WO2020223900A1

WO2020223900A1 - Method of classifying and counting cells in a sample, bioassay kit, and micropore array plate

Info

Publication number: WO2020223900A1
Application number: PCT/CN2019/085861
Authority: WO
Inventors: Haochen CUI; Zhan Zhang; Jing Li
Original assignee: Boe Technology Group Co., Ltd.
Priority date: 2019-05-07
Filing date: 2019-05-07
Publication date: 2020-11-12
Also published as: CN110476058B; CN110476058A

Abstract

A method of classifying and counting cells(CT1, CT2, CT3) in a sample having the cells(CT1, CT2, CT3) suspended in an electrolyte is provided. The method includes introducing a plurality of cells(CT1, CT2, CT3) respectively into a plurality of micropores(MP), wherein, in at least 80% of the plurality of micropores(MP), no more than one cell(CT1, CT2, CT3) is introduced into a respective one of the plurality of micropores(MP); individually applying a first current to the plurality of cells(CT1, CT2, CT3) respectively in the plurality of micropores(MP); detecting first impedance values of the plurality of cells(CT1, CT2, CT3) respectively in the plurality of micropores(MP) applied with the first current; classifying a first group of the plurality of cells(CT1, CT2, CT3) as a first type based on a first distribution of the first impedance values; and counting a total number of the first group of the plurality of cells(CT1, CT2, CT3).

Description

METHOD OF CLASSIFYING AND COUNTING CELLS IN A SAMPLE, BIOASSAY KIT, AND MICROPORE ARRAY PLATE

TECHNICAL FIELD

The present invention relates to bioassay technology, more particularly, to a method of classifying and counting cells in a sample, a bioassay kit, and a micropore array plate.

BACKGROUND

Red blood cells, platelets, and white blood cells are suspended in the plasma of peripheral blood. Specimens are frequently examined since a great deal of clinical information can be obtained by blood analyses that examine these cells. For example, normal white blood cells are classified into five types: lymphocyte, monocyte, basophil, eosinophil, and neutrophil. In normal peripheral blood, these types of blood cells exist at certain ratios. However, if a subject has a disease, there may be cases where the number of blood cells of a specific type increases or decreases. Therefore, in the field of clinical laboratory tests, information highly useful for disease diagnosis can be obtained by classifying and counting white blood cells.

SUMMARY

In one aspect, the present invention provides a method of classifying and counting cells in a sample comprising the cells suspended in an electrolyte, comprising introducing a plurality of cells respectively into a plurality of micropores, wherein, in at least 80%of the plurality of micropores, no more than one cell is introduced into a respective one of the plurality of micropores; individually applying a first current to the plurality of cells respectively in the plurality of micropores; detecting first impedance values of the plurality of cells respectively in the plurality of micropores applied with the first current; classifying a first group of the plurality of cells as a first type based on a first distribution of the first impedance values; and counting a total number of the first group of the plurality of cells.

Optionally, the first current is a direct current or a low-frequency alternating current having a frequency no more than 100 kHz.

Optionally, the method further comprises individually applying a second current to the plurality of cells respectively in the plurality of micropores, the second current being a high-frequency alternating current; detecting second impedance values of the plurality of cells respectively in the plurality of micropores applied with the second current; classifying a second group of the plurality of cells as a second type based on a second distribution of the second impedance values; and counting a total number of the second group of the plurality of cells.

Optionally, the second distribution of the second impedance values identifies the second group of the plurality of cells and a third group of the plurality of cells; the method further comprises classifying the third group of the plurality of cells as a third type based on the second distribution of the second impedance values; and counting a total number of the third group of the plurality of cells.

Optionally, the high-frequency alternating current has a frequency greater than 1 MHz.

Optionally, each of the plurality of micropores has a diameter in a range of 15 μm to 40 μm, a depth in a range of 25 μm to 40 μm; and a distance between two adjacent micropores of the plurality of micropores is in a range of 25 μm to 40 μm.

Optionally, introducing the plurality of cells respectively into the plurality of micropores comprises introducing the sample comprising the cells suspended in the electrolyte onto a micropore array plate comprising the plurality of micropores; and applying an electric field to the sample to drive the plurality of cells respectively into a plurality of micropores.

Optionally, the electric field is applied through a first conductive layer and a second conductive layer respectively on two opposite sides of the micropore array plate, the plurality of micropores being sandwiched between the first conductive layer and the second conductive layer.

Optionally, the cells comprise white blood cells comprising one or a combination of lymphocyte, neutrophil, monocyte, eosinophil, and basophil.

Optionally, in at least 95%of the plurality of micropores, no more than one cell is introduced into a respective one of the plurality of micropores.

In another aspect, the present invention provides a bioassay kit for classifying and counting cells in a sample comprising the cells suspended in an electrolyte, comprising a micropore array plate; and instructions for methods of using the bioassay kit to classify and count cells in the sample comprising the cells suspended in an electrolyte; wherein the micropore array plate comprises a base substrate; a micropore wall layer on the base substrate defining a plurality of micropores; and an electrode layer comprising a plurality of pairs of electrodes on the base substrate; wherein a respective pair of the plurality of pairs of electrodes is in a respective one of the plurality of micropores and comprises a first electrode and a second electrode.

Optionally, the bioassay kit further comprises one or more power source regulators for individually applying a current to a plurality of cells respectively in the plurality of micropores; wherein the one or more power source regulators are capable of generating at least two types of currents having different frequencies.

Optionally, the at least two types of currents having different frequencies comprise a first type of current and a second type of current; the first type of current is a direct current or a low-frequency alternating current having a frequency no more than 100 kHz; and the second type of current is a high-frequency alternating current having a frequency greater than 1 MHz.

Optionally, the micropore array plate further comprises a first conductive layer on the base substrate; a passivation layer on a side of the first conductive layer away from the base substrate; and a second conductive layer on a side of the micropore wall layer away from the first conductive layer; wherein the plurality of micropores and the micropore wall layer are sandwiched between the first conductive layer and the second conductive layer.

Optionally, the bioassay kit further comprises a data analyzer configured to counting cells of different types based on signals detected respectively from the plurality of micropores upon applications of the at least two types of currents having different frequencies.

Optionally, the bioassay kit is a kit for classifying and counting white blood cells in a sample comprising one or a combination of lymphocyte, neutrophil, monocyte, eosinophil, and basophil.

In another aspect, the present invention provides a micropore array plate, comprising a base substrate; a micropore wall layer on the base substrate defining a plurality of micropores; and an electrode layer comprising a plurality of pairs of electrodes on the base substrate; wherein a respective pair of the plurality of pairs of electrodes is in a respective one of the plurality of micropores and comprises a first electrode and a second electrode; and a respective one of the plurality of micropores has a diameter sufficient for holding a single cell and insufficient for holding two or more cells in a sample.

Optionally, the micropore array plate further comprises a spacer layer between the micropore wall layer and the second conductive layer.

BRIEF DESCRIPTION OF THE FIGURES

The following drawings are merely examples for illustrative purposes according to various disclosed embodiments and are not intended to limit the scope of the present invention.

FIG. 1 is a plan view of a micropore array plate in some embodiments according to the present disclosure.

FIG. 2 is zoom-in view of a circled region in FIG. 1.

FIG. 3 is a cross-sectional view of a portion of a micropore array plate in some embodiments according to the present disclosure.

FIG. 4 is an equivalent circuit diagram inside a micropore in some embodiments according to the present disclosure.

FIG. 5 illustrates a process of introducing cells into a plurality of micropores in some embodiments according to the present disclosure.

DETAILED DESCRIPTION

The disclosure will now be described more specifically with reference to the following embodiments. It is to be noted that the following descriptions of some embodiments are presented herein for purpose of illustration and description only. It is not intended to be exhaustive or to be limited to the precise form disclosed.

In conventional methods of classifying and counting cells of different types in a sample, typically a flow cytometry technique is used to form a flow of single cells. The cells in the flow of single cells are then detected when the flow passes through a detector. The conventional methods require bulky instruments which are typically expensive and require regular maintenance from specialized technicians. The conventional methods are not suitable for point-of-care use or home use.

Accordingly, the present disclosure provides, inter alia, a method of classifying and counting cells in a sample, a bioassay kit, and a micropore array plate that substantially obviate one or more of the problems due to limitations and disadvantages of the related art. In one aspect, the present disclosure provides a method of classifying and counting cells in a sample comprising the cells suspended in an electrolyte. In some embodiments, the method includes introducing a plurality of cells respectively into a plurality of micropores, wherein, in at least 80%of the plurality of micropores, no more than one cell is introduced into a respective one of the plurality of micropores; individually applying a first current to the plurality of cells respectively in the plurality of micropores; detecting first impedance values of the plurality of cells respectively in the plurality of micropores applied with the first current; classifying a first group of the plurality of cells as a first type based on a first distribution of the first impedance values; and counting a total number of the first group of the plurality of cells.

As used herein, the term “micropore” refers to pores having cross-sectional dimensions in the range of approximately 1 nm to approximately 1000 μm, e.g., approximately 1 nm to approximately 50 nm, approximately 50 nm to approximately 100 nm, approximately 100 nm to approximately 1 μm, approximately 1 μm to approximately 10 μm, approximately 10 μm to approximately 100 μm, approximately 100 μm to approximately 200 μm, approximately 200 μm to approximately 400 μm, approximately 400 μm to approximately 600 μm, approximately 600 μm to approximately 800 μm, and approximately 800 μm to approximately 1000 μm. The term “cross-sectional dimension” may relate to height, width and in principle also to diameter. When a wall (including a side wall of the pore) of the pore is irregular or curved, the terms “height” and “width” may also relate to mean height and mean width, respectively. A micropore may have any selected cross-sectional shape, for example, U-shaped, D-shaped, rectangular, triangular, elliptical, oval, circular, semi-circular, square, trapezoidal, pentagonal, hexagonal, etc. cross-sectional geometries. Optionally, the micropore has an irregular cross-sectional shape. The geometry may be constant or may vary along the length of the micropore. Further, a micropore may have any selected arrangement or configuration, including linear, non-linear, merging, branching, looped, twisting, stepped, etc. configurations. Optionally, the micropore may have one or more open ends.

As used herein, a "sample" refers to any substance containing or presumed to contain target cells (e.g., from a bacteria, virus, tissue biopsy etc. ) . The sample can be an amount of tissue or fluid, or a purified fraction thereof, isolated from an individual or individuals, including, but not limited to, for example, skin, plasma, serum, whole blood, spinal fluid, saliva, peritoneal fluid, lymphatic fluid, aqueous or vitreous humor, synovial fluid, urine, tears, blood cells, blood products, semen, seminal fluid, vaginal fluids, pulmonary effusion, serosal fluid, organs, bronchio-alveolar lavage, tumors, paraffin embedded tissues, etc. Samples also can include constituents and components of in vitro cell cultures, including, but not limited to, conditioned medium resulting from the growth of cells in the cell culture medium, recombinant cells, cell components, etc. In some embodiments, the sample is a blood sample, e.g., a white blood cell sample. In one example, the white blood cell sample includes lymphocyte, neutrophil, monocyte, eosinophil, and basophil.

FIG. 1 is a plan view of a micropore array plate in some embodiments according to the present disclosure. FIG. 2 is zoom-in view of a circled region in FIG. 1. As shown in FIG. 1 and FIG. 2, the micropore array plate includes a plurality of micropores MP arranged in an array form. The micropore array plate may include any appropriate numbers of micropores. Optionally, the micropore array plate includes at least 10,000 micropore, e.g., at least 50,000, at least 100,000, or more micropores. Each of the plurality of micropores MP may have any appropriate shape or dimension. Optionally, each of the plurality of micropores MP has a cylinder shape.

In some embodiments, the plurality of micropores MP may be fabricated so that a respective one of the plurality of micropores MP has a diameter sufficient for holding a single cell and insufficient for holding two or more cells in a sample. For example, for analyzing a white blood cell sample, each of the plurality of micropores MP may have a diameter in a range of 15 μm to 40 μm, e.g., 15 μm to 20 μm, 20 μm to 25 μm, 25 μm to 30 μm, 30 μm to 35 μm, or 35 μm to 40 μm. Optionally, each of the plurality of micropores MP may have a depth in a range of 25 μm to 40 μm, e.g., 25 μm to 30 μm, 30 μm to 35 μm, or 35 μm to 40 μm. Optionally, a distance between two adjacent micropores of the plurality of micropores MP is in a range of 25 μm to 40 μm, e.g., 25 μm to 30 μm, 30 μm to 35 μm, or 35 μm to 40 μm.

A monocyte cell has an average diameter in a range of 15 μm to 40 μm. A lymphocyte cell has an average diameter in a range of 4 μm to 10 μm. A basophil has an average diameter in a range of 12 μm to 15 μm. An eosinophil has an average diameter in a range of 10 μm to 12 μm. A neutrophil has an average diameter in a range of 10 μm to 12 μm. The plurality of micropores MP may be fabricated to have a dimension sufficient for holding a single cell and insufficient for holding two or more cells in a sample.

In some embodiments, a plurality of cells respectively are introduced into a plurality of micropores MP. Optionally, in at least 80%of the plurality of micropores MP, no more than one cell is introduced into a respective one of the plurality of micropores MP. Optionally, in at least 85%of the plurality of micropores MP, no more than one cell is introduced into a respective one of the plurality of micropores MP. Optionally, in at least 90%of the plurality of micropores MP, no more than one cell is introduced into a respective one of the plurality of micropores MP. Optionally, in at least 95%of the plurality of micropores MP, no more than one cell is introduced into a respective one of the plurality of micropores MP. Optionally, in at least 99%of the plurality of micropores MP, no more than one cell is introduced into a respective one of the plurality of micropores MP. For example, typically a total number of white blood cells per micro liter blood sample is in a range of 4500 to 10000. In one example, the micropore plate can be designed to have around 100,000 micropores, and to ensure that, in at least 99%of the 100,000 micropores, each micropore contains zero or one cell.

FIG. 3 is a cross-sectional view of a portion of a micropore array plate in some embodiments according to the present disclosure. Referring to FIG. 3, the micropore array plate in some embodiments includes a base substrate 10; a micropore wall layer 60 on the base substrate 10 defining a plurality of micropores MP; and an electrode layer including a plurality of pairs of electrodes on the base substrate 10. A respective pair of the plurality of pairs of electrodes is in a respective one of the plurality of micropores MP. The respective pair of the plurality of pairs of electrodes includes a first electrode 81 and a second electrode 82. A respective one of the plurality of micropores MP has a diameter sufficient for holding a single cell and insufficient for holding two or more cells in a sample. As shown in FIG. 3, a cell of a first type CT1, a cell of a second type CT2, and a cell of a third type CT3 are respectively distributed into three different micropores of the plurality of micropores MP.

In some embodiments, the micropore array plate further includes a first conductive layer 30 on the base substrate 10; a passivation layer 50 on a side of the first conductive layer 30 away from the base substrate 10; and a second conductive layer 40 on a side of the micropore wall layer 60 away from the first conductive layer 30. Optionally, the plurality of micropores MP and the micropore wall layer 60 are sandwiched between the first conductive layer 30 and the second conductive layer 40.

In some embodiments, the micropore array plate further includes a spacer layer 70 between the micropore wall layer 60 and the second conductive layer 40. The spacer layer 70 is on a peripheral portion of the micropore wall layer 60, spacing apart the first conductive layer 30 from the plurality of micropores MP and the micropore wall layer 60.

The electrode layer enables one to individually applying a current to the plurality of cells respectively in the plurality of micropores MP. For example, in a respective one of the plurality of micropores MP, the first electrode 81 and the second electrode 82 are configured to apply a current to the respective one of the plurality of micropores MP (and the cell therein) .

The first electrode 81 and the second electrode 82 are configured to apply different types of currents having different frequencies to the respective one of the plurality of micropores MP, respectively at different stages of the analysis. For example, the first electrode 81 and the second electrode 82 are configured to apply at least two types of currents having different frequencies to the respective one of the plurality of micropores MP, respectively at different stages of the analysis. Optionally, the at least two types of currents having different frequencies include a first type of current and a second type of current. Optionally, the first type of current is a direct current or a low-frequency alternating current. Optionally, the second type of current is a high-frequency alternating current. Optionally, the low-frequency alternating current has a frequency no more than 500 kHz, e.g., no more than 400 kHz, no more than 300 kHz, no more than 200 kHz, or no more than 100 kHz. Optionally, the high-frequency alternating current has a frequency greater than 500 kHz, e.g., greater than 1 MHz, greater than 2 MHz, greater than 3 MHz, greater than 4 MHz, greater than 5 MHz, or greater than 6 MHz. Optionally, the first electrode 81 and the second electrode 82 are configured to apply more than two (e.g., 3, 4, 5) types of currents having different frequencies to the respective one of the plurality of micropores MP, respectively at different stages of the analysis.

FIG. 4 is an equivalent circuit diagram inside a micropore in some embodiments according to the present disclosure. Referring to FIG. 4, when the first electrode 81 and the second electrode 82 apply a direct current or a low-frequency alternating current to the respective one of the plurality of micropores MP containing a cell, the impedance of the cell is mostly determined by resistance of the membrane R _membrane, resistance of the cytoplasm R _cytoplasm, and resistance of the nucleoplasm R _nucleoplasm. The circuit can be considered as R _cytoplasm and R _nucleoplasm connected in parallel then connected to the in R _membrane series. Because R _membrane is far greater than R _cytoplasm and R _nucleoplasm, the impedance of the cell is mostly determined by R _membrane. The resistance of the membrane R _membrane is correlated to the size of the cell. Referring to FIG. 3, the cell of the first type CT1 (e.g., a lymphocyte cell) is smaller than the cell of the second type CT2 (e.g., a neutrophil cell) and smaller than the cell of the third type CT3 (e.g., a monocyte cell) . The resistance of the membrane R _membrane of the cell of the first type CT1 is also smaller than the resistance of the membrane R _membrane of the cell of the second type CT2 and the resistance of the membrane R _membrane of the cell of the third type CT3. In a respective one of the plurality of micropores MP that does not contain a cell, the impedance is much lower, and can be used as a background or as a control.

In some embodiments, the method includes introducing a plurality of cells respectively into a plurality of micropores MP, wherein, in at least 80%of the plurality of micropores, no more than one cell is introduced into a respective one of the plurality of micropores; individually applying a first current to the plurality of cells (e.g., the cell of the first type CT1, the cell of the second type CT2, and the cell of the third type CT3) respectively in the plurality of micropores MP; and detecting first impedance values of the plurality of cells respectively in the plurality of micropores MP applied with the first current. Optionally, the first current is a direct current or a low-frequency alternating current. Due to the differences in R _membrane among the cells of different types (e.g., the differences in R _membrane among the cell of the first type CT1, the cell of the second type CT2, and the cell of the third type CT3) , a first distribution of the first impedance values can be determined based on the results of detection. Optionally, the method further includes classifying a first group of the plurality of cells as a first type based on a first distribution of the first impedance values; and counting a total number of the first group of the plurality of cells. For example, based on the first distribution of the first impedance values, the cell of the first type CT1 (e.g., a lymphocyte cell) can be classified as the first group, and a total number of the cells of the first type CT1 can be counted.

Referring to FIG. 4 again, when the first electrode 81 and the second electrode 82 apply a high-frequency alternating current to the respective one of the plurality of micropores MP containing a cell, the capacitances of the cell components become non-negligible or even important factors in determining the impedance of the cell. For example, upon application of a high-frequency alternating current to the respective one of the plurality of micropores MP containing a cell, in some embodiments, the impedance of the cell is mostly determined by capacitance of the membrane C _membrane, resistance of the nucleoplasm R _nucleoplasm, and capacitance of the nucleoplasm C _nucleoplasm. The circuit can be considered as C _membrane, R _nucleoplasm, and C _nucleoplasm connected in series, and capacitances of the membrane C _membrane and the nucleoplasm C _nucleoplasm become important factors in determining the impedance of the cell. Because membranes of cells of different types have different membrane structures and different membrane constituents, capacitances of the membranes of cells of different types are also quite different capacitances. Based on the differences among the capacitances of the membranes of cells of different types, cells of different types can be further differentiated upon application of a high-frequency alternating current. Referring to FIG. 3, the cell of the second type CT2 (e.g., a neutrophil cell) and the cell of the third type CT3 (e.g., a monocyte cell) may have similar sizes, thus not easily differentiated upon application of a direct current or a low-frequency alternating current. However, due to the differences in the capacitances of the membranes of the cell of the second type CT2 and the cell of the third type CT3, they can be further differentiated upon application of a high-frequency alternating current.

In some embodiments, the method includes introducing a plurality of cells respectively into a plurality of micropores MP, wherein, in at least 80%of the plurality of micropores, no more than one cell is introduced into a respective one of the plurality of micropores; individually applying a second current to the plurality of cells (e.g., the cell of the first type CT1, the cell of the second type CT2, and the cell of the third type CT3) respectively in the plurality of micropores MP; and detecting second impedance values of the plurality of cells respectively in the plurality of micropores MP applied with the second current. Optionally, the second current is a high-frequency alternating current. Due to the differences in C _membrane (as well as C _nucleoplasm and R _nucleoplasm) among the cells of different types (e.g., the differences in C _membrane between the cell of the second type CT2 and the cell of the third type CT3) , a second distribution of the second impedance values can be determined based on the results of detection. Optionally, the method further includes classifying a second group of the plurality of cells as a second type based on a second distribution of the second impedance values; and counting a total number of the second group of the plurality of cells. For example, based on the second distribution of the second impedance values, the cell of the second type CT2 (e.g., a neutrophil cell) can be classified as the second type and the cell of the third type CT3 (e.g., a monocyte cell) can be classified as a third type, a total number of the cells of the second type CT2 can be counted, and a total number of the cells of the third type CT3 can be counted. Optionally, the second distribution of the second impedance values identifies the second group of the plurality of cells and a third group of the plurality of cells. Optionally, the method further includes classifying the third group of the plurality of cells as a third type based on the second distribution of the second impedance values; and counting a total number of the third group of the plurality of cells. Similarly, other cells of different types (e.g., eosinophil, and basophil) can also be differentiated by this method.

Optionally, the method further includes individually applying a third current to the plurality of cells respectively in the plurality of micropores MP; detecting third impedance values of the plurality of cells respectively in the plurality of micropores MP applied with the third current; classifying an additional group of the plurality of cells as an additional type based on a third distribution of the third impedance values; and counting a total number of the additional group of the plurality of cells. Optionally, the third current is a high-frequency alternating current, frequencies of the third current and the second current being different from each other. Cells of different types may exhibit different response profiles upon applications of high-frequency alternating currents of different frequencies. By using high-frequency alternating currents of different frequencies, cells of a target type may be best differentiated from other cells under a particularly high-frequency alternating current of a selected frequency, thereby classifying and counting the cells of the target type with higher accuracy.

The present method can be used for classifying and counting various cells of different cell types. Examples of cells of different types include blood cells (e.g., various white blood cells of different types, red blood cells, platelets) , sperm cells (e.g., active sperm cells vs. dead or inactive sperm cells) , liver cells (e.g., hepatocytes) , lung cells, spleen cells, pancreas cells, colon cells, skin cells, bladder cells, eye cells, brain cells, esophagus cells, cells of the head, cells of the neck, cells of the ovary, cells of the testes, prostate cells, placenta cells, epithelial cells, endothelial cells, adipocyte cells, kidney/renal cells, heart cells, muscle cells, central nervous system (CNS) cells, tumor cells, the like and combinations of the foregoing.

FIG. 5 illustrates a process of introducing cells into a plurality of micropores in some embodiments according to the present disclosure. Referring to FIG. 5, in some embodiments, the method includes introducing a sample including the cells (e.g., the cell of the first type CT1, the cell of the second type CT2, and the cell of the third type CT3) suspended in the electrolyte onto a micropore array plate having the plurality of micropores MP; and applying an electric field E to the sample to drive the plurality of cells respectively into a plurality of micropores MP, thereby introducing the plurality of cells respectively into the plurality of micropores MP. For example, as shown in FIG. 5, the electric field E is applied through a first conductive layer 30 and a second conductive layer 40 respectively on two opposite sides of the micropore array plate, the plurality of micropores MP being sandwiched between the first conductive layer 30 and the second conductive layer 40. By applying the electric field E to the sample, it can ensure that the plurality of cells can be driven into the plurality of micropores MP, respectively.

Various appropriate materials may be used for making the first conductive layer 30 and the second conductive layer 40. Examples of materials appropriate for making the first conductive layer 30 and the second conductive layer 40 include various metals and alloys, and semiconductive materials such as indium tin oxide. Various appropriate materials may be used for making the first electrode 81 and the second electrode 82. Examples of materials appropriate for making the first electrode 81 and the second electrode 82 include various metals and alloys such as gold, platinum, and silver (e.g., inert metals and alloys) . Each of the first electrode 81 and the second electrode 82 may be made to have an appropriate size. In one example, each of the first electrode 81 and the second electrode 82 has a height in a range of 50 nm to 150 nm (e.g., 100 nm) and a width in a range of 1 μm to 10 μm (e.g., 2-3 μm) . Optionally, a distance between the first electrode 81 and the second electrode 82 in a respective one of the plurality of micropores MP is in a range of 10 μm to 20 μm. Various appropriate materials may be used for making the micropore wall layer 60. For example, the micropore wall layer 60 may be made using a hydrophobic material, followed by treating an inner wall and a bottom surface of a respective one of the plurality of micropores P with a hydrophilic material. The micropore wall layer 60 can be fabricated to maintain hydrophobicity between adjacent micropores of the plurality of micropores P while keeping inner walls and bottom surfaces of the plurality of micropores P hydrophilic, to facilitate the cells entering the plurality of micropores P.

In another aspect, the present disclosure further provides a bioassay kit for classifying and counting cells in a sample having the cells suspended in an electrolyte. In some embodiments, the bioassay kit includes a micropore array plate; and instructions for methods of using the bioassay kit to classify and count cells in the sample having the cells suspended in an electrolyte. In some embodiments, the micropore array plate includes a base substrate; a micropore wall layer on the base substrate defining a plurality of micropores; and an electrode layer comprising a plurality of pairs of electrodes on the base substrate. Optionally, a respective pair of the plurality of pairs of electrodes is in a respective one of the plurality of micropores and includes a first electrode and a second electrode.

In some embodiments, the bioassay kit further includes one or more power source regulators for individually applying a current to the plurality of cells respectively in the plurality of micropores. The one or more power source regulators are capable of generating at least two types of currents having different frequencies. Optionally, the at least two types of currents having different frequencies includes at least a first type of current and a second type of current. The first type of current is a direct current or a low-frequency alternating current having a frequency no more than 100 kHz. The second type of current is a high-frequency alternating current having a frequency greater than 1 MHz. Optionally, the low-frequency alternating current has a frequency no more than 500 kHz, e.g., no more than 400 kHz, no more than 300 kHz, no more than 200 kHz, or no more than 100 kHz. Optionally, the high-frequency alternating current has a frequency greater than 500 kHz, e.g., greater than 1 MHz, greater than 2 MHz, greater than 3 MHz, greater than 4 MHz, greater than 5 MHz, or greater than 6 MHz.

Optionally, the one or more power source regulators is a power source regulator capable of adjusting the frequency of output current. Optionally, the one or more power source regulators is a power source regulator capable of converting an alternating current into a direct current. Optionally, the one or more power source regulators is a power source regulator capable of converting an alternating current of a higher frequency into an alternating current of a lower frequency. Optionally, the one or more power source regulators is a power source regulator capable of converting an alternating current of a lower frequency into an alternating current of a higher frequency. Optionally, the one or more power source regulators use a domestic power source (e.g., a wall power source) as an input power source, and are capable of converting the domestic power source into a direct current or an alternating current of a different frequency. Optionally, the one or more power source regulators use a battery as an input power source, and are capable of converting the input current into an alternating current.

In some embodiments, the instructions for methods of using the bioassay kit include a reference chart of percentages of cells of different types in a control sample (e.g., in a sample from a healthy subject) . In one example, the bioassay kit may be used for classifying and counting different types of white blood cells in a sample. Table 1 is an exemplary reference chart listing percentages of different types of white blood cells in a control sample.

Table 1: Percentages of different types of white blood cells in a control sample.

Cells	Percentages
neutrophil	55-73%
lymphocyte	20-40%
monocyte	2-8%
eosinophil	1-4%
basophil	0.5-1%

In some embodiments, the micropore array plate further includes a first conductive layer on the base substrate; a passivation layer on a side of the first conductive layer away from the base substrate; and a second conductive layer on a side of the micropore wall layer away from the first conductive layer. Optionally, the plurality of micropores and the micropore wall layer are sandwiched between the first conductive layer and the second conductive layer.

In some embodiments, the micropore array plate further includes a spacer layer between the micropore wall layer and the second conductive layer. The spacer layer is on a peripheral portion of the micropore wall layer, spacing apart the first conductive layer from the plurality of micropores and the micropore wall layer.

In some embodiments, the bioassay kit further includes a data analyzer configured to counting cells of different types based on signals detected respectively from the plurality of micropores upon applications of the at least two types of currents having different frequencies. Optionally, the data analyzer is configured to classifying a first group of the plurality of cells as a first type based on a first distribution of the first impedance values, and counting a total number of the first group of the plurality of cells, as discussed above. Optionally, the data analyzer is further configured to classifying a second group of the plurality of cells as a second type based on a second distribution of the second impedance values, and counting a total number of the second group of the plurality of cells. Optionally, the data analyzer is further configured to classifying the third group of the plurality of cells as a third type based on the second distribution of the second impedance values, and counting a total number of the third group of the plurality of cells. Optionally, the data analyzer includes a memory and one or more processors.

Optionally, each of the plurality of micropores has a diameter in a range of 15 μm to 40 μm, e.g., 15 μm to 20 μm, 20 μm to 25 μm, 25 μm to 30 μm, 30 μm to 35 μm, or 35 μm to 40 μm. Optionally, each of the plurality of micropores has a depth in a range of 25 μm to 40 μm, e.g., 25 μm to 30 μm, 30 μm to 35 μm, or 35 μm to 40 μm. Optionally, a distance between two adjacent micropores of the plurality of micropores is in a range of 25 μm to 40 μm, e.g., 25 μm to 30 μm, 30 μm to 35 μm, or 35 μm to 40 μm.

In some embodiments, the bioassay kit further includes one or more reagents (e.g., one or more electrolytes) . Optionally, the bioassay kit further includes one or more containers for receiving the one or more reagents.

In some embodiments, the bioassay kit further includes one or more syringes for taking samples from a subject (e.g., a human or a mammal) . Optionally, the bioassay kit further includes one or more sample processors for processing a raw sample. In one example, the one or more sample processors include a separator for separating white blood cells from other cells of other types (e.g., red blood cells and platelets) .

In another aspect, the present disclosure further provides a micropore array plate. In some embodiments, the micropore array plate includes a base substrate; a micropore wall layer on the base substrate defining a plurality of micropores; and an electrode layer comprising a plurality of pairs of electrodes on the base substrate. A respective pair of the plurality of pairs of electrodes is in a respective one of the plurality of micropores and comprises a first electrode and a second electrode. A respective one of the plurality of micropores has a diameter sufficient for holding a single cell and insufficient for holding two or more cells in a sample.

The foregoing description of the embodiments of the invention has been presented for purposes of illustration and description. It is not intended to be exhaustive or to limit the invention to the precise form or to exemplary embodiments disclosed. Accordingly, the foregoing description should be regarded as illustrative rather than restrictive. Obviously, many modifications and variations will be apparent to practitioners skilled in this art. The embodiments are chosen and described in order to explain the principles of the invention and its best mode practical application, thereby to enable persons skilled in the art to understand the invention for various embodiments and with various modifications as are suited to the particular use or implementation contemplated. It is intended that the scope of the invention be defined by the claims appended hereto and their equivalents in which all terms are meant in their broadest reasonable sense unless otherwise indicated. Therefore, the term “the invention” , “the present invention” or the like does not necessarily limit the claim scope to a specific embodiment, and the reference to exemplary embodiments of the invention does not imply a limitation on the invention, and no such limitation is to be inferred. The invention is limited only by the spirit and scope of the appended claims. Moreover, these claims may refer to use “first” , “second” , etc. following with noun or element. Such terms should be understood as a nomenclature and should not be construed as giving the limitation on the number of the elements modified by such nomenclature unless specific number has been given. Any advantages and benefits described may not apply to all embodiments of the invention. It should be appreciated that variations may be made in the embodiments described by persons skilled in the art without departing from the scope of the present invention as defined by the following claims. Moreover, no element and component in the present disclosure is intended to be dedicated to the public regardless of whether the element or component is explicitly recited in the following claims.

Claims

A method of classifying and counting cells in a sample comprising the cells suspended in an electrolyte, comprising:

introducing a plurality of cells respectively into a plurality of micropores, wherein, in at least 80%of the plurality of micropores, no more than one cell is introduced into a respective one of the plurality of micropores;

individually applying a first current to the plurality of cells respectively in the plurality of micropores;

detecting first impedance values of the plurality of cells respectively in the plurality of micropores applied with the first current;

classifying a first group of the plurality of cells as a first type based on a first distribution of the first impedance values; and

counting a total number of the first group of the plurality of cells.
The method of claim 1, wherein the first current is a direct current or a low-frequency alternating current having a frequency no more than 100 kHz.
The method of claim 1, further comprising:

individually applying a second current to the plurality of cells respectively in the plurality of micropores, the second current being a high-frequency alternating current;

detecting second impedance values of the plurality of cells respectively in the plurality of micropores applied with the second current;

classifying a second group of the plurality of cells as a second type based on a second distribution of the second impedance values; and

counting a total number of the second group of the plurality of cells.
The method of claim 3, wherein the second distribution of the second impedance values identifies the second group of the plurality of cells and a third group of the plurality of cells;

the method further comprises classifying the third group of the plurality of cells as a third type based on the second distribution of the second impedance values; and

counting a total number of the third group of the plurality of cells.
The method of claim 3, wherein the high-frequency alternating current has a frequency greater than 1 MHz.
The method of any one of claims 1 to 5, wherein each of the plurality of micropores has a diameter in a range of 15 μm to 40 μm, a depth in a range of 25 μm to 40 μm;and

a distance between two adjacent micropores of the plurality of micropores is in a range of 25 μm to 40 μm.
The method of any one of claims 1 to 6, wherein introducing the plurality of cells respectively into the plurality of micropores comprises:

introducing the sample comprising the cells suspended in the electrolyte onto a micropore array plate comprising the plurality of micropores; and

applying an electric field to the sample to drive the plurality of cells respectively into a plurality of micropores.
The method of claim 7, wherein the electric field is applied through a first conductive layer and a second conductive layer respectively on two opposite sides of the micropore array plate, the plurality of micropores being sandwiched between the first conductive layer and the second conductive layer.
The method of any one of claims 1 to 8, wherein the cells comprise white blood cells comprising one or a combination of lymphocyte, neutrophil, monocyte, eosinophil, and basophil.
The method of any one of claims 1 to 9, wherein, in at least 95%of the plurality of micropores, no more than one cell is introduced into a respective one of the plurality of micropores.
A bioassay kit for classifying and counting cells in a sample comprising the cells suspended in an electrolyte, comprising:

a micropore array plate; and

instructions for methods of using the bioassay kit to classify and count cells in the sample comprising the cells suspended in an electrolyte;

wherein the micropore array plate comprises:

a base substrate;

a micropore wall layer on the base substrate defining a plurality of micropores; and

an electrode layer comprising a plurality of pairs of electrodes on the base substrate;

wherein a respective pair of the plurality of pairs of electrodes is in a respective one of the plurality of micropores and comprises a first electrode and a second electrode.
The bioassay kit of claim 11, further comprising one or more power source regulators for individually applying a current to a plurality of cells respectively in the plurality of micropores;

wherein the one or more power source regulators are capable of generating at least two types of currents having different frequencies.
The bioassay kit of claim 12, wherein the at least two types of currents having different frequencies comprise a first type of current and a second type of current;

the first type of current is a direct current or a low-frequency alternating current having a frequency no more than 100 kHz; and

the second type of current is a high-frequency alternating current having a frequency greater than 1 MHz.
The bioassay kit of any one of claims 11 to 13, wherein the micropore array plate further comprises:

a first conductive layer on the base substrate;

a passivation layer on a side of the first conductive layer away from the base substrate; and

a second conductive layer on a side of the micropore wall layer away from the first conductive layer;

wherein the plurality of micropores and the micropore wall layer are sandwiched between the first conductive layer and the second conductive layer.
The bioassay kit of any one of claims 12 to 14, further comprising a data analyzer configured to counting cells of different types based on signals detected respectively from the plurality of micropores upon applications of the at least two types of currents having different frequencies.
The bioassay kit of any one of claims 11 to 15, wherein each of the plurality of micropores has a diameter in a range of 15 μm to 40 μm, a depth in a range of 25 μm to 40 μm; and

a distance between two adjacent micropores of the plurality of micropores is in a range of 25 μm to 40 μm.
The bioassay kit of any one of claims 11 to 16, wherein the bioassay kit is a kit for classifying and counting white blood cells in a sample comprising one or a combination of lymphocyte, neutrophil, monocyte, eosinophil, and basophil.
A micropore array plate, comprising:

a base substrate;

a micropore wall layer on the base substrate defining a plurality of micropores; and

an electrode layer comprising a plurality of pairs of electrodes on the base substrate;

wherein a respective pair of the plurality of pairs of electrodes is in a respective one of the plurality of micropores and comprises a first electrode and a second electrode; and

a respective one of the plurality of micropores has a diameter sufficient for holding a single cell and insufficient for holding two or more cells in a sample.
The micropore array plate of claim 18, further comprising:

a first conductive layer on the base substrate;

a passivation layer on a side of the first conductive layer away from the base substrate; and

a second conductive layer on a side of the micropore wall layer away from the first conductive layer;

wherein the plurality of micropores and the micropore wall layer are sandwiched between the first conductive layer and the second conductive layer.
The micropore array plate of claim 19, further comprising a spacer layer between the micropore wall layer and the second conductive layer.