High-specificity multi-level electrical impedance cell immunoassay chip and counting and extraction method

Abstract

The present invention relates to a high-specificity multi-stage electrical impedance cell immunoassay chip and a counting and extraction method. The chip structure includes at least one cavity, the at least one cavity is provided with a test sample inlet channel and a test sample outlet channel, the test sample inlet channel is provided with a first electrode, the test sample outlet channel is provided with a second electrode, the at least one cavity contains microbeads, the structure of the microbeads includes a microbead body and a capture structure modified on the surface of the microbead body, and the microbead body is a magnetic bead; it also includes an electrical impedance analysis system, which is connected to the first electrode and the second electrode. The counting method is to obtain the number of corresponding target cells captured in each cavity based on the number of pulse signals detected by the first electrode and the second electrode of each cavity and calculate the difference in the number of pulse signals. The target cells can be extracted and the microbeads can be reconstructed for the next detection. The present application realizes the simultaneous non-labeled counting and extraction of multiple cells.

Description

Technical Field

[0001] The present invention relates to the field of cell detection and counting, in particular to a high-specificity multi-stage electrical impedance cell immune analysis chip and a counting and extraction method. Background Art

[0002] Counting and analyzing cells in the blood can not only reflect the growth and development of an organism, but also reveal information such as the occurrence, development, and severity of related diseases, helping to provide a basis for disease prevention, clinical diagnosis, and treatment. There are two main types of existing cell counting technologies: one is the flow cytometer based on optical signals. When flowing cells pass through a focused laser beam, they scatter the laser light, so the cell properties can be analyzed based on the scattered light information. The other is the Coulter counter based on electrical signals. When cells pass through electrodes, an impedance peak is generated at the signal receiving end, enabling cell counting. However, these counting devices are large, expensive, and require long processing times. Some also require labeling of the target sample, which greatly consumes counting time and increases counting costs, making it difficult to quickly count specific cells.

[0003] When detecting and extracting cells, it is difficult to distinguish cells by appearance due to the small differences in cell size and morphology, so most existing cell detection systems are based on the principle of immunoassay. Due to the different origins of cells, there are differences in their surface specific antigens. By modifying specific antibodies on the solid surface and utilizing the binding of antibodies to specific antigens on the cell surface, specific cells can be captured. The existing cell detection method is mainly immunofluorescence: a specific fluorescent pigment is labeled on the antibody. When it binds to the corresponding antigen on the cell surface, it will show a specific fluorescent reaction under a fluorescence microscope, realizing the detection and extraction of specific cells. However, this method has the disadvantages of complex procedures, slow detection speed, and easy damage to cells, and cannot quickly and accurately realize cell detection and extraction. Summary of the Invention

[0004] In view of the shortcomings of the existing technology, the present invention provides a highly specific multi-stage electrical impedance cell immunoassay chip and a counting and extraction method, which can realize the non-labeling counting and extraction of one or more specific target cells.

[0005] The technical solution adopted in the present invention is as follows:

[0006] A high-specificity multi-stage electrical impedance cell immunoassay chip comprises at least one cavity, wherein the at least one cavity is provided with a test sample inlet channel and a test sample outlet channel, wherein the test sample inlet channel is provided with a first electrode, and the test sample outlet channel is provided with a second electrode, wherein the at least one cavity contains microbeads, wherein the microbeads comprise a microbead body and a capture structure modified on the surface of the microbead body, and wherein the microbead body is a magnetic bead;

[0007] Also included is an electrical impedance analysis system connected to the first electrode and the second electrode.

[0008] Further technical solutions are:

[0009] The at least one cavity is further provided with a magnetic pole for stacking the microbeads layer by layer, and the gap between adjacent microbead layers is larger than the volume of the cells to be detected.

[0010] The capture structure is a specific antibody.

[0011] The at least one cavity is connected in series through the sample inlet channel and the sample outlet channel to be tested, and the capture structures of the microbeads contained in different cavities are of the same or identical type to achieve the capture of different types of target cells.

[0012] The structure of the microbeads is formed in the following manner: the microbead body is placed in a hydroxylation reagent or a carboxylation reagent, and then a corresponding specific antibody surface modification reagent is added.

[0013] The volume of the at least one cavity is three to five times the equivalent volume of the microbeads contained therein.

[0014] The at least one cavity is further provided with a microbead inlet channel and a microbead outlet channel.

[0015] A cell counting method based on the high-specificity multi-stage electrical impedance cell immunoassay chip comprises:

[0016] The microbeads are placed in a hydroxylation reagent or a carboxylation reagent, and then the corresponding specific antibody surface modification reagent is added to obtain the corresponding microbeads;

[0017] introducing microbeads containing different surface-modified specific antibodies into the at least one cavity and encapsulating them;

[0018] Introducing the sample solution to be tested into the cavity from the sample inlet channel to be tested of the first cavity;

[0019] During the movement of the test sample solution from the test sample inlet channel of the first cavity to the test sample outlet channel of the last cavity in the cavity, the target cells are captured by the corresponding microbeads and retained in the corresponding cavity;

[0020] The number of pulse signals detected by the first electrode and the second electrode of each cavity is recorded and the difference in the number of pulse signals is calculated. The electrical impedance analysis system calculates the number of corresponding target cells captured in each cavity based on the difference in the number of pulse signals.

[0021] The cell counting method according to the high-specificity multi-stage electrical impedance cell immunoassay chip further comprises:

[0022] The microbeads that have captured the target cells are guided out of the cavity, the captured target cells are eluted, the target cells are extracted, and the microbead bodies are obtained, and new microbeads are prepared;

[0023] The chamber is cleaned and new microbeads are introduced for the next detection.

[0024] The beneficial effects of the present invention are as follows:

[0025] The present application realizes the simultaneous non-labeled counting and extraction of one or more cells, with simple operation and high detection accuracy.

[0026] The microbeads of this application are surface-modified with antibodies that bind to specific cell surface antigens. The electrical impedance system utilizes the cell capture function of the microbeads to achieve electrical impedance counting of specific cells. The target cells captured by the microbeads can then be specifically extracted by introducing a specific solution to dissolve the immune interaction between the magnetic beads and the cells. The microbeads can then be reconstructed for subsequent testing.

[0027] Other features and advantages of the present invention will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Schematic diagram of the chip structure of an embodiment of the present invention.

[0029] Figure 2 Schematic diagram of the cellular immune detection principle involved in the technical method of an embodiment of the present invention.

[0030] In the figure: 1. first electrode; 2. cell; 3. first cavity; 4. microbead; 5. microbead inlet channel; 6. shell; 7. second test sample outlet channel; 8. microbead outlet channel; 9. microbead body; 10. capture structure; 11. target cell; 12. specific antigen; 13. magnetic pole; 14. second cavity; 15. first test sample inlet channel; 16. first test sample outlet channel; 17. second test sample inlet channel; 18. second electrode; 19. third electrode. DETAILED DESCRIPTION

[0031] The specific embodiments of the present invention are described below with reference to the accompanying drawings.

[0032] The high-specificity multi-stage electrical impedance cell immunoassay chip of the present application comprises at least one cavity, wherein at least one cavity is provided with a test sample inlet channel and a test sample outlet channel, wherein the test sample inlet channel is provided with a first electrode, and the test sample outlet channel is provided with a second electrode, and at least one cavity contains microbeads, wherein the structure of the microbeads comprises a microbead body and a capture structure modified on the surface of the microbead body, wherein the microbead body is a magnetic bead;

[0033] Also included is an electrical impedance analysis system connected to the first electrode and the second electrode.

[0034] See also Figure 1 The high-specificity multi-stage electrical impedance cellular immunoassay chip of this embodiment includes a housing 6, within which a first cavity 3 and a second cavity 14 are formed. The left end of the first cavity 3 is connected to a first sample inlet channel 15 to be tested, and the right end is connected to a first sample outlet channel 16 to be tested. The left end of the second cavity 14 is connected to a second sample inlet channel 17 to be tested, and the right end is connected to a second sample outlet channel 7 to be tested. The first cavity 3 and the second cavity 14 are connected in series through the first sample outlet channel 16 to be tested and the second sample inlet channel 17 to be tested. The first electrode 1, the second electrode 18, and the third electrode 19 are provided on the first sample inlet channel 15, the first sample outlet channel 16 (the second sample inlet channel 17), and the second sample inlet channel 17 to be tested. A microbead inlet channel 5 and a microbead outlet channel 8 are also provided on the two cavities, respectively.

[0035] The cavity can be used to accommodate microbeads 4. The structure of microbeads 4 is formed by placing microbead bodies 9 in a hydroxylation reagent or a carboxylation reagent, and then adding corresponding specific antibody surface modification reagents to obtain capture structures 10 on the surface of microbead bodies 9. Microbead bodies 9 are magnetic beads.

[0036] See also Figure 2 By combining the capture structure 10 with the specific antigen 12 on the surface of the target cell 11, the cell can be captured.

[0037] Depending on the target cell type, different types of specific antibodies can be used in the capture structure 10. The two cavities can be used to accommodate microbeads 4 with different capture structures 10, respectively, for capturing different types of target cells.

[0038] Magnetic poles 13 are also provided on the two cavities to enable the microbeads 4 to be stacked layer by layer, with the gap between adjacent microbead layers being larger than the volume of the cells to be detected.

[0039] Specifically, the magnetic pole 13 is square or cylindrical and is fixed to the bottom of the chip housing 6 .

[0040] The volume of each cavity is three to five times the equivalent volume of the microbeads contained therein.

[0041] The electrical impedance analysis system is used to monitor changes in the electrical impedance of the sample being tested within the chip in real time. Three electrodes are used to apply a DC or AC detection signal to each channel. The peak value of the AC detection signal is approximately 1V, and the frequency is 3KHz to 3MHz. The three excitation electrodes have a three-dimensional structure, which is used to construct an excitation electric field with multiple spatial layers. This simultaneously enhances the electric field strength in the detection area within the chip, thereby improving detection sensitivity and spatial resolution. Three pairs of electrodes can detect three locations. When cells pass through the electrodes in the channel, current pulses are generated. The number of current pulses can be analyzed to determine the number of cells being tested. Because the target cells are captured within the cavity, the number of cells passing through the entrance channel differs from the number of cells passing through the exit channel. The difference in number can be calculated using the pulse signal to determine the number of captured target cells.

[0042] During chip manufacturing in this embodiment, the housing 6 is fabricated using photolithography. The housing 6 is then engraved with the first sample inlet 15, the first cavity 3, the first sample outlet 16, the second sample inlet 17, the second cavity 14, and the second sample outlet 7. Electrodes and magnetic poles are embedded in the chip housing 6. The electrodes are made of polymer or silver.

[0043] The cell counting analysis method of the high-specificity multi-stage electrical impedance cell immunoassay chip of this embodiment includes:

[0044] The microbeads are placed in a hydroxylation reagent or a carboxylation reagent, and then the corresponding specific antibody surface modification reagent is added to obtain the corresponding microbeads;

[0045] Introduce microbeads 4 with different surface-modified specific antibodies into the first cavity 3 and the second cavity 14 respectively and encapsulate them;

[0046] A sample solution is introduced into the first cavity 3 from the first sample inlet channel 15, wherein the solution contains cells 2, and the cells 2 include target cells and non-target cells;

[0047] The sample solution flows from the first cavity through the second cavity and out of the second sample outlet channel 7. During the flow, the two target cells are captured by the corresponding microbeads in the two cavities and retained in the corresponding cavities.

[0048] The number of pulse signals detected by the first electrode 1 and the second electrode 18 is recorded and the difference in number is calculated. The number of pulse signals detected by the second electrode 18 and the third electrode 19 is recorded and the difference in number is calculated. The electrical impedance analysis system calculates the number of corresponding target cells captured in each cavity based on the difference in number.

[0049] Specifically, the electrical impedance analysis system collects data on electrical signals, analyzes their amplitude and phase, and modulates and demodulates the AC signals to obtain the number of cells entering and exiting the two cavities. The difference between the two is the number of target cells, thus realizing the counting of specific cells in the sample to be tested.

[0050] The cell counting analysis method of the high-specificity multi-stage electrical impedance cell immunity analysis chip of this embodiment further includes:

[0051] After the detection is completed, the microbeads that captured the target cells are guided out of the cavity, and the captured target cells are eluted to extract the target cells for further analysis. At the same time, the microbeads are obtained and re-prepared;

[0052] The cavity is cleaned and new microbeads are introduced before the next detection.

[0053] Specifically, the microbeads can be treated with an elution buffer to eliminate the immune interaction between the magnetic beads and the cells, thereby achieving specific extraction of the target cells.

[0054] By increasing the number of cavities and corresponding electrodes, the counting of two or more target cells can be achieved.

[0055] The cell counting method of this embodiment is based on electrical impedance cell counting technology and cell immune detection technology, and realizes non-labeled counting and extraction of specific target cells. It is simple to operate and has high detection accuracy, and the microbeads can be recycled.

[0056] Those skilled in the art will understand that the foregoing descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art will be able to modify the technical solutions described in the foregoing embodiments or substitute equivalents for some of the technical features. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. A high-specificity multi-level electrical impedance cell immune analysis chip, characterized in that: The method comprises at least one cavity, wherein the at least one cavity is provided with a sample inlet channel and a sample outlet channel to be tested, the sample inlet channel to be tested is provided with a first electrode, the sample outlet channel to be tested is provided with a second electrode, and microbeads are accommodated in the at least one cavity, wherein the structure of the microbeads includes a microbead body and a capture structure modified on the surface of the microbead body, and the microbead body is a magnetic bead; The at least one cavity is further provided with a magnetic pole for causing the microbeads to stack layer by layer, with the gap between adjacent microbead layers being larger than the volume of the cells to be detected; Also included is an electrical impedance analysis system connected to the first electrode and the second electrode; The number of pulse signals detected by the first electrode and the second electrode of each cavity is recorded and the difference in the number of pulse signals is calculated. The electrical impedance analysis system calculates the number of corresponding target cells captured in each cavity based on the difference in the number of pulse signals.

2. The high-specificity multi-stage electrical impedance cell immune analysis chip according to claim 1, characterized in that: The capture structure is a specific antibody.

3. The high-specificity multi-stage electrical impedance cell immune analysis chip according to claim 1, characterized in that: Two adjacent cavities are connected in series through the sample outlet channel of the upper cavity and the sample inlet channel of the lower cavity. The capture structures of the microbeads contained in different cavities are different in type to achieve the capture of different types of target cells.

4. The high-specificity multi-stage electrical impedance cell immune analysis chip according to claim 1, characterized in that: The structure of the microbeads is formed in the following manner: the microbead body is placed in a hydroxylation reagent or a carboxylation reagent, and then a corresponding specific antibody surface modification reagent is added.

5. The high-specificity multi-stage electrical impedance cell immune analysis chip according to claim 1, characterized in that: The volume of the at least one cavity is three to five times the equivalent volume of the microbeads contained therein.

6. The high-specificity multi-stage electrical impedance cell immunity analysis chip according to claim 1, characterized in that: The at least one cavity is further provided with a microbead inlet channel and a microbead outlet channel.

7. A cell counting method using the high-specificity multi-stage electrical impedance cell immunoassay chip according to any one of claims 1 to 6, characterized in that: include: The microbeads are placed in a hydroxylation reagent or a carboxylation reagent, and then the corresponding specific antibody surface modification reagent is added to obtain the corresponding microbeads; introducing microbeads containing different surface-modified specific antibodies into the at least one cavity and encapsulating them; Introducing the sample solution to be tested into the cavity from the sample inlet channel to be tested of the first cavity; During the movement of the test sample solution from the test sample inlet channel of the first cavity to the test sample outlet channel of the last cavity in the cavity, the target cells are captured by the corresponding microbeads and retained in the corresponding cavity; The number of pulse signals detected by the first electrode and the second electrode of each cavity is recorded and the difference in the number of pulse signals is calculated. The electrical impedance analysis system calculates the number of corresponding target cells captured in each cavity based on the difference in the number of pulse signals.

8. The cell counting method of the high-specificity multi-stage electrical impedance cell immunoassay chip according to claim 7, characterized in that: Also includes: The microbeads that have captured the target cells are guided out of the cavity, the captured target cells are eluted, the target cells are extracted, and the microbead bodies are obtained, and new microbeads are prepared; The chamber is cleaned and new microbeads are introduced for the next detection.

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