membrane micro-fluidic chip, which comprises the following components:
step 1: through investigation, a reliable microfluidic automated control system is developed, and a combined system comprising a CTCs porous filter membrane chip filtration separation platform and a microfluidic automated control method is built, as shown in FIG. 1.
Step 2: designing and processing a porous filter membrane micro-fluidic chip, developing a CTCs filtration and separation experiment, and performing theoretical analysis. Determining the hole passing time of a circulating tumor cell by comparing experimental and theoretical results, and respectively adding safety factors of 0.8 and 1.5 to the hole passing time of the cell under the condition of determining parameters such as flow rate, fluid viscosity and the like of an injection pump to obtain a system control parameter, namely positive flow injection time T1And reverse flow flush time T2。
And step 3: blood containing CTCs is extracted from a patient, anticoagulant is added into the blood, the blood is put into a syringe of a syringe pump 1, and a saline flushing fluid is put into a syringe of a syringe pump 2.
And 4, step 4: starting the system, initializing the system, and determining the injection time T obtained in step 21Time of flushing T2Flow rate Q of syringe pump 11Flow rate Q of injection pump 22And the electromagnetic valve name corresponding to each microfluidic channel is written into a register (the flow rate satisfies Q)1＝Q2). Assuming the blood inlet valve is CV-01, the normal saline flushing liquid inlet valve is CV-02, the filtered blood outlet valve is CV-03, and the normal saline flushing liquid outlet valve is CV-04.
And 5: and (4) starting the system at the same time, opening the injection pump 1, introducing the blood containing the CTCs of the patient into the microfluidic chip, opening the blood valves CV-01 and CV-03, and implementing the CTCs filtration and capture process controlled by the computer program. When the filtering process time reaches T from 01In the process, the automatic control system of the micro flow controls the injection pump 1 and the valves CV-01 and CV-03 to be closed, and simultaneously opens the injection pump 2 and the flushing valves CV-02 and CV-04. And (3) introducing a normal saline flushing fluid into the chip, and flushing the CTCs captured on the porous filter membrane by using the normal saline controlled by the computer program, wherein the waste liquid containing the CTCs is introduced into a waste liquid pool. During this time, blood that is free of CTCs is recovered and stored using a specially made disposable blood storage bag. When the flushing progress time reaches T from 02In the process, the micro-flow automatic control system controls the injection pump 2 and the flushing valves CV-02 and CV-04 to be closed, and simultaneously opens the injection pump 1 and the valves CV-01 and CV-03 to enter the next round of process. After repeated many times, the blood without CTCs is returned to the patient, thereby achieving the purpose of clearing the circulating tumor cells by physical means.
Wherein, the whole operation process involved in the steps is carried out in an aseptic and low-temperature environment.
Wherein, the hole passing time of a circulating tumor cell is respectively added with a safety factor of 0.8 and 1.5 to obtain a system control parameter, namely positive flow injection time T1And reverse flow flush time T2The aim is to ensure one hundred percent capture and wash away of the CTCs.
The device comprises a micro-flow automatic control system and a CTCs porous filter membrane chip filtering and separating platform. The micro-flow automatic control system comprises a computer, a singlechip AT89C52 control module, an electromagnetic valve driving module, a pressure regulator, a driving air source and the like. The CTCs porous filter membrane chip filtering and separating platform comprises an injection pump, an injector, a porous filter membrane microfluidic chip, a polyethylene catheter, blood containing CTCs, physiological saline flushing fluid, a micro valve and the like.
The method comprises the steps of designing and processing a porous filter membrane micro-fluidic chip, carrying out CTCs filtration and separation experiments, and carrying out theoretical analysis on cell via holes, so as to obtain the via hole time of circulating tumor cells. Specifically, a CTCs filtration separation experiment is carried out through a porous filter membrane chip to obtain the capture efficiency of CTCs; by performing dynamic analysis on the cell via hole, establishing a theoretical model on the basis of reasonable assumption and simplification to obtain a relational expression of the cell via hole time and the capture efficiency; and then the experimental result and the theoretical result are combined to determine the via hole time of the circulating tumor cell.
Wherein, the micro-flow automatic control system is a pneumatic micro-valve driving system based on single-chip microcomputer control. A series of control signals are output through the program setting of the single chip microcomputer platform, and the instructions are converted into the opening and closing actions of the electromagnetic valve through the electromagnetic valve driving circuit. The gas in the gas tank passes through the regulator to obtain a driving gas source with adjustable pressure, and the driving gas source provides driving force for the micro-pump pneumatic film, so that the micro-channel and the injection pump are controlled to be opened and closed, and the purpose of controlling whether liquid flows in the micro-channel or not is achieved.
In addition, the main principle of the micro-flow automatic control system is that the pressure difference between the air flow channel and the liquid flow channel is utilized to deform and rebound the middle PDMS layer, so that the liquid path is controlled to be cut off and conducted. When appropriate pressure is provided for the gas control channel, the middle elastic PDMS film layer is subjected to bending deformation, so that the fluid is blocked; when the pressure of the control channel is cancelled, the elastic PDMS film rebounds, and the liquid channel restores the flow state to play the role of a micro valve.
The micro-flow automatic control system comprises a singlechip control part, an electromagnetic valve drive circuit and a micro-flow control chip. The hardware part of the singlechip control part is designed by adopting an AT89C52 control module, generates a control signal through an I/O port of the AT89C52 and sends the control signal to the input end of the electromagnetic valve driving circuit, and the overall hardware design is detailed as shown in FIG. 2. The software part and the program design of the single chip microcomputer are mainly written in the keil C, the action signals of the valve are output according to the set time, and the whole program design is shown in detail in figure 3. In the design of the electromagnetic valve driving circuit, a signal output signal of the singlechip is connected with a base electrode of the triode, an anode of the electromagnetic valve is connected with a 12V power supply, and a cathode of the electromagnetic valve is connected with a collector electrode of the triode. In addition, after the voltage stabilizing diode is connected with the fast recovery rectifier diode in series, the voltage stabilizing diode is reversely connected with the electromagnetic valve in parallel. The output signal of the single chip microcomputer controls the state of the electromagnetic valve through an electromagnetic valve driving circuit, and the design of the single-way electromagnetic valve driving circuit is shown in figure 4. The microfluidic chip in the microfluidic automatic control system is designed to be a four-layer structure consisting of a glass chip, a Polydimethylsiloxane (PDMS) film and a Polycarbonate (PC) filter membrane. Wherein the upper and lower layers are glass chips, the upper layer glass chip is provided with a gas micro-channel, the lower layer glass chip is provided with a liquid micro-channel, the middle 2 layers of PDMS film and the 3 layers of PC filter membrane are provided with through holes for connecting the inlets of the upper and lower micro-channels, and the structure is shown in figures 5 and 6.
The micro-flow automatic control system comprises the following operation steps: firstly, two groups of inlet ends and outlet ends of the chip are respectively inserted into polyethylene catheters, blood of a patient containing CTCs flows in through the catheter 1, saline flushing fluid flows in through the catheter 2, blood without CTCs flows out through the catheter 3, and waste liquid containing CTCs flows out through the catheter 4. The inlet and outlet switches of the chip are controlled by a pneumatic micro valve. Secondly, turning on a system switch, initializing the system and setting the preset injection time T1Time of flushing T2And the electromagnetic valve names corresponding to all the microfluidic channels are written into a register, wherein the patient blood inlet valve containing CTCs is CV-01, the physiological saline flushing fluid inlet valve is CV-02, the filtered blood outlet valve without CTCs is CV-03, and the physiological saline flushing fluid outlet valve containing CTCs is CV-04. And starting the timer, and simultaneously opening the injection pump 1, the electromagnetic valve CV-01 and the electromagnetic valve CV-03. When the filtering process time reaches T1When the filtration process is finished, the injection pump 1 and the valves CV-01 and CV-03 are closed. Fourthly, when the process is finished, the injection pump 2, the electromagnetic valves CV-02 and CV-04 are opened. Introducing a normal saline flushing fluid into the chip, reversely flushing the CTCs captured on the porous filter membrane, and enabling the CTCs to be containedIs led via a conduit 4 to a waste reservoir. When the flushing progress time reaches T from 02When the flushing process is finished, the injection pump 2, the valves CV-02 and CV-04 are closed; simultaneously, the injection pump 1 and the valves CV-01 and CV-03 are opened, and the next round of process is started. And fifthly, continuously and circularly executing the step three and the step four until the filtering work of all blood samples is finished so as to realize the automatic process of capturing and separating the CTCs from the blood.
The research of carrying out the experiments of capturing and separating the CTCs by the microfluidic porous filter membrane also comprises designing and preparing the CTCs capturing microfluidic chip containing the porous filter membrane. The chip is prepared into a sandwich structure of two PDMS layers, and a PC filter membrane is arranged in the middle of the sandwich structure. The cavity and the channel of the PDMS layer are prepared by a photoetching method, and after the upper and lower PDMS layers are prepared, a hole is punched by a puncher to form a pre-designed inlet hole and a pre-designed outlet hole. The PDMS layer and the filter membrane are bonded into a whole after being processed by plasma.
The specific operations of carrying out the microfluidic porous filter membrane separation and capturing CTCs experimental research are as follows: CTCs in blood were fluorescently stained with a staining agent (e.g., CellTracker Green or CellTracker Red), and then a volume of blood containing a known number of CTCs was injected into the porous filter chip at a flow rate (e.g., 5mL/h) using a syringe pump, as shown in FIG. 7. And (3) carrying out an observation experiment by using a fluorescence microscope provided with a CCD camera, and shooting after a period of time to obtain a capture result of the stained CTCs on the porous filter membrane. The captured cells are counted using digital image processing. Then, the capturing efficiency was determined according to the ratio between the number of captured CTCs and the total number of injected CTCs.
Through the technical scheme, compared with the prior art, the invention discloses an automatic control system for filtering and separating circulating tumor cells by using a porous filter membrane microfluidic chip. The method comprises the following steps: through investigation, a reliable micro-flow automatic control system is developed, and a combined system comprising a CTCs porous filter membrane chip filtration separation platform and a micro-flow automatic control method is built. Designing and processing a porous filter membrane micro-fluidic chip, developing a CTCs filtration and separation experiment, and performing theoretical analysis. Determined by comparing experimental and theoretical resultsA hole passing time for circulating tumor cells, and under the condition of determining parameters such as flow rate and fluid viscosity of the injection pump, respectively adding safety factors of 0.8 and 1.5 to the hole passing time of the cells to obtain a system control parameter, namely forward flow injection time T1And reverse flow flush time T2. Blood containing CTCs is extracted from a patient, anticoagulant is added into the blood, the blood is put into a syringe of a syringe pump 1, and a saline flushing fluid is put into a syringe of a syringe pump 2. Starting the system, initializing the system, and determining the injection time T obtained in step 21Time of flushing T2Flow rate Q of syringe pump 11Flow rate Q of injection pump 22And the electromagnetic valve name corresponding to each microfluidic channel is written into a register (the flow rate satisfies Q)1＝Q2). Suppose the blood inlet valve is CV-01, the saline flushing fluid inlet valve is CV-02, the filtered blood outlet valve is CV-03, and the saline flushing fluid outlet valve is CV-04. And (4) starting the system at the same time, opening the injection pump 1, introducing the blood containing the CTCs of the patient into the microfluidic chip, opening the blood valves CV-01 and CV-03, and implementing the CTCs filtration and capture process controlled by the computer program. When the filtering process time reaches T from 01In the process, the automatic control system of the micro flow controls the injection pump 1 and the valves CV-01 and CV-03 to be closed, and simultaneously opens the injection pump 2 and the flushing valves CV-02 and CV-04. And (3) introducing a normal saline flushing fluid into the chip, and flushing the CTCs captured on the porous filter membrane by using the normal saline controlled by the computer program, wherein the waste liquid containing the CTCs is introduced into a waste liquid pool. During this time, blood that is free of CTCs is recovered and stored using a specially made disposable blood storage bag. When the flushing progress time reaches T from 02In the process, the micro-flow automatic control system controls the injection pump 2 and the flushing valves CV-02 and CV-04 to be closed, and simultaneously opens the injection pump 1 and the valves CV-01 and CV-03 to enter the next round of process. After repeated many times, the blood without CTCs is returned to the patient, thereby achieving the purpose of clearing the circulating tumor cells by physical means. The invention combines the technology of filtering and separating CTCs by a porous filter membrane chip with a microflow automatic control method, and providesThe system is simple and easy to implement, low in cost and strong in practicability, and has important significance for eliminating CTCs in a blood circulation system, preventing cancer cell diffusion and treating cancers.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following is a brief description of the drawings used in the description of the embodiments or the prior art. It is obvious that the drawings in the following description are only embodiments of the invention, and that for a person skilled in the art, other drawings can be obtained from the provided drawings without inventive effort.
FIG. 1 is a schematic view of a combined system of CTCs filtration separation and microfluidic automation control according to the present invention;
FIG. 2 is a diagram of the overall hardware design of the control part of the single chip microcomputer in the microfluidic automation control system device according to the present invention;
FIG. 3 is a flowchart of the software programming of the control portion of the single chip computer in the microfluidic automated control system device according to the present invention;
FIG. 4 is a diagram of a single solenoid valve driving circuit in the microfluidic automated control system of the present invention;
FIG. 5 is a plan view of the design of a microfluidic chip in the microfluidic automated control system device of the present invention;
FIG. 6 is a three-dimensional structure diagram of the design of the microfluidic chip in the microfluidic automated control system device of the present invention;
FIG. 7 is a diagram of an apparatus for performing experimental studies on separation and capturing of CTCs by a microfluidic porous filter membrane according to the present invention;
FIG. 8 is a flow chart of an automated control system for filtering and separating circulating tumor cells by using the porous filter membrane microfluidic chip of the present invention.
Detailed description of the invention
The technical solutions in the embodiments of the present invention will be described clearly below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only some embodiments, not all embodiments, of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without inventive step, are within the scope of the present invention.
The invention discloses an automatic control system for filtering and separating circulating tumor cells by a porous filter membrane micro-fluidic chip. The method comprises the following steps: through investigation, a reliable micro-flow automatic control system is developed, and a combined system comprising a CTCs porous filter membrane chip filtration separation platform and a micro-flow automatic control method is built. Designing and processing a porous filter membrane micro-fluidic chip, developing a CTCs filtration and separation experiment, and performing theoretical analysis. Determining the hole passing time of a circulating tumor cell by comparing experimental and theoretical results, and respectively adding safety factors of 0.8 and 1.5 to the hole passing time of the cell under the condition of determining parameters such as flow rate, fluid viscosity and the like of an injection pump to obtain a system control parameter, namely positive flow injection time T1And reverse flow flush time T2. Blood containing CTCs is extracted from a patient, anticoagulant is added into the blood, the blood is put into a syringe of a syringe pump 1, and a saline flushing fluid is put into a syringe of a syringe pump 2. Starting the system, initializing the system, and determining the injection time T obtained in step 21Time of flushing T2Flow rate Q of syringe pump 11Flow rate Q of injection pump 22And the electromagnetic valve name corresponding to each microfluidic channel is written into a register (the flow rate satisfies Q)1＝Q2). Suppose the blood inlet valve is CV-01, the saline flushing fluid inlet valve is CV-02, the filtered blood outlet valve is CV-03, and the saline flushing fluid outlet valve is CV-04. And (4) starting the system at the same time, opening the injection pump 1, introducing the blood containing the CTCs of the patient into the microfluidic chip, opening the blood valves CV-01 and CV-03, and implementing the CTCs filtration and capture process controlled by the computer program. When the filtering process time reaches T from 01In the process, the automatic control system of the micro flow controls the injection pump 1 and the valves CV-01 and CV-03 to be closed, and simultaneously opens the injection pump 2 and the flushing valves CV-02 and CV-04. Introducing a normal saline washing solution into the chip, and implementing the process of washing away the CTCs captured on the porous filter membrane by the normal saline controlled by the computer program, wherein the process comprises the steps ofAnd introducing the waste liquid containing the CTCs into a waste liquid pool. During this time, blood that is free of CTCs is recovered and stored using a specially made disposable blood storage bag. When the flushing progress time reaches T from 02In the process, the micro-flow automatic control system controls the injection pump 2 and the flushing valves CV-02 and CV-04 to be closed, and simultaneously opens the injection pump 1 and the valves CV-01 and CV-03 to enter the next round of process. After repeated many times, the blood without CTCs is returned to the patient, thereby achieving the purpose of clearing the circulating tumor cells by physical means. The invention combines the technology of filtering and separating CTCs by the porous filter membrane chip with the micro-flow automatic control method, the proposed system is simple and easy to implement, has low cost and strong practicability, and has important significance for eliminating CTCs in a blood circulation system, preventing cancer cells from diffusing and treating cancers.
The instruments and materials used in the process of the method of the invention are: the system comprises a micro-flow automatic control system, an injection pump, an injector, a porous filter membrane micro-fluidic chip, a polyethylene catheter, blood containing CTCs, physiological saline flushing fluid, a micro valve and the like.
Referring to fig. 8, the present invention discloses an automatic control system for filtering and separating circulating tumor cells by using a porous filter membrane micro-fluidic chip. The method comprises the following steps:
step 101: through investigation, a reliable micro-flow automatic control system is developed, and a combined system comprising a CTCs porous filter membrane chip filtration separation platform and a micro-flow automatic control method is built;
step 102: designing and processing a porous filter membrane micro-fluidic chip, developing a CTCs filtration and separation experiment, and performing theoretical analysis. Determining the hole passing time of a circulating tumor cell by comparing experimental and theoretical results, and respectively adding safety factors of 0.8 and 1.5 to the hole passing time of the cell under the condition of determining parameters such as flow rate, fluid viscosity and the like of an injection pump to obtain a system control parameter, namely positive flow injection time T1And reverse flow flush time T2；
Step 103: blood containing CTCs in a patient body is extracted, anticoagulant is added into the blood, the blood is put into a syringe of a syringe pump 1, and saline flushing fluid is put into the syringe of a syringe pump 2;
step 104: starting the system, initializing the system, and determining the injection time T obtained in step 21Time of flushing T2Flow rate Q of syringe pump 11Flow rate Q of injection pump 22And the electromagnetic valve name corresponding to each microfluidic channel is written into a register (the flow rate satisfies Q)1＝Q2). Assuming that a blood inlet valve is CV-01, a physiological saline flushing fluid inlet valve is CV-02, a filtered blood outlet valve is CV-03 and a physiological saline flushing fluid outlet valve is CV-04;
step 105: and (4) starting the system at the same time, opening the injection pump 1, introducing the blood containing the CTCs of the patient into the microfluidic chip, opening the blood valves CV-01 and CV-03, and implementing the CTCs filtration and capture process controlled by the computer program. When the filtering process time reaches T from 01In the process, the automatic control system of the micro flow controls the injection pump 1 and the valves CV-01 and CV-03 to be closed, and simultaneously opens the injection pump 2 and the flushing valves CV-02 and CV-04. And (3) introducing a normal saline flushing fluid into the chip, and flushing the CTCs captured on the porous filter membrane by using the normal saline controlled by the computer program, wherein the waste liquid containing the CTCs is introduced into a waste liquid pool. During this time, blood that is free of CTCs is recovered and stored using a specially made disposable blood storage bag. When the flushing progress time reaches T from 02In the process, the micro-flow automatic control system controls the injection pump 2 and the flushing valves CV-02 and CV-04 to be closed, and simultaneously opens the injection pump 1 and the valves CV-01 and CV-03 to enter the next round of process. After repeated many times, the blood without CTCs is returned to the patient, thereby achieving the purpose of clearing the circulating tumor cells by physical means.
In conclusion, the invention discloses an automatic control system for filtering and separating circulating tumor cells by using a porous filter membrane micro-fluidic chip. The method comprises the following steps: through investigation, a reliable micro-flow automatic control system is developed, and a combined system comprising a CTCs porous filter membrane chip filtration separation platform and a micro-flow automatic control method is built. Designing and processing a porous filter membrane micro-fluidic chip, developing CTCs filtration and separation experiments,and theoretical analysis is carried out. Determining the hole passing time of a circulating tumor cell by comparing experimental and theoretical results, and respectively adding safety factors of 0.8 and 1.5 to the hole passing time of the cell under the condition of determining parameters such as flow rate, fluid viscosity and the like of an injection pump to obtain a system control parameter, namely positive flow injection time T1And reverse flow flush time T2. Blood containing CTCs is extracted from a patient, anticoagulant is added into the blood, the blood is put into a syringe of a syringe pump 1, and a saline flushing fluid is put into a syringe of a syringe pump 2. Starting the system, initializing the system, and determining the injection time T obtained in step 21Time of flushing T2Flow rate Q of syringe pump 11Flow rate Q of injection pump 22And the electromagnetic valve name corresponding to each microfluidic channel is written into a register (the flow rate satisfies Q)1＝Q2). Suppose the blood inlet valve is CV-01, the saline flushing fluid inlet valve is CV-02, the filtered blood outlet valve is CV-03, and the saline flushing fluid outlet valve is CV-04. And (4) starting the system at the same time, opening the injection pump 1, introducing the blood containing the CTCs of the patient into the microfluidic chip, opening the blood valves CV-01 and CV-03, and implementing the CTCs filtration and capture process controlled by the computer program. When the filtering process time reaches T from 01In the process, the automatic control system of the micro flow controls the injection pump 1 and the valves CV-01 and CV-03 to be closed, and simultaneously opens the injection pump 2 and the flushing valves CV-02 and CV-04. And (3) introducing a normal saline flushing fluid into the chip, and flushing the CTCs captured on the porous filter membrane by using the normal saline controlled by the computer program, wherein the waste liquid containing the CTCs is introduced into a waste liquid pool. During this time, blood that is free of CTCs is recovered and stored using a specially made disposable blood storage bag. When the flushing progress time reaches T from 02In the process, the micro-flow automatic control system controls the injection pump 2 and the flushing valves CV-02 and CV-04 to be closed, and simultaneously opens the injection pump 1 and the valves CV-01 and CV-03 to enter the next round of process. After repeated many times, the blood without CTCs is returned to the patient, thereby achieving the purpose of clearing the circulating tumor cells by physical means. The invention filters and separates the CT with the porous filter membrane chipThe technology of the Cs is combined with the micro-flow automatic control method, the provided system is simple and easy to implement, low in cost and strong in practicability, and has important significance for eliminating CTCs in a blood circulation system, preventing cancer cells from diffusing and treating cancers.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (10)
1. The invention relates to an automatic control system for filtering and separating circulating tumor cells by a porous filter membrane micro-fluidic chip, which comprises the following components:
step 1: through investigation, a reliable micro-flow automatic control system is developed, and a combined system comprising a CTCs porous filter membrane chip filtration separation platform and a micro-flow automatic control method is built.
Step 2: designing and processing a porous filter membrane micro-fluidic chip, developing a CTCs filtration and separation experiment, and performing theoretical analysis. Determining the hole passing time of a circulating tumor cell by comparing experimental and theoretical results, and respectively adding safety factors of 0.8 and 1.5 to the hole passing time of the cell under the condition of determining parameters such as flow rate, fluid viscosity and the like of an injection pump to obtain a system control parameter, namely positive flow injection time T1And reverse flow flush time T2。
And step 3: blood containing CTCs is extracted from a patient, anticoagulant is added into the blood, the blood is put into a syringe of a syringe pump 1, and a saline flushing fluid is put into a syringe of a syringe pump 2.
And 4, step 4: starting the system, initializing the system, and determining the injection time T obtained in step 21Time of flushing T2Flow rate Q of syringe pump 11Flow rate Q of injection pump 22And for each microfluidic channelThe electromagnetic valve is named and written into a register (the flow satisfies Q)1＝Q2). Suppose the blood inlet valve is CV-01, the saline flushing fluid inlet valve is CV-02, the filtered blood outlet valve is CV-03, and the saline flushing fluid outlet valve is CV-04.
And 5: and (4) starting the system at the same time, opening the injection pump 1, introducing the blood containing the CTCs of the patient into the microfluidic chip, opening the blood valves CV-01 and CV-03, and implementing the CTCs filtration and capture process controlled by the computer program. When the filtering process time reaches T from 01In the process, the automatic control system of the micro flow controls the injection pump 1 and the valves CV-01 and CV-03 to be closed, and simultaneously opens the injection pump 2 and the flushing valves CV-02 and CV-04. And (3) introducing a normal saline flushing fluid into the chip, and flushing the CTCs captured on the porous filter membrane by using the normal saline controlled by the computer program, wherein the waste liquid containing the CTCs is introduced into a waste liquid pool. During this time, blood that is free of CTCs is recovered and stored using a specially made disposable blood storage bag. When the flushing progress time reaches T from 02In the process, the micro-flow automatic control system controls the injection pump 2 and the flushing valves CV-02 and CV-04 to be closed, and simultaneously opens the injection pump 1 and the valves CV-01 and CV-03 to enter the next round of process. After repeated many times, the blood without CTCs is returned to the patient, thereby achieving the purpose of clearing the circulating tumor cells by physical means.
2. The automated control system for filtration and separation of circulating tumor cells with a porous filter membrane microfluidic chip of claim 1, wherein the whole process involved in the above steps is performed in a sterile, low temperature environment.
3. The automated control system for filtration and separation of circulating tumor cells of claim 1, wherein a safety factor of 0.8 and a safety factor of 1.5 are respectively added to the time of passing through the hole of a circulating tumor cell to obtain a system control parameter, i.e., forward flow injection time T1And reverse flow flush time T2In order to ensureOne hundred percent of the CTCs can be captured and washed away.
4. The automated control system for filtration and separation of circulating tumor cells with a porous filter membrane microfluidic chip as claimed in claim 1, wherein the inventive device comprises a microfluidic automated control system and a CTCs porous filter membrane chip filtration and separation platform. The micro-flow automatic control system comprises a computer, a singlechip AT89C52 control module, an electromagnetic valve driving module, a pressure regulator, a driving air source and the like. The CTCs porous filter membrane chip filtering and separating platform comprises an injection pump, an injector, a porous filter membrane microfluidic chip, a polyethylene catheter, blood containing CTCs, physiological saline flushing fluid, a micro valve and the like.
5. The automated control system for filtration and separation of circulating tumor cells of claim 1, wherein the system is designed to process a porous membrane microfluidic chip, perform CTCs filtration and separation experiments, and perform theoretical analysis on cell via holes, so as to obtain a via hole time for circulating tumor cells. Specifically, a CTCs filtration separation experiment is carried out through a porous filter membrane chip to obtain the capture efficiency of CTCs; by performing dynamic analysis on the cell via hole, establishing a theoretical model on the basis of reasonable assumption and simplification to obtain a relational expression of the cell via hole time and the capture efficiency; and then the experimental result and the theoretical result are combined to determine the via hole time of the circulating tumor cell.
6. The automated control system for filtration and separation of circulating tumor cells with a porous filter membrane microfluidic chip of claim 1, wherein the automated microfluidic control system is a pneumatic micro valve driving system based on single chip microcomputer control. A series of control signals are output through the program setting of the single chip microcomputer platform, and the instructions are converted into the opening and closing actions of the electromagnetic valve through the electromagnetic valve driving circuit. The gas in the gas tank passes through the regulator to obtain a driving gas source with adjustable pressure, and the driving gas source provides driving force for the micro-pump pneumatic film, so that the micro-channel and the injection pump are controlled to be opened and closed, and the purpose of controlling whether liquid flows in the micro-channel or not is achieved.
In addition, the main principle of the micro-flow automatic control system is that the pressure difference between the air flow channel and the liquid flow channel is utilized to deform and rebound the middle PDMS layer, so that the liquid path is controlled to be cut off and conducted. When appropriate pressure is provided for the gas control channel, the middle elastic PDMS film layer is subjected to bending deformation, so that the fluid is blocked; when the pressure of the control channel is cancelled, the elastic PDMS film rebounds, and the liquid channel restores the flow state to play the role of a micro valve.
7. The automated control system for filtration and separation of circulating tumor cells with a porous filter membrane microfluidic chip of claim 1, wherein the automated control system comprises a single chip microcomputer control part, a solenoid valve driving circuit and a microfluidic chip. The hardware part of the singlechip control part adopts an AT89C52 control module, generates a control signal through an I/O port of the AT89C52 and sends the control signal to the input end of the electromagnetic valve driving circuit. The software part and the program design of the single chip microcomputer are mainly written in the keil C, and the action signals of the valve are output according to set time. In the design of the electromagnetic valve driving circuit, a signal output signal of the singlechip is connected with a base electrode of the triode, an anode of the electromagnetic valve is connected with a 12V power supply, and a cathode of the electromagnetic valve is connected with a collector electrode of the triode. In addition, after the voltage stabilizing diode is connected with the fast recovery rectifier diode in series, the voltage stabilizing diode is reversely connected with the electromagnetic valve in parallel. The output signal of the singlechip controls the state of the electromagnetic valve through the electromagnetic valve driving circuit. The microfluidic chip in the microfluidic automatic control system is designed to be a four-layer structure consisting of a glass chip, a Polydimethylsiloxane (PDMS) film and a Polycarbonate (PC) filter membrane. Wherein the upper and lower layers are glass chips, the upper layer glass chip is provided with a gas micro-channel, the lower layer glass chip is provided with a liquid micro-channel, and the middle 2 layers of PDMS film and the 3 layers of PC filter membrane are provided with through holes for connecting the inlets of the upper and lower micro-channels.
8. The method of claim 1The automatic control system for filtering and separating the circulating tumor cells by the porous filter membrane micro-fluidic chip is characterized by comprising the following operation steps: first, two sets of inlet and outlet ports of the chip are inserted into polyethylene tubes, respectively, wherein blood of a patient containing CTCs flows in through the tube 1, saline flush fluid flows in through the tube 2, blood containing no CTCs flows out through the tube 3, and waste fluid containing CTCs flows out through the tube 4. The inlet and outlet switches of the chip are controlled by a pneumatic micro valve. Secondly, turning on a system switch, initializing the system and setting the preset injection time T1Time of flushing T2And the electromagnetic valve names corresponding to all the microfluidic channels are written into a register, wherein the patient blood inlet valve containing CTCs is CV-01, the physiological saline flushing fluid inlet valve is CV-02, the filtered blood outlet valve without CTCs is CV-03, and the physiological saline flushing fluid outlet valve containing CTCs is CV-04. And starting the timer, and simultaneously opening the injection pump 1, the electromagnetic valve CV-01 and the electromagnetic valve CV-03. When the filtering process time reaches T1When the filtration process is finished, the injection pump 1 and the valves CV-01 and CV-03 are closed. Fourthly, when the process is finished, the injection pump 2, the electromagnetic valves CV-02 and CV-04 are opened. And introducing a normal saline flushing fluid into the chip, reversely flushing the CTCs captured on the porous filter membrane, and introducing the waste liquid containing the CTCs into a waste liquid pool through a conduit 4. When the flushing progress time reaches T from 02When the flushing process is finished, the injection pump 2, the valves CV-02 and CV-04 are closed; simultaneously, the injection pump 1 and the valves CV-01 and CV-03 are opened, and the next round of process is started. And fifthly, continuously and circularly executing the step three and the step four until the filtering work of all blood samples is finished so as to realize the automatic process of capturing and separating the CTCs from the blood.
9. The automated control system for filtration and separation of circulating tumor cells with a porous filter membrane microfluidic chip of claim 1, wherein the development of experimental studies on the capture and separation of CTCs with a microfluidic porous filter membrane also includes the design and preparation of a CTCs-capture microfluidic chip with a porous filter membrane. The chip is prepared into a sandwich structure of two PDMS layers, and a PC filter membrane is arranged in the middle of the sandwich structure. The cavity and the channel of the PDMS layer are prepared by a photoetching method, and after the upper and lower PDMS layers are prepared, a hole is punched by a puncher to form a pre-designed inlet hole and a pre-designed outlet hole. The PDMS layer and the filter membrane are bonded into a whole after being processed by plasma.
10. The automated control system for filtration and separation of circulating tumor cells with a porous filter membrane microfluidic chip according to claim 1, wherein the specific operations for developing the experimental research on the separation and capture of CTCs with the microfluidic porous filter membrane are as follows: CTCs in blood are fluorescently stained with a staining agent (e.g., CellTracker Green or CellTracker Red), and then a volume of blood containing a known number of CTCs is injected into the porous filter chip at a flow rate (e.g., 5mL/h) using a syringe pump. And (3) carrying out an observation experiment by using a fluorescence microscope provided with a CCD camera, and shooting after a period of time to obtain a capture result of the stained CTCs on the porous filter membrane. The captured cells are counted using digital image processing. Then, the capturing efficiency was determined according to the ratio between the number of captured CTCs and the total number of injected CTCs.
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