WO2014142754A1 - Microtamis - Google Patents

Microtamis Download PDF

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Publication number
WO2014142754A1
WO2014142754A1 PCT/SG2014/000122 SG2014000122W WO2014142754A1 WO 2014142754 A1 WO2014142754 A1 WO 2014142754A1 SG 2014000122 W SG2014000122 W SG 2014000122W WO 2014142754 A1 WO2014142754 A1 WO 2014142754A1
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WIPO (PCT)
Prior art keywords
microsieve
cell
membrane
cells
micropore
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PCT/SG2014/000122
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English (en)
Inventor
Mo-Huang Li
Wai Chye Cheong
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Cellsievo Pte Ltd
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Publication of WO2014142754A1 publication Critical patent/WO2014142754A1/fr

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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D69/00—Semi-permeable membranes for separation processes or apparatus characterised by their form, structure or properties; Manufacturing processes specially adapted therefor
    • B01D69/02—Semi-permeable membranes for separation processes or apparatus characterised by their form, structure or properties; Manufacturing processes specially adapted therefor characterised by their properties
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D63/00—Apparatus in general for separation processes using semi-permeable membranes
    • B01D63/08—Flat membrane modules
    • B01D63/088—Microfluidic devices comprising semi-permeable flat membranes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D2325/00—Details relating to properties of membranes
    • B01D2325/02—Details relating to pores or porosity of the membranes
    • B01D2325/021—Pore shapes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M47/00—Means for after-treatment of the produced biomass or of the fermentation or metabolic products, e.g. storage of biomass
    • C12M47/02—Separating microorganisms from the culture medium; Concentration of biomass

Definitions

  • the present invention relates to a microsieve.
  • a microsieve having an improved micropore structure for highly efficient and rapid detection of circulating tumor cells (CTCs) from whole blood samples.
  • CTCs circulating tumor cells
  • Circulating tumor cells are tumour cells shed into the bloodstream from the primary tumour site.
  • CTCs are also the fundamental entities responsible for spawning metastatic disease. The number of CTCs has been shown to correlate with disease development, and represents a potential alternative to invasive biopsies for cancer metastasis analysis.
  • CTCs provide real-time information about a cancer patient, reflecting the time-sensitive status of the cancer progression and imminent relapse of a patient. Furthermore, procurement of 5-10 mL of peripheral blood which is required for CTC-based analysis would be a relatively non-invasive procedure, and blood is easier to obtain than bone marrow or primary tumor tissues.
  • CTCs-based assays represent a long-awaited prognostic and therapeutic tool for the detection of high-risk patients.
  • CTCs-based biomarkers Besides enumeration of CTCs, there has also been intensive search for CTC-based biomarkers to provide molecular characterization of the metastatic potential of the cancers. Thus CTCs-based diagnostics has great potential in revolutionizing the current invasive tissue biopsy-based cancer diagnostics. CTCs could also play an important role in the development of novel cancer drugs.
  • CTCs are exceedingly rare in early stage of cancer patients, where as few as one CTC exists per ⁇ hematologic cells in the blood of cancer patients. Thus, their isolation and differentiation from the other cells found in peripheral blood have posed a tremendous challenge.
  • the myriad of technologies developed to-date to isolate the CTCs from patients' blood can be broadly classified into immuno-capture- and size-based methods.
  • the immuno-capture methods utilize antibodies targeting epithelial-specific markers (such as EpCAM and cytokeratin) expressed on the CTC surface to capture CTCs.
  • epithelial-specific markers such as EpCAM and cytokeratin
  • immuno-capture methods using epithelial markers- targeting antibodies could give false negative results, as the invasive CTCs tend to lose their epithelial antigens via the epithelial to mesenchymal transition (EMT) process.
  • EMT epithelial to mesenchymal transition
  • EpCAM expression in primary tumor tissues and metastases an immunohistochemical analysis, J Clin Pathol, 64, 415-420, 2011) reported that EpCAM is highly expressed in most tumors of gastrointestinal origin and in some carcinomas of the genitourinary tract. However, hepatocellular carcinomas, clear cell renal cell cancer, urothelial cancer and squamous cell cancers were frequently EpCAM-negative. Also, they observed that more than 50% of patients with invasive ductal and lobular breast cancers showed no or weak EpCAM expression. These results spur further concern over the reliability of utilizing the EpCAM antibody for CTC isolation.
  • the diameter of the circular holes are designed to allow most of WBCs to freely flow through, and to capture CTCs with dimension larger than the hole diameter. These methods utilize the size differences between cancer cells and leukocytes to extract CTCs from whole blood samples.
  • cell isolation techniques include the use of microsieves made of silicon, parylene or polycarbonate.
  • microsieve materials suffer disadvantages such as incompatibility with cell culture reagents, high material costs, and low pore density, to name a few. Therefore, there is a need to develop a microsieve to overcome or at least alleviate the above problems for rapid cell and particle filtration.
  • a microsieve membrane for retaining an object of interest in a fluid sample, the microsieve comprising a plurality of micropore structures, wherein each micropore structure is shaped and sized so that the object of interest having a size equal to or larger than the defined size of the micropore structure is retained on the microsieve membrane without covering the entire micropore structure.
  • microsieve membrane it is meant to include any microsieves that are produced using micromachining and offer the possibility in microflltration technology.
  • micropore structure it is meant to include any pore on the microsieve membrane that may be well defined by photolithographic methods or anisotropic etching that allow for the accurate separation of particles by size.
  • the microsieve membrane thickness is usually smaller than the size of the micropore structure in order to keep the flow resistance small.
  • the micropore structure is non-circular in shape.
  • the shape of the micropore structure is any shape selected from the group: a square, a rectangle, a triangle and a cross.
  • the micropore structure is irregular in shape.
  • “irregular” it is meant to include any shape that may not be even or balanced in shape or arrangement. Such shapes maybe asummetrical, non-uniform, uneven, crooked, misshapen, lopsided, and/or the like.
  • the micropore structure further includes at least one branch.
  • the term “branch” may be used synonymous with the term “arm”.
  • “branch” or “arm” it is meant to include any offshoot that comes out of the non-circular micropore structure.
  • the entire structure (including the branch) is taken to be the micropore structure.
  • the shape of the at least one branch is any shape selected from the group: a square, a rectangle, a circle, and a triangle.
  • the at least one branch is irregular in shape.
  • the plurality of micropore structures define a plurality of different shapes.
  • the micropore structure is elongate.
  • elongate it is meant to include any micropore structure that may be longer or slender, especially unusually so in relation to its width. More preferably, the width of the widest portion of the micropore structure is between 5-10 um and the length of the micropore structure is between 5-5000 ⁇ .
  • the microsieve membrane has a thickness of about ⁇ . ⁇ to about ⁇ .
  • the microsieve membrane has a micropore structure density from about 100 to 6000 micropore structure per square millimetre.
  • the microsieve membrane has a micropore structure having an opening area ratio of about 5% to 70%.
  • the object of interest is a cell and the patient's fluid sample is whole blood or processed blood cells.
  • the whole blood may be processed in any way known in the skilled person for analysis of its contents.
  • the cell is a circulating tumour cell.
  • a microsieve comprising: (a) a microsieve membrane according to the first aspect of the invention; and (b) a membrane support layer for supporting the microsieve membrane, the membrane support layer having a plurality of openings, wherein the support layer has a thickness of about 50 ⁇ to about 1500 ⁇ .
  • the membrane support layer is formed of a silicon material.
  • the opening of the membrane support layer is at least 5 times larger than the micropore structure.
  • a device for analysing a patient's fluid sample comprising: (a) an inlet for receiving the fluid sample; (b) an outlet for discharging the fluid sample; and (c) a microsieve membrane according to the first aspect of the invention or a microsieve according to the second aspect of the invention, the microsieve membrane or microsieve intermediate the inlet and outlet.
  • the inlet, the outlet and microsieve membrane or microsieve are positioned on the same lateral so that the fluid flows in a lateral direction.
  • lateral direction it is meant that the inlet, outlet and microsieve membrane or microsieve are not arranged in a vertical / stacked manner. But rather, they are arranged alongside each other (either on the same plane or not), i.e. the inlet and outet may be positioned left and right of the microsieve membrane or microsieve respectively.
  • the device further comprises a window above the microsieve to allow imaging of the cells retained in the microsieve membrane or microsieve.
  • a window above the microsieve to allow imaging of the cells retained in the microsieve membrane or microsieve.
  • the entire device may be enclosed in a housing wherein the window allows a person to view the cells that are retained in the microsieve membrane or microsieve.
  • the device further comprises a pump for pumping the fluid sample through the device.
  • the pump may be a peristaltic pump.
  • a method for retaining an object of interest in a fluid sample comprising filtering the fluid sample through an inlet of the device according to the third aspect of the present invention.
  • the object of interest is a cell and the patient's fluid sample is whole blood or processed blood cells.
  • the cell is a circulating tumour cell.
  • a method method for determining the presence of a circulating tumour cell in a fluid sample comprising: (a) filtering the fluid sample through an inlet of the device according to the third aspect of the invention; (b) staining the cells retained on the microsieve with a readily detectable moiety; (c) imaging the stained cells; and (d) determining the presence of the circulating tumour cell based on the image obtained.
  • staining it is meant that the cell is contacted with the readily detectable moiety and the presence of the retained cell is detected by detecting the moiety.
  • the readily detectable moiety includes an antibody. More preferably, the detectable moiety is a fluorescent-probe conjugated antibody. Still more preferably, the retained cell is detected by contacting the retained cell with a tissue-specific or cell-type specific antibody having a label. Alternatively, the retained cell may be contacted with a reagent capable of detecting a tumour associated antigen or a nucleic acid encoding the tumour associated antigen.
  • the step of imaging the stained cells includes imaging a plurality of images and stitching the plurality of images.
  • the microsieve membrane's diameter may be more than l mm.
  • one may have to stitch several fluorescence images together before using a software to automatically count the retained cells.
  • the morphology of the retained cells may not be uniform, and the fluorescence signal may be weak.
  • bright field image of the microsieve structure is used for stitching purposes.
  • imaging capturing device Any suitable imaging capturing device may be used.
  • the imaging capturing device may be used together with a microscope.
  • fluorescence cell counting may be conducted directly onto the microsieve membrane surface.
  • the method may further comprises the step of centrifuging the device after step (a) and prior to step (b) set out above.
  • the centrigufal force is used to transfer the retained cells from the surface of the microsieve membrane to the glass window of the device according to the third aspect of the present invention.
  • FISH fluorescence in-situ hybridisation
  • the fluid sample is whole blood or processed blood cells.
  • the fluid sample is whole blood or processed blood cells.
  • Figure 1 Operational principle of circulating tumor cell (CTC) enrichment by microsieve-defined differential size and deformability separation, (b) Various non-limiting embodiments of microsieve device with various hole (or micropore) structures.
  • Figure 2 (a) Illustration of cell isolation on a microsieve with circular holes, (b) Relative positions of cells on top of a circular-hole microsieve. The Fp and Fs represents the pressure and shear forces applied on the cells respectively, and Fc is the reaction force generated from the microsieve surface, (c, d) Relative positions of cells on top of a rectangle, and cross-shape holes, respectively.
  • Figure 3 Top view of microsieve devices with: (a) cross-shape holes and (b) slot holes.
  • the cross-hole includes four arms with arms dimension of about 3-10 ⁇ in width (h 2 ), and about 5-50 ⁇ in length (hi).
  • the dimension of the slot holes is about 3-10 ⁇ in width (s 2 ), and about 10-50 ⁇ in length (si).
  • the spaces between the cross-shape holes and slot holes are about 2-20 ⁇ (x), about 1-30 ⁇ (y) and about 1-30 ⁇ (z), respectively.
  • the space between holes define the porosity and the mechanical strength of a microsieve. Reducing the space distance increases the porosity, while reduces the mechanical strength of the microsieve. In addition, higher porosity reduces the fluid resistance of a microsieve, shortening the filtration of samples processing.
  • Figure 4 The schematic diagram of a microsieve device, (a) Top view of the microsieve device with a two-layer structure and on-device marks, (b) Side view of the microsieve device showing the two-layer structure and on-device marks.
  • Figure 5 illustrates cell isolation on a microsieve with circular holes
  • the Fp and Fs represents the pressure and shear forces applied on the cells respectively, and Fc is the reaction force generated from the microsieve surface.
  • Figure 6 Mechanisms of cell isolation with a microsieve device having cross-shape holes, (a) Schematic of cell isolation with cell sizes either smaller or much larger than the hole size. (b) Cell "isolation with cell size at the same range of pore size. (c,d,e) Mechanism of cell isolation. Cell movement due to the unbalance of shear force (F s ). (f-i) The illustration of cell deformation due to the shear forces around the cell surface. (j,k) Schematic of cell deformation for same-size cells with different nucleus dimensions.
  • Figure 7 Fabrication process of microsieve devices, (a) Pattern photoresist to define the holes and marks structure, (b) Etch the front-side silicon layer (5-50 ⁇ thick).
  • the on- device marks are also created at this step, (c) Pattern photoresist to define the supporting structure of microsieve device, (d) Etch the backside silicon layer (0.25-1 mm thick), (e) Etch the insulator layer (1 ⁇ -thick Si0 2 layer), (f) Pattern and define the spacer layer (0.5-1 mm thick), (g) Bond the microsieve devices with the spacer layer, (h) Bond the device with a glass window (0.13 - 1 mm thick). Processes (a-e) are utilized to fabricate microsieve device without an integrated chamber (Device at Figure 9). Processes (a-h) are conducted to fabricate microsieve device with an integrated chamber (Device at Figure 8).
  • Figure 8 An exploded and a perspective views of an embodiment of a microsieve device with co-fabricated chamber, respectively.
  • Figure 9 Images of fabricated devices. Silicon microsieve filter with: (a) circle holes, (b) cross-shape holes, and (c) rectangular (slot) holes, (d) The backside of the microsieve membrane showing the supporting rings, (e) The dimension of the silicon microsieve and membrane area is 7 mm in square and 5 mm in diameter, respectively, (f) The structure and dimension of cross-shape holes.
  • Figure 10 (a) Schematic of the filter unit with the microsieve filter embedded within a plastic plate and sandwiched between the blood reservoir and plate holder, (b) The plastic plate with an embedded microsieve filter, (c) The workstation setup for CTC separation. Freshly drawn whole blood is filtered through the microsieve filter unit with the fluid flow rate controlled by a peristaltic pump. Captured cells is released from microsieve into the blood reservoir by a back flush controlled by a syringe pump.
  • Figure 11 (a) Schematic of a filter unit used for on-microsieve cell imaging, (b) The workstation setup for real-time CTC isolation and cell imaging. Freshly drawn whole blood is filtered through the microsieve filter unit with the fluid flow rate controlled by a peristaltic pump.
  • Figure 12 Comparison of shear-force enhanced cancer cell isolation utilizing microsieves with slot holes (width of 5- ⁇ ( ⁇ ) and 7- ⁇ ( ⁇ ), respectively) and cross-shape holes (o).
  • the holes dimension in respect to the cell size The microsieve dimension is 5 mm in diameter with membrane thickness of 15 ⁇ .
  • the cross-shape hole has arms dimension of 5 ⁇ in width, and 30 ⁇ in length (Structure 2), while the slot holes are 30 ⁇ in length with a width of 5 ⁇ (Structure 1) and 7 ⁇ (Structure 3), respectively.
  • Figure 13 Capture of white blood cells and MB231 on a microsieve membrane with cross- shape holes (Structure 2). The cells were stained with DAPI (b) and fluorescence-labeled antibodies targeting WBCs (c). The merged image (d) shows the boundary of cells and pore array.
  • Figure 14 Capture of MB231 and WBCs cells on a microsieve membrane with slot holes which are 30- ⁇ long and 7- ⁇ wide (Structure 3). The cells were stained with DAPI (b) and fluorescence-labeled antibodies targeting WBCs (c). The merged image (d) shows the boundary of cells and pore array.
  • Figure 15 Capture of WBCs cells on a microsieve membrane with 8-um diameter circular holes. The cells were stained with DAPI (a) and fluorescence-labeled antibodies targeting WBCs (b). The merged image (c) shows the boundary of cells and pore array.
  • Figure 16 The effects of membrane thickness on the tumor cell isolation by comparison of capture efficiency versus flow rate using microsieves with 15- ⁇ (o) and 40- ⁇ ( ⁇ ) thick cross-shape holes, respectively.
  • the microsieve dimension is 3.8 mm in diameter.
  • the cross- shape hole has arms dimension of 5 ⁇ in width, and 30 ⁇ in length (Structure 2 in Figure 9b). The results indicate that the thick microsieve is more sensitive to flow rate than that of a thin one.
  • Figure 17 Comparison of microsieve with cross-shape (Structure 2: o) and circular (Structure 4: ⁇ ) holes for MB231 (a) and HepG2 (b) tumor cell isolation.
  • the microsieve membrane is 3.8mm in diameter with membrane thickness of ⁇ or 4 ⁇ . The results indicate that a microsieve with cross-shape holes is more sensitive to flow rate than that of one with circular holes.
  • Figure 18 Comparison of WBCs ( ⁇ ) and MB231 (o) cancer cells isolation using microsieve with cross-shape holes.
  • the microsieve dimension is 5 mm in diameter with membrane thickness of 15 ⁇ . The results indicate that WBCs are more sensitive to flow rate than that of MB231 cancer cell.
  • Figure 19 Comparison of cancer cell isolation using microsieve with cross-shape (Structure 2) and slot holes (Structure 3), respectively. Briefly, 1000 of MB231 cells was spiked into 2 ml of healthy donor's whole blood and filtered through the microsieve at a flow rate of 2 mL/min, in triplicates, (a) Number of co-captured white blood cells, (b) Capture yield of MB231 for cross-shape and slot holes, respectively.
  • Figure 20 Fluorescence images of DAPI-only cells and WBCs on microsieve surface obtained from a breast cancer patient.
  • Figure 21 A perspective view of an embodiment of a cartridge with a window
  • Figure 22 An exploded view of an embodiment of a cartridge with a window
  • Figure 23 A perspective view of another embodiment of a cartridge without a window
  • Figure 24 An exploded view of another embodiment of a cartridge without a window
  • Figure 25 A perspective view of another embodiment of a cartridge with an integrated microsieve.
  • Figure 26 An exploded view of another embodiment of a cartridge with an integrated microsieve.
  • Figure 27 Parts for elution. Plug (A) and collect tube (B).
  • Figure 28 An approach for fluid manipulation using two peristaltic pumps for microsieve pre-wetting (peristaltic pump 1) and blood filtration (peristaltic pump 2).
  • Figure 29 An embodiment for cell elution using a lateral centrifugal force.
  • Figure 30 Mechanism of centrifugal cell elution. (a) Forces encountered by cells, (b) Cells are on top of the microsieve surface, (c) Cells are within the channel. Ppi and Pp2 are the centrifugal pressures generated from the fluid within the bottom and top channels, respectively. Fm is the buoyancy force, and Fd is the viscosity (Stokes) force.
  • Figure 31 An embodiment for cell transportation
  • Figure 32 Mechanism of centrifugal cell transportation, (a) Forces encountered by cells with the cartridge in upward direction and the microsieve facing outward, (b) Cells are on top of microsieve surface.
  • P pv is the centrifugal pressure generated from the fluid underneath the microsieve chamber.
  • Fm is the buoyancy force.
  • Figure 33 A schematic diagram of a cartridge positioned in an optical platform with three light sources. 1-3: light sources 1-3; 4: lens; 5: beam splitter, 6: excitation filter; 7: emission filter; 8 CCD camera; 9: microsieve cartridge; 10: motorized x-y stage.
  • Figure 34 Schematic diagrams of images stitching, (a) Cells on top of circular-hole microsieve surface. The squares represent the areas captured by CCD camera.
  • FIG. 35 The process of image stitching using bright field images as references, (a) Cells on top of microsieve surface. The square area represents the CCD capturing area, (b) The sequence of image scanning. The dimension of each capturing area is XxY. (c) The process flow of image scanning and capture.
  • Figure 36 Representations of images of cells obtained using an analyte-detection system, (a-e) Fluorescence images with captured cells stained with various fluorescent biomarkers. The biomarkers may stain on elements at the cell surface or inside the cells, (f) Bright field image showing the microsieve structure.
  • Figure 37 Schematic diagrams using on-device marks as references for image alignment and overlapping.
  • (a,b) Cell images of the whole microsieve area. Cell imaging performed on cells captured on the same microsieve, but at different times,
  • (c) The method using on-device marks for image alignment of cell images captured at two different experiments.
  • Figure 39 The method and process as applied to the detection and differentiation of multiple types of CTCs
  • the present invention relates to a microsieve that is highly efficient and may be used for the rapid detection of circulating tumor cells (CTCs) from whole blood samples. It also relates to a device incorporating the microsieve for the rapid detection of circulating tumor cells (CTCs) from whole blood samples.
  • the device utilizes a microfabricated silicon microsieve with a densely pack pore array to rapidly separate tumor cells from whole blood, utilizing both the size and deformability differences between the CTCs and normal blood cells.
  • the silicon microsieve consists of a two-layer structure with a thin membrane to minimize the whole-blood fluid resistance and a thick underneath-membrane structure to enhance the mechanical property of the thin membrane.
  • the microsieve employs a unique pore structure which provides extra space for cell to deform and additional shear force to differentiate cancer cells and blood cells, superior to conventional microsieves with circular holes.
  • the whole process including the tumor cell capture, antibody staining, removal of unwanted contaminants and immunofluorescence imaging, was performed directly on the microsieve within an integrated microfluidic unit, interconnected to a peristaltic pump for fluid regulation and a fluorescence microscope for cell counting.
  • a high recovery rate of > 80% was achieved with defined numbers of MB231 and HepG2 cancer cells spiked into human whole blood and filtered at a rapid flow rate of 1 mL/ min.
  • Fluid samples from patient could contain white blood cells (WBCs), red blood cells, palates, and CTCs. Most of WBCs has a cell size smaller than 8 ⁇ in diameter while CTCs are normally having a cell size larger than 8 ⁇ .
  • the cell structure of CTCs and WBCs includes a cell nucleus surrounding by cytoplasma and a cell membrane.
  • the mechanical property of a cell can be modeled as a deformable ball with a core (nucleus) surrounding by a soft shell (cytoplasma and cell membrane). Tumor cells are known to possess larger nucleus/ cytoplasmic ratio and non-homogeneous texture than that of WBCs.
  • the elastic (Young's) modulus of cancer cells and white blood cells (WBCs) is > 0.5 kPa (S.E. Cross, et al., Nanomechanical analysis of cells from cancer patients, Nature Nanotechnology, 780-783, 2007.) and 20-50 Pa (G.W. Schmid-Schonbein, et al., Passive mechanical properties of human leukocytes, Biophys. J., 36, 243-256, 1981), respectively. Therefore, CTCs are stiffer and less deformable in comparison with WBCs of the same size.
  • the present invention utilizes a microsieve filter with unique micropore structure for CTCs isolation using the difference of cell size and deformability difference between WBCs and CTCs for CTCs isolation.
  • Figure 1 illustrates the operational general principle of the present invention which utilizes the combination of the deformability difference, and the distinct morphology and size differences between cancer cells and leukocytes to extract CTCs from whole blood samples taken from patients.
  • WBCs and CTCs are normally have a circular cell morphology. Thus these cells can wholly cover the conventional microsieve with circular holes.
  • the present microsieve contains holes (or micropore structures) with a structure whereby objects of interest (i.e.
  • target cells in the present case do not fully cover the micropore structure area, which creates an unoccupied open area around cell (See “A” in Fig. lb).
  • These micropore structures provide two advantages over the circular holes in providing extra-space for cell to deform, to allow softer WBCs to squeeze through the hole.
  • additional shear forces are created when fluid flows through the open area, which also assists to drag softer WBCs through holes.
  • our microsieve allow small WBCs with diameter smaller than the pore size to free go through. Whereas for those WBCs with size similar to CTCs, one can control the shear force to separate them by exploring the difference of cell stiffness.
  • Figure 2 illustrates the difference between using circular micropore structures and the non- circular micropore structure (for example, as shown in Figure 2, a cross-shaped, rectangular micropore structure) for CTCs isolation.
  • cells When cells were captured on top of the microsieve surface, they will be subjected to several forces including the pressure generated from the blood flow (Fp), the shear stress generated from fluid flow through the cell surface (Fs), and the microsieve reaction force (Fc).
  • the pressure force is linearly proportional to the volume flow rate of whole blood, while the shear force is related to the fluid velocity at the cell surface with the force direction along the direction of fluid velocity.
  • WBCs and CTCs are normally have a circular cell morphology. Thus these cells can wholly cover the conventional microsieve with circular holes.
  • FIG. 3 shows the microsieve having micropore structures that are cross-shaped and rectangular for CTCs enrichment.
  • the cross-shape design includes branches or arms and the center part of the cross-area.
  • the cross-shape microsieve has similar mechanism as that of the circular-hole microsieve for the isolation of small and large cells. It allows the small cells having cell sizes smaller than the opening area of the cross-shape hole to freely go through, but will capture the large cells. The small cells could either flow through along the branches or arms or the center part of the hole.
  • the cells will only occupy part of the hole.
  • fluid can flow along unoccupied area, which generates a shear flow on the cell surface at the direction perpendicular to the microsieve surface.
  • the rectangular micropore structure can also be used to generate the shear force for cell separation.
  • the microsieve may comprise a two-layered structure with an upper thin porous microsieve membrane 5 for cell filtration and a lower layer of thick honeycomb rings for membrane support.
  • the two-layer structure balances the flow resistance and the mechanical strength of the microsieve (Figure 4).
  • the microsieve also includes four marks 15 at the four corners of the device. These markers are to facilitate device-to-device image alignment, and automated focusing of optical detection for cell identification.
  • the thickness of the top-layer is about 0.5-100 ⁇ , which is defined by the device layer of the silicon-on-insulator wafer, used for microsieve fabrication.
  • the top-layer may also be created by depositing a thin layer of silicon dioxide, silicon nitride, silicon carbide or even metal (such as nickel or copper) on top of a carrier silicon wafer.
  • the top layer defines the micropore thickness and pore dimension for cell isolation, while the bottom layer enhances the mechanical strength of the micropore, preventing it from breaking as the microsieve device is subjected to an applied fluid force.
  • the thickness of the bottom-layer is about 50 ⁇ - l mm, and the diameter of the supporting ring (d as indicated in Fig.
  • the shape of supporting ring may be a circle, a triangle, a rectangle, a hexagonal, or other irregular shape.
  • a fluid force is applied to the microsieve membrane for cell filtration, which generates a force on the microsieve membrane, and a corresponding stress at the junctions of microsieve/supporting ring.
  • the stress force depends on the shape of the supporting ring.
  • a supporting ring includes sharper corners that are more fragile, thus a hexagonal or honeycomb structure are utilized, in providing a uniform stress across the microsieve-supporting rings.
  • the microsieve may also include but is not limited to four marks at the four corners of the device.
  • the design of alignment mark may include +, J j-
  • the marks are co-fabricated and embedded on the microsieve surface to provide references for image focusing and downstream image process.
  • the dimension of the marks is about o.ioo - l mm in both x and y directions.
  • Micropore structures can be utilized to generate the shear forces for cell separation, as long as the micropore has the following properties: l) The width of the widest area of the micropore is between 5-10 ⁇ , and it has a length of 10-500 ⁇ , ⁇ 2) The shape of the micropore may be a rectangle, a triangle, or an irregular shape; 3) The micropore may be non-circular; 4) The micropore may contain branches or arms with a width of the widest area of 5-10 ⁇ , a length of 5-500 ⁇ ; and the arm/branch may be square, rectangle, circular, triangle or irregular shapes (Fig. lb).
  • a micropore may comprise a generally elongate opening (or slot, which may resemble a rectangle) wherein the width of the generally elongate opening is smaller than the smallest possible width (or the smallest possible diameter) of a cell being retained on the micropore, and the length of the generally elongate opening is larger than the largest possible length (or the largest possible diameter) of the cell being retained on the micropore.
  • the micropore may comprise a plurality of elongate openings, jointly arranged with one another.
  • the joint arrangement of the plurality of elongate openings may form arms/branches of the slot as mentioned above.
  • the smallest possible width and the largest possible length of the cell may occur when the cell deforms from its equilibrium state in which the cell is typically circular.
  • the width of the generally elongate opening may refer to the effective width of the generally elongate opening or the smallest width of the generally elongate opening.
  • the length of the generally elongate opening may refer to the effective length of the generally elongate opening or the largest length of the generally elongate opening.
  • the width of the generally elongate opening may be less than 7 ⁇ and the length of the generally elongate opening may be more than 15 ⁇ .
  • the width of the generally elongate opening may be less than 15 ⁇ and the length of the generally elongate opening may be more than 30 ⁇ . If a target cell is about 25 ⁇ , the width of the generally elongate opening may be less than 18 ⁇ and the length of the generally elongate opening may be more than 40 ⁇ .
  • the micropore has a shape (so termed "irregular shape") which may be designed and dimensioned in such a way that a cell having a size equal to or larger than a defined size may be retained on the micropore without covering the whole (or entire) micropore or with at least a gap provided through the micropore when the cell is retained to allow sample fluid that was used to carry the cell to flow through the gap.
  • the microsieve may contain the following features: (a) A microsieve device with a two-layer structure, fabricated using silicon as substrate.
  • a shear-stress hole may comprise a generally elongate opening (or slot) wherein the width of the generally elongate opening is smaller than the smallest possible width (or the smallest possible diameter) of a cell being retained on the micropore, and the length of the generally elongate opening is larger than the largest possible length (or the largest possible diameter) of the cell being retained on the micropore.
  • the microsieve device having an arrangement of rectangular and irregular-shaped holes/slots, or an arrangement of triangular, crossed-shaped and rectangular holes/slots.
  • the holes/slots of different shapes may be uniformly distributed or randomly distributed.
  • a microsieve device with or without on-device markers is preferred to have on-device markers for: 1) Automated focusing for optical detection. 2) High content immunofluorescence detection for multi- image alignment.
  • the direction and the magnitude of the shear force depends on the cell size and the density of pores.
  • the shear force (Figs. 5b,e) occurs at the out- skirt of cell surface.
  • the shear force will push the out-skirt of the cell down, while the inert area of the cell is supported by the microsieve surface. Under this situation, the shear force will push the cell closer to the microsieve surface.
  • the shear force will assist in pushing cells into pores. Higher shear force will be generated for dense pores, where the space between the cell and surrounding holes is reduced. In this situation, the cell encounters higher fluid velocity when the fluid flows through the empty surrounding holes.
  • the cross-shaped microsieve utilizes the size and deformability differences of WBCs and CTCs for CTCs enrichment.
  • the cross-shaped design includes branches or arms and the center part of the cross-area.
  • the cross-shape microsieve has similar mechanism as that of the circular-hole microsieve for the isolation of small and large cells. It allows the small cells having cell sizes smaller than the opening area of the cross-shape hole to freely go through, but will capture the large cells (Fig. 6a). The small cells could either flow through along the arms or the center part of the hole. For cells having sizes at the range of the arm-size (5-10 ⁇ ), the cells will only occupy part of the hole.
  • fluid can flow along unoccupied area, which generates a shear flow on the cell surface at the direction perpendicular to the microsieve surface.
  • the magnitude of a shear force is proportional to the shear stress and the area subjected to shear stress.
  • the cross-shape hole has four arms.
  • a cell may encounter uneven strengths of shear force depending on the cell location related to holes (Figs. 6c-e). For example, the bottom part of cell in Fig. 6c has more area exposed to the through-hole flow than the top part of cell, thus the bottom part will experience a larger shear force than the top part.
  • the uneven shear forces can generate a lateral force to drag cells to the center cross area where the cell is subjected to even shear forces generated from fluid flow through the four arms.
  • the cross-shape hole can generate larger shear force than that of circular hole, as the fluid can flow close to the cell surface instead of through the surrounding holes for the circular-hole microsieve.
  • the microsieve of the present invention is different from a microsieve having pores which are circular or of any shapes that is dimensioned to be covered wholly or in its entirety by a cell.
  • FIG. 6f A cell will encounter four shear forces when the cell is located at the center of the cross- area (Fig. 6f).
  • a cell may only encounter three shear forces at different strengths when the cell partially covers a hole (Figs. 6i, g).
  • Shear forces can also be generated for a cell located at the long arm, where the cells will counter two shear forces at both the out-skirt of the cells (Fig. 6h).
  • Figures 6f,g shows that when a cell is soft, the surrounding shear forces can drag the cell through the micropore, whereas a stiff cell (such as CTCs) is retained on top of the pore.
  • the WBCs are less stiff than CTCs (Fig. 6j) as the latter ones are known to possess larger nucleus/ cytoplasmic ratios.
  • the viscous (Stokes) shear force for a cell subjected to a flow velocity of v is given by:
  • ⁇ is the viscosity of liquid
  • R is the radius of the cell
  • v is the velocity of cell with respect to the surrounding medium.
  • the slot membrane provides higher shear-pressure force ratio than that of the circular one at the same membrane porosity and thickness.
  • the ratio of shear-pressure force increases as the membrane thickness decreases.
  • the shear force depends only on the fluid velocity, while the pressure force depends on both the membrane resistance and fluid velocity. Reducing the membrane thickness will minimize the membrane fluid resistance and the corresponding pressure across a membrane, while the volume flow rate is kept constant.
  • a thinner membrane would provide a better performance for CTCs isolation on utilizing the shear force and the deformability difference of CTCs and WBCs.
  • Table 1 Comparison of pressure and shear forces for membranes with circular and slot holes.
  • the microsieve was fabricated with a 6" silicon-on-insulator (SOI) wafer, using a deep reactive ion etching (DRIE) process.
  • the fabrication process was as follows: A 2 ⁇ m-thick Si0 2 film was first deposited on top of the handle layer of the SOI wafer by plasma-enhanced chemical vapor deposition (PECVD), which served as a hard mask for creating the honeycomb structure. Then, the pattern of the microsieve was created by etching through the device layer of the SOT wafer using a photoresist mask (Figs. 7a,b).
  • the handle layer of the processed wafer was etched through using patterned photoresist/Si0 2 as a DRIE mask to create the honeycomb structure (Figs. 7c,d).
  • the microsieve was finally released from the SOI wafer by dielectric reactive ion etching to remove the exposed Si0 2 embedded layer of the SOI wafer (Fig. 7e).
  • three more process steps were added.
  • a 6" silicon wafer was etched using photoresist/Si02 as a DRIE mask which defines the chamber area (Fig. f .
  • the processed wafer was then bonded to the process microsieve wafer (Fig. 7g), and the glass wafer (Fig. ) using anodic bonding.
  • the rigid silicon material property and highly reproducible silicon microfabrication technology provides a microsieve device with thin silicon membrane and highly porous structure.
  • the rigid silicon structure enables CTC capture, cell staining and fluorescence imaging directly on the microsieve.
  • the non-autofmorescence silicon material offers additional imaging benefit for on-microsieve fluorescence imaging, with high-quality fluorescence images.
  • This microsieve includes a co-fabricated chamber with the chamber structure defined by the middle layer.
  • the top layer provides a glass or transparent window for cell imaging using a microscope.
  • Figure 9 shows the images of fabricated devices with circular, cross-shape, and rectangular holes, and the backside supporting structure.
  • the size of the square microsieve is 7 mm wide with an effective porous membrane area of 5 mm in diameter, and a thin microfabricated silicon membrane (15-40 ⁇ thickness).
  • the pore size of circular hole is 8 ⁇ in diameter with 15- ⁇ pitch, corresponding to a densely packed pore array of -5000 pores/mm 2 or ⁇ 105 pores per device or ⁇ 40% porosity (percentage of hole area).
  • the cross-shape hole includes four arms with arms dimension of 5 ⁇ in width (h 2 ), and 30 ⁇ in length (hi).
  • the space between hole is 5 ⁇ , which corresponds to a pore array of -1975 pores/mm 2 , -3.88x104 pores per device or ⁇ 42% porosity.
  • the dimension of the slot holes is 7 ⁇ in width (s 2 ), and about 30 ⁇ in length (si).
  • the space between hole is 5 ⁇ , which corresponds to a pore array of -2381 pores/mm 2 , -4.6x104 pores per device or -50% porosity.
  • CTC Filtration system 1 Vertical flow system A fluid drive system was specifically designed to control the fluid manipulation.
  • the micro- fabricated microsieve was mounted on a plastic plate and sandwiched between two clamps with integrated fluid connectors and the blood reservoir (Fig. 10a).
  • the plastic plate acts as a support for the silicon microsieve, which serves for CTC capture and separation from the other blood cells. It also facilitates the additional feature of a detachable microsieve that can be transferred to a nearby microscope for cell imaging (Fig. 10b).
  • a gasket between the microsieve and the top insert reservoir prevents leaks and ensures that the entire sample is delivered into the flow cell and filtered through the microsieve.
  • the effective filter area is self-defined by the opening of the plastic plate (diameter: 5 mm) while the fluidic flow of whole blood is controlled by a peristaltic pump connected to the filter unit through a plate holder at the base.
  • the wash buffer is introduced through a syringe pump connected to the plate holder.
  • the filtered waste of whole blood sample is collected by a waste bottle 120.
  • Whole blood filtration, sample wash, and fluorescence cell staining were performed directly on the microsieve within the integrated filtration unit (Fig. 10c) linked to a peristaltic and syringe pump fluid drive.
  • a fluid system was specifically designed for real-time monitoring CTC isolation under a microscope, with a lateral flow approach (Fig. 11).
  • the microsieve was fixed on a metal holder base using a adhesive tape and sandwiched between a structure including gaskets (Gasket 1 and Gasket 2), glass window and two clamps with integrated fluid connectors.
  • the gasket (Gasket 2) defines the fluidic channel, and connects the inlet and the microsieve.
  • the glass window prevents leaks and ensures that the sample is delivered into the flow cell and filtered through the microsieve.
  • the effective filter area is self-defined by the opening of the adhesive tape (diameter: 5 mm) while the fluidic flow of whole blood is controlled by a peristaltic pump connected to the filter unit through a metal holder at the base.
  • the filtered waste of whole blood sample is collected by a waste bottle 120.
  • a cartridge that incorporates the microsieve of the present invention.
  • An example is shown in Figure 21.
  • This cartridge includes or consists of 4 layers.
  • the exploded view of the cartridge may be seen in Figure 22.
  • the cartridge contains a whole blood introduction inlet 20, a microsieve pre-wet inlet 25, a blood waste outlet 30, a cell elution outlet 35, a microsieve 5, 10 and a glass window 40 on top of the microsieve area.
  • the microsieve 5, 10 may either be the microsieve membrane 5, or a microsieve having both the microsieve membrane 5 and membrane support layer 10.
  • the glass window 40 is created by sandwiching a glass slide 45 between Chamber Clamp 50 (at Layer 3) and Chamber Recessed (at Layer 4).
  • a glass slide is first bonded to the Chamber Clamp at Layer 3, which seals the microsieve chamber. Then Layer 3 (with the glass slide) is further bonded with Layer 4, to seal the glass window.
  • the microsieve is fixed to the Microsieve-Recess at Layer 2 at a separated process, and then sandwiched by Layer 1 and the processed Layers 3-4 component.
  • Layer 1 is for sealing the bottom channels and chambers of Layer 2.
  • Layer 2 consists of or comprises structures for Cell Elution Outlet (which may also be herein referred to as Elution Outlet), Microsieve- Recess 80, Vias 90, Channels 75 and a chamber underneath the microsieve device.
  • the channel 85 for blood waste outlet is located at the bottom of Layer 3, which is connected to the Waste Outlet through a via 55.
  • the Pre-Wet inlet is connected to the bottom of the microsieve chamber through a vertical zigzag structure, to prevent the processed blood entering the channel for pre-wet.
  • the Layer 3 contains structures for Cell Elution Outlet, Blood introduction Inlet, a channel connecting Blood Introduction Inlet and Microsieve Chamber, Waste Outlet, Pre-wet Inlet, and a recess for Chamber Clamp.
  • Layer 4 contains the structures of an Open Window 40, a Recessed Clamp 65, and Open Holes 1, 2 70. Open Holes 1, 2 are utilized to facilitate the bonding of Layer 3 and Layer 4. All four Layers also include Alignment Notches 60 to facilitate layer-to-layer bonding.
  • the cartridge may include or consist of 3 layers, as shown in Fi. 23.
  • This fluidic structure is shown in Fig. 24.
  • the cartridge is very similar to the one in Fig. 22 except for the glass window. It contains a whole blood introduction inlet, a microsieve pre-wet inlet, a blood waste outlet, a microsieve and a cell elution outlet.
  • the microsieve is fixed to the Microsieve-Recess at Layer 2, and then sandwiched by Layers 1 and 3.
  • Layer 1 is for sealing the bottom channels and chambers of Layer 2.
  • Layer 2 consists of or comprises structures for Cell Elution Outlet, Microsieve-Recess, Vias, Channels and a chamber underneath the microsieve device.
  • the channel for blood waste outlet is located at the bottom of Layer 3, which is connected to the Waste Outlet through a via.
  • the Pre-Wet inlet is connected to the bottom of the microsieve chamber through a vertical zigzag structure, to prevent the processed blood entering the channel for pre-wet.
  • the Layer 3 contains structures for Cell Elution Outlet, Blood introduction Inlet, a channel connecting Blood Introduction Inlet and Microsieve Chamber, Waste Outlet, and Pre-wet Inlet.
  • the shape and dimension of microsieve chamber is defined at Layer 3. All three Layers also include Alignment Notches to facilitate layer-to-layer bonding.
  • there is included another cartridge that may include or consist of 4 layers (see Fig. 25) with structures similar to the one shown in Fig. 22.
  • the cartridge contains a whole blood introduction inlet, a microsieve pre-wet inlet, a blood waste outlet, a cell elution outlet, and an integrated microsieve.
  • the microsieve is fixed to the Microsieve- Recess at Layer 2, and then sandwiched by Layers 1, and 3. After that the processed component is bonded with Layer 4, to seal the integrated microsieve, and to prevent any leakage between the inlet channel, the inlet of integrated microsieve.
  • Layer 1 is for sealing the bottom channels and chambers of Layer 2
  • Layer 2 consists of or comprises structures for Cell Elution Outlet, Microsieve-Recess, Vias, Channels and a chamber underneath the microsieve device.
  • the channel for blood waste outlet is located at the bottom of Layer 3, which is connected to the Waste Outlet through a via.
  • the Pre-Wet inlet is connected to the bottom of the microsieve chamber through a vertical zigzag structure, to prevent the processed blood entering the channel for pre-wet.
  • the Layer 3 contains structures for Cell Elution Outlet, Blood introduction Inlet, a channel connecting Blood Introduction Inlet and Microsieve Chamber, Waste Outlet, Pre-wet Inlet, and Insert Opening (Opening 1 in Fig. 26) for integrated microsieve.
  • Layer 4 contains the structures of an Open Window, a Recessed Clamp, and Open Holes 1, 2. Open Holes 1, 2 are utilized to facilitate the bonding of Layer 3 and Layer 4, and the Recessed Clamp is for sealing the integrated microsieve device. All four Layers also include Alignment Notches to facilitate layer-to-layer bonding.
  • Figure 27 shows a plug and elution collection tube.
  • the plug is inserted into the port of Cell Elution Outlet during cell isolation process.
  • the plug is removed from the port of Cell Elution Outlet and then the port of Cell Elution Outlet is connected with the collection tube for cell elution.
  • Peristaltic pumps 100 are used for fluid manipulation within the cartridge. This is shown in Figure 28.
  • Figure 28 shows the device (or cartridge) 110 according to an embodiment of the present invention.
  • a peristaltic pump 100 uses a rotor to compress a flexible tube for fluid pumping.
  • a peristaltic pump can either withdraw or pump fluid depending on the rotation direction of the rotor of a peristaltic pump.
  • the fluidic channels 115, chambers 125 and the microsieve are first pre-wet with pre-wetting solution.
  • the pre-wetting solution is withdrawn from the pre-wetting bottle by the peristaltic pump 1 (rotating in clockwise) and introduced into the cartridge through the Pre-Wet Inlet.
  • the pre-wet solution is filled up to the bottom of the syringe reservoir which is used for whole blood introduction.
  • Peristaltic pump 2 is at ideal mode, which stops the pre-wet solution going into the waste blood fluid pathway.
  • Disposable syringe is utilized as a reservoir for reagents introduction, which is inserted into the inlet of the cartridge.
  • peristaltic pump 2 After the cartridge is pre-wetted, whole blood sample (1-20 ml) is pipetted into the syringe. The blood solution is withdrawn through the microsieve with flow rate controlled by peristaltic pump 2 (peristaltic pump 1 is switched off). The required steps of blood residual wash, fluorescence antibodies staining, etc. are all conducted using peristaltic pump 2 for fluidic control.
  • the microsieve 5, 10 is arranged to be in fluidic communication with the channel 115 that is, in turn, in fluidic communication with the (syringe 105) reservoir containing the sample fluid.
  • the channel 115 is positioned along a plane of or parallel to the plane or lateral of the microsieve 5, 10.
  • the device 110 may include only the microsieve membrane 5 or both the microsieve membrane 5 and the microsieve membrane support layer 10.
  • the (syringe 105) reservoir is positioned laterally offset from (i.e., not above) the microsieve. This way, the sample fluid is fed from the (syringe 105) reservoir through the channel and into the microsieve in a lateral (or transverse) direction.
  • the microsieve is arranged in this way to allow direct cell imaging, and to facilitate cell elution by centrifugal force, independent of the micropore shape.
  • a centrifugal method is used to elute the captured cells from the microsieve and to collect the eluted cells for downstream molecular analysis.
  • the cell-elution plug is removed from the cartridge, and a cell collection tube is connected to the Cell Elution port. After that, the cartridge is mounted on a centrifuge with the Cell-Elution port facing outward (Fig. 29).
  • a cell will be subjected to various forces depending on its location within a cartridge (Fig. 30).
  • F m radial buoyancy
  • P pl centrifugal pressure
  • the combination of these two forces assists the release of captured cells from the microsieve surface (Fig. 30).
  • F m buoyancy force
  • Stokes viscous drag force
  • P p2 centrifugal pressure
  • the buoyancy force is given by: Where m is the mass of a cell, ⁇ is the angular rotation frequency, r is the distance between the cell and the central axis, and pi and p c represent the densities of the fluid and the cell, respectively.
  • the drag force is given by:
  • n is the viscosity of fluid
  • R is the radius of the cell
  • v is the velocity of the cell with respect to the surrounding medium.
  • the centrifugal pressure generated by the liquid underneath the microsieve is given by:
  • the buoyancy force is given by:
  • ⁇ is the angular rotation frequency
  • r is the distance between the cell and the central axis
  • p represents the densities of the fluid and the cell, respectively.
  • This centrifugal pressure will generate a force to push captured cells out of the microsieve surface.
  • This magnitude of centrifugal pressure can be easily controlled by adjusting the angular frequency of a centrifuge.
  • a centrifugal method to transport captured cells from the microsieve surface to the microsieve window for cell imaging (Fig. 31).
  • the pre- wet inlet and waste outlet of a cartridge are capped after cell capture.
  • the cartridge is mounted on a centrifuge with the cartridge standing vertically, the elution outlet pointing upward, and the microsieve surface facing outward.
  • a cell will be subjected to a buoyancy force (Fm) generated from the cell itself under the centrifugal spinning, and a pushing force generated from the centrifugal pressure (P p ) of the liquid underneath the microsieve (Fig. 32).
  • Fm buoyancy force
  • P p centrifugal pressure
  • the buoyancy force is given by:
  • pi is the density of the liquid
  • ⁇ is the angular velocity
  • r avg is about the distance of the microsieve to the center of the centrifuge
  • Ar is the length of the liquid sample underneath the microsieve.
  • This centrifugal pressure will generate a force to push captured cells out of the microsieve surface. In addition, it can be easily controlled by adjusting the angular frequency of the centrifuge.
  • Table 1 shows the calculated centrifugal forces for a cartridge in lateral (F p i) and vertical (F pu ) positions respectively, with centrifugal frequencies of 1-50 Hz.
  • the cartridge contains the following features: - A cartridge with an analyte introduction inlet, elution outlet, waste outlet, pre-wet inlet, and a microsieve.
  • a cartridge contains a window on top of the microsieve area.
  • Another embodiment of a cartridge contains an integrated microsieve.
  • the cartridge contains an elution outlet for analyte elution.
  • the elution outlet is located at the opposite side of pre-wet inlet and waste outlet.
  • the pre-wet inlet contains a vertical zigzag structure made of channels and vias.
  • Fluid manipulation is conducted using two peristaltic pumps for analyte-solution manipulation and device pre-wetting.
  • a centrifugal approach is utilized to release captured cells from the microsieve surface, and to elute them out of the cartridge.
  • a cartridge is positioned laterally with the elution outlet facing outward.
  • a centrifugal approach is utilized to transport captured cells to the microsieve window.
  • a cartridge is positioned vertically with the elution outlet pointing upward, and the microsieve window facing outward.
  • a centrifugal approach is utilized to lift off captured cells from the microsieve surface using the liquid packet underneath the microsieve and channels. Identifying the captured objects of interests (cells)
  • the captured cells are stained with various fluorescent-probe conjugated antibodies.
  • the captured cells are normally stained with fluorescence biomarkers (such as DAPI (nucleus), anti-CD45 (targeting WBCs), anti-EpCAM and anti-pan-cytokeratin (both for CTCs)).
  • fluorescence biomarkers such as DAPI (nucleus), anti-CD45 (targeting WBCs), anti-EpCAM and anti-pan-cytokeratin (both for CTCs)
  • a fluorescence microscope is normally utilized to scan across the whole microsieve area and to capture the fluorescence images of each color/biomarker.
  • a microscope normally has limited viewing area in order to obtain high spatial resolution of cell images. Thus, one often utilizes a software program to stitch individual images together into a full image before cell identification.
  • the subsequent image is overlapped slightly with the precedent image to obtain a common area/image which is utilized as references for image stitching.
  • This approach usually works well for images with rich pattern information where the common area contains sufficient information to facilitate image stitching. However, this approach will fail when there is no pattern within the common area. This situation may occur for the rare cell identification where the captured images contain zero cell, generating a blank image (Fig. 34c).
  • Fig. 34b we utilize a novel approach on using the bright field image of microsieve structure as references for cell stitch. In our approach, we capture the bright field image first, followed by the fluorescence images of each fluorescence biomarker, then we overlap the fluorescence images with the bright field one (Fig. 34d). As the microsieve holes have very consistent structure, this approach greatly simplifies the image stitching, and works for the situation of zero-cell within the imaging area.
  • Figure 35 illustrates the process of image stitching utilizing the bright field image as references, where the CCD imaging area is XxY mm.
  • the microscope first captures the bright-field image followed by the fluorescence images of each antibody. This process captures a set of images at the same position. After that, the microscope moves to the next position and repeats the same imaging process.
  • the overlapping distance between the precedent and subsequent images are ⁇ and ⁇ in the X and Y directions, respectively.
  • the overlapping in the X and Y direction assists in the lateral and vertical image stitching, respectively.
  • Fig. 36 illustrates the approach for CTCs identification.
  • the captured cells are stained with five different fluorescent biomarkers (Colors 1-5), which may bind to elements on the cell surface or inside the cells.
  • the fluorescence images are captured using the optical system depicted in Fig. 33, by employing the image stitching approach discussed at Figs. 34 and 35.
  • DAPI morphology of cell nuclear
  • EpCAM epithelial cell
  • CD45 leukocytes
  • the segmented images were further filtered based on the size cut-off values for DAPI-stained nuclei (DAPI+: 8-22 ⁇ ), EpCAM-stained epithelial CTCs (EpCAM + : 10-30 ⁇ ) and CD45-stained leukocytes (CD45 + : 8-18 urn). Only cells which were DAPI + CD45 EpCAM + were counted as CTCs, while cells which stained up as DAPI + CD45 + EpCAM" were counted as leukocytes and those cells/objects showing DAPI + EpCAM + CD45 + or DAPI + EpCAM CD45- were recorded as contaminants and unknowns, respectively.
  • the microsieve is co-fabricated with on-device marks to assist in: 1) device-to-device fluorescence image overlapping, and 2) automated image focusing.
  • the approach and application of device-to-device image overlapping is discussed in Fig. 37 while the automated image focusing is illustrated in Fig. 38.
  • CTCs Human whole blood is known to contain various blood cells, and circulating cells (such as endothelial progenitor cells (EPCs), circulating endothelial cells (CEC)) which may also be co-captured by the size separation approach.
  • EPCs endothelial progenitor cells
  • CEC circulating endothelial cells
  • CTCs are heterogeneous and may contain various phenotypes such as cancer stem cells, cells under the epithelial- mesenchymal transition and/or mesenchymal-epithelial transition, etc. As the CTCs are released from primary tumor cells, the phenotypes of CTCs are known to correlate with primary tumor cells at certain levels. It is known that cancer has various sub-types. For example, Blows (F.M.
  • Blows, et al., 2010 has classified the breast cancer into six major subtypes using five immunohistochemical markers: the estrogen receptor and the progesterone receptor (two hormone receptors expressed by luminal cells), the human epidermal growth factors receptor-2 (HER2, a protein marker used to select specific adjuvant therapies), and C 5/ 6 and EGFR (proteins expressed by basal cells).
  • the estrogen receptor and the progesterone receptor two hormone receptors expressed by luminal cells
  • HER2 human epidermal growth factors receptor-2
  • C 5/ 6 and EGFR proteins expressed by basal cells.
  • the current microscope has limited capability to detect more than 5 different fluorescence biomarkers in one experiment due to the overlapping of the emission spectrum of fluorescence probes/dyes.
  • NA the objective numerical aperture
  • ⁇ the wavelength of illuminating light
  • n the refractive index of the medium between the cover slip and the objective lens
  • e the pixel size of a CCD camera
  • M magnification of the objective lens
  • Fig. 38 illustrates the methods of automated focusing adjustment using the on-device marks as references. We first measure the focusing conditions of the four on-device marks which are located at the four corners of the microsieve device.
  • the x n , and y n are the positions in x and y axis, while the z n is the focus distance.
  • the z is set to o.
  • the constant of a, b, c and d can be determined from the data points of Pi- P3.
  • the focus distance (z) can then be automatically compensated by moving the objective lens or the motorized stage holding the microsieve device (Fig. 38c) as the microscope scans across the microsieve surface.
  • Another approach is to adjust the focus plan of the microsieve device using a x-y-z tilting stage (Fig. 38d) before cell imaging.
  • the microsieve device is mounted on the tilting stage where the plan of the tilting stage is adjustable with adjustment screw.
  • Figure 39 illustrates the process flow chart for CTCs detection using our microsieve- cartridge. CTCs are first captured on the microsieve device surface using the microsieve cartridge. Captured cells are then stained with a set of fluorescent biomarkers directly on the microsieve-cartridge. The fluorescent cell images are then analyzed using a fluorescence microscope.
  • the microscope first measures the focus condition of the microsieve, by measuring the focus distances derivation at the four on-device marks, and then compensates the focus distance variation of the microsieve device before cell imaging. After that, the fluorescence microscope captures the fluorescence cell images based on the number of fluorescence biomarkers and stitch images together using the bright field images as a stitching reference. If one would like to stain captured cells with additional fluorescence biomarkers, the existing fluorescence biomarkers are then photo- bleached using the fluorescence microscope. After that the captured cells are further stained with another set of fluorescence biomarkers and repeating the image capturing process.
  • the captured cell images of all biomarkers are aligned and overlapped into one image file which contains the fluorescence information from all fluorescence biomarkers.
  • the captured cells are then sub-typed and grouped based on existing information of fluorescence biomarkers.
  • the effective membrane area is either 3.8 mm or 5 mm in diameter, and the membrane thickness could be either 15 ⁇ or 40 ⁇ , for all four structures.
  • Structure 1 consists slot (rectangular) micropore structures with dimension of 5 ⁇ in width (s 2 ), and 30 ⁇ in length (si). The space between hole is 5 ⁇ (y and z in Fig. 3), which corresponds to a pore array of -2857 pores/mm 2 , ⁇ 5.6xio4 pores per device or -43% porosity.
  • Structure 2 consists cross-shape micropore structures with branches or arms dimension of 5 ⁇ in width (h 2 ), and 30 ⁇ in length (hi). The space between each micropore structure is 5 ⁇ , which corresponds to a pore array of -1975 pores/mm 2 , -3.88x1 ⁇ 4 pores per device or ⁇ 42% porosity (Fig. 9b).
  • Structure 3 consists slot (rectangular) micropore structures of 7 ⁇ in width (s 2 ), and 30 ⁇ in length (s .
  • the space between each micropore structure is 5 ⁇ , which corresponds to a pore array of ⁇ 238i pores/mm 2 , -4.6x1 ⁇ 4 pores per device or -50% porosity (Fig. 9c).
  • Structure 4 consists circular micropore structures of 8 ⁇ in diameter with 15- ⁇ pitch, corresponding to a densely packed pore array of -5000 pores/mm 2 or ⁇ ios pores per device or - 40% porosity (percentage of hole area) (Fig. 9a). 2.
  • HepG2/GFP liver cancer
  • MDA-MB231 breast cancer
  • American Type Culture Collection, Manassas, VA, USA cell lines were cultured on 75 cm 2 tissue culture flasks (Corning Life Sciences, Lowell, MA, USA).
  • HepG2 cells were transfected with a green fluorescent protein (GFP) reporter plasmid (pMax-GFP, Amaxa, Lonza, Switzerland).
  • GFP green fluorescent protein
  • the HepG2/GFP cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) containing 10% fetal bovine serum (FBS, Gibco, Invitrogen, USA), 1% (v/v) penicillin/ streptomycin (Gibco), and 0.8% G418 sulphate solution (PAA Laboratories GmbH, Austria) for 3-4 days at 37°C with 5% C0 2 supplementation.
  • MB231 cells were cultured in a similar manner as described above, in DMEM and RPMI (Gibco, Invitrogen) containing 10% FBS, respectively. Prior to each experiment, cells grown to confluence were trypsinized and re-suspended in their respective growth medium.
  • MB231 and HepG2/GFP cells were used for cancer cell spiking experiments.
  • MB231 cells were pre-stained with Hoechst (Invitrogen, USA), while HepG2/GFP cells were used without antibody pre-staining.
  • Cell concentrations were first determined by manual counting using a hemocytometer (Hausser Scientific, Horsham, PA, USA), and serially diluted with Dulbecco's phosphate-buffered saline (DPBS; PAA Laboratories GmbH) to an approximate concentration of 600 cells ⁇ L.
  • DPBS Dulbecco's phosphate-buffered saline
  • the cell suspension volume amounting to 3000 cells was spiked into 2 mL of PBS/EDTA/BSA buffer (DPBS with 2 mM EDTA (ethylenediaminetetraacetic acid; Promega, USA) and 0.5% BSA (bovine serum albumin; Sigma-Aldrich, USA)).
  • PBS/EDTA/BSA buffer DPBS with 2 mM EDTA (ethylenediaminetetraacetic acid; Promega, USA) and 0.5% BSA (bovine serum albumin; Sigma-Aldrich, USA)
  • Fluorescence imaging was performed on a Carl Zeiss Axiolmager Zi with a motorized xy stage, a Colibri LED light source (Carl Zeiss) and a AxioCam MRm CCD camera (Carl Zeiss, Germany).
  • the MetaMorph software (Molecular Devices, Sunnyvale, CA, USA) was used to acquire images - to control the motorized xy stage to scan the entire microsieve area.
  • Automated cell counting was performed by using MetaMorph Multi Wavelength Cell Scoring module (Molecular Devices) to analyze the stitched images. The number of MB-231, HepG2/GFP and white blood cells is counted based on the Hoechst dye (blue) and GFP (green), and AlexaFluor-488 dye (green) respectively.
  • the shear force enhanced tumor cell isolation was first studied by comparison of capture efficiency versus flow rate using microsieves with slot (rectangular) micropore structures (width of 5- ⁇ and 7- ⁇ , respectively) and cross-shape holes.
  • the microsieve dimension is 5 mm in diameter with membrane thickness of 15 ⁇ .
  • the cross-shape micropore structures have branches/arms dimension of 5 ⁇ in width, and 30 ⁇ in length (Structure 2 in Fig. 9b), while the slot (rectangular) micropore structures are 30 ⁇ in length with a width of 5 ⁇ (Structure 1) and 7 ⁇ (Structure 3), respectively.
  • MB231 cells are spiked into 2 ml of PBS/EDTA/BSA buffer, and then filtered through the microsieve using the lateral flow system (Fig. 11) at a very low flow rate of 0.1 ml/min to gently capture the cancer cells on top of microsieve surface.
  • the number of capture cells is then automatically counted using a Carl Zeiss fluorescence microscope and MetaMorph software, which serves as the baseline cell number (termed initial cell count).
  • captured cells are washed with 2 ml of PBS/EDTA/BSA buffer with various flow rate of 1-4 ml/min, to introduce pressure and shear force on the captured cells.
  • the cell number (termed remaining cell count) is counted after every 2-ml buffer wash step using the Carl Zeiss fluorescence microscope and MetaMorph software.
  • Figure 12 shows the relationship of the ratio of remaining MB231 cancer (remaining cell count / initial cell count) versus the flow rate for the slots and cross-hole microsieves. The results clearly indicate that MB231 cancer cell (-15-20 ⁇ in diameter) can gradually deform and squeeze through the microsieve-micropore structures at elevated flow rates (> 2 ml/min), and the ratio of remaining cells depends on the flow rate and hole structures.
  • Figures 13-15 shows the images of cells on top of cross-shape micropore structure (Fig. 13), slot (rectangular) micropore structure (Fig. 14) and circular micropore structure (Fig. 15).
  • the images clearly show that the white blood cells and MB231 cancer cells only partially cover the micropore structure areas for the cross-shape and slots (rectangular) micropore structure, while wholly cover the circular micropore structure.
  • Experiment 2 The effect of microsieve thickness on cancer cell isolation was further studied by comparison of capture efficiency versus flow rate using microsieves with 15- ⁇ and 40- ⁇ thick cross- shape micropore structure, respectively.
  • the microsieve dimension is 3.8 mm in diameter.
  • the cross-shape hole has arms dimension of 5 um in width, and 30 ⁇ in length (Structure 2 in Fig. 9b).
  • the cancer cell isolation and cell counting process are the same with Experiment 1.
  • the results (Fig. 16) indicate that a thick microsieve is more sensitive to flow rate than that of a thin one. Cells encounter both of fluidic pressure and shear force when they are located on the microsieve surface.
  • the shear force is proportional to the flow rate and unrelated to the membrane thickness at a fixed flow rate, while the pressure is proportional to the membrane thickness.
  • a thicker membrane has a higher fluidic resistance, and generates a corresponding higher pressure than that of a thin one at the same flow rate.
  • captured cells encounter a higher force as filtered with a thick membrane at the same flow rate than that of a thin one.
  • the microsieve performance of cross-shape (Structure 2) and circular (Structure 4) micropore structure for MB231 and HepG2 cancer cell isolation with various flow rates were compared.
  • the microsieve dimension is 3.8 mm in diameter with membrane thickness of ⁇ 5 ⁇ or 40 ⁇ .
  • the cancer cell isolation and cell counting process are the same with Experiment 1. Briefly, 3000 Hoechst-stained MB231 and HepG2/GFP cells respectively are added in 2 ml PBS and filtered through the microsieves at a flow rate of 0.1 ml/min and then washed with 2 ml of PBS buffer repeatedly at flow rates of 1-4 ml/min. The remaining cell number is counted after each wash steps.
  • Figure 17 shows the results which clearly indicates that microsieve with cross-shape micropore structure is more sensitive to the flow rate than that of circular micropore structure. More cancer cells are dragged through the cross-shape microsieve than that of circular one at the same flow rate.
  • the MB231 (diameter: -15-20 ⁇ ) and HepG2 (Diameter: ⁇ 12-20 ⁇ ) are rounded shape with size larger than the circular micropore structure.
  • cancer cells can wholly cover the 8- ⁇ circular holes, and sit on top of the hole surface. Cells need to deform and squeeze into/through the micropore structure in responding to an increased flow rate (pressure).
  • cross-shape micropore structure with 4 arms, fluid can flow through the un-occupied arms area which provides additional shear force on cells. Moreover, the cross-shape micropore structure provide extra space for cell to deform. Both of these effects makes the cross-shape micropore structure more sensitive to the flow rate.
  • the performance of cross-shape microsieve in isolation of MB231 and white blood cells were compared.
  • the microsieve dimension is 5 mm in diameter with membrane thickness of 15 ⁇ .
  • 3000 Hoechst-stain MB231 cancer cells are mixed with 3000 AlexaFlour-488 labeled WBCs (through anti-CD45 antibody), and added into 2 ml PBS.
  • the mixture was filtered through the microsieve at a flow rate of 0.1 ml/min and then washed with 2 ml of PBS buffer repeatedly at flow rates of 1-5 ml/min.
  • the remaining cell number of MB231 and WBCs is counted after each wash steps.
  • Fig. 18 demonstrates that WBC is more sensitive to flow rate than MB231 cancer cells. The remaining WBC count drops more rapidly than that of MB231 as flow rate increased. The results indicate one can utilize the shear force (flow rate) to increase the purity of captured cells population or the ratio of cancer cells versus WBCs.
  • Fig. 19 The results indicate that the microsieve with cross-shape micropore structure has better cancer cell capture yield (average ⁇ 100%) than that of the slot (rectangular) micropore structure ( ⁇ 88%).
  • the cross-shape micropore structure capture more white blood cells than that of the slots (rectangular) micropore structure.
  • Figure 20 shows an example of the fluorescence cell images of cells captured on top of microsieve surface, with whole blood from a breast cancer patient. These images clearly demonstrated the capability of the microsieve membrane in enrichment of circulating tumor cells from whole blood. We found that some of CTCs did not stain with EpCAM and CD45 (DAPI + CD45'EpCAM ) antibodies, which demonstrated that our device is capable to capture both EpCAM-negative and -positive CTCs releasing from the cancer tissues.
  • Blood is a non-Newtonian fluid that is primarily made of highly deformable red blood cells (RBCs; 8- ⁇ diameter and 2- ⁇ thickness; >io9 cells/mL), spherical white blood cells (WBCs; ⁇ 10 ⁇ diameter; >io 6 cells/mL), and discoid platelets ( ⁇ 2 ⁇ diameter) suspended in plasma.
  • RBCs red blood cells
  • WBCs spherical white blood cells
  • ⁇ 10 ⁇ diameter >io 6 cells/mL
  • discoid platelets ⁇ 2 ⁇ diameter suspended in plasma.
  • the mechanism of human whole blood filtration through a microsieve is controlled by a combination of externally applied pressure/force, structure and dimension of the micropores and the fluid resistance of the microsieve membrane.
  • the present invention utilizes a microsieve filter with unique micropore structure for CTCs isolation, where the WBCs and CTCs are unable to wholly cover the individual pore area. This innovative pore structure provides additional space for cell to
  • the rigid silicon structure enables CTC capture, cell staining and fluorescence imaging directly on the microsieve, with a simple peristaltic pumping regulating sample filtration rates.
  • the relative small device footprint (5 mm in diameter) provides additional advantage for efficient fluorescence imaging and cell identification and classification.
  • the microsieve device was fabricated using matured silicon fabrication technology, which will greatly facilitate high-throughput device production and enable larger-scale clinical studies at a reasonable cost.
  • One 6" wafer is capable of producing ⁇ 200 devices per wafer.
  • the non-autofluorescence silicon material offers additional imaging benefit for on-microsieve fluorescence imaging.
  • the present invention provides the following unique advantages:
  • the cartridge integrates the microsieve device with fluid channels, and chambers for fluid manipulation. Isolated CTCs are captured on top of the microsieve surface, which can be either enumerated directly on microsieve using a fluorescence microscope, or be eluted from the microsieve into a collection tube utilizing a centrifugal approach.
  • a microsieve device with high CTC recovery rate and purity includes a unique pore structure which effectively separates CTCs from WBCs based on the size and deformability difference of these two types of cells.
  • the device has a cross-shape or slot pore, which is capable of recovering > 85% of the CTCs, better than that of a circular-pore device.
  • the technology uses microfabricated membrane to isolate CTCs. This is relatively inexpensive as compared to other CTC isolation technologies.
  • the microsieve structure is utilised as a reference to facilitate the stitching of irregular and rare events of cell images, which eliminates the miss-stitching issue.
  • the microsieve device is co-fabricated with marks which provide on-device references for microscope focusing and automated focus plan adjustment.
  • the co-fabricated marks provide the reference for overlapping of fluorescence images performed at subsequent immuno- fluorescence experiments. This feature enables one to detect > 20 antibody-antigen reactions of CTCs for CTC sub-typing.

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  • Dispersion Chemistry (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)

Abstract

La présente invention concerne un microtamis. En particulier, elle concerne un microtamis présentant une structure des micropores améliorée pour la détection extrêmement efficace et rapide de cellules tumorales circulantes (CTC) dans des échantillons de sang total. Plus particulièrement, elle concerne une membrane de microtamis comprenant une pluralité de structures à micropores, chaque structure à micropores ayant une forme et une dimension telles que l'objet d'intérêt ayant une dimension égale ou supérieure à la dimension définie de la structure à micropores est retenu sur la membrane du microtamis sans recouvrir toute la structure à micropores. L'invention concerne des dispositifs et des procédés intégrant cette membrane de microtamis.
PCT/SG2014/000122 2013-03-13 2014-03-13 Microtamis WO2014142754A1 (fr)

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JP2016180753A (ja) * 2015-03-23 2016-10-13 アークレイ株式会社 稀少細胞を分離又は検出する方法
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US11717670B2 (en) 2017-06-07 2023-08-08 Shifamed Holdings, LLP Intravascular fluid movement devices, systems, and methods of use
CN107271440A (zh) * 2017-07-01 2017-10-20 中山大学 一种寄生虫虫卵的便携式检测装置及方法
US11511103B2 (en) 2017-11-13 2022-11-29 Shifamed Holdings, Llc Intravascular fluid movement devices, systems, and methods of use
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US10722631B2 (en) 2018-02-01 2020-07-28 Shifamed Holdings, Llc Intravascular blood pumps and methods of use and manufacture
US11964145B2 (en) 2019-07-12 2024-04-23 Shifamed Holdings, Llc Intravascular blood pumps and methods of manufacture and use
US11654275B2 (en) 2019-07-22 2023-05-23 Shifamed Holdings, Llc Intravascular blood pumps with struts and methods of use and manufacture
US11724089B2 (en) 2019-09-25 2023-08-15 Shifamed Holdings, Llc Intravascular blood pump systems and methods of use and control thereof
CN114514309A (zh) * 2019-09-26 2022-05-17 京瓷株式会社 细胞检测装置和细胞检测方法
CN114514309B (zh) * 2019-09-26 2024-05-24 京瓷株式会社 细胞检测装置和细胞检测方法

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