TWI703212B - Cell screen, its making method, and method of using the cell screen to screen and measure cells - Google Patents

Abstract

The present invention relates to a cell sieve, its manufacturing method, and a method of using the cell sieve for cell screening and measurement. The surface of the cell screen has specific antibodies that can bind to specific target cells. The sample flows through the cell screen through the fluid device to grab the target, and other non-target impurities such as white blood cells will follow the liquid through the cell screen and finally bind Electrochemical detection uses changes in current and impedance after capturing cells for quantitative analysis; therefore, the cell screen of the present invention and the method of using the cell screen to screen cells and cell measurement are not limited to circulating tumor cells. It can also be applied to the screening and quantification of highly infectious disease sources (such as Yersinia pestis).

Description

Cell screen, its manufacturing method, and method for cell screening and measurement using the cell screen

The present invention relates to a cell screening technology, in particular to a cell screen capable of screening specific target cells, and the use of the cell screen for cell screening and measurement.

For a long time, people have recognized that the spread of cancer is due to the infiltration of cancer cells into the circulatory system from the original site, and then extravasation and reproduction, which eventually leads to metastatic disease. This view was first discovered by Thomas Ashworth in 1869 from patients with metastatic tumors. He observed from the inside of the patient’s blood vessels that cancer cells that broke away from the original tumor and entered the bloodstream. These cancer cells are also called circulation. Tumor cells (circulating tumor cells, CTCs).

The most frightening thing about cancer is its ability to metastasize, which causes 90% of cancer-related deaths. Cancer metastasis is a multi-step process in which tumor cells break away from the primary cells and enter the bloodstream, extravasate and proliferate at secondary sites to form secondary tumor colonies. In the process of passing through the primary tissue and infiltrating into the blood, cancer cells undergo several changes. Increased fluidity and decreased adhesion proteins enable the cells to pass through the extracellular matrix and basement membrane at their original location; therefore, circulating tumor cells also It is often used as an index for prognostic judgment or efficacy evaluation of cancer patients.

The collection methods of circulating tumor cells can be roughly divided into two categories: physical methods or biological immunoaffinity methods. The method of collecting circulating tumor cells using physical properties is to capture the cells with the characteristics of circulating tumor cells, such as the density, size, deformability and charge of circulating tumor cells. Biological immunoaffinity methods can be divided into forward collection and reverse collection. In forward grasping, circulating tumor cells use antibodies to interact with cell surface markers such as EpCAM. In reverse grabbing, the white blood cell surface marker CD45 is used to remove the signal of non-circulating tumor cells.

The above methods have their own advantages, but the biggest disadvantage is that they cannot achieve standardized detection and are easily affected by various factors such as reagent components, operating procedures, and interpretation processes. Especially in every milliliter of blood, there are usually only a small number of circulating tumor cells, but there are millions to billions of blood cells. If you want to screen out circulating tumor cells from a large number of blood cells, it will often appear Find a needle in a haystack.

For example, if a blood sample is filtered to separate living cells, the filter may be blocked due to a large number of blood cells during the filtration process, which will affect the screening of rare circulating tumor cells. Repeated testing of the same sample is very difficult. The results are prone to deviation; in addition, biological immunoaffinity methods usually use fluorescent cursors to locate the target and use a fluorescent microscope for image interpretation, which has the disadvantages of long observation time and high fluorescent cost.

Therefore, the key technical challenge is how to effectively capture and analyze circulating tumor cells in a suitable way, so that doctors can better help and manage and track cancer patients.

In view of this, the inventors of the present invention through painstaking research and searching for various possible solutions to solve the above-mentioned problems of traditional technology, and then developed a novel cell sieve, the cell sieve can be combined with specific target cells The specific antibody, through the fluid device, flows the sample through the cell screen to capture the target, and other non-target impurities such as white blood cells will follow the liquid through the cell screen, and finally combine with electrochemical detection to use the current after the cell is captured Quantitative analysis of changes in impedance and impedance; therefore, the cell screen and the screening measurement method are not limited to circulating tumor cells, and can also be applied to the screening of highly infectious pathogens (such as Yersinia pestis); in addition, based on the cell screen It has the characteristics of grabbing target cells, so it can also be set in a hemodialysis device, and the cell mesh is used to grab or remove specific cells in the patient's body during hemodialysis, such as circulating tumor cells. In addition, in addition to screening and quantifying specific target cells, the cell screen can also be used to cultivate cells and monitor cell growth in real time.

In other words, the present invention can provide a cell sieve, which is a laminated structure composed of at least a sieve substrate, an electrode layer, and a polymer layer; wherein the sieve substrate is composed of filamentous materials in straight rows and rows. Woven into a mesh structure, and the average pore size of the mesh substrate is in the range of 15-30 μm, preferably in the range of 20-30 μm; the electrode layer is arranged on the surface of the mesh substrate, and The surface has amine groups; the polymer layer is arranged on the surface of the electrode layer, and the surface has streptavidin and specific antibodies that can bind to specific target cells.

In addition, the present invention can also provide a method for making a cell screen, which includes the following steps:

The metal material is formed on the outer surface of the mesh substrate by physical vapor deposition to form an electrode layer and then activated, so that the surface of the electrode layer has hydroxyl groups; the outer surface of the electrode layer is contacted with the aminosilane solution The reaction is carried out so that the surface of the electrode layer is provided with amine groups; the surface of the electrode layer with amine groups is contacted with the polymer binder for reaction, so that the surface of the electrode layer forms a polymer layer; After the surface is contacted with the streptavidin for reaction, it is then contacted with the specific antibody that can bind to the specific target cell to react, so that the surface of the polymer layer carries the streptavidin and the specific antibody, and then obtains the ability to screen the specific Cell mesh of the target cell.

According to an embodiment of the present invention, the polymer binder includes a coupling agent and a copolymerized polymer; wherein the coupling agent is selected from 1-ethyl-3-(3-dimethylaminopropyl) carbazide Amine (1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide, also known as EDC), N-Hydroxysulfosuccinimide sodium salt (also known as Sulfo-NHS), N-hydroxysuccinimide (N-Hydroxysuccinimide, also known as NHS), sulfosuccinimidyl 4-(N-maleimidophenyl) butyrate, also known as Sulfo- SMPB), 4-(N-maleimidomethyl)cyclohexane-1-carboxylate sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate, and Called Sulfo-SMCC), and any one of its combinations; preferably selected from any one of EDC, Sulfo-NHS, NHS, and combinations thereof; most preferably selected from EDC, NHS, and combinations thereof Any kind.

According to an embodiment of the present invention, the copolymer is obtained by copolymerizing a carboxyl-containing polymer and a zwitterionic polymer, and the carboxyl-containing polymer (X) is relative to the zwitterionic polymer The ratio of (Y) (X:Y) is in the range of 1:9-9:1; preferably in the range of 3:7-9:1; more preferably in the range of 1:1-9:1; The best is in the range of 7:3~9:1.

According to an embodiment of the present invention, the carboxyl group-containing high molecular polymer is any one of polyacrylic acid, polymethacrylic acid, sodium polyacrylate, polyacrylamide, sodium polymethacrylate, polymaleic acid and combinations thereof.

According to an embodiment of the present invention, the zwitterionic polymer is a high molecular polymer containing phosphocholine (PC), sultaine (SB), and/or carboxybetaine (CB).

In addition, the present invention can also be a method for screening cells and cell measurement, which includes the following steps: cell screening step: the sample to be tested is passed through the cell mesh at a specific flow rate, so that specific target cells in the sample to be tested Is adsorbed on the surface of the cell mesh; cell measurement step: electrically connect the cell mesh after cell screening to an electrochemical meter, and use the electrochemical meter to apply an alternating voltage of a specific amplitude in the circuit The signal is then scanned in the frequency range of 0.01 to 70,000 Hz to obtain the impedance change value of the cell screen, and then the specific target cell concentration in the test sample is calculated based on the impedance change value of the cell screen.

According to an embodiment of the present invention, the method for screening cells and cell measurement further comprises a calibration curve establishment step, and the calibration curve establishment step includes: passing a standard with a known concentration of the specific target cell through the cell at the specific flow rate Screen, so that the specific target cells in the standard product are adsorbed on the surface of the cell screen; the cell screen after cell screening is electrically connected to the electrochemical instrument, and the electrochemical instrument is used for The circuit is applied to the AC voltage signal of the specific amplitude, and then scanned in the frequency range of 0.01 to 70,000 Hz to obtain the impedance change value of the cell mesh; repeat the above steps to obtain the cell mesh using different concentrations of the specific target cells The impedance change value of the standard product establishes the concentration calibration curve of the specific target cell, and the determination coefficient (R ² ) of the concentration calibration curve is greater than 0.9.

According to an embodiment of the present invention, the specific flow rate is in the range of 0.5~4.0ml/hr; preferably in the range of 0.5~3.5ml/hr; more preferably in the range of 0.5~2.5ml/hr; most preferably It is in the range of 0.5~1.5ml/hr.

According to an embodiment of the present invention, the specific amplitude is in the range of 5-30 mVRMS; preferably, it is in the range of 5-20 mVRMS.

According to an embodiment of the present invention, the cell measurement step further comprises: after obtaining the impedance change value of the cell sieve, using the concentration calibration curve to calculate the content of the sample to be tested based on the impedance change value of the cell sieve The specific target cell concentration.

Furthermore, the present invention can also provide a cell screening device, which includes the above-mentioned cell screen and a syringe; wherein the cell screen is arranged at the bottom of the syringe, and the surface of the cell screen is capable of interacting with specific target cells. The bound specific antigen; when the cell screening device is used to extract blood from the subject, the blood will pass through the cell screen so that the specific target cell is adsorbed on the surface of the cell screen.

In the following, different specific embodiments are listed and explained in more detail for the implementation of the present invention, so as to make the spirit and content of the present invention more complete and easy to understand; however, those with ordinary knowledge in this art should understand Of course, the present invention is not limited to these examples, and other same or equal functions and sequence of steps can also be used to achieve the present invention.

In this article, the scientific and technical terms used here have the same meanings as understood and used by those with ordinary knowledge in the technical field of the present invention. In addition, without conflict with context, the singular nouns used in this specification cover the plural nouns; and the plural nouns also cover the singular nouns.

In this article, the values and parameters used to define the scope of the present invention inevitably contain standard deviations due to individual test methods, and therefore are mostly expressed as approximate quantitative values. However, in specific embodiments, Relevant values presented as accurately as possible. In this article, "about" usually depends on the considerations of those with ordinary knowledge in the technical field of the present invention, and generally means that the actual value falls within the acceptable standard error of the average value. For example, the actual value is a Within ±10%, ±5%, ±1%, or ±0.5% of a specific value or range.

In summary, in the above-listed embodiments, examples have been given to specifically illustrate the content of the present invention, but the present invention is not limited to these embodiments. Those with ordinary knowledge in the technical field to which the present invention belongs should understand that various changes and modifications can be made without departing from the spirit and scope of the present invention; for example, combining or combining various technical contents illustrated in the foregoing embodiments. It is changed to become a new embodiment, and these embodiments are of course regarded as the content of the present invention. Therefore, the scope of protection in this case also includes the scope of patent application and the scope defined by it.

First, please refer to FIG. 1, which is a schematic diagram showing the cross-sectional structure of the cell screen of the present invention. The cell screen 1 is a laminated structure including a screen substrate 11, an electrode layer 12, and a polymer layer 13. .

The screen substrate 11 is a mesh structure with a uniform pore size; preferably, the screen substrate 11 can be made of a woven wire material, and the pore size is in the range of 15 μm-30 μm. The electrode layer 12 is made of conductive metal, and is arranged on the surface of the mesh substrate 11, and the surface is chemically modified with amine groups. The polymer layer 13 is arranged on the surface of the electrode layer 12, and its surface is chemically modified with streptavidin and a specific antibody that can bind to specific target cells.

According to the technical idea of the present invention, the screen substrate 11 and the electrode layer 12 can be made of a polymer material as the screen substrate and then plated with metal, for example, a nylon screen is plated with a gold electrode layer; or The mesh substrate 11 and the electrode layer 12 are directly woven directly from the wire-like material, thereby saving the time of plating the electrode layer. In addition, in addition to nylon, the polymer material that can form the base of the screen can also be polyimide, aliphatic polyamide, aromatic polyamide, polyimide, cellulose, cellulose acetate, and polyether. , Polyurethane, poly (urea urethane), polybenzimidazole, polyetherimide, polyacrylonitrile, polyethylene terephthalate, polypropylene, polyaniline, polyethylene oxide, Polyethylene naphthalate, polybutylene terephthalate, styrene-butadiene rubber, polystyrene, polyvinyl chloride, polyvinyl alcohol, polyvinylidene fluoride, polyvinyl butene , Any one of its derivatives, and combinations thereof; and the wire-like material may be silver wire, platinum wire, stainless steel wire, or tungsten wire; preferably, stainless steel wire.

According to the technical idea of the present invention, the polymer layer 3 is composed of a copolymerized polymer obtained by copolymerizing a carboxyl-containing polymer and a zwitterionic polymer, wherein the carboxyl-containing polymer may be polyacrylic acid , Polymethacrylic acid, sodium polyacrylate, polyacrylamide and polymethacrylate sodium, polymaleic acid and any combination thereof. And, the zwitterionic polymer is a high molecular polymer containing phosphocholine (PC), sultaine (SB), and/or carboxybetaine (CB).

Next, specific examples are given below to illustrate the method of making the cell screen of the present invention and the method of using the cell screen for cell screening and quantification. "Preparation Examples 1 to 6" (synthetic copolymer polymer)

(I) Add 0.5 mL of Poly[2-(Methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammoniumhydroxide, PSBMA) and Polyacrylic acid (PAA) in the proportions shown in Table 1 below. After mixing the distilled water with an ultrasonic shaker, add 0.3mmole of initiator ammonium persulfate (APS) and catalyst tetramethylethylenediamine (N,N,N',N'-Tetramethylethylenediamine, TMEDA). ) 0.05 mmole, and then the container was sealed and placed at 37° C. for reaction for 24 hours to obtain copolymers S1 to S5 (copolymer S6 was 100% PAA). The reaction formula is shown in I:

(I)

Then, the IR spectrum and NMR spectrum were used to confirm whether SBMA and AA were successfully polymerized into copolymers (poly(SBMA-co-AA)) S1 to S5.

Please refer to FIG. 2, which shows the IR spectra of acrylic acid monomer (AA), sultaine methacrylate monomer (SBMA), and copolymer (poly(SBMA-co-AA)). First we see wherein AA monomer peak respectively -C = C stretching vibration peak at 1637cm ^-1, 1711cm ^-1 with a C = O stretching vibration of 3600 cm ^-1 and have -OH ~ 2500 of stretching vibration peak. Followed by the characteristic peaks of SBMA monomer, at 1040/1170~1200cm ^-1 is the characteristic peak quality of -SO, 1483cm ^-1 is the characteristic peak of quaternary amine, 1645cm ^-1 is the C=C stretching vibration peak and 1720cm ^-1 has a stretching vibration peak of C=O. Finally, after seeing the polymerized copolymer polySBMA-co-AA, it can be clearly seen that it contains the characteristic peaks of the above two monomers, but because the two monomers are copolymerized, the double bond is used to polymerize to form a long chain, so It can be clearly seen that the peak quality at the position of 1640cm-1 has disappeared after polymerization, which proves that PAA and PSBMA have successfully polymerized into copolymers.

In addition, please refer to Figure 3A and Figure 3B; Figure 3A shows the NMR spectra of acrylic acid monomer (AA), sultaine methacrylate monomer (SBMA), and copolymer S5, and Figure 3B shows the high copolymerization NMR spectra of molecules S1 to S5. Use D ₂ O (δ=4.7ppm) solvent as the calibration chemical shift.

As shown in Figure 3A, the chemical shift δ=1.1, 2.3, 2.9, 3.2, 3.5, 3.8, and 4.5 ppm of PSBMA are labeled a, h, i, f, g, e, and d, respectively. The chemical shift of PAA is δ=1.3~2.1, 2.66 is b and c. From the poly(SBMA10-co-AA90) spectrum, it can be found that there are characteristic peaks of PSBMA and PAA, which can prove that the two polymers have been successful. polymerization.

Then, using the result shown in Figure 3B, convert the peak integral area of the characteristic peak δ=2.3 of PSBMA and the characteristic peak δ=1.3~2.1 of PAA into the molar ratio of SBMA and AA in the copolymer, and the result Recorded in Table 1.

Table 1 Preparation Example 1 Preparation Example 2 Preparation Example 3 Preparation Example 4 Preparation Example 5 Preparation Example 6 PSBMA(mol) 0.9 0.7 0.5 0.3 0.1 0 PAA(mol) 0.1 0.3 0.5 0.7 0.9 1.0 Distilled water (ml) 5.0 5.0 5.0 5.0 5.0 5.0 APS(mmol) 0.3 0.3 0.3 0.3 0.3 0.3 TMEDA(mmol) 0.05 0.05 0.05 0.05 0.05 0.05 Copolymer S1 S2 S3 S4 S5 S6 Qualitative analysis SBMA/PAA 87/13 68/32 53/47 36/67 11/89 -

From the results in Table 1, it can be found that the ratios of SBMA and PAA of the synthesized copolymers S1 to S5 are approximately the same as the ratios of PSAMA and PAA used in the reaction, indicating that the copolymers of different ratios have been successfully synthesized. "Examples 1 to 6" (making cell sieve)

In this embodiment, a nylon mesh prepared by melt electrospinning technology is selected as the mesh substrate, which is a single-layer mesh spinning, and its mesh morphology is arranged neatly. The mesh aperture is 20μm.

Cut the prepared screen substrate into 6 test pieces with an area of 1 cm×2 cm, and remove surface impurities with nitrogen gas, and then use Physical Vapor Deposition on the surface of the screen substrate The electrode layer is made by plating gold, the operating current is 20 mA, and the operating time is three minutes. Then, use X-Ray Photoelectron Sprectroscpoe (XPS) (brand: British VGESCA, model: Scientific Theta Probe) to perform elemental analysis on the surface of the test piece, and confirm that the gold has been successfully removed by XPS spectroscopy. Plated on the surface of the screen substrate.

After the electrode layer is made, put the test piece into the oxygen plasma machine (brand: Mory Egnieering. Co., LTD, model: CV-e300) for 8 minutes under the condition of 100 watts (W). Activation treatment makes the surface of the electrode layer carry hydroxyl radicals. Next, immerse the test piece in an aqueous solution of (3-Aminopropyl)triethoxysilane ((3-Aminopropyl)triethoxysilane, APTES) with a concentration of 0.2% (v/v) and let it stand for about ten minutes. Distilled water to wash away excess unreacted reagents. Then, elemental analysis was performed on the surface of the test piece using an X-ray photoelectron spectrometer, and it was confirmed through XPS spectrogram that the surface of the electrode layer had an amine group.

Next, the polymer binder is prepared. The preparation method is as follows:

Add 10 mL of 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and 15 mL of 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide to every 100 mL of PBS. N-Hydroxysuccinimide (N-Hydroxysuccinimide, NHS) was added to 10% (v/v) of copolymerized polymers S1 to S6 and mixed uniformly to obtain polymer binders M1 to M6.

Then, the 6 test pieces were immersed in polymer binders M1 to M6, and reacted at room temperature for 24 hours to bond the electrode layer with amino groups and the copolymer polymer with carboxyl groups, and then the electrode layer A polymer layer is formed on the surface. Then, an X-ray photoelectron spectrometer was used to perform elemental analysis on the surface of the test piece, and it was confirmed through the XPS spectrum that the polymer layer was successfully formed on the surface of the electrode layer.

After the above-mentioned test piece with completed surface modification was washed with distilled water to remove more than the unreacted reagent on the surface, the electrode surface now has carboxyl groups and is co-coated with Streptavidin with amine groups using EDC/NHS. Valence bond fixation method.

Add 10mLEDC and 15mLNHS to every 100mL of PBS and add 25mL of Streptavidin to mix evenly, soak the test piece in the solution for 1 hour, and then add the biotinylated EpCAM antibody (AlexaFluor488anti-humanCD326 (EpCAM) Antibody, brand: Biolegend), because streptavidin has a strong affinity for biotin, antibodies can be successfully modified on the electrode surface to obtain cell meshes X1 to X6.

Whether the electrode surface is successfully modified with streptavidin can be observed and judged by fluorescence microscope. First, the surface of the nylon electrode is modified with Streptavidin-FITC, which emits green fluorescence as shown in Figure 4a. Then the test piece with streptavidin on the surface was grafted with Biotin-Cy5 and then showed red fluorescence, which can prove its specificity, as shown in Figure 4b. After forming the overlay image of Figure 4a and Figure 4b on Figure 4d, it can be observed that the area of fluorescence emitted after the overlay image overlaps, which proves that the streptavidin has been successfully modified onto the surface.

Next, the following describes the method of cell screening and quantitative analysis using the cell screen of the present invention.

First, the sample preparation method (cell line culture and blood sample preparation), the standard method for cell screening, and the electrochemical impedance spectroscopy analysis method for cell measurement will be explained. "Cell Line Culture"

The cell lines used in the examples of the present invention are human colorectal cancer cells HCT-116, HT-29, DLD1, and human cervical cancer cells Hela cell (provided by Taipei Medical University).

Add 1 mL of 0.5% Trypsin-EDTA to each of the above cell lines and shake to detach the cells. Take 1 mL to a centrifuge tube for centrifugation, remove the supernatant, and clean Trypsin-EDTA. Then, add 1 mL of 4% Paraformaldehyde to fix the cells for 10 minutes, wash with PBS, centrifuge, remove the supernatant, and fix the cells. Add anti-EpCAM conjugated FITC for staining for 1 hour, and finally add DAPI for staining for 10 minutes, wash with PBS and centrifuge, remove the supernatant, and re-dissolve it with PBS to 1 mL to count with a cell counter. "Blood Test Preparation"

The clinical blood samples used in the examples of the present invention were taken from patients with colorectal cancer, and have passed the review of the Joint Human Research Ethics Committee of Taipei Medical University and Affiliated Hospital, project number: (TMU-JIRB): N201701082.

Since the white blood cells in the blood do not show EpCAM protein reaction, it can be used for non-specific adhesion test and to simulate the number of white blood cells in the human body; the white blood cell separation method is explained as follows:

(1) Dispense 10 mL of whole blood sample into two 50 mL centrifuge tubes, add RBC lysis buffer to 40 mL, and shake evenly for 10 minutes to destroy red blood cells.

(2) Put it in a centrifuge, centrifuge at 450g for 12 minutes, remove the supernatant.

(3) Add 40mLRBC lysis buffer again and shake it evenly for five minutes to destroy the remaining red blood cells.

(4) Put it in a centrifuge and centrifuge at 340g for 6 minutes, then remove the supernatant.

(5) After re-dissolving to 1 mL with PBS, wash and centrifuge to remove the supernatant.

(6) After fixing the cells with 1 mL of 4% Paraformaldehyde for 10 minutes, they were washed with PBS and centrifuged to remove the supernatant.

(7) After adding DAPI working concentration for staining for 10 minutes, wash with PBS, centrifuge, remove the supernatant, re-dissolve it with PBS to 1 mL, and then count with a cell counter. "Cell Screening Standard Methods"

The cell screening method of the present invention passes the test sample through the cell mesh at a specific flow rate, so that specific target cells in the test sample are adsorbed on the surface of the cell mesh.

The fluid device used in the embodiment of the present invention is shown in Figure 5, which is to fill the sample (blood) to be tested into the syringe, fix the cell screen on the front end of the syringe, and then fix the syringe to the injection aid. Syringe pump injects the sample to be tested at a specific flow rate, so that the sample to be tested passes through the cell mesh. "Electrochemical Impedance Spectroscopy Analysis Method"

Use the cell sieve to be tested as a working electrode to electrically connect with an electrochemical analyzer (brand: METEK; model: VersaSTAT 4 Series potentiostat/galvanostat), and use electrochemical impedance spectroscopy (Electrical Impedance Spectroscopy, EIS) to detect The change in impedance caused by grabbing the analyte on the surface of the electrode is used for quantitative detection of cells. The electrolyte ratio is 5 mM Potassium ferrocyanide (Potassium ferrocyanide), 5 mM Potassium ferricyanide (Potassium ferricyanide) Configured in 0.1 M PBS buffer solution, electrochemical impedance parameter setting: Scanning frequency is 70,000 ~ 0.01Hz The range is Hz, and the amplitude is (Amplitude) 10 mVRMS. "Examples 7 to 12" (Analysis of cell screen capture rate)

Fix the cell screens X1 to X6 obtained in the above Examples 1 to 6 respectively in front of the needle of the injection pump, and make 1 ml of human colorectal cancer cell HCT-116 at a flow rate of 1 mL/hr. The sample to be tested passes through the cell meshes X1 to X6. After all the samples to be tested pass through, the number of HCT-116 cells on the surface of the cell meshes X1 to X6 is observed with an optical microscope and converted into a grabbing rate. The crawl rate is calculated as follows: Grab rate (%) = (number of target cells on the surface of the cell screen/number of target cells in the sample) x 100%

Table 2 Example 7 Example 8 Example 9 Example 10 Example 11 Example 12 Cell screen X1 X2 X3 X4 X5 X6 Sample flow rate (ml/hr) 1 1 1 1 1 1 Crawl rate (%) 3.3 6.67 18.83 37 52.5 57.1

Refer to Figures 6A to 6F, and Figure 7; Figures 6A to 6F are fluorograms showing the screening of HCT-116 cells using cell screens X1 to X6, and Figure 7 shows the screening of HCT-116 cells using cell screens X1 to X6. Comparison chart of cell capture rate.

It can be seen from the results in Figures 6A to 6F and Figure 7 that when the same number of HCT-116 cells are used for grasping, it can be seen from the optical microscope that the number of cells on the surface of the cell mesh X1 to X6 gradually increases. It shows that as the proportion of AA in the polymer layer in the cell screen increases, the capture rate of circulating tumor cells also increases, because the more AA, the more carboxyl groups involved in the modification reaction on the surface, enabling modification As the area of antibodies increases, the crawl rate gradually increases.

"Examples 13 to 18" (Analysis of the anti-leukocyte adhesion effect of cell mesh)

The cell screens X1 to X6 obtained in the above Examples 1 to 6 were respectively clamped in front of the needle of the injection pump, and the test sample containing a fixed number of white blood cells was passed through the cell screen X1 to the cell screen at a flow rate of 1 mL/hr. X6, after all the samples to be tested have passed, use an optical microscope to observe the number of white blood cells on the surface of the cell mesh X1 to X6, and convert it into an anti-sticking rate.

The calculation method of anti-sticking rate is as follows: Anti-sticking rate (%)=[1-(the number of white blood cells on the surface of the cell screen/the number of white blood cells in the sample)] x 100%

table 3 Example 13 Example 14 Example 15 Example 16 Example 17 Example 18 Cell screen X1 X2 X3 X4 X5 X6 Sample flow rate (ml/hr) 1 1 1 1 1 1 Anti-sticking rate (%) 99.913 99.908 99.895 99.905 99.888 58.7

Refer to Figures 8A to 8F and Figure 9; Figures 8A to 8F respectively show the fluorescence images after the cell screen X1 to X6 are used to capture and screen white blood cells, and Figure 9 shows the anti-staining effect of the cell screen X1 to X6 in the screening of white blood cells. Viscosity comparison chart.

It can be seen from the results in Figures 8A to 8F and Figure 9 that when the same number of white blood cells is used for screening, it can be seen from an optical microscope that the number of white blood cells adhered on the surface of the cell mesh X1 to X5 gradually increases. It shows that when the proportion of PSBMA in the polymer layer in the cell screen gradually decreases, the number of white blood cells adhered to the surface of the cell screen gradually increases, but they are maintained above 99%. The reason is that after synthesizing the copolymer, the molecular weight is about 300,000 after the gel permeation chromatograph is used to determine the molecular weight. Presumably because of the large molecular weight, although the ratio is low, there is still a certain amount of PSBMA on the surface.

In contrast, when using a cell mesh (cell mesh X6) without PSBMA at all, the adhesion resistance of white blood cells dropped to 58.7%, which means that a large number of white blood cells will stick to the cell mesh when they pass through the cell mesh through the fluid device. The hole 洞 is blocked because of the large number of white blood cells sticking to the surface of the cell screen, which will greatly reduce the subsequent capture rate of circulating tumor cells in colorectal cancer. In addition to affecting the interpretation of fluorescence, it also leads to the use of electrochemical impedance In the part of the spectrum to detect circulating tumor cells in a small amount, the signal is unstable due to a large amount of non-specific substances adhered to it.

In addition, it can be seen from the above analysis of the cell screen capture rate and the analysis of the anti-leukocyte adhesion effect that the cell screen X5 has a relatively good capture rate and has a considerable anti-adhesion effect, showing that the cell screen of the present invention The best ratio of PSBMA to PAA in the polymer layer is 1:9. "Examples 19-22" (Analysis of the influence of fluid flow rate on the capture rate of circulating tumor cells in colorectal cancer)

The cell mesh X5 obtained in Example 5 was clamped in front of the needle of the injection pump, and the cells containing a fixed number of human colorectal cancer cells HCT-116 were made at flow rates of 0.5, 1, 2, and 4 mL/hr. The sample to be tested passes through the cell screen X5. After all the samples to be tested have passed, use an optical microscope to observe the number of HCT-116 cells on the surface of the cell screen X5, and convert it into a capture rate.

Table 4 Example 19 Example 20 Example 21 Example 22 Cell screen X5 X5 X5 X5 Sample flow rate (ml/hr) 0.5 1 2 4 Crawl rate (%) 53 51 12 8

Refer to Figure 10, which is a graph showing the relationship between different flow rates and capture rates. From the results in Figure 10, it can be seen that when the flow rate is 0.5 and 1 mL/hr, the capture rate is above 50%, and when the flow rate reaches 2 and 4 mL/hr, the capture rate drops below 20%. It is shown that when the flow rate is faster, the time of the experiment can be saved and non-specific adhesion can flow with the solution, but the relative accuracy and capture rate are lower; on the contrary, when the flow rate is slower, it can be improved The grasping of the target is efficient but time-consuming. Therefore, 1mL/hr, which has a good gripping rate and is more time-saving, can be selected as the flow rate condition of the fluid device. "Examples 23 to 27" (selective analysis of cell screens)

In Examples 23-27, human colorectal cancer cells HCT-116, HT-29, and DLD-1, which are specific for EpCAM2 protein, were used with non-specific white blood cells and human cervical cancer cells Hela cells. Screen X5 for cell screening, and the operating conditions are shown in Table 4.

After the screening, the number of cells on the surface of the cell mesh in different embodiments was observed by an optical microscope, and converted into a grabbing rate, which is recorded in Table 4.

Next, the aforementioned electrochemical impedance spectroscopy analysis method is used to measure the impedance change on the surface of the cell mesh X5. Then, based on the impedance value of the cell screen before screening and the impedance value after screening, the changing rate of impedance was calculated and recorded in Table 4.

Table 4 Example 23 Example 24 Example 25 Example 26 Example 27 Cell screen X5 X5 X5 X5 X5 Cell to be tested HCT-116 HT-29 DLD-1 leukocyte Hela cell Sample flow rate (ml/hr) 1 1 1 1 1 Crawl rate (%) 50 50 52.76 1.11 2.5 Impedance change rate (%) 115.3 107.9 101.3 9.6 7.2

Please refer to FIGS. 11 and 12; FIG. 11 is a comparison diagram showing the grabbing rate of the cell screen X5 for screening different cells, and FIG. 12 is a comparison diagram showing the impedance change of the cell screen X5 for screening different cells.

From the results shown in Figure 11, it can be seen that the cell screen X5 has a grasping rate of more than 50% for human colorectal cancer cells HCT-116, HT-29, and DLD-1, while for white blood cells and human cervical cancer cells Hela The crawl rate of cells is below 5%.

In addition, from the results shown in Figure 12, it can be seen that the cell screen X5 screened for human colorectal cancer cells HCT-116, HT-29, and DLD-1, and the impedance change rate was all above 100%, while for white blood cells and The impedance change rate of the human cervical cancer cell Hela cells after screening is below 10%. This result corresponds to the result of the grabbing rate. It shows that the cell screen of the present invention can be used for cell screening and electrochemical detection and detection. The cells on the cell sieve are quantified. "Examples 28 to 32" (Quantitative Analysis of Circulating Tumor Cells in Trace Colorectal Cancer)

In Examples 28 to 32, the cell screen X5 was used to screen samples containing 5, 10, 20, 40, and 80 colorectal cancer cells HCT-116 in 1 ml. The operating conditions are shown in Table 5.

After screening, use an optical microscope to observe the number of cells on the surface of the cell screen in different examples and photograph them into optical micrographs, as shown in Figure 13; then convert it into a grabbing rate and record it in Table 4 and draw the value as Figure 14.

Then, electrically connect the cell sieve to the electrochemical analyzer, and use cyclic voltammetry to measure the current signal on the surface of the cell sieve. The scanning speed is 0.1V/sec and the applied voltage is -0.4V→+0.8 V→-0.4V to scan (working electrode is cell mesh, auxiliary electrode is platinum, reference electrode is silver-silver chloride), and the response current during scanning is drawn into a cyclic voltammetry wave pattern. As shown in Figure 15.

table 5 Example 28 Example 29 Example 30 Example 31 Example 32 Cell screen X5 X5 X5 X5 X5 Cell to be tested HCT-116 HCT-116 HCT-116 HCT-116 HCT-116 Sample flow rate (ml/hr) 1 1 1 1 1 Number of cells to be tested before screening 5 10 20 40 80 Number of cells on the surface of the cell screen after screening 3 5 11 20 41 Crawl rate (%) 60 50 55 50 51 R _ct 6 twenty two 34 58 77

From the results shown in Fig. 13 and Fig. 14, it can be seen that the fluorescent colorectal cancer cell HCT-116 can be seen in the photos taken by the optical microscope, indicating that the target can still be caught even with a small amount of cells, and The grabbing rate of each cell number is similar to the stable grabbing rate (52.5%) made in the previous experiment, which shows that the cell screen of the present invention has good stability for cell screening and is suitable for quantitative analysis.

In addition, it can be seen from the results shown in Figure 15 that when the cells are grabbed by the cell mesh, the electrolytic solution and the surface of the cell mesh are obstructed, which makes the electron transfer effect worse, so the current signal will increase with the number of cells. There is a downward trend.

Then, the electrochemical impedance spectroscopy analysis method is used to draw the electrochemical impedance spectrum for quantitative cell analysis, as shown in FIG. 16.

Using the electrochemical impedance spectrum of different cell numbers to calculate the corresponding impedance value Rct (charge-transfer resistance) and draw it into a calibration curve, as shown in Figure 17, it can be found that the coefficient of determination (R ² ) is as high as 0.945, showing a high degree of Positive correlation means that electrochemical impedance spectroscopy is feasible for quantitative analysis of circulating tumor cells in colorectal cancer. "Examples 33 to 40" (Imitated clinical trials)

According to the reference, in normal human blood, there will be about 4-10 million white blood cells per 1 mL of whole blood, and there are other substances in the blood that may stick to the cell mesh. This causes errors in the interpretation of electrochemical signals.

Therefore, in Examples 34 to 41, this part uses 4 million white blood cells, 10 million white blood cells, and 1 mL of healthy people’s whole blood to add a small amount of colorectal cancer circulating tumor cells HCT-116 to simulate various situations. For cell screening, the sample composition and screening operating conditions are shown in Table 6.

Table 6 Example 33 Example 34 Example 35 Example 36 Example 37 Example 38 Example 39 Example 40 Cell screen X5 X5 X5 X5 X5 X5 X5 X5 Sample composition HCT-116 (number of pieces) 0 0 0 5 10 20 40 80 Human whole blood (ml) 0 0 1 1 1 1 1 1 White blood cells (number/ml) 4×10 ⁶ 1×10 ⁷ 0 0 0 0 0 0 Sample flow rate (ml/hr) 1 1 1 1 1 1 1 1 R _ct 11 10 10 11 12 twenty three 31 51

Next, the electrochemical impedance spectroscopy analysis method was used to plot the electrochemical impedance spectra of Examples 34 to 41, as shown in FIG. 18. Then use the electrochemical impedance spectrum to convert the corresponding impedance value Rct (charge-transfer resistance) and draw it as a calibration curve. As shown in Figure 19, it can be found that the coefficient of determination (R2) is as high as 0.949, showing a high degree of positive correlation.

In addition, it can be seen from the result of comparing the impedance value Rct that when there are 10 colorectal cancer circulating tumor cells, the error value overlaps with 0 colorectal cancer circulating tumor cells, which may cause errors in judgment. Obviously there can be a difference. It can be seen from this that after the cell screen of the present invention is used for cell screening, more than 10 colorectal cancer circulating tumor cells can be detected in 1 mL of whole blood by electrochemical impedance spectroscopy. "Example 41" (clinical test)

In this embodiment, the cell screen of the present invention is used to confirm the success of the surgery for patients with colorectal cancer undergoing tumor resection. There are 27 subjects in total, and the number of stages of colorectal cancer suffered by each subject is shown in Table 7.

Before the operation, a certain amount of blood sample is taken, and the blood sample is passed through the cell sieve and the number of cells on the cell sieve is calculated by electrochemical impedance spectroscopy analysis; then one month after the operation After passing the blood sample of the subject through the cell sieve, the number of cells on the cell sieve is calculated by electrochemical impedance spectroscopy analysis, and the results are recorded in Table 7.

Table 7 Subject number Number of periods Preoperative testing Postoperative testing result Examination volume (cc) Total CTC Examination volume (cc) Total CTC 1 Ⅰ 5 12 8 0 No recurrence 2 Ⅲ 5 27 3.5 2 No recurrence 3 Ⅲ 6 31 6 3 No recurrence 4 Ⅲ 8 twenty one 8 3 No recurrence 6 Ⅰ 7 10 7 1 No recurrence 7 Ⅰ 7 14 7 0 No recurrence 8 0 6 0 8 0 No recurrence 9 0 5 0 6 0 No recurrence 10 Ⅰ 8 16 7 0 No recurrence 11 Ⅰ 6 12 8 1 No recurrence 15 IV 5 42 9 8 No recurrence 16 Ⅰ 7 11 9 1 No recurrence twenty one Ⅰ 6 11 7 0 No recurrence twenty two Ⅰ 6 10 6 2 No recurrence twenty three Ⅰ 7 9 8 0 No recurrence twenty four Ⅲ 7 33 8.5 4 No recurrence 25 Ⅰ 7 18 8.5 0 No recurrence 26 Ⅰ 3 16 9 2 No recurrence 27 Ⅰ 6.5 11 8 1 No recurrence

From the results in Table 7, it can be seen that the number of circulating tumor cells (CTC) in the blood of patients with colorectal cancer stage 1 to stage 4 undergoing tumor resection has been significantly reduced, indicating that the tumor in the subject has been Successfully removed; in addition, the follow-up postoperative follow-up can also use the cell screen and measurement method of the present invention to confirm whether the subject's tumor has recurred. "Example 42" (cell culture monitoring test)

Cultivate a known number of HCT-116 live cells on the cell mesh, add the culture medium for 1 hour after the cells are attached and culture for 0, 3, 6, 12 hours, observe the changes of the cells on the substrate with a microscope and use Electrochemical impedance spectroscopy quantifies the results of its changes. When the cell is growing, it will gradually open the pseudopod and attach it to the growing surface. As the growth time, the area of the pseudopod attached to the cell mesh becomes larger, which affects the change of electrode impedance. This can monitor the growth of cells.

Please refer to FIG. 20, which is an SEM image showing the observation of cell culture using a scanning electron microscope at 3, 6, and 12 hours. From the results shown in Fig. 20, it is obvious that as the growth time is longer, the cells are clearly attached to the electrode.

Next, the cell meshes attached to cells that grew at different times were drawn into electrochemical impedance spectra using the aforementioned electrochemical impedance spectroscopy analysis method, and the result is shown in Figure 21; and the impedance change rate was measured by electrochemical analysis, and the result is shown in Figure 22 Shown.

From the results shown in Figure 21 and Figure 22, it can be seen that at 0 hours, that is, the cells to be tested are not attached to the cell mesh, and only a few non-test cells stick to the surface, causing slight impedance changes (impedance The rate of change was 2.1%); at 3 and 6 hours after the growth and attachment, the pseudopods were slightly stretched out, but not fully opened, so the impedance of the cell screen surface increased slightly, and the impedance changes were 27.36% and 39.19%, respectively After 12 hours of growth and attachment, the pseudopods were completely opened, the area attached to the cell screen surface increased greatly, and the impedance change was 141.63%.

It can be seen from this that if cells are cultured on the surface of the cell mesh of the present invention, the growth of the cells can be effectively monitored immediately after the pseudopodia are extended by the growth of the cells.

Therefore, the use of the cell screen of the present invention for cell screening and quantitative analysis reduces the amount of blood collected, reduces blood pretreatment time, uses electrochemical detection to be faster and more convenient, and does not require fluorescent dyes and greatly reduces costs. A large number of health or patient samples can be distinguished in a short time, saving a lot of time, manpower and cost.

For example, the cell screen of the present invention can also be combined with a conventional blood sampling syringe to form a cell screening device. As shown in FIG. 23, the cell screening device P includes a cell screen 1, a syringe 2 and a needle 3 with a specific antigen capable of binding to specific target cells on the surface; one end of the syringe 2 and the needle 3 Are connected to each other, and the cell screen 1 is arranged between the syringe 2 and the needle 3; therefore, when the cell screening device P is used to extract blood from the subject, the blood will pass through the cell screen 1, so that the Specific target cells are adsorbed on the surface of the cell mesh. In addition, the cell screen of the present invention can also be installed in a hemodialysis device, and the cell screen is used to capture specific cells (such as circulating tumor cells) in the patient during the process of hemodialysis, thereby reducing The amount of specific cells in the patient’s blood.

In addition, it can be confirmed from the above examples that the cell screen of the present invention can be used for cell culture, which is beneficial to real-time monitoring of cell growth.

It should be understood that the above-mentioned embodiments and examples are only examples, and those familiar with the art can make various modifications. The purpose of the above description, examples and data is to complete the structure of this description and to serve as an example of implementing the present invention. Although the content of this disclosure has been disclosed in the above manner, it is not intended to limit the content of this disclosure. Anyone who is familiar with this technique can make various changes and modifications without departing from the spirit and scope of this disclosure. Therefore, this The scope of protection of the disclosed content shall be subject to the scope of the attached patent application.

1: Cell screen

11: Screen substrate

12: Electrode layer

13: polymer layer

2: Syringe

3: needle

P: Cell screening device

Figure 1 is a schematic diagram showing the cross-sectional structure of the cell screen of the present invention. Figure 2 shows the IR spectra of acrylic acid monomer (AA), methacrylic acid sulfobetaine monomer (SBMA) and copolymer (poly(SBMA-co-AA)). Figure 3A shows the NMR spectra of acrylic acid monomer (AA), methacrylic acid sulfobetaine monomer (SBMA), and copolymer S5; where a is methacrylic acid sultaine monomer (SBMA), b is acrylic acid monomer (AA), and c is copolymer S5. Figure 3B shows the NMR spectra of copolymers S1 to S5; where a is copolymer S1, b is copolymer S2, c is copolymer S3, d is copolymer S4, and e is copolymer S5. Figure 4 is a fluorescence microscope image showing whether the electrode surface was successfully modified with streptavidin in Examples 1 to 6; where a is the modification of streptavidin-FITC on the electrode surface, and b is the surface with streptavidin. Biotin-Cy5 was grafted onto the test piece, c is a bright filed, and d is a superimposed image of 4a and 4b. Whether the electrode surface is successfully modified with streptavidin can be observed and judged by fluorescence microscope. Fig. 5 shows the fluid device used in the embodiment of the present invention. Figure 6 shows the luminescence after screening HCT-116 cells using cell meshes X1 to X6; where a is the use of cell mesh X1, b is the use of cell mesh X2, c is the use of cell mesh X3, and d is the use of cells The screens X4 and e use the cell screen X5, and f use the cell screen X6. Fig. 7 is a graph showing the comparison of the grabbing rate of HCT-116 cells using the cell screens X1 to X6. Figure 8 is a fluorogram showing the use of cell screens X1 to X6 to grab and screen white blood cells; where a is the cell screen X1, b is the cell screen X2, c is the cell screen X3, and d is the cell screen The screens X4 and e use the cell screen X5, and f use the cell screen X6. Fig. 9 is a graph showing the comparison of the anti-adhesion rate of leukocytes screened using cell screens X1 to X6. Fig. 10 is a comparison diagram showing the relationship between different flow rates and grabbing rates in Examples 19-22. Figure 11 is a graph showing the comparison of the grabbing rate of the cell screen X5 used to screen different cells in Examples 23-27 Figure 12 is a graph showing the comparison of impedance changes of the cell mesh X5 in Examples 23-27 in screening different cells. Figure 13 is an optical micrograph showing the surface of the cell screens in Examples 28 to 32 after screening; where a is Example 28, b is Example 29, c is Example 30, d is Example 31, and e is Example 32. a is Example 28. 14 is a graph showing the comparison of the grabbing rates of samples with different cell numbers in Examples 28 to 32 after screening. 15 is a graph showing the cyclic voltammetry waveforms obtained by cyclic voltammetry after the cell screens in Examples 28 to 32 screen samples with different cell numbers. FIG. 16 shows the electrochemical impedance spectrogram obtained by electrochemical impedance analysis after the cell screens in Examples 28 to 32 screen samples of different cell numbers. Fig. 17 shows the calibration curve obtained by electrochemical impedance analysis after the cell screens in Examples 28 to 32 screen samples with different cell numbers. Fig. 18 shows the electrochemical impedance spectrogram obtained by electrochemical impedance analysis after screening different samples with the cell screens in Examples 33-40. Figure 19 shows the calibration curve obtained by electrochemical impedance analysis after screening different samples with the cell screens in Examples 33-40. Fig. 20 is a scanning electron microscope image showing the use of a cell mesh to culture cells in Example 42 for 3, 6, and 12 hours; where a is 3 hours, b is 6 hours, and c is 12 hours. Fig. 21 shows the electrochemical impedance spectrogram obtained by electrochemical impedance analysis after 3, 6, and 12 hours of culturing cells using a cell mesh in Example 42. Figure 22 shows the calibration curve obtained by electrochemical impedance analysis after 3, 6, and 12 hours of cell culture using a cell mesh in Example 42. Figure 23 is a schematic diagram showing the structure of the cell screening device of the present invention.

1: Cell screen

11: Screen substrate

12: Electrode layer

13: polymer layer

Claims (10)

A cell sieve, which at least includes a layered structure composed of a sieve substrate, an electrode layer, and a polymer layer; wherein the sieve substrate is a mesh structure woven from filamentous materials, and the The average pore size of the mesh substrate is in the range of 15-30 μm; the electrode layer is arranged on the surface of the mesh substrate, and the surface of the electrode layer has amine groups; and the polymer layer is arranged on the electrode On the surface of the layer, and the surface of the polymer layer has streptavidin and specific antibodies capable of binding to specific target cells. The cell screen described in claim 1, which is used to screen out and quantify the specific target cells from a sample to be tested, for cell culture and detection of cell growth, or for use in the process of hemodialysis Remove the specific target cell. A method for manufacturing a cell screen, which includes the following steps: forming an electrode layer on the outer surface of a screen substrate by physical vapor deposition of a metal material and then performing activation treatment to make the surface of the electrode layer carry hydrogen and oxygen Base; contact the outer surface of the electrode layer with an aminosilane solution to react, so that the surface of the electrode layer has amine groups; contact the surface of the electrode layer with amine groups with a polymer binder to react, so that the A polymer layer is formed on the surface of the electrode layer; and after the surface of the polymer layer is contacted with a streptavidin for reaction, it is then contacted with a specific antibody capable of binding to specific target cells for reaction, so that the surface of the polymer layer carries the Streptavidin and the specific antibody to obtain a cell sieve capable of screening the specific target cells; wherein the average pore size of the sieve substrate is in the range of 15-30 μm; the polymer binding agent includes a coupling agent and a copolymer Polymer; The coupling agent is selected from any one of EDC, Sulfo-NHS, NHS, Sulfo-SMPB, Sulfo-SMCC, and combinations thereof; And the copolymer polymer is obtained by copolymerizing a carboxyl group-containing polymer and amphoteric ion polymer. The method for manufacturing a cell mesh as described in claim 3, wherein in the copolymer, the molar ratio (X:) of the carboxyl-containing polymer (X) to the zwitterionic polymer (Y) is Y) is in the range of 1:9~9:1. The method for manufacturing a cell screen as described in claim 3, wherein the carboxyl-containing polymer is polyacrylic acid, polymethacrylic acid, sodium polyacrylate, polyacrylamide, sodium polymethacrylate, polymaleic acid, Any one of its combinations. The method for manufacturing a cell screen as described in claim 3, wherein the zwitterionic polymer is a polymer polymer containing phosphocholine (PC), sultaine (SB), and/or carboxybetaine (CB) Things. A method for cell screening and measurement, which includes the following steps: cell screening step: the sample to be tested is passed through the cell screen described in any one of claims 1 to 2 at a specific flow rate, so that the sample to be tested The specific target cells in the cell are adsorbed on the surface of the cell mesh; and the cell measurement step: the cell mesh after the cell screening is electrically connected to an electrochemical meter, and the electrochemical meter is applied to the circuit in the circuit An AC voltage signal of a specific amplitude is then scanned in the frequency range of 0.01~70,000 Hz to obtain the impedance change value of the cell screen, and then the specific target in the test sample is calculated based on the impedance change value of the cell screen Cell concentration; wherein the surface of the cell mesh has streptavidin and specific antibodies capable of binding to the specific target cells; the specific flow rate is in the range of 0.5~4.0ml/hr; and the specific amplitude is in the range of 5~ The range of 30mVRMS. The method for cell screening and measurement as described in claim 7, further comprising a calibration curve establishment step, the calibration curve establishment step comprising: passing a standard with a known specific target cell concentration through the cell at the specific flow rate Screen, so that the specific target cells in the standard product are adsorbed on the surface of the cell screen; the cell screen after cell screening is electrically connected to the electrochemical instrument, and the electrochemical instrument is used for The circuit is applied to the AC voltage signal of the specific amplitude, and then scanned in the frequency range of 0.01 to 70,000 Hz to obtain the impedance change value of the cell screen; and repeat the above steps to obtain the cell screen using different specific target cell concentrations The impedance change value of the standard is established to establish the concentration calibration curve of the specific target cell, and the determination coefficient (R ² ) of the concentration calibration curve is greater than 0.9. The method for cell screening and measurement according to claim 8, wherein the cell measurement step further comprises: after obtaining the impedance change value of the cell screen, calculating the concentration calibration curve based on the impedance change value of the cell screen The concentration of the specific target cell in the sample to be tested. A cell screening device comprising the cell screen as described in any one of claims 1 to 2, a syringe, and a needle; wherein one end of the syringe is connected to the needle, and the cell screen is set Between the syringe and the needle; the surface of the cell screen has a specific antigen that can bind to specific target cells; and when the cell screening device is used to extract blood from the subject, the blood will pass through the cell screen Mesh so that the specific target cells are adsorbed on the surface of the cell mesh.

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