TW201217781A - Microfluidic system for detecting a biological entity in a sample - Google Patents

Abstract

According to various embodiments, a microfluidic system for detecting a biological entity in a sample volume is provided. The microfluidic system may include: a chamber configured to receive the sample volume, wherein the chamber includes a detection region for detecting the biological entity; a first port in fluid communication with the chamber; and a second port including a filter in fluid communication with the chamber; and wherein a fluid provided to the first port or the second port flows between the first port and the second port through the chamber.

Description

201217781 VI. Description of the invention: [Related application of the cross-reference] This application claims the priority of the application of the Singapore application No. 200606999-8, which is filed on October 20, 2009, the entire contents of which are hereby Use as a reference. TECHNICAL FIELD Various embodiments are directed to a microfluidic system for detecting a living body in a sample volume and a method of forming the microfluidic system. Embodiments are further directed to methods for detecting organisms using the microfluidic system. [Prior Art] The number of circulating cells in the blood of a patient is a biological indicator for routine use in clinical diagnosis. However, current systems for detecting cell counts are large, expensive, time consuming and require an external sample preparation procedure. In addition, microfluidic devices have been utilized to provide a compact and inexpensive solution that provides the promise of moving the test toward patients in a bedside environment. Unfortunately, these techniques often require a certain amount of sample preparation (especially when using a label-free method) to achieve good performance. Recent developments have included the use of specific subpopulations as biomarkers for various diseases. The subtype of circulating cells has been used as a diagnostic biomarker for various conditions (eg, circulating tumor cells for cancer (circulating 201217781 tumor cells, CTCs) ("Circulating" by Mocellin S. et al. Tumor cells.: the 4 leukemic phase' of solid cancers j, published in "TRENDS in Molecular Medicine", 2006, 12 (3), 13 0-139 ), lymphocytes CD4 for HIV (Jokerst JV· et al. "Integration of semiconductor quantum dots into nano-bio-chip systems for enumeration of. CD4+ T cell counts at the point-of-need", published in Lab Chip, 2008, 8, 2079-2090), and for cardiovascular Endothelial progenitor cells (EPCs) (Increased circulating hematopoietic and endothelial progenitor cells (EPCs) in the early phase of acute myocardial infarction, published by Blood, 2005, l〇5 : 199-206 ). These biomarkers can be used for early diagnosis, prediction, treatment monitoring, or Minimal residual disease (MRD) control. Their number in gold is quite low (for example, less than 5 cells per 7.5 ml for CTC) to relatively abundant (for example, 200 cells/pl for cd4 T lymphocytes) ): .epc is about 0.01%-2% of peripheral blood mononuclear cells (pBMC), and the blood contains about 7000-10000 white blood cells and more than 1〇〇〇 of red blood cells. This makes it very low. Biosensors that measure limits to detect biological indicators in the blood face significant challenges. The number of circulating cells has been used as a diagnostic indicator in conventional procedures, such as white blood cell counts routinely performed in clinical laboratories. EPC has also been used as a biological indicator of cardiovascular status. The circulating Epc is 201217781 bone marrow-derived stem cells, which have the ability to differentiate into vascular endothelial cells for vascular lining repair, and their number in the blood can be used as a biological indicator (Rosenzweig A. "Circulating Endothelial Progenitors - Cells As Biomarkers, The New England Journal of Medicine, 2005, 353 (10), 1055-1057; JM Hill et al., "Circulating Endothelial Progenitor Cells,"

Vascular Function, and Cardiovascular Risk", The New England Journal of Medicine, 2003, 348, 593-600; PE Szmitko "Endothelial progenitor cells: new hope for a broken hear", Circulation, 2003, 107, 3093-100) The 〇EPC level can be used for health surveillance because their level in the blood is associated with coronary artery disease or cardiovascular status and its risk factors. EPC is also used in therapeutic surveillance to monitor the effects of primary and secondary prevention strategies, with known specific drugs (such as statin', a hydroxymethyl quinone coenzyme A reductase inhibitor) increasing EPC counts. In addition, EPC can be transplanted for tissue regeneration. However, it is not trivial to analyze the specific type of circulating cells, and it has not been used as a usual practice due to technical limitations and relatively high costs. The efficient and selective extraction of rare target cells from whole blood is very challenging for the Microcr〇 Total Analysis System (pTAS). The μ1 whole blood sample can contain approximately 4 million to 5 million red blood cells (RBC) and approximately 4000 to 11 peripheral blood mononuclear cells (pBMC). Assuming that CD34+ is detected at the level of 0.1% PBMC, this would mean as few as 7 cells in the whole blood of i μ1 201217781. Conventionally, in order to separate this low concentration of EPC from blood, a sample preparation method for cell purification is required. The general ·_ procedure includes: (1) culturing the sample with RBC lyse buffer, (2) removing the cardiac cell suspension and removing the supernatant, and (3) marking with magnetic beads, attached to the magnetic beads (tag) antigen-specific antibodies, and (4) re-centrifuging and removing unbound beads from the solution. The total time for the sample preparation procedure can be about 1 to 2 hours' and in addition the procedure requires a large centrifuge and those skilled in the art. Results 'The limitations of the use of these conventional sample purifications test the application of the key care test (p〇int>〇f._care). Cell subtypes are usually defined by the expression of their specific surface markers. For example, detection of CTC is generally based on the presence of a specific epithelial index (EpCAM) on the surface, while EPC can be derived from its CD34 or CD133 protein or endothelial marker protein (VEGFR2/KDR or a combination of these proteins). ) Definition. In order to detect these specific cells, the conventional technique is flow cell technology (Khan S. S. et al., Factory Detection of Circulating Endothelial Cells and Endothelial Progenitor Cells by

Flow Cytometry", Clinical Cytometry '2005, 64B, 1-8), such as the Fluorescent Cell Sorter (FACS), which optically reads cells that are stained by thin capillaries with specific indicators. Fluorescent. • However, this technique is cumbersome and time consuming (used for dyeing procedures and analysis for approximately 4 to 5 hours) requiring a larger sample size (mi), requiring a highly skilled person and generally performing off-site execution. In the advent of microfluidics, a variety of approaches have emerged to overcome the shortcomings of the 201217781 FACS (ie, the time and skill involved) and to enhance portability. Flow-through system (Taek Dong Chung, Hee Chan Kim, "Recent advances in miniaturized microfluidic flow cytometry for clinical use", Electrophoresis, 2007, 28,, 45 11-4520) directly miniaturizes the classification concept and uses cells The specific nature of the guide to direct them to the counter for optical or unlabeled hands (Roeser T's "Lab-on-chip for the Isolation and Characterization of Circulating Tumor Cells", Proceedings Of the 29th Annual International Conference of the IEEE EMBS, 2007 ' 6446-6448 ) or both (Wang YN et al. '"〇n-chip counting the number and the percentage of CD4+ T lymphocytes", Lab Chip, 2008, 8 , 309-315) detected. These systems are capable of accurately calculating the number of cells passed' but require preparative off-wafer sample preparation' for example, involving fluorescent or magnetic staining' and which may also include isolating PBMC. Furthermore, the circulation system may cause cell loss and may not be suitable for large sample processing, which will affect cost and sensitivity and specificity issues. The process of marking or staining cells is also time consuming. It is also possible to use the "fl〇w_stop" system, which uses the specific binding of cells on the surface of the micro-device to directly purify the sample from whole blood on the wafer (Nagrath S. et al. "Isolati〇 n 〇f rare circulating tumour cells in cancer patients by microchip technology»,

Natu%2〇〇7'45()' 1235_1239). However, 'detection is performed optically after camping staining and requires complex optical analysis 201217781 system to automate' and has relatively poor performance. Additional care has been used to prevent known components from reaching critical care test detection in a light manner, at a rate that is responsive to the heart, and at a rate of disease (eg, for an acute cardiovascular condition, less than one hour). The chamber system has ..., 屺 ,, coupled with surface-specific cell selection to avoid the use of markers during the detection phase. Most of these systems rely on samples, such as PBMC, which are pre-purified (Nel SY et al., "Label-free Impedance of Low Levels of Circulating Endothelial Progenitor Cells for Diagnosis",

Biosensors and Bioelectronics, 2010, 25, 1095-1 101). However, their dependence on surface-specific capture limits the occurrence of paralysis, as extremely abundant red blood cells can mask surface access for other cells. The conventional preparation method for PBMC is based on the difference in density and size compared to its counterpart (such as red and spherical cells). For cell-based debt testing, it is not necessary to separate the remaining components (such as plasma) from the cells, although the techniques used typically operate as such. There are many conventional methods for preparing PBMC, the most widely used method being centrifugation, in which after the centrifugation procedure, the cells are recovered in the skin layer of the skin color in a specific tube containing different density portions. The blood sample required is usually in the milliliter (ml) grade and is indexed by a syringe and requires laboratory facilities for preparation. Therefore, off-wafer preparation of PBMCs greatly reduces the benefits of using microfluidic devices for critical care testing applications, which require processing a small volume on the patient side (eg, 'a blood sample from a finger thorn (finger 201217781 prick) is about 5〇μ1). The concept of centrifugation has also been applied to wafers to provide a sample through the channel and chamber. It is also applied to the separation of cells from other blood components (Kang D-R et al., "Blood micro-separator", US 2006/0263265), but requires a rotating mechanism and a sophisticated detection integration strategy. Dimensional transition of microporous membranes (microfabricated) (Myangbrane microfiilter device for selective capture, electrolysis and genomic analysis of human circulating tumor cells), Journal of

Chromatography A, 2007, 1162, 154-161) or paper (V〇na G. et al., "Isolation by Size of Epithelial Tumor Cells", American Journal of Pathology, 2000, 15 6 (1) > 57-63 *» Illert W· '"Methods of preparing peripheral stem cells from leukocytes reduction filters", EP1484390)) has been used to prepare and detect rare circulating cells (mostly CTCs). In these systems, the sample passes through a membrane containing pores of a specific size that allow small cells (e.g., red blood cells) to pass while leaving larger cells (e.g., CTCs and/or white blood cells). The achieved efficacy is relatively high and the cells are directly analyzed on the membrane by optical or biomolecular detection. However, such techniques cannot be easily integrated into a label-free, focused care inspection system that requires the calculation of cells on the membrane or the transfer of the melted sample to a specific detector, such as an instant PCR machine, which does not provide an accurate level. Size filters have also been microfabricated in fully sealed systems (MaUez〇s G. et al., "Fluorescence detector, filter device and re" 10 s 201217781 methods" US2008/0013092; Battr ell CF et al., "Method and System for microfluidic manipulation, amplification and analysis of fluids, for example, bacteria assays and antiglobulin testing"' US74 1 6892 ). However, such systems are commonly used to capture large undesired particles and to detect small (molecular) materials. Another disadvantage of these systems is the planar manufacturing technique (mainly the 矽 process), which rushes to reduce the area captured in the system. SUMMARY OF THE INVENTION According to one embodiment, a microfluidic system for detecting an organism in a sample volume is provided. The microfluidic system can include a chamber configured to receive the sample volume, wherein the chamber Include a detection area for detecting the living body; a first port in fluid communication with the chamber; and a second port including a filter and in fluid communication with the chamber, and wherein A fluid of the first port or the second port flows through the chamber between the first port and the second port. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The following detailed description refers to the accompanying drawings in the claims The embodiments are described in sufficient detail to enable those skilled in the art to practice the invention. Other embodiments may be utilized and structural, logical, and electronic changes may be made without departing from the scope of the invention. The various embodiments are not necessarily mutually exclusive, and some embodiments may be combined with one or more other real forms to form new embodiments. Embodiments may provide a microfluidic system and method for detecting organisms (such as cells or biological indicators) with relatively improved performance and performance, with or without at least some of the disadvantages associated with current systems. Embodiments may provide a microfluidic system and methods for detecting organisms from relatively small volumes of blood samples, such as cellular or biological indicators, such as peripheral blood mononuclear cells (PBMC) or circulating endothelial progenitors. Cells (EPC). In various embodiments, the microfluidic system can include an open chamber for detecting organisms. The microfluidic system can be disposed on or in the microchip to form a microfluidic wafer having an integrated open chamber. Embodiments may provide an integrated microfluidic system for label-free detection of peripheral blood mononuclear cell (PBMC) or circulating endothelial progenitor cells (EPC) in the blood of a patient to provide an integrated bedside diagnostic test. The microfluidic system provides sample preparation functions, such as isolation of peripheral blood mononuclear cells (PBMC) or circulating endothelial progenitor cells (EPC), specific antigen-based cells based on antibody antigens in a relatively compact system. Capture, and relatively highly sensitive, unmarked impedance detection. In various embodiments, the microfluidic system can provide the ability to prepare cells (e.g., PBMC or EPC) from a relatively small amount of whole blood sample and to detect such cells. Preparation of the cells can include isolating, enriching, or concentrating the cells to be detected. Embodiments may provide a sample-to-answer integration system that includes a microfluidic wafer to detect PBMC or 12 201217781 EPC from a whole blood sample. Samples can be loaded into the system, and information or responses can be projected from the sample in a relatively short time. The integrated system can be based on a mark-free impedance spectroscopy-like detection platform that can detect relatively small numbers of cells. The pBMC or EPC is isolated from other blood cells and delivered to the open chamber for detection. The system can exclude other blood cells based on the difference in size between PBMC, EPC and other blood cells. In various embodiments, specific cells, such as EPC, can be magnetically labeled (e. g., with magnetic beads) for further isolation and concentration. This helps magnetically isolate the magnetically labeled Epc from the pMBC to detect EPC. Various embodiments may provide methods and microfluidic systems for obtaining PBMC or EPC solutions from relatively small amounts of blood samples (less than 100 μΐ) with high efficiency, and integrating with open chambers to detect subtypes of specific cells ( Such as PBMC or EPC) to reduce the loss of rare cells. Embodiments can provide a sample response result to a system capable of critical care testing with very microfluidic, unmarked detection. Embodiments may provide a microfluidic system and method for providing preparation and detection of cells in a chamber (such as pBMC or Epc or other types of cells having distinguishable surface indicators) in a single microfluidic wafer. The wafer can tolerate unlabeled cell detection. PBMC or EPC or other types of cells can be in solution in whole blood. Embodiments may provide a microfluidic system and method for preparing a pBMC or EPC sample purified from a whole blood sample in an open chamber in a microfluidic wafer, the microfluidic system being integrated with labelless detection technology to provide # Near the detection area of the sample preparation area. The detection region can include a 13 201217781 capture surface that is functionalized with specific capture molecules. The integration of cell preparation, filtering and detection in the open cells of the microfluidic wafers of the various embodiments eliminates the need for transfer procedures (eg, transfer between different modules for filtering and detection), thereby minimizing sampling procedures Loss of cells during, for example, at the interconnect or along the walls of the microchannel. The microfluidic system can include a semi-permeable membrane having a pore size between about 3 microns and about 8 microns for filtration purposes. The semipermeable membrane filter can be integrated with the microfluidic system to provide an integrated method based on size separation. Semi-permeable membranes provide a relatively large transition area and relatively high cell recovery. Embodiments can provide a method and microfluidic system having an open chamber that includes a detection zone to detect organisms in a sample volume, wherein the sample volume can be directly loaded into the microfluidic system from the top opening of the open chamber Open chamber. This eliminates the need for a transfer procedure or a flow-through fluid method to deliver a biological object to the detection zone, thereby reducing cell loss, for example, at fluid interconnections. In various embodiments, a small sample volume can be used in conjunction with a sequential batch loading into the open chamber of the microfluidic system. In various embodiments, the sample can be purified by passing the sample through a filter (such as a semi-permeable membrane) in a microfluidic system to isolate and retain the organism to be detected (eg, PBMC4 Epc) through the filter while Remove any organisms that pass through the filter that are not to be detected. The fish object to be detected (for example PBMC or EPC) is then removed from the filter, the sheet and transferred to the chamber where the prediction can be performed. In various embodiments, the detection zone can be located at the bottom of the open chamber, with a capture surface functionalized with a specific capture molecule. The capture surface can be connected to any sensor or detector that detects the presence of the organism. The microfluidic system can include two steps in fluid communication with the chamber or capture surface, "the port can be lightly connected or connected to the cavity by relatively short microchannels such that fluid such as a buffer solution can pass through the ports Anyone is supplied to the open cavity for cleaning of the sample processing and detection area. The two ports may be coupled to a pump or a syringe to move the buffer solution along the fluid microchannel and the chamber. For example, it may be used to buffer The solution is input into the microfluidic system or the sample and/or the buffer solution is extracted from the microfluidic system. In each of the embodiments, a fluid such as a buffer solution can pass through the empty chamber containing the sample (the organism to be tested). The sample flow transition piece, so that the organism to be detected is left in the filter, and the undetected organism and the buffer solution are removed and discarded. The reflux of the fluid such as a buffer solution can be filtered in the opposite direction. a sheet for removing and transferring the organism to be detected remaining in the filter to the chamber, the detection area of the chamber, or the array of microelectrodes in the chamber. In various embodiments, the capture molecules can be set and / Attached to the surface of the microelectrode array to capture the organism to be detected. The capture is an antibody that is specific for the organism to be detected. In various embodiments, a microelectrode array can be installed to generate Non-uniform electric field induces dielectrophoresis to assist in capturing and culturing the organism to be detected. In various embodiments, the microfluidic system may further comprise a movably arranged magnetic element disposed in the detection zone or A magnetic field is provided or generated near the microelectrode array. The magnetic field element can be a permanent magnet or an electromagnet. The generated magnetic field can help capture organisms that have been magnetically labeled at the detection region or microelectrode array. In a further embodiment A plurality of magnets may be provided so that two, three, or four magnets may be provided. In each embodiment, the organism may be captured by a capture molecule, a dielectrophoresis, or a magnetic field in the detection region of the microfluidic system, or the foregoing. Captured by combination. Embodiments can provide surface index identification. This can be achieved by providing specific capture molecules such as antibodies. For example, it can be provided on top of ctc A skin cell attachment molecule (EpCAM), or a CD34 or CD33 protein, or an EPC endothelial marker protein (VEGFR2/KDR) specific antibody. Further examples may provide for multiple indicator separations. For example, a first antibody can be coupled to a target Cells and providing a second antibody for magnetic capture while being specific to the target cell can be functionalized on the surface of the electrode. After removal of other non-target cells (eg, after a flushing procedure), only the first and the coupling are coupled Specific cells of both secondary antibodies remain on the electrodes. Embodiments may provide methods and microfluidic systems for integrated cell detection for relatively rapid separation of small volumes (less than 丨〇〇) Endothelial precursor cells (EPC), which are sparsely populated in the blood, and their selective capture or immobilization and their detection by immunochemical chemistry coupled to electrochemical impedance sensing (such as microelectrode arrays) on localized detection regions Measurement. The whole blood sample can be a blood sample from the patient's finger stick. Each embodiment can tolerate the potential for detecting EPC as low as G.1% directly from a small blood sample on a lab-on-chiP facility. Peripheral blood mononuclear, 'field cell (PBMC). This can provide screening tests, bedside surveillance systems, or key care testing applications for health care surveillance, medication, and vascular care. Embodiments may allow the door to be in the time of full detection. The information in such a time can be provided to the doctor for a quick decision, especially in an emergency. This relatively rapid separation procedure, identification of field surface indicators, and ugliness of target cells (eg, EPC) can result in a wide range of: cell-based applications performed on micro-integrated analysis systems (μ τ A) s ). Embodiments can provide red blood cell cells for immunomagnetic separation

(RBC) method of melting and microfluidic systems. Liquid samples containing EPC and RBC can be initially prepared and mixed with a melting buffer solution of certain chemicals, which are included in the chlorination of the melting effect (NH4C1, which is used as a solvent), as a pH. Sodium carbonate (NaHC〇3) in buffer and ethylenediaminetetraacetic acid (EdTA) as an anti-agglomerating agent. In each of the examples, sodium carbonate in the dissolution buffer solution may also serve as a blocker to suspend the Na+_K+_ATp enzyme. The melting buffer solution may further comprise a magnetic element such as a magnetic bead. The magnetic beads can be magnetic beads attached to the antibody (ie, the magnetic beads are coupled to the antibody) to couple with Epc, which can facilitate selective capture of EPC and detection by immunochemistry in the microfluidic systems of various embodiments. In each of the examples, selective cell solvation (such as dissolving RBC) may occur in a solution containing EPC, however, it may also occur in a & EPC-free melting process. This preparation procedure performs simultaneous migration in a relatively short period of time. In addition to red blood cell (RBC) and cell surface index markers (requires about 10 minutes) 'can cause completion of the preparation and debt testing procedures in less than 1 hour. Each example can result in relatively higher potency after melting (> 90% EPC). 17 201217781 In each of the examples, the dissolution buffer solution may include about 150 mM NH4C1, 10 mM NaHC03, 0.1 rnM EDTA, and about 2000 magnetic beads. However, it is to be understood that the concentration of NH4C1 may range from about 10 mM to about 150 mM 'eg, from about 1 〇 to about 100 mM, from about 10 mM to about 50 mM, or from about 50 mM to about 150 mM. Thus, the concentration of NH4C1 can be about 1 mM, about 20 mM, about 50 mM, about 100 mM, about 120 mM, or about 150 mM. It will be appreciated that the concentration of NaHC〇3 can range from about 1 mM to about 100 mM, such as in the range of about 10 mM to about 50 mM, or in the range of about 10 mM to about 20 mM 'so' the concentration of NaHC03 can be About 1 mM, about 20 mM, about 30 mM, about 50 mM, about 80 mM, or about 100 mM. It will be appreciated that the concentration of EDTA may range from about 〇〇ι to about 1.0 mM, such as from about 0.01 mM to about 5.5 mM, from about 〇.〇i mM to about 0.1 mM, or about 〇丨 mM. A range of up to about io mM can be such that the concentration of EDTA can be about 1 mM, about 5 mM, about 〇 mM, about 2 2 mM, about 5 5 mM, or about 1 . In each of the examples, the blood sample can be mixed with the dissolution buffer solution such that ΝΗ4 <:1 to blood volume ratio (NHUCl has a concentration of about 150 mM) ranging from 1:1 1 1:10. In each of the examples, the total volume of the mixed solution may be less than 100 μΐ. It will be appreciated that the number of magnetic beads varies in the various embodiments depending on the number of cells to be transferred to the magnetic beads. In various embodiments, the ratio of two magnetic beads per cell is provided. For example, in a blood sample of about 2 〇 μ1, the number of (d) can be about 〇. The range of Μ% of PBMC is equivalent to approximately 18 201217781 ΗΗΜ000 EPCs. Therefore, approximately 2 magnetic beads may be required. In a further embodiment, a relatively higher ratio of magnetic beads per cell can be provided. Depending on the size of the magnetic beads (for example, micron-sized magnetic beads or nano-sized magnetic beads) can provide approximately 2 〇〇〇 2 磁 magnetic beads. Embodiments may provide methods and systems for relatively rapid separation and detection of rare endothelial progenitor cells (EPCs) based on selective melting red blood cells (RBC) and immunomagnetic richness to separate Epc from blood (such as CD34+). cell). A sample containing CD34+ cells can be prepared and mixed with a solution containing NaHC〇3, EDTA, and magnetic beads attached to the antibody. This solution can help to dissolve or eradicate RBC in the sample, and also label the magnetic beads attached to the antibody to the CD34+ cell index in a relatively short period of time (about 丨〇 minute). This time limit meets the needs of CD34+ detection. After culturing the sample, the sample and mixed solution containing CD34+ cells can be loaded or supplied to the microfluidic system of each example. The microfluidic system filters the sample to concentrate and concentrate CD34+ cells by removing other non-target cells. Then, a localized magnetic field can be applied to the vicinity of the microelectrode region at the detection region to selectively isolate and concentrate the immunomagnetically labeled CD34+ cells (coupled with the magnetic beads) on the microelectrode, thereby capturing and immobilizing the CD34+ cells. Micro-electricity and above to detect and quantify measurements. This facilitates the specific extraction of CD34+ cells from blood samples, while other remaining cells that do not express CD34 antigen (such as undissolved RBC and pBMc) can be removed from the sample by washing it away. Embodiments may provide an unmarked detection method. Such an unmarked detection method can be performed by impedance measurement or impedance spectroscopy as described in WO 20 丨 0/050898 filed on Sep. 19, 2009, the entire disclosure of which is incorporated herein by reference. For reference. With this detection mechanism, the capture surface in the open chamber is patterned with a gold electrode on the microchip, and the gold electrode is connected or electronically communicated with the measurement or detection system. The electrodes can be arranged as a microelectrode array. The electrodes may be specifically provided with a capture molecule (such as an antibody) for a specific target cell or organism' while the remaining portion of the microchip that is not covered by the electrode may be passivated by a repellent material, such as polyethylation Glycol (PEG). The electrodes can also be used to perform dielectrophoresis (DEp) to attract and concentrate cells on the electrodes to accelerate cell capture and increase efficacy. In other embodiments, other detection mechanisms may be used, and the detection area may be modified or set in a suitable form correspondingly based on the detection mechanism. For example, a refractive index-based sensing mechanism (such as surface-plasma resonance (SPR), or a light-applied resonator (0pticai ring res〇nat〇r), or a dry-source instrument) can be used as a detection mechanism. Wherein the capture surface can be patterned with a gold layer that contains spots of capture molecules such as antibodies. Light can be passed through the channel to the capture area using photons or through direct illumination. It is also possible to use localized SPR. Another possible detection mechanism is a field effect sensor, such as a nanowire array, which can be placed and patterned on the capture surface and functionalized with specific capture molecules such as antibodies. The array of nanowires can be coupled to a measurement system' to measure the resistance of the nanowires, which can change when a biological object, such as a cell, is captured on a surface. Conventional fluorescence measurements can also be used to detect organisms. The captured cells can be stained with various dyes, which can be excited using an external light source, and the 201217781 emission can be detected by, for example, a charge coupled device (CCD) camera or a photomultiplier tube (PMT). . In a further embodiment, a shear flow controlled cleaning scheme can be implemented that is accomplished by flowing a fluid, such as a buffer solution, through the microelectrode array and chamber to facilitate specific cell selection and removal on the microelectrode Any organism that may exist but is not detected. Various embodiments may alleviate the challenge of sample loss during transfer between different systems or the challenge of a sample at the interface between different systems by providing a microfluidic system in a single microfluidic package or in a cavity This is achieved by providing a microfluidic wafer for sample preparation and detection in the chamber. The chamber can be an open chamber that provides a small sensing area for highly sensitive sensing and provides a relatively high performance level of cellular response during the detection procedure. Embodiments may provide a microfluidic system based on fluid movement or flow in a microchannel and chamber from one section of the microfluidic system to another, thereby allowing seamless detection with no marks Integration. Embodiments may provide: diagnosis of cells such as detection of peripheral blood mononuclear cells (PBMC) or rare circulating cells (eg, EPC:); label-free for highly sensitive hands-free integration systems Detection; automated systems for relatively low-cost processing; and diagnostics, prognosis, and therapeutic surveillance, for example, for cancer, cardiovascular disease, and transplant surveillance. Each embodiment may provide bedside or critical care of an organism, such as a rare circulating tumor cell (CTC), such as endothelial progenitor cells (EPC), from a relatively small amount of blood sample (less than 100 μιη) in less than one hour. Check 21 201217781 (POC) processing and detection for diagnosis. Embodiments may provide an integrated microfluidic system for label-free detection of EPC in a patient's blood to provide an integrated bedside diagnostic test for integrating samples that are made from one or more patients A hand-pointed thorn (each finger is needled with about 50 μΐ of blood) is obtained. Microfluidic systems provide the following functions in relatively tight systems: sample preparation from blood samples, specific cell capture based on antibody antigen recognition, and relatively high sensitivity label-free impedance detection (eg, in PBMC samples) . 1% EPC). This may help, for example, to make decisions on the type of stent used by a patient, such as a heart patient. In various embodiments, microfluidic systems and methods provide relatively high sensitivity, relatively high throughput, and relatively low cost cell detection. The microfluidic system can include a tantalum wafer and a plastic fixture. Embodiments may provide a microfluidic system and method including an open chamber to separate white blood cells (including circulating cells) based on differences in size of different cells or organisms within the open chamber. The microfluidic systems of the various embodiments can be produced at relatively low cost and can be disposable. In the context of the various embodiments, the term "microfluidic system" may mean a fluid system comprising channels (also referred to as microchannels) in one or more micrometer ranges, wherein a sample volume is provided to flow into the microchannel based on fluid motion. And flowing along the microchannel. In various embodiments, a microfluidic system can be formed on a microchip to form a microfluidic wafer. In the context of the various embodiments, the term "detection area" may mean an area in which an object can be detected 22 201217781. In various embodiments, the detection zone can be located in the chamber, such as at the bottom of the chamber. In various embodiments, the chamber is an open chamber. Detection can be performed based on an unmarked guessing method such as impedance measurement or sensing. The detection region can include an electrode, a pair of electrodes, or a microelectrode array comprising more than one electrode or more than a pair of electrodes. Each pair of electrodes can include an inner electrode and an outer electrode. The outer electrode has a complementary shape that substantially surrounds the inner electrode. Implementation. In this example, the electrode, a pair of electrodes, or a microelectrode array can be positioned at the bottom of the detection area. In various embodiments, the organism can be captured in the detection zone by dielectrophoresis or capture of capture molecules (e. g., antibodies). The capture molecules can be disposed on the surface of the electrode and/or attached to the surface of the electrode. The term "open chamber" in the context of the various embodiments may mean that the solution may flow or pass through or remain in a chamber or passage in the chamber or passage. In various embodiments, the open chamber has a top opening. In other words, the open chamber does not have a top cover. In each of the embodiments, the term "chamber" means "open chamber". The term "fluid communication" associated with different sections of a microfluidic system may mean communication between two sections of a microfluidic system. In this embodiment 'this communication may be a direct connection or a direct path between two sections of the microfluidic system' or may include one or more zones in between the two sections of the microfluidic system segment. In the context of the various embodiments, the "port" - word may mean - an opening, a recess or a cavity to provide a conduit through which fluid passes. In various embodiments, the microbody system can include at least two ports in fluid communication with the chamber. The micro-peach system can include an inlet port that provides access to or introduction of work 23 201217781 , . The fluid system can include an outlet conduit. In each of the embodiments, each of the ports has a hollow cylindrical structure which is provided to leave or exit the hollow cylindrical structure. It is possible to have a continuous knot cylindrical structure that is early in the range of about 1 mm ^ / in the embodiment, hollow in the length of about 1 mm to about 2 from > about 3 mm, for example, such that the county sound · mm to, The force of 3 mm can be about 1 mm in length, kiss, and c is about 2. 5 mm, or about 3 々 by 2 mm, secondary, force 3 mm. In each of the embodiments, the range is about 〇. 6mm to about the middle back column, the structure can be 5,, , 01 straight, such as in the range of about 6 _ to about 1 or at about i 6 so that the diameter can be about ,6, to, force Range, test 4 rugged · 6 mrn, about 〇 , , Λ U · 66 _, about 〇 · 8 mm, about 1 · 〇 mm, or about 25 mm. In the embodiment of the -^ t step, each port having a Φ* cylindrical structure may include a first portion and a second portion, and the first -° smash has about 0. 66 mm (diameter) xi s two slightly, and 仫) Xl. 5mm (height) size, and the second part has about 1 > 5 mm (straight, and also j xl. 5 mm (height) size. The first part can be on the second τ knife item 0卩, or vice versa. The bran should be understood that each of the ports or per-ports may have a different 'structure and configuration' and may have different dimensions. In various embodiments, a third port or more ports may be provided in communication with the cavity body, having a suitable configuration, configuration and dimensions. The term "organism" in each embodiment may mean a biological indicator, a cell, a cell, a virion, a biopolymer, or a combination of the foregoing. The term "cell" can include eukaryotic cells and prokaryotic cells. The term "cell" may also include peripheral blood mononuclear cells, cells including the white blood cells, tau cells, and the immune system of the helper tau cells, including circulating tumor cells, lymphocytes, and 24 201217781 CD4 lymphocytes and endothelial progenitor. Biological indicator of animal cells or yeast cells. "Eukaryotic cells. The term "mammalian cells" can include tumor cells, blood cells, immune system cells, precursor cells, and embryonic cells. "Biopolymer" - words may include polypeptides, nucleic acids, lipids, and sugar collection. The organisms in various embodiments may have anchors for culture and capture on the surface of the microelectrode array. In various embodiments, the sample volume can be a blood sample volume. The blood sample volume can be a whole blood sample volume. In various embodiments, the chamber or open chamber can have a volume ranging from about ΐμ1 to about 500 μΐ, such as in the range of about 1 μ1 to about 5 〇〇 y, in the range of about _μ1, about 1 μl to about 2〇〇μΐ range, from about 1 μm to about 100 μΐ, about! a range of μ1 to about 5〇μΐ, a range of about i μ! to about 2〇μ1, a range of about 2〇〇μ1 to about 〜, a range of about 5〇μΐ to about 500 μΐ, or about 5〇μι to about a range of 2 〇μΐ such that the chamber can have about 24, about 5 μ, about 1 〇, about 2 〇 4, about 5 〇 μl, about 100 μl, about 2 〇〇 μ, about 3 〇〇 μ ΐ, or about 5 〇〇 ^ volume. In various embodiments, the detection region may include a microelectrode array, which may include one or more pairs of electrodes, such as two pairs of electrodes, four pairs of electrodes, six pairs of electrodes, eight pairs of electrodes, twelve pairs of electrodes, sixteen pairs of electrodes Or twenty pairs of electrodes, and they may be arranged in a 2x1 array, a 1Χ4 array, a 2χ2 array, a 1χ6 array, a 2×3 array, a 3χ2 array, a 2χ4 array, a 4χ2 array, a j" array, a 4×3 array, a 2χ8 array, a 4χ4 array, a 4χ6ρ Train, or 3χ8 array. Each pair of electrodes may comprise an inner electrode and an outer electrode, the outer electrode having a substantially complementary shape to the periphery of the electrode. In each of the embodiments, one pole may be made of gold, or other metal or conductive material. For each implementation, the inner electrode can be a dish electrode, a shoe electrode. However, it should be understood that the inside is a triangle, a square, a rectangle, such as a rectangle or a diamond, and the outer electrode may have a complementary shape substantially surrounding the periphery of the corresponding inner electrode. The inner electrode can be a working electrode and the outer electrode can be a reference electrode. In various embodiments, the outer electrode can be connected (sh (med) to provide a relatively large surface area to increase sensitivity" while the inner electrode can be individually controlled for relatively high sensitivity impedance measurements, or - Shorting to generate a non-uniform electric field to induce dielectrophoresis (DEP). In various embodiments, specific capture molecules can be provided on and/or attached to the surface of the microelectrode array. Electrophoresis is a technique often used to separate microparticles, which is achieved by inducing a spatially varying non-uniform field (dielectrophoretic field) that produces a non-uniform polarized dipole to a neutral dielectric particle (for example including In the cell, thus, the dielectric force of the medium. The dielectric properties of the medium surrounding the particle can affect the dielectric ice force experienced by the particle. The more polarizable particles than the surrounding medium will experience the net force toward the high electric field region ( Positive DEP), and particles that are less polarizable than the surrounding medium will experience a net force (negative DEP) toward the low electric field region. In various embodiments, the electric field generated by dielectrophoretic capture may have an internal electrode. central The field is extremely small, thus directing the cells toward the capture molecules. In each embodiment, the generated dielectrophoretic field can be a negative dielectrophoretic field 'so that the target organism can be concentrated at the value of the minimum electric field occurring at the center of the inner electrode 26 201217781, Thus, the impedance detection sensitivity is enhanced without labeling the sample. In each of the embodiments, the electronic signal applied to the microelectrode array to induce the dielectrophoretic field may have a peak-to-peak range ranging from about 〇·1 v to about 20 V. Amplitude, for example about 0. A range of from 1 V to about 1 〇 v, a range of from about 1 v to about 5 v, about 0. 1 V to about 1. The range of 5 V, the range of about ι·5 V to about 1 〇 v, about 1. The range of 5 V to about 20 V makes the electronic signal have about 0. 1 V, about 0. Peak-to-peak amplitude of 5 V, about 1·0 ν, about 丨5 ν, about 5 V, about 10 V, or about 20 V. In various embodiments, the electronic signal applied to the microelectrode array to induce the dielectrophoretic field can have a frequency ranging from about 10 kHz to about 1 kHz, such as from about 1 kHz to about 5 kHz, about 10 kHz. Up to a range of about 1 〇 MHz, a range of about i 〇 kHz to about 1 MHz, a range of about 1 MHz to about 1 〇〇 MHz, a range of about ι 〇 m Hz to about 100 MHz, such that the frequency of the electronic signal can be about Work kHz, about 1 kHz, about 1 MHz, about 5 MHz, about 1 〇 MHz, about 20 MHz, about 50 MHz, or about 100 MHz. In various embodiments, the electronic signal applied to the microelectrode array to induce the dielectrophoretic field can have about 1. Peak-to-peak amplitude of 5 V and frequency of approximately 1 MHz. In various embodiments, in order to obtain high sensitivity in the detection area, relatively small sensing electrodes or microelectrode arrays may be arranged on the detection area. Each of the electrodes in each embodiment may have a size of about 1 〇〇 μπι. In various embodiments, the filter may be a filter paper, a fibrous screen, a polymeric filter, or a functional filter having a coated antibody or charge. In various embodiments, the filter may be made of parylene, polystyrene, polyethylene, polymethylmethacrylate (polymethylmethacrylate) or polyamethylene phthalocyanine 27 201217781 oxyalkylene (PDMS). In various embodiments, the filter can be a semipermeable membrane filter. It is well known that the filter can be a semi-permeable membrane of Sterlitech polycarbonate. In various embodiments, the filter may have a size ranging from about 2 mm to about 4 mm in diameter, such as from about 2 mm to about 3 mm, such that the diameter of the sheet may be about 2 mm, about 3 _, or About 4 _. The filter area of the filter can have substantially the same dimensions as the filter. In various embodiments, the filter may have a pore size ranging from about 3 μηη to about 50 μηη, for example, a force ranging from 3 μm to about 30 μηη, ranging from about 3 μm to about 20 μηη, from about 3 μηι to about 1 The range of 〇μιη, a range of about 5 丨 coffee to about 5 〇 μηη, a range of about 10 μηι to about 50 μηι, or a range of about 1 〇 pm to about 30 μηη, so as to provide a pore size of about 3 μm, about 5 μηι, about 8 μπι, about 1 μμΓη, about 15 μπι, about 2 〇 array, about 3 〇μηι, about 40 μηη, or about 50 μηι filter. "However, it should be understood that the organism to be filtered or allowed to pass The filter may have any pore size depending on the body or cell. In various embodiments, the microchannels of the microfluidic system that couples the first port to the chamber or the second port to the chamber may have a width ranging from about 5 μm to about 200 μm, such as about 50 μm to A range of about 1 μm to about 200 μm to about 200 μm, such that the channel can have a width of about 5 μm, about 1 μm, about 15 μm, or about 2 Å. In each embodiment, The microchannels can have a height ranging from 20 μm to about 1 μm, such as in the range of from about 50 μm to about 100 μηη, in the range of from about 2 μηη to about 50 μηη, or from about 2 μm μη to A range within about 30 μηη such that the microchannel can have a height of about 20 μΐη, about 4 〇μηι, about 6 〇μιη′ 28 201217781 about 80 μηι, or about ι〇〇μηι. In each of the examples, the flow rate or sample is filtered. And the flow rate of the fluid (eg, buffer solution) through the * sheet to retain the target organism in the filter and to filter other organisms may range from about 3 (microliters per minute) to about 600 μΐ/min 'eg, A range of from 3 μΐ/min to about 400 μΐ/ηήη, a range of from about 3 μΐ/min to about 200 μΐ/min, a range of from about 50 μΐ/min to about 600 μΐ/min, from about 5 μμΐ/min to about 400 The range of μΐ/min, the range of about 100 μΐ/min to about 200 μΐ/min, and the range of about 200 μΐ/min to about 4〇0 μΐ/min, so that the filtration flow rate can be about 3 μ1/ηιίη, about 10 Μΐ/min, about 30 μΐ/min, about 50 μΐ/min, 100 μΐ/min, about 200 μΐ/min, about 300 ΐ / min, about 400 μΐ / min, about 500 μΐ / min, or from about 600 μΐ / min. In various embodiments, the flow rate of the retentate or fluid (e.g., buffer solution) through the filter in the opposite direction relative to the filtration process to remove the organism retained at the filter can range from about 200 μΐ/min to about 1 Torr. The range of 〇μΐ/min, for example, in the range of about 200 μΐ/min to about 800 μΐ/min, or in the range of about 400 μΐ/min to about 800 μΐ/min, such that the reflux rate can be about 2 μμΐ/min. , about 400 μΐ/min, about 600 μΐ/min, about 800 pl/min, or about 1 000 μΐ/min. The flow rate of the rinsing rate or fluid (eg, buffer solution) through the first or second port to remove other non-specific organisms in various embodiments may range from about 15 μΐ/min to about 400 μΐ/ The range of min, for example, in the range of about 15 μΐ/min to about 300 μΐ/min, in the range of about 15 μ1/πιίη to about 2〇〇μΐ/min, in the range of about 50 μΙ/min to about 400 μΐ/miri, Approximately 29 201217781 ranges from 50 μΐ/min to about 300 μΐ/min, from about 100 μΐ/min to about 300 μl/ιηιη, or from about 1 μμΐ/min to about 200 μΐ/min. The rate can be about 15 μΐ/min, about 50 μΐ/min, about 1 μμΐ/min, about 200 μΐ/min, about 300 μΙ/min, or about 400 μΐ/min. In each embodiment, the 'target organism or specific organism can be cultured on the surface of the electrode array for a period of time ranging from about 5 minutes to about 6 minutes per hour, for example, from about 5 minutes to 40 minutes. , a range of about 5 minutes to 20 minutes, a range of about 5 minutes to 1 minute, a range of about 1 minute to 60 minutes, or a range of about 2 minutes to 6 minutes - so that the culture duration can be about 5 minutes , about 1 minute, about 2 minutes, about 30 minutes, about 40 minutes, about 50 minutes, or about 60 minutes. In various embodiments, a method for fabricating a microfluidic system to detect organisms in a sample volume is provided. The method can include providing a chamber configured to receive the sample volume, wherein the chamber can include a detection region to detect the organism; providing a first port in fluid communication with the chamber; Providing a second port in fluid communication with the chamber, the second port including a sheet, and wherein a fluid supplied to the first port or the second port is at the first port and the The chamber passes between the second ports. In various embodiments, 'providing a method for detecting an organism in a sample volume using a microfluidic system for detecting an organism in a sample volume, the microfluidic system comprising: a body configured to receive the sample volume a chamber, wherein the chamber can include a detection area to detect the living body; a first port in fluid communication with the cavity; and a second port in fluid communication with the chamber, the second The port includes a filter; and a fluid provided to the first or second port of 30 201217781 flows through the chamber between the first port and the second port. The method can include: providing the sample volume to the chamber; providing the fluid to the first port to pass the sample volume through the filter to retain the organism; removing the organism from the filter to the The detection area of the chamber; and detecting the organism. 1A through 1C are perspective views of a microfluidic system 100 in accordance with various embodiments. The microfluidic system 102 can be provided with or integrated with the microchip 104 to form an integrated microfluidic system 100 for preparing and detecting organisms in the sample volume, such as peripheral blood samples in a blood sample. Nuclear cells (PBMC). The microchip 1 〇4 can be a germanium microchip. The microfluidic system 102 can have a size of about 7 mm X 6 mm X 3 mm in various embodiments. In various embodiments, the microchip i 〇 4 may have about 1 melon X 1 mm X 0. Size of 75 mm. The microfluidic system 102 can include a pair of chambers 1A, 6a, 6b that are configured to receive a sample volume. The chambers 106a, 106b can be open chambers. In other words, in addition to other organisms, in particular the sample volume containing the organism to be detected is provided through the chambers 106a, 1bb to the microfluidic system 102 of the integrated microfluidic system 100. Each of the chambers 1〇6a, 1Ci6b may include individual debt measurement areas 1〇8a, 1〇8b (having individual capture surfaces) to detect organisms' the individual detection areas are located in individual chambers 1 〇 6a , 底部 6b at the bottom. In each embodiment, the detection regions 10a, 8b, 8b may include a microelectrode array (not shown). The surface may be a microelectrode array on the surface of the microelectrode array, wherein the detection regions 108a, 10b Capture face. The capture molecules can be set up and/or attached to capture the target organism for detection 31 201217781. The capture molecule can be specific to the target organism being detected. In various embodiments, each chamber 1 0 6 a, 1 0 6 b can be an open chamber. The microfluidic system 102 can further include a first port 1] and a second port 112, the first port 110 being connectable to the chambers 16a, 6b, such that the first port 110 can be coupled to the chamber 106a Each of the ports 6b is in fluid communication, and the second port 112 is connectable to the chambers 106a, 106b' such that the second port 112 is in fluid communication with each of the chambers 16a, 106b. The first port 110 and the second port 112 may be arranged substantially perpendicular to the capture surface of the detection regions 108a, 108b. In various embodiments, the microfluidic system 102 can further include a filter 114 disposed in the second port 112. The filter 114 can be integrated with the second port 112. Filter 114 can be a semipermeable membrane. In various embodiments, the filter 114 can be disposed horizontally in the second port 112 or, in other words, horizontally in the second port 112. The 'microchannels (not shown) may be arranged in various embodiments to couple the first port 11 0 to each of the chambers 1 〇 6a, j 06b. In various embodiments, a microchannel (not shown) may be provided to couple the second port U2 to each of the chambers 106a, 106b. In a further embodiment, more than one microchannel (such as two, three, or four microchannels) may be disposed to couple the first port 11 每一 to each of the chambers 〇6a, i 〇 6b, and More than one microchannel (such as two, three, or four microchannels) is provided to couple the second port 12'' to each of the chambers 16a, 6b, 6b. In each of the embodiments, the first port 11 〇, the chambers iOh'j 〇 6b, and the first port 112 are in fluid communication with each other such that the fluid supplied to the first port 110 32 201217781 or the first port 丨 12 is provided. (for example, a buffer solution) can be used in the first port and the second port. Between the 112 flows through the chambers l〇6a, l〇6b. According to this, the fluid supplied to the first port 110 can flow from the first port 110 to each of the chambers 16a, 106b, and through each of the chambers 1 to 1 to the second port 112. . Similarly, in the opposite direction, the body provided to the second port mountain may flow from the second port 112 to each of the chambers i〇6a, and through each of the chambers 106a, 1〇6b to the first port Ιι〇. In various embodiments, the integrated microfluidic system can further include a plurality of contact tips 116 formed therein or formed on the microchip. A plurality of contact pads 116 can be in electrical communication with the microelectrode array disposed in the detection regions 2 〇 8a, 〇 8b. A plurality of contact pads 116 are connectable to the microelectrode array through a plurality of electrical connectors (not shown). In various embodiments, the number of the plurality of contact pads 116 may correspond to the number of electrodes of the microelectrode array (not shown) at the detection area 1 〇 8 a, 1 〇 8 。. In various embodiments, the plurality of contact pads 116 can be made of gold, titanium, platinum, or other metal or conductive material. In the ic diagram, a metal hollow needle 118 is shown which is inserted into the second port ι 2 as an outlet. The metal hollow needle 118 is further connected to a tubing (not shown) to remove any sample. In various embodiments, the microfluidic system 1A can further include a removable magnetic element (not shown, such as a magnet) configured to provide or generate a magnetic field adjacent the detection regions 108a, 108b. The magnets can be positioned below the detection zones 108a, 108b. The generated magnetic field can assist in capturing magnetically labeled organisms at detection regions 108a, 108b. In various embodiments, the magnet can be a permanent magnet or an electromagnet. 33 201217781 In the embodiments, a third port or more ports may be provided in fluid communication with the chambers 106a, 106b to mitigate the filtration process by increasing the filtration area and fluid in and out. The third port or more ports may or may not have a pass. In a further embodiment, the filter 114 can be implemented in a vertical configuration at the second port 112, or in other words, vertically in the second port 112, which can affect the flow regime and provide certain advantages. In an alternative embodiment, only one chamber is provided. In a further embodiment, more than two chambers may be provided such that three, four, or five, or even more chambers may be provided. In various embodiments, the microfluidic system 102 can be made of polycarbonate, polyethylene, polymethacrylate (PMMA), or polybismethyl decane (pDMS). In a further embodiment, the microfluidic system 1〇2 can be made of metal, such as aluminum or stainless steel. The operation of the microfluidic system 1〇2 based on the detection of organisms using impedance measurements will now be described by way of example and not limitation. The microfluidic system 102 can be used to prepare and detect peripheral blood mononuclear cells (PBMC) in a blood sample. Preparing peripheral blood mononuclear cells (pBMc) may mean concentrating peripheral blood mononuclear cells (PBMC) or filtering or isolating peripheral blood mononuclear cells (PBMC) from other cells or organisms in the blood sample. Initially, a buffer solution can be provided directly to the chambers l〇6a, 106b prior to providing the blood sample. A buffer solution may be provided to fill the chamber "Μ, the entire volume of the cut, which may have a volume of about 4 μl. In an alternative embodiment, 34 201217781 may provide a buffer solution to fill half the volume of chambers 106a, 1〇6b Or fully provided to cover or overlay the microelectrode array. Impedance measurements can be taken as background signals for normalization purposes. The buffer solution in the chamber can then be removed. However, the chamber 1 is maintained. It is beneficial to dilute a volume of the buffer solution supplied to the chambers l6a, lG6b in a volume of 6a, 168b to relax the treatment at the detection area n 1 〇 8 b and reduce the satiety (4) # 红 血 血 血 血 血 血 血 血 血 血 血 血 血 血 血 血 血 血 血 血 血 血 血 血 血 血 血 血 血 血 血 血 血 血 血 血 血 血 血 血 血 血 血 血 血 血 血 血 血 血A volume of buffer solution in chambers 1〇6a, 1〇6b can be removed'. The volume can be about 2μ1, which is half of the volume of chamber 1〇6a, 1〇讣. The blood sample can then be supplied to the chamber 1〇 ( 5&, 1 〇. At this stage, blood The sample may contain various organisms other than pBMc to be treated, such as red blood cells, white blood cells, and other cells. A filtering procedure may then be performed. The sample may be extracted through the second port]12 (the sample is diluted with a buffer solution of blood) Sample), which is achieved, for example, by means of a pump attached to the second port 112 to discard the sample through the filter and the second port 112. The overall volume of the sample can be removed, or the chamber 106a can be removed, A sample of a volume within 6〇, for example about 2, at this stage, the PBMC can remain in the filter 114 while the red blood cells can be removed and discarded. Since the second port 112 is generally available for passage of the sample through the filter 114 After the sample is output, the second port 112 can also be defined as an outlet or a discharge port. The filter program can be executed several times, for example, two times, three times, 35 201217781 or four times to maximize the retention of the PBMC. And (iv) W ΐ 4 removes other organisms such as red blood cells. Additional buffer solution is provided for the mouth η. (At this time, the second pass 2 is filled to fill the chambers 106a, 1 () 6b, and the internal sample is further diluted. Subsequently Further filtering is performed by extracting the sample through the second pass α 112. A volume of the sample in the chambers 106a, 106b can be removed by the filter ι 4 and the second port 112 and discarded. In each embodiment, A port can be generally used as an input tool for a buffer solution, and the first port can also be defined as an inlet or an inlet port. In various embodiments, the organism can exist in the detection area during the filtering process. On the capture surface of l〇8a, l〇8b, these regions are located at the bottom of the chambers l〇6a, 1〇6b. Therefore, culture and dielectrophoresis (as part of the detection scheme) can be implemented during the filtration process. To capture the PBMC on the detection areas 1〇8a, l〇8b. At the end of the filtration procedure, most white blood cells or pBMC may remain in the filter 114 while most of the red blood cells are removed and discarded. In order to recover the portion of the PBMC from the cells remaining in the filter 114 and to cause capture and detection of the PBMC, the buffer solution is provided through the second port 112 and the first port 1 10 is closed, thus reversing the detection of the liquid flow. (compared to liquid flow during the filtration process). In other words, the buffer solution flowing through the second port 112 is recirculated or reversed, thereby removing the cells from the filter 114 and transferring the cells to the chambers 106a, 1 〇 6b so that the cells can be transferred to the chamber 106a, The detection area of 106b is 1〇8a, 1〇8b. After the buffer solution is refluxed and the cells are transferred to the chambers a6a and 丨〇6b, culture and/or dielectrophoresis and rinsing (as part of the detection protocol) may be performed at 36 201217781 to detect regions 108a, 108b (eg, The cells are concentrated at the microelectrode array of the detection regions 108a, 108b. During the cleaning procedure, fluid can be flowed through the first port 11 〇 and/or the second port 112 to remove non-specific organisms that are not captured on the surface of the microelectrode array. Then, the detection of the PBMC at the detection areas i 〇 8a, i 〇 8b of the chambers 〇 6a, i 〇 6b can be performed. PBMC can be detected using a non-marking method such as impedance measurement. In each of the embodiments, the reflow procedure can be repeated after the PBMC is captured in the detection areas 108a and 8b to increase the recovery performance. In various embodiments, the reflow procedure can be performed for a third time or a fourth time, each time after capturing Pbmc at the detection regions 108a, 108b. Accordingly, in various embodiments, a fluid such as a buffer bath provided to the first port 11〇 may be provided to flow through the chamber 丨〇6a, 〇6b and the filter 丨丨4, so that the living body is filtered. 114 is retained, and a fluid such as a buffer solution provided to the second port 112 may be provided with a flow filter 114 such that the organism is removed from the filter 114 to the chambers 106a, 106b for attachment through the microelectrode array. Capture of capture molecules on the surface. In various embodiments, it is also possible to implement a sequential batch processing scheme that allows different volumes of blood samples to be processed sequentially. After processing the program, the PBMc captured on the array of the electrode electrodes can be detected. Figure 2 shows a flow chart 2A illustrating a method for fabricating a microfluidic system in accordance with various embodiments. At 202, a chamber is provided for receiving a sample volume, wherein the chamber 37 201217781 includes a detection area for detecting the organism β. • At 204, a first port in fluid communication with the chamber is provided. • providing a second port comprising a filter and in fluid communication with the chamber at 206'; and wherein the fluid supplied to the first port or the port of the flute is at the first port and the second port Flow through the chamber. Manufacturing and Experimental Materials The fabrication of the microfluidic systems of the various embodiments will now be described by way of example and not limitation. Microfluidic System Design Figures 3 through 3D show schematic views of a microfluidic system 102 in accordance with one embodiment. The microfluidic system 1〇2 can include a bottom germanium microchip 104 comprising a microelectrode array 3 (for unmarked cross-sectional measurement). The microfluidic system 102 also includes a microfluidic chamber 6a, L〇6b, first port 110 and second port m (having an integrated microfilter semi-permeable membrane 114). Each of the pair of microfluidic chambers i 〇 6a, 106b may have a volume of about 4 μΐ. Membrane filter 114 can have a pore size of about 3 μηη. The microelectrode array 300 can be formed on or in the germanium microchip 1 4 or integrated on the germanium microchip 104 to form a microelectrode array (me A ) • wafer. Thus, the detection area of the microfluidic system 102 can be integrated into the micro-fluid.  On the wafer 104. Figure 3A shows a schematic bottom view of a microfluidic system 102 in accordance with various embodiments, for the purpose of illustration, a 32 201217781 microchip 104 comprising a microelectrode array 300 is removed. Fig. 3B is a schematic cross-sectional view showing the microfluidic system 1〇2 taken along the line A A of Fig. 3A. As shown in FIG. 3B, the microfluidic system 102 can include a plastic chamber structure 302 with two tape layers (tape Iayer) 304 and a bottom tape layer. According to FIGS. 3A and 3B, the microelectrode array 3 can be set. Covering the portion of the microchip 1G4 - this portion corresponds to the chamber inspection, the secret and the bottom surface of the plastic chamber 3〇2, as shown by the dashed box 3〇8 in Figure 3a. In various embodiments, the plastic chamber structure 302 can be attached to the microchip 104 via the two layers of adhesive tape 3〇4 and the bottom kneeband. In various embodiments, the two center belt layers and the bottom belt layer 306 can be double sided tape layers. 3C and 3D respectively show schematic top views of the tape layers 3〇4 and 306 according to the respective embodiments. The centering tape layer has just included openings 31a, 310b, each corresponding to the first port and the second port 112. Centering tape layer 3 (Μ进_舟句红 „ Progress includes opening 312, so that 302 and chamber 106a, 1〇, version to,. The microchip 104 is referred to as a mouth. ...contacting the microelectrode array 3. . In each of the embodiments, the bottom tape layer 3〇6 includes openings Μ, ·, corresponding to the first port 11 〇 and the second one, the sentence mouth 112. The bottom tape layer 306 advancement includes an opening 316 such that the plastic chamber 106a' 1 Of\h hh ±.  The surface of the chamber 302 is in contact with the surface of the chamber. The microelectrode array 300 can be second-passed aa. The sheets, or in other words, vertically arranged in the second port, perform filtering and provide certain advantages. Figure 3E shows a microflow of a root-to-flow regime. The schematic view of the body system 3 1 8 has a vertically disposed filter 320 » in addition to the filter configuration, the 3E circle The embodiment is similar to the embodiment of Figure 3B, including the microfluidic component or part. In the embodiment of Figure 3B, the filter 114 is implemented in a horizontal configuration in the second port 丨丨2. In the embodiment of Fig. 3E, the filter 32 is implemented in a vertical configuration in the second port 322. Therefore, the filter 32 is disposed parallel to the wall of the second port 322. This configuration is relatively less prone to blockages caused by 32 〇 drops on the filter. It will be appreciated that the microfluidic system 3 18 having the vertically disposed filter 32 可以 can be implemented in any suitable design for the application. It should be understood in the various embodiments that any number of centering tape layers 3〇4 and bottom tape layers 306 can be provided. As is known in the art, surface chemistry methods can be performed to attach a linker such as thiol to the microelectrode array 300. The microfluidic system 1 〇 2 including the plastic chamber structure 302 can then be mounted to the Shixi microchip 1 〇4. A capture molecule (such as an antibody) for specific cell capture can be attached via EDAC/NHS ( 1 - (3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride/N-hydroxy succinimide) coupled to a linker. The surface of the germanium microchip 1 〇4 not covered by the microelectrode array 300 can be deactivated by material, such as polyethylene glycol (PEG). Thus, there may be a coating layer comprising a linking agent, a capture molecule, and a repellency material. Subsequently, the microfluidic system 102 mounted on the microchip 1 〇 4 can be stored in the refrigerating compartment to preserve or maintain the functionality of the coating. A buffer solution can be provided in the microfluidic system 丨02 to protect the coating layer. In alternative embodiments, other storage means known in the art 40 201217781 may be used, such as dry, cold, or vacuum storage. The use of the repellency material on the surface of the device not covered by the microelectrode array 300 reduces the non-specific attachment of the organism and adds specific detection of the target organism, which is quite helpful when the number of target organisms is relatively small. The figure shows the technical diagram of the plastic chamber structure according to the embodiment. Figure 4 shows the top, bottom and cross-sectional views of the plastic chamber 3〇2, while the dimensions of the plastic chamber structure 3〇2 are mm—). Figure 5A shows a top view of a microchip 5 according to one embodiment, which may be provided in the microfluidic system of various embodiments. The microchip 500 can include a microelectrode array 502 and a plurality of electrical interconnects, such as 5〇4a, 5〇4b 504c, 504d, 504e' for connection to a plurality of contact pads 5〇6. A plurality of contact pads 506 can be coupled to a printed circuit board (pcB) for electrically controlling the microelectrode array 502 and processing. The microchip 5 can be a germanium microchip. The use of PCB-based signal processing reduces the processing and detection time of the organism to less than one hour, which allows the microfluidic system of each embodiment to be used in the focus care detection (p〇C) setting. For diagnosis and surveillance of acute and chronic diseases such as heart disease and progression of atherosclerosis. Fig. 5B shows a top view of the microelectrode array 5〇2 according to one embodiment provided on the microchip 500 of Fig. 5A. As shown in Fig. 5E; the microelectrode array 502 includes 24 pairs of electrodes arranged in a 4x6 array. The number of 'multiple contact pads 506' in each embodiment may correspond to the number of electrode pairs. In various embodiments, each pair of electrodes can be identified by each of its column numbers (as indicated on the left side of Figure 5B) and each row number (as indicated by the 201217781 indicator at the top of Figure 5B). The pair of 24 pairs of electrode wipes can have internal electrodes (for example, 5_, 5_, 508 (;, 5〇8〇 and external electrodes (for example, 5 hearts, ·, 51〇c, 51〇d) Each of the embodiments t, such as 鸠, 卜, 5, the inner electrode of the side may be a working electrode and is r-shaped, and the electrodes may be A, for example, 5 〇 a, 5 j 〇 b, 510c, 510d |+ kg is a reference electrode and is shaped like a horseshoe that substantially surrounds the inner electrodes (eg, 508a, 5〇8b, MSb, 508c, 5〇8d). External electrodes (eg 51〇a, 510b, 51〇uuc, 51) 〇d) may be short-circuited or electrically connected to each other to act together as a tape n to collect the electrodes with horses, while providing a relatively large surface area to increase sensitivity, while internal electrodes (eg, 508a, 508b, 508c, 5〇) 8d) may be individually controlled 'for relatively highly sensitive impedance measurements, or - shorted or in electrical communication with each other to act as a collecting electrode to generate a spatially non-uniform electric field to induce dielectrophoresis (DEp), thus Causing dielectrophoretic force. Figure 6A shows a simulated plot 600 of a non-uniform electric field generated by a microelectrode array according to various embodiments. The non-uniform electric field causes a dielectrophoretic field. As shown in Fig. 6A, the generated electric field has a minimum value of the electric field occurring at the inner electrode of the inner electrode, which can guide and concentrate the target cell when used for negative dielectrophoresis (DEp). The center of the inner electrode is for impedance measurement. Fig. 6B shows an optical microscope image 602 of a microelectrode array with cells according to various embodiments, the peak-to-peak amplitude of the applied electronic signal is about 1. 5V, and the frequency is about 1MHz. Fig. 6B shows that a plurality of cells 604 due to negative DEp are concentrated in the center of the inner electrodes 6〇6 of the microelectrode array 6〇2. Referring to Figure 5, in various embodiments, the inner electrode 42 201217781 of the first column of electrode pairs can connect a plurality of contact pads 5 〇 6 through interconnects 504a, 504e. The inner electrodes of the second and third columns of the electrode pair may be connected to a plurality of contact pads 506 through interconnections 504b, 504d. The inner electrodes of the fourth column of electrode pairs can be connected to a plurality of contact ports 506 via interconnects 504c. The outer electrodes (which are shorted and in electrical communication with one another) can connect a plurality of contact pads 506 through interconnects 504b, 504d. In various embodiments, surface chemistry can be performed to functionalize the microelectrode array 502 with a linker such as a thiol. Internal electrodes (eg, 508a, 508b, 508c, 508d) and external electrodes (eg, 51〇a, 51〇b, 51〇c, 51〇d). Capture molecules (such as antibodies) for specific cell capture can be attached via EDAC/NHS coupled to a linker. The surface of the ruthenium microchip 104 (which may be ruthenium oxide) that is not covered by the microelectrode array 3 can be cell passivated, such as polyethylene glycol (PEG). Embodiments will now be described by way of the following illustrative but non-limiting examples.

Jurkat cells were used to aid in the development and testing of the microfluidic systems of the various examples, using a sample solution of cultured cells (such as Jurkat cells of T lymphocytes) having the same concentration as PBMC in the blood (ie, about 6 X). 1 〇 6 cells/ml (cells per ml)), and the volume is sufficient to saturate the detection area, for example, about 2 ΐ cells in about 2 μΐ. The cells were provided to the microfluidic systems of the various examples using the procedures of the various embodiments previously described and filtered at different flow rates and backflushed or refluxed at different flow rates of 43 201217781. The cells are then cultured in DEP to establish surface coverage of the microelectrode array. Figures 7A through 7C show optical microscopy images of microelectrode arrays with cells according to various embodiments. Optical microscope images are obtained through the Olympus BX51 Upright High Power Microscope. Figure 7A shows an optical microscopy image of cells 700 on a microelectrode array 702 after filtration at a flow rate of about 400 μΐ/min (i.e., over flow rate) and incubation with DEP for about 5 minutes. Figure 7B shows an optical microscopy image of cells 704 on the microelectrode array 702 after subsequent reflux at a flow rate of about 800 μΐ/min (i.e., reflux flow rate) and incubation with DEP for about 5 minutes. Figure 7C shows an optical microscopy image of cells 706 on microelectrode array 702 after refluxing at a flow rate of about 800 μΐ/min and culturing with DEP for about 16 minutes. As can be seen from Figure 7A, after the filtration procedure, cells 700 are present on the surface of the microelectrode array 702, which means that not all cells move toward the semipermeable membrane filter and pass through the semipermeable membrane filter, and thus can be in DEP Settling during cultivation. In various embodiments, a multi-pass filtration procedure can be performed to allow a sample (which can be a blood sample) to flow toward the semipermeable membrane filter. In addition, reflux performance is determined by staining the cells on a filter membrane using a fluorescent dye (e.g., Calcein AM from Invitrogen), which stains the living cells and produces green fluorescence. 7D and 7E, respectively, shows a fluorescent microscope image of the cells 708 remaining at the semipermeable membrane filter 710 and the cells 712 in the waste after filtration according to various embodiments. Fluorescence microscopy images were obtained with an Olympus BX61 Upright Fluorescent Microscope with FITC (fluorescent isothiocyanate) filter 44 201217781. The estimated number of cells 708 remaining in the semipermeable membrane filter is about 20 1 cells. It will be appreciated that other conventional methods can be performed to establish the number of dead cells and the number of cells passing through the filter. The number of cells (about 2 cells) left on the membrane filter is relatively large, and may affect the detection of rare cells, which may be present in relatively low numbers, such as below one cell. Furthermore, as shown in Figures 7 and 7C, after reflow, the cells 7〇4, 7〇6 can be affected by the reflux rate and can be cultured on one side of the microelectrode array 7〇2, rather than substantially uniformly The cells were cultured on the surface of the microelectrode array 7〇2. Fig. 8 shows an optical microscope image of cells which were cultured without DEP and filtered on a microelectrode array 8 以 at a flow rate of about 2 〇〇 μ1/ιηίη. Fig. 8 shows an optical micromirror image of cells which were subsequently refluxed at a flow rate of about 600 μl/ιηίη and cultured on the surface of the microelectrode array 8〇0 after about 10 minutes of incubation. Fig. 8C shows a close-up cross-sectional view of Fig. 8B showing that a relatively large number of cells are present on the microelectrode array 8〇〇, which may affect the detection efficiency of specific cells for the microfluidic system of each embodiment. Thus, in various embodiments, it is not necessary to provide a filtered flow rate and a reflow rate such that a substantially optimal level of cells can be provided on the microelectrode array for detection. Figure 9 shows a graph 900 of sample preparation performance in accordance with various embodiments. Graph 900 is based on the number of cells on the filter (as a percentage of 12〇〇〇 45 201217781 cells input) 902 from the blood sample preparation and filtration pBMc and at a relative reflux rate of 9〇4. According to various embodiments, the number of cells on the filter refers to the number of cells remaining on the filter after filtration and reflux treatment. Circular data points (represented by 906) were obtained with a flow rate of approximately 2 〇〇 pi/min, and square data points (represented by 908) were obtained with a flow rate of approximately 400 μΐ/min. . Figure 10A shows a flow rate transition of about 200 μΐ/min and an optical micrograph of PBMC 1000 on the surface of the microelectrode array 1002 after about 5 minutes of dep incubation. Fig. 10B shows an optical micrograph of PBMC 10 04 on the surface of the microelectrode array 1 〇〇6 after reflux at a flow rate of about 600 μΐ/min and after incubation with DEP for about 10 minutes. As shown in Fig. 10B, the cells 1 〇〇 4 after the reflow were substantially uniformly cultured on the surface of the microelectrode array 1006. Preparation of doped ik solution Approximately 750 CD34 cells (or endothelial progenitor cells, EPC) were doped into approximately 1 μΐ of blood and CD34 cells were provided to the microfluidic system of each example', as previously described The process of each embodiment is processed. In addition, a pure blood sample is also prepared and processed according to the various embodiments previously described. CD34+ cell specific expression was captured on the microelectrode array using negative dielectric electrophoresis (DEP), specific antibody recognition, and microfluidic methods for washing non-specific cells. Figure 11 shows an optical microscopy image of a microelectrode array with PBMC from a pure blood sample after purification of PbmC, the purification involving a transition at a flow rate of about 50 μΐ/min for about 2 minutes and a flow of about 600 μΙ/min 46 201217781 rate The one-person return machine (_ PBMc is transferred into the open chamber), and the 11B chart shows that the flow rate of the spoon 5〇μι/ηιίη is performed for about 2 minutes for further rinsing procedures 彳i and 丨丨Α An optical microscope image of the same microelectrode array. Figure 11C shows an optical microscopy image of a microelectrode array of cells with a white blood sample, and a flow of about 50 μl/ιηίη with the r 1 and 6 β丄 brothers. Rate filtration, about 2 minutes and 6 Λ η 丨 / . X 4 600 pl / min flow rate to perform a secondary reflux (transfer the cells into the beginning of swelling, Ί enemy L to) ' and the 11D circle shows An optical microscope image of the same microelectrode array as in Fig. 11C after performing a 2 minute feed rinsing procedure at a flow rate of 50 μl/min. Although a relatively large number of cells are present after grasping the order (i i Α and i (1)), after rinsing the private sequence, most of the cells are removed, leaving cD34+ cells on the surface of the microelectrode array. A separate sample of CD34-cells and CD34+ cells doped in about 1 μl of blood is taken from the microfluidic system of each of the fine examples, and according to the procedures of the various embodiments described above Handle it. Cell capture was performed using a specific capture molecule for CD34+ cells using 贞 guess. Figure 1 and Figure 12 show the optical display of the microelectrode array 1200 before and after the rinsing procedure with a sample containing cd34_ cells at a flow rate of about 400 μm/ηιίη for 2 minutes. J兀* pre-nitrogen microscopy image. Fig. 12C and Fig. 12D show optical microscope images of a ruthenium electrode array 1202 having a sample containing CD34 、 and performing a rinsing procedure on the order of about 400 μΐ/min for about 2 minutes. As shown in Figure 12 and Figure 12C, there is a T's private sequence pair. A relatively large number of cells of 201217781 appear on the microelectrode arrays 1 200, 1 202. After the flushing procedure, most cells including CD34-cells are from the microelectrode array ι2. 〇〇 离 (as shown in Figure 12B), while CD34 + cells remain on the microelectrode array 12 〇 2 (as shown in Figure 12D) 'This is due to the capture molecules through the microelectrode array 12 〇〇, 1202 Specific capture of CD34+ cells. Doped blood with integrated impedance detection using the above described embodiments for preparing and processing a doped blood sample 'executable impedance measurement or impedance spectroscopy to detect capture on a microelectrode array Cell. Cells are detected by impedance spectroscopy in a batch mode of operation, which is capable of detecting relatively small amounts of cells (<1 cells) with relatively significant clinically significant cuts (cut_〇ff) iiL Approximately 0.5% EPC has better sensitivity (Hill jm, "Circulating Endothelial Progenitor Cells, Vascular Function, and Cardiovascular Risk" 'N. Engl. J, Med. '2003, 348(7), 593). Figure 13A shows a system 1300 for measuring impedance in accordance with various embodiments. System 13A can be an integrated Pcb-based electronic system for detecting and measuring impedance based on 24 pairs of electrodes of a microelectrode array of microfluidic systems 13〇4. System 13A includes a pCB based measurement system 1302' which includes 24 channels corresponding to 24 pairs of electrodes. The PCB based measurement system 1 302 can be connected to a computer and controlled using software such as Labview. System 1300 further includes pumps 1308a, 1308b, each coupled to a first port and a second port of microfluidic system 1304. Pumps n〇8a, 13 48 48 201217781 are used to supply a buffer solution to the microfluidic system 丨 304 and move the buffer solution along the microchannels of the fluid and the chamber of the microfluidic system 1 304. Figure 13B shows a schematic diagram illustrating the PCB-based interconnection of the embodiment of Figure 13A. The PCB-based measurement system 1302 is coupled to the (fpga) board 1306 via an electrical interconnect 1310. The FPGA board 1306 can include a connector 1312' that is adapted to mate with the connector 1314 of the Pcb card (subcard) 1316. The PCB card 1316 further includes a connector 1318 for connecting the microelectrode array of the microfluidic system 1 304 (Fig. 13A). For detection of a target organism or cell (e. g., CD34 cells), impedance measurements are initially recorded in the presence of a buffer solution but lacking organisms or cells to measure background values. Next, after filtration, reflux, and rinsing procedures in various embodiments, the organisms in most of the samples are removed, leaving the target cells on the surface of the microelectrode array. Impedance measurements were then recorded and the changes in impedance were summed over all of the electrodes of the microelectrode array. Figure 13C shows a graph U2 of the impedance measurement, the pBS solution containing cells for the channels of the system of the Figure 13A example. For comparison, a circle 1322 of impedance measurements of the system according to conventional settings is also shown. Figures 1320, 1322 show relatively good correlations for different systems for the channel. The dotted line indicated by 1324 indicates the frequency at which the impedance change or sensitivity is the highest. Figure 14 shows an impedance measurement chart 1400 for some samples in accordance with various embodiments. Graph 1400 is displayed as a percentage change in impedance "" versus frequency (in logarithmic scale) 1404. Figure 14 shows the results of cell-free phosphate buffered saline (pBs) buffer solution 49 201217781 (as represented by data point 1406), and the results of cell-free pre-cleaning with pBs (represented by circle data point) And the results of a sample of cells after the rinsing protocol according to various embodiments (as represented by triangle data point 1410). At the data point 14〇6, the percentage change in impedance observed on the work is just low, because there is only PBs and no cells are added, so it is used as a background measurement. Figure 14 shows that cells are specifically attached to the electrodes, causing an increase in impedance realities, peak 1412 being at about 380 kHz (according to a logarithmic scale, corresponding to a value of about 5.58). The frequency of peak 1412 indicates the frequency of the highest sensitivity observed for the cells' and thus serves as the basis for detecting the frequency of such cells during the measurement. Figure 15 shows a graph 1500 for impedance measurements of some samples in accordance with various embodiments. Graph 1500 is obtained after chemugination and specific cell capture, and is shown by the impedance change 丨5〇2 to the percentage of CD34 cells doped in the sample of 15〇4. Figure 15 is based on performing impedance measurements or spectroscopy at a frequency of approximately 380 kHz. The detection time for this sample is approximately 20 minutes per batch. Figure 15 shows the result of a negative control of 15 〇 6 sample, which is a PBS buffer solution with a volume of approximately 4 μΐ, without cells. The measurement for the negative control ι5〇6 indicates the background signal of the corresponding buffer solution, and this measurement is removed from the measurement results of other cell-like samples, so that the measurement results represent the changes induced by the cells in each sample' and the buffer is excluded. The effect of dissolving Wen. Figure 15 further shows the following results: about 1 μ1 of blood is doped with about 20% of CD34 (or EPC) sample 1508, PBS buffer solution is mixed 50 201217781 about 330/〇 of CD34 cells of about 4 μΐ sample 151〇 (about 990,000 CD34 cells per ml in pBs), about 4 μM of about 8 μ% of CD34 cells in PBS buffer solution, 1S12 (about 240,000 CD34 cells per ml), and PBS buffer The solution was doped with about 4 μl of a sample of about 14 μl of CD34 cells, 1514 (about 340,000 CD34 cells per ml in PBS). Figure 15 shows that the observed signal correlates with the number of cells present in the sample. Figure 16 shows an impedance measurement chart 1 600 for some samples in accordance with various embodiments. Figure 1 6 is obtained after cell purification and specific cell capture, and is shown as a total change in impedance of 22 pairs of electrodes (%) 丨 6 〇 2 for sample type 1604. Graph 1600 is obtained based on impedance measurements or spectroscopy performed at a frequency of about 3 80 kHz. The detection time for the sample is approximately 20 minutes per batch. Figure 16 shows the following results: cell-free PBS buffer solution sample 1606, Jurkat cell pure sample 1 608 (Jurkat cells are 3 million cells per ml, approximately 4 μM), with approximately 1% CD34 + cells Sample 1610 (approximately 4 μΐ of sample, taken from approximately 5 μΐ of CD34+ cells (113,000 cells per ml) and approximately 10 μΐ of jurlcat cells (4.44 million cells per ml)), approximately 5% Sample U12 of CD34+ cells (approximately 4 μΐ of sample, taken from approximately 5 μΐ of CD34 + cells (563,000 cells per ml) and approximately 1 μ〇 of jurkat cells (4.22 million cells per ml)) and Pure sample 1614 of CD34+ cells (CD34+ cells are 300 cells per ml, 4 μΐ). Figure 17 shows a chart 1 700 for batch processing impedance measurements of some samples 51 201217781, in accordance with various embodiments. Graph 1 700 was obtained after cell purification and specific cell capture, and is shown as a total impedance change (%) 1702 versus sample type 1704 for 22 pairs of electrodes. Graph 1700 is based on impedance measurements or spectroscopy performed at a frequency of about 3 80 kHz. Figure 17 shows the results of sample 1706a (batch 1) of about 24,000 PBMC cells and the result of sample 1708a of about 24,000 PBMC cells doped with about 240 CD34+ cells (batch 1). Figure 17 further shows that when approximately 24,000 PBMC cells or approximately 24,000 PBMC cells doped with approximately 240 CD34 + cell batch samples are correspondingly supplied to the Batch 1 sample, the total impedance change is increased. Sample 1706b (batch 2), such as approximately 24,000 PBMC cells, and sample 1708b (batch 2) of approximately 24000 PBMC cells doped with approximately 240 CD34+ cells. The results of the third batch treatment showed sample 1706c (batch 3) of approximately 24,000 PBMC cells and sample 1708c (batch 3) of approximately 24000 PBMC cells doped with approximately 240 CD34+ cells. Figure 17 shows that about 720 CD34+ cells doped in the PBMC solution can be detected. The detection time for the three batches is approximately 90 minutes. For Figure 17, each sample of approximately 24,000 PBMC cells contained approximately 4 μΐ of 6 million cells/ml (per liters per cell) of PBMC, while approximately 240 CD34 + cells were dosed with approximately 24,000 PBMC cells. Each sample contained approximately 4 μΐ of sample taken from approximately 5 μΐ of CD3 4 + cells (113,000 cells per ml) and approximately 10 μΐ of PBMC cells (8.88 million cells per ml). 52 201217781 Selective cell solubilization and speculation A magnetic bead of 1 μm in size and pre-coated with an anti-CD34 antibody was prepared and diluted in a dissolution buffer at an appropriate concentration. The prepared magnetic beads-containing melting buffer solution has a volume of about 4 μ1 and contains a final concentration of about 150 mM of NH4C1, 1 〇 mM NaHC 〇 3 , ed 福 ed edta and about 2000 magnetic beads in a ratio of 2 beads per i cell, suitable for 50-1000 CD34 + cells. The blood sample was then mixed with the dissolution buffer solution to form a mixture having a volume ratio of 4:1 of the dissolution buffer liquid to the blood, and then a mixed solution of about 10 μM was prepared. The mixture was incubated for about 丨〇 minutes. The mixture solution in each of the examples can be prepared in the range of about 5 to 1 μl. The microfluidic system (e.g., the embodiment in Figure 1) is then used for filtering and detection. Approximately one minute of pBS buffer solution is provided at a flow rate of about 30 μΐ/min to perform preloading and initiation of the chamber of the microfluidic system. The chamber was then maintained half filled with PBS solution. Approximately 5 μΐ of the prepared 1 μ μl mixture solution was loaded directly into the chamber or through the inlet port. The filtration and reflow procedures according to various embodiments are then performed. A PBS solution having a filtration flow rate of about 3 μΐ/min is supplied through the inlet port (ie, the first port) for about 6 minutes to remove non-target organisms or cells, such as RBC. At the end of the filter program, the chamber is half filled and held for about 5 minutes. Most of the Epc remains in the ^, while most of the red blood cells and other cells are removed to be discarded through the outlet port (ie, the second port). In order to recover the EPC remaining in the filter and to capture and detect Epc, 53 201217781 In the reflow procedure, the pBS solution is supplied through the outlet port to push out the sputum. The cells on the 'throws' are removed from the tablets and the cells are transferred. The area to be tested in the chamber, chamber or chamber. The PBS solution was supplied through the outlet port at a reflux rate of about 600 μl/πιηη until the chamber was completely filled. The transition procedure was repeated by flowing the pBS solution through the inlet pass and the scoop 2 at a flow rate of about 3μ1/ηιίη. At the end of the filter program, the chamber is half filled. The filtration procedure was repeated by flowing the PBS solution through the outlet port at a reflux flow rate of about 6 〇〇μ1/ΐΠίη until the chamber was completely filled. It should be appreciated that the filter can be executed any number of times to maximize the residence of the Epc and remove other organisms through the filter, and the reflow procedure can be performed any number of times to increase the recovery performance. Samples with EPC were then incubated in the chamber for approximately 15 minutes. The EPC can be captured by a specific cell capture method based on antigen recognition based on antigen-antibody recognition in the defect region of the chamber and on the microelectrode array. In order to enhance the capture efficiency of the EPC on the microelectrode array, a movable array of magnets (provided to provide or generate a magnetic field) is placed adjacent to the microelectrode array. The resulting magnetic field assists in capturing the magnetically labeled Epc on the microelectrode array. The magnet is then removed and the flushing procedure is performed at a flush rate of about i 5 μi / m i n for about 3 minutes' which is achieved by passing the pBS solution through the outlet port into the chamber and exiting the inlet port. Next, the preparation of the Epc can be performed using the impedance measurement method according to each embodiment. Figure 18 shows a schematic cross-sectional view illustrating selective capture of skin precursor cells (EPC) 1800 according to various embodiments. Selective capture of Epc 18〇〇 occurs by binding to a CD34 antigen (eg, 1802) on Epc 18〇〇 with a CD34 antibody (eg, 18〇4) deposited or coated on a gold (Au) electrode 1 806 The upper Epc is further coupled to a magnetic bead (eg, 18〇8) to which the antibody is attached. As shown in Figure 8, gold electrode 1 806 and microchip! 8 i 〇Set up or integrate. The microchip j 8 i 〇 can be a Si/Si 〇 2 microchip. The remainder of the microchip 181 that is not covered by the electrodes 18 〇 6 can be stripped of material 1 8 12 (e.g., polyethylene glycol (Peg)) passivation. The microfluidic system is provided with a movable array of magnets 丨8丨4 (e.g., permanent magnets) located adjacent the electrodes 1806 to facilitate selective magnetic capture of the EPC 1800. As shown in Figure 18, the white blood cells are not captured by the α)34 antibody 1804. Compared to the non-magnetic capture mode, the use of immunomagnetic force can enhance the average EPC capture performance by about 80%-1%. Further, the capture force can be enhanced by near-field flow and/or oscillating flow. In various embodiments, near field flow refers to the flow of the sample such that magnetically labeled cells can be provided as close as possible to the magnets in the vicinity of the microelectrode array to enhance capture of the magnetically labeled cells. This can be achieved, for example, by reducing the height of the open chamber. In each embodiment, 'oscillation flow refers to repeated sample flow, or reciprocating through the microelectrode array or detection region, for example, by performing the filtration and reflow procedures of the various embodiments, to enhance the magnetically labeled cells captured in the micro-electrode. Probability on the electrode array. The embodiments described herein can be used for multiple indicator separations. For example, a first antibody can be coupled to a target cell and provided for magnetic capture, while a second antibody specific for the target cell can be functionalized on the electrode surface. After removal of other non-target cells (eg, after the rinsing procedure), only specific cells coupled to the first and second antibodies will remain on the electrodes. In various embodiments, multiple marker separation can be performed based on different markers CD34 and CI) 133. The effect of NHW1 on RBC and CD34+ cells in the lysis buffer solution was examined. On average, about 2.2 million cells per ml of CD34+ cells were mixed with the lysis buffer solution and cultured for about 丨〇, 2 〇, or 3 〇 minutes, or without culture. CD34+ cells cultured in PBS solution were used for control measurements. After the incubation, CD34+ cells were diluted and counted using a hemocytometer. Fig. 19 shows a graph of cell counts of 19 〇〇, and is shown by the number of cells 1902 versus the culture time of 19 〇 4. The first! 9 _ shows that for 丄〇 min and 20 min incubation time, no substantial loss of CD34+ cells was observed in the solubilization buffer solution. However, when the culture time was 3 minutes, the cell viability of CD34+ decreased by about 5%, indicating that the time for culturing with the lysis buffer solution was 2 minutes. In individual measurements (no results shown), whole blood samples with RBC were incubated in a lysis buffer for about 10 minutes, and the results showed that about 99.9% of RB C was dissolved. Comparative measurements were performed by applying about 3000 magnetic beads to a range of blood-doped CD34+ cells (approximately 1000, 2000, and 3 cells) to determine no magnetic compared to the corresponding number The improvement of capture efficiency of the bead CD34+ cells using immunomagnetic separation of magnetic beads. Figure 20A shows a graph of cell technology 2〇〇〇 and it is shown in the blood sample doped epc 56 201217781 20〇4 according to the number of EPCs on the electrodes 2002. This result shows an increase in the number of CD34 + cells captured according to the immunomagnetic method, and an average capture efficiency improvement of about 800/0-1 〇〇〇 / compared to the non-magnetically labeled CD34 + cells. . Figure 20B (left image) shows the optical microscope image of the microelectrode array 2〇〇6, its EPC is non-magnetic & and the second 〇B diagram (right image) shows the optical microscopy image of the microelectrode array 2008 The EPC is magnetically marked. Figure 20B shows that a substantially higher number of magnetic labels Epc are captured on the microelectrode array. Also, it can be observed that the EPC is primarily captured in the gold microelectrode region, rather than the microchip surface, thus suggesting minimal signal loss for subsequent impedance measurements. Fig. 21 shows a graph 2100' of the cell count on the electrodes before and after the washing procedure and is shown by the EPC 2102 on the electrode for the culture time 2丨〇4. Culture time 2104 refers to the time provided for attaching the magnetic beads to the Epc. Approximately 2000 magnetic beads were applied to the doped CD34+ cells in the blood, and blood samples were then provided to the microfluidic systems of the various examples for processing. The movably arranged magnets are used to assist in capturing CD34+ cells on the electrodes (e.g., microelectrode arrays), and then the magnetic beads are removed prior to cleaning. The results show that many magnetically labeled EPCs can be washed away by a cleaning procedure. The results also indicate that for a culture time of 10 minutes and 2 minutes, the residence time of the EPC on the microelectrode (ie, the number of cells remaining on the microelectrode array) is extremely small, indicating that the culture time of about 10 minutes is enough. Performing comparative measurements by applying about 2000 magnetic beads to a range of blood-doped CD34+ cells (approximately simplifications, ·, and 3 细胞 cells) to obtain magnetism after removal of the magnet and after final cleaning The retention rate of CD34+ cells of the standard 201257 201217781 (compared to the corresponding number of non-magnetic beads of CD34+ cells). Table 1 shows the retention of EPC on the microelectrode array at different concentrations of Epc with magnetic beads and Epc without magnetic beads. This result shows that, in the absence of magnetic beads, about 50% of CD34+ cells are washed away during the final washing step. However, in the case of magnetic beads, when the cell-to-magnetic ratio is 1:1 and 1:2, respectively, the respective retention ratios are improved to approximately 72.1 / 〇 and 8 0.8 / 〇. The results of 5H indicate that the number of preferred beads is 2〇〇〇 for the concentration of cd34+ cells in a single cell (pBMc based on about 0.1 μM/〇_1% of pBMc in 20 μΐ blood). Magnetic beads. Table 1: Retention rate of EPC Retention rate EPC number No magnetic beads Magnetic beads 1000 57.3% 80.8% 2000 57.6% 72.1% 3000 50.7% 50.3% Perform full range characterization to determine the detection limits of the various examples. About 5 〇-i 〇〇 (m solid blood doping & CD34+ cells were cultured and filtered using the procedures of the respective examples, and the final cell number was examined by counting CD34+ cells on the gold electrode region using a microscope. The graph 2200' showing the filtration and capture performance is shown by the number of cells captured on the electrode 22〇2 for the input cell count 22〇4, while the 22B shows the linear graph and linear fit of the transition and capture performance results of the map The results shown in /, 22B indicate that the detection limit can be about a total of 58 201217781 heterozygous CD34 + cells (about ο"% pbmc), which has a good linear relationship with the full range of capture performance (〆 = 〇 96). The capture efficiency is about 15-20 ° / 〇 (for example, can capture about 150-200 cells per 1 input cell) also perform comparative measurements 'to obtain magnetically labeled CD34 + cells for Capturing performance of multiple batches of treatment (compared to the corresponding number of non-magnetic beads of CD34+ cells). Table 2 shows Epc capture efficiency of EPC and non-magnetic beads of different batches of magnetic beads on the microelectrode array. For measurement, every The batch EPC contains about 1000 EPCs. The results show that after performing the filtration procedure according to each example for the first 1000 EPCs, for the EPC without magnetic beads, 62 cells out of 1 cell were Captured on the microelectrode array, and for Epc with magnetic beads, 丨53 cells were captured. Then, add about 1 E Epc of the second batch, followed by a filtering procedure. The result shows no magnetic The Epc of the bead and the Epc with the magnetic bead were captured to 68 £1> and 214 Epc. The third batch of approximately 1000 EPCs was added, followed by a filtering procedure. The results showed that the EPC without the magnetic beads had The EPCs of the magnetic beads were each captured with 71 EPOs involving three batches, totaling about 3 Ερ (: after the filter program, the reflow procedure according to each embodiment was imposed to retain the filter The EPC was transferred to the microelectrode array. The results showed that EPC without magnetic beads and EPC with magnetic beads were captured to 78 Epcs and 35: Epcs. Magnetic beads Table 2: Number of EPCs processed in multiple batches Magnetic Beads 59 201217781 First 62 153 Second 68 214 Third 71 310 Reflow 78 355 23A to 23D An optical microscopy image of the purified microelectrode array with cd34 cells is shown. The initial sample of the dissolution buffer solution of each example contains about 1000 CD34 cells, 2 magnetic beads and 3500 RBCs in a volume of about 5 μl. Purification and extraction of CD34 involves filtration at a flow rate of about 3 μl/ηύη for about 6 minutes and incubation for about 5 minutes. Then two reflow processes are performed, the flow rate is about 6〇〇μ1/ιηίη, and the EPC is transferred to The chamber was opened and cultured for about 5 minutes. The cleaning procedure was then performed at a flow rate of approximately 15 μΐ/min for approximately 3 minutes. Fig. 23 and Fig. 23 show optical microscope images of the microelectrode array with CD34 cells before the cleaning procedure, and Fig. 23C and Fig. 23D show optical microscope images of the microelectrode array with CD34 cells after the cleaning procedure. While the invention has been particularly shown and described with reference to the specific embodiments of the present invention, it will be understood And category. The scope of the invention is therefore intended to be limited by the scope of the appended claims. 60 201217781 [Simple description of the drawings] In the drawings, like reference numerals generally refer to the same parts throughout the different views. The drawings are not necessarily to scale and, in the alternative, are generally exaggerated to illustrate the principles of the invention. In the specification, embodiments of the invention are described with reference to the following drawings, wherein: Figures 1A through 1C show perspective views of a microfluidic system in accordance with various embodiments. Figure 2 shows a flow diagram illustrating a method for fabricating a microfluidic system in accordance with various embodiments. Figures 3A through 3D show schematic views of a microfluidic system in accordance with one embodiment. Figure 3E shows a schematic side view of a microfluidic system in accordance with one embodiment. Figure 4 shows a technical diagram of a microfluidic system in accordance with one embodiment. The scale in Figure 4 is millimeters (mm). Figure 5A shows a top view of a microchip in accordance with one embodiment. Figure 5B shows a top view of a microelectrode array according to one embodiment, which is disposed on a microchip of the embodiment of Figure 5A. Figure 6A shows a simulated plot of a non-uniform electric field generated by a microelectrode array in accordance with various embodiments. Figure 6B shows an optical microscope image of a microelectrode array with cells in accordance with various embodiments. Figures 7-8 through 7C show optical microscopy images of the micro-electric 201217781 polar array of the root embodiment. Fig. 7D and Fig. 7E show the fluorescence microscope image of the cells in the membrane filter and the waste according to the respective examples.

Fig. 8A to Fig. 8C show optical microscope images of the microelectrode array having cells of the respective embodiments of roots &, s, ^ L. Figure 9 shows a graph of sample transition performance in accordance with various embodiments. Fig. 10A and Fig. 1B show an optical microscope image of a microelectrode array having cells according to each embodiment. Figure 11A and Figure 1 show an optical microscopy image of a microelectrode array with cells from a sample of pure blood in accordance with various embodiments. Figures 11C and 11D show optical microscopy images of microelectrode arrays with cells from a mixed gold sample according to various embodiments. Figures 12A and 12B show optical microscopy images of microelectrode arrays with CD34- cells according to various embodiments. Fig. 12C and Fig. 12D are diagrams showing the optical display of the microelectrode array with CD3 4 + cells according to the respective examples (4) (d). Fig. 13A shows a system for measuring impedance according to various embodiments. Fig. 13B is a diagram showing an outline of a PCB-based interconnection illustrating an embodiment of a flute 1 > A ^ ° 兄明第13Α. Fig. 13C is a graph showing the resistance measurement based on the system channel of the embodiment of Fig. 13A. Fig. 14 shows the impedance measurement chart of the rootcasting & Fig. 15 is a graph showing the impedance measurements of some peppers in various embodiments of the roots. 62 201217781 Figure 16 Sg Dan ", a graph of impedance measurements for some samples not according to various embodiments. Figure 17 shows a graph of some of the impedance measurements for batch processing in accordance with various embodiments. Figure 18 - Bie's schematic cross-sectional view of selective capture of endothelial pro-driver cells in accordance with the teachings of the various examples. Figure 19 gg - h ., , is not according to the cell count round table of each embodiment. The second 〇A round batch _ shows a graph of cell counts on the electrodes not according to the respective examples. Figure 20 shows an optical microscope image of a microelectrode array with cells according to various embodiments. Figure 21 shows a graph of cell counts on the electrodes according to one embodiment. Figures 22A and 22B show graphs of filtering and capture performance in accordance with various embodiments. Figures 23A through 23D show optical microscopy images of microelectrode arrays with cells in accordance with various embodiments. [Main component symbol description] 10〇 integrated microfluidic system 102 microfluidic system 104 microchip l〇6a, i〇6b chamber l〇8a, i〇8b detection area 63 201217781 110 first port 112 second port 114 filter 116 contact pad 118 metal hollow needle 200 flow chart 202-206 step 3 00 microelectrode array 3 02 plastic chamber structure 304 centering tape layer 306 bottom tape layer 308 dashed squares 310a, 3 10b opening 3 12 opening 314a ' 314b Opening 316 opening 3 1 8 microfluidic system 320 filter 322 second port 500 microchip 502 microelectrode array 504a-e electrical interconnect 506 contact pad 508a, 508b, 508c, 508d inner electrode 64 201217781 5 10a ' 5 10b, 5 10c, 5 10d 电极 电极 electrode 600 simulation drawing 602 optical microscope image 604 cells 606 internal electrodes 700, 704, 706, 708, 712 cells 702 microelectrode array 71 0 semipermeable membrane 800 microelectrode array 900 chart 902 filter Cell number 904 reflow rate 906, 908 data point 1000, 1004 PBMC 1002, 1006 microelectrode array 1200, 1202 microelectrode array 1300 system 1302 measurement system 1304 microfluidic system 1306 (FPGA) plate 1308a, b pump 1310 Interconnects 1312, 1314, 1318 Connector 1316 PCB Card 65 201217781 1320, 1322 Chart 13 24 Dotted Line 1400 Chart 1402 Impedance Change Percentage 1404 Frequency 1406, 1408, 1410 Data Point 1500 Chart 1 502 Impedance Change 1504 Doped CD34 in Sample Percentage of cells 1506 Negative control 1508-15 14 Sample 1600 Chart 1 602 Total impedance change of 22 pairs of electrodes (%) 1604 Sample type 1606-1614 Sample 1700 Chart 1 702 Total impedance change of 22 pairs of electrodes (°/〇) 1704 sample Type 1706a-c, 1708a-c Sample 1800 Endothelial precursor cell 1802 CD34 Antigen 1804 CD34 Antibody 1806 Electrode 1808 Antibody-linked magnetic beads 66 201217781 1 8 1 0 Microchip 1 8 12 Releasing material 1 8 14 Removable magnet 1 8 1 6 White blood cells 1900 Chart 1902 Cell number 1904 Incubation time 2000 Chart 2002 EPC number on the electrode

EPC 2006 optical microscope image doped in blood samples in 2004 2100 Chart

EPC 2104 incubation time on 2102 electrode 2200 chart 2202 number of cells captured on the electrode 2204 input cell count <: ί 67

Claims (1)

201217781 VII. Patent Application Range: 1. A microfluidic system for detecting an organism in a sample volume. The microfluidic system comprises: a chamber configured to receive the sample volume, wherein the chamber comprises a chamber Detecting an area for detecting the living body; - a first port in fluid communication with the chamber; and - a second port including a filter and in fluid communication with the chamber; and wherein the A fluid of a port or the second port flows through the chamber between the first port and the second port. 2. The microfluidic system of claim 1 , further comprising: at least one microfluidic channel configured to couple the first port to the chamber; and at least one to set the second port A microfluidic channel coupled to the chamber. The microfluidic system of claim 1, further comprising: - a third port or more ports in fluid communication with the chamber. 4. The microfluidic system of any of claims 1 to 3, wherein the detection zone is configured to detect the biological object by a markerless detection method. The method of claim 1, wherein the detection region comprises a microelectrode array. 6. The microfluidic system of clause 5, further comprising a capture molecule configured to attach a surface of the array of microelectrodes. The microfluidic system of claim 6, wherein the fluid supplied to the first port is configured to flow through the chamber and the filter such that the organism is retained; The fluid provided to the second port flows through the filter to move the organism from the filter to the cavity to the capture molecules attached to the surface of the microelectrode array Captured. 8. The microfluidic system of any one of clauses 5 to 7, wherein the microelectrode array is configured to generate a dielectrophoretic force. The microfluidic system of any of claims 5 to 7, further comprising a microchip formed on the microelectrode array or in the microelectrode array. The microfluidic system of any of claims 1 to 3, wherein the §Hail chamber comprises an open chamber. A microfluidic system according to any one of the preceding claims, wherein the sheet comprises a half membrane. The microfluidic system of any one of clauses 3 to 3, wherein -rf* ', -V comprise a movable magnetic element arranged to provide a magnetic field adjacent to the detection area . The microfluidic system according to any one of the items 1 to 3, wherein the organism is selected from the group consisting of a biological indicator, a cell, a prokaryotic cell, and a true Nuclear cells, a mammalian fine yeast cell, a tumor cell, a circulating tumor cell, a blood cell, a peripheral blood mononuclear cell, a cell of an immune system, a white blood cell, a T cell, an auxiliary T Cell, lymphocyte, CD4 lymphocyte, a precursor cell, an endothelial progenitor cell, an embryonic cell, a cell, a virion, a biopolymer, a polypeptide, a nucleic acid, a lipid, a An oligosaccharide, and a combination of any of the foregoing. 14. A method for manufacturing a microfluidic system for detecting an organism of the same volume, the method comprising the steps of: providing a chamber configured to receive the sample volume, wherein the chamber contains a Detector Measuring a region to detect the living body; providing a first port in fluid communication with the chamber; and providing a second port in fluid communication with the chamber, the second 70 201217781 port including a filter; And a fluid provided to the first port or the second port flows through the chamber between the first port and the second port. 15. A method of detecting an organism in the same volume using a microfluidic system for detecting the organism in the sample volume, the microfluidic system comprising: configured to receive the sample volume a chamber, wherein the chamber includes a detection area to detect the living body; a first port in fluid communication with the chamber; and a second port in fluid communication with the chamber, the The two port includes a ferrite; and a fluid provided to the first port or the second port flows through the chamber between the first port and the second port; The following step: providing the sample volume to the chamber; providing the fluid to the first port to pass the sample volume through the filter to retain the living body; removing the living body from the filter to the cavity The detection area of the chamber; and detecting the organism. 16. The method of claim 15, further comprising the step of: 71 201217781 capturing the organism near the detection area by a movable magnetic element. 17. The method of claim 15 or claim 16, wherein the step of providing the fluid to the first port such that the sample volume passes through the filter to retain the organism is repeated at least once. 18. The method of claim 15 or 16, wherein the step of withdrawing the organism to the detection region of the chamber is repeated at least one time. 19. The method of claim 15 or claim 16, wherein the step of removing the living body to the chamber comprises the steps of: providing the fluid to the second port 20. The step of the organism of item 15 or item 16 comprises the steps of: detecting the culture of the organism; and performing a measurement to detect the organism. 2 1. If the item is item 5 or the 16th-containing process, the process is selected from the group consisting of dielectric:; two methods, further impedance, and the foregoing; any combination of the seized molecules The group that makes up. The method of claim 15 or claim 16, wherein the fluid comprises a buffer solution. The method of claim 15 or claim 16, wherein the sample volume comprises: - a blood sample; and - a dissolution buffer solution; and wherein the dissolution buffer solution comprises: a solvent; a pH buffer ; and a primary coagulant. The method of claim 23, wherein the dissolution buffer solution advance comprises a plurality of magnetic beads to couple the organism. The method of claim 23, wherein the solvating agent comprises a gas 匕 ~ pH buffer comprising sodium carbonate, and the anti-agglomerating agent comprises ethylenediaminetetraacetic acid. 26. The method of claim 25, wherein the concentration of the vaporized ammonium is from about 1 mM to about 50 mM. 2 7 The method of claim 25, wherein the volume ratio of the vaporized ammonium to the blood to the blood is between 1:1 and 1:1 〇. The method of claim 25, wherein the sodium carbonate has a concentration of from about 10 mM to about 100 mM. 29. The method of claim 25, wherein the concentration of the ethylenediaminetetraacetic acid is from about 0.01 mM to about 1.0 mM or about 0.1 mM. 74