WO2016019401A1 - Microfluidic device with smooth surface for enrichment of rare cells and biomarkers from a biological fluid - Google Patents

Microfluidic device with smooth surface for enrichment of rare cells and biomarkers from a biological fluid Download PDF

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Publication number
WO2016019401A1
WO2016019401A1 PCT/US2015/053337 US2015053337W WO2016019401A1 WO 2016019401 A1 WO2016019401 A1 WO 2016019401A1 US 2015053337 W US2015053337 W US 2015053337W WO 2016019401 A1 WO2016019401 A1 WO 2016019401A1
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Prior art keywords
cell
capture
micro fluidic
fluidic device
microfluidic device
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PCT/US2015/053337
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French (fr)
Inventor
Yi Dong
Chunsheng Jiang
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Medvisionus Llc
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Priority to CN201580053017.6A priority Critical patent/CN107148468B/en
Publication of WO2016019401A1 publication Critical patent/WO2016019401A1/en

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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502761—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads, for physically stretching molecules
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/06—Fluid handling related problems
    • B01L2200/0647—Handling flowable solids, e.g. microscopic beads, cells, particles
    • B01L2200/0668—Trapping microscopic beads
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0848—Specific forms of parts of containers
    • B01L2300/0851—Bottom walls
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/08—Regulating or influencing the flow resistance
    • B01L2400/084—Passive control of flow resistance
    • B01L2400/086—Passive control of flow resistance using baffles or other fixed flow obstructions

Definitions

  • the invention relates to the fields of micro fluidic devices, cell detection, cell separation, and molecular biomarkers.
  • Circulating tumor cells originate from primary tumors and perhaps from metastatic sites. Cancer cells disseminate from a primary tumor into the circulatory system as a first step toward metastasis in distant organs. An average tumor may release an estimated million cells per day into the bloodstream. Butler et al., "The Physical and functional behavior of capture antibodies adsorbed on polystyrene. Butler et al., "" L
  • CTCs do not survive, but those that do survive pose a risk of further cancer to the host organism. Metastasis is the leading cause of death in cancer patients. For solid tumors, the presence of CTCs is evident late in disease and is most apparent when metastatic disease sites are already established. Identification and characterization of CTCs offers an opportunity to study, monitor, and, ultimately, alter the metastatic process.
  • CTCs are exceedingly rare cells, being present at 1-10 CTCs per milliliter (mL) of whole blood.
  • Ordinary blood cell types are present in tremendous numbers by comparison, and patients with cancer may have altered levels of other blood cell types to consider. These other cell types may comprise leukocytes (about 7 million/mL blood) and red blood cells (about 5 billion/mL blood).
  • One prior CTC-chip is a silicon chamber etched with 78,000 microposts in staggered arrangement with 50- ⁇ spacing. The posts are coated with anti-EpCAM antibodies. The CTC capture yield is limited at about 60% (Nagrath et al., Isolation of rare circulating tumour cells in cancer patients by microchip technology," Nature, 450: 1235- 1239 (2007).
  • the "post” like structures have major disadvantages due to short cell-post contact length, high shear stress, and low capture rate.
  • cfDNA circulating cell-free DNA
  • ctDNA circulating tumor D A
  • a microfluidic device in one aspect of the invention, includes an inlet; an outlet; an enclosed fluid channel and, in operation, a biological fluid flowing within the channel, the biological fluid containing a rare amount of whole biological cell analyte; and a cell capture module in fluid communication between the inlet and the outlet via the channel, the cell capture module comprising at least one cell capture chamber comprising a smooth- featured surface on the bottom of the chamber, the smooth- featured surface characterized by a smooth topographic surface profile that simulates the topographic surface profile of a naturally-occurring water body.
  • the cell capture module comprises greater than one cell capture chamber having a smooth- featured surface, each capture chamber in fluid communication with the inlet through the channel, and the smooth-featured surface characterized by a smooth topographic surface profile that simulates the topographic surface profile of a naturally-occurring water body.
  • each of the cell capture chambers is of equivalent dimensions.
  • the cell capture modules have equal flow rates.
  • the microfluidic device has at least one of the cell capture chambers having dimensions which differ from dimensions of another cell capture chamber.
  • the microfluidic device of the invention can include a cell capture chamber which further comprises a smooth- featured surface on the top of the chamber.
  • All of the micro fluidic devices disclosed herein are improved from prior microfluidic devices by having surfaces within one, some, or all of the one or more cell capture chambers which are smooth featured surfaces.
  • the smooth featured surfaces are designed to simulate, or to mimic, the topographic features of the bottom surface of a naturally-occurring water body, i.e., the naturally-occurring topographic features of a sea floor, an ocean floor, or a river bed or stream.
  • the smooth-featured surface includes an array of smooth features, each of the smooth features characterized by a profile.
  • the smooth features can be designed by simulation can be based on a
  • the surface profile includes surface features with a half peak width between 10 and 5000 micrometers.
  • the surface profile which is characteristic of the surfaces of microfluidic devices of the invention enhance cell rolling on the surface. This surface profile prolongs cell-surface interactions, promotes affinity based rare-cell isolation, and reduces shear stress.
  • the surface profile which is characteristic of the surfaces of microfluidic devices of the invention also can include surface features of optimal peak height
  • trough depth of "valley”
  • slope and curvature between peak and trough Such surfaces promote local blood mixing in the cell capture chamber, and facilitate easy release of captured rare cells while maintaining cell viability and integrity.
  • the microfluidic device of the invention has a sample throughput of 1 to 20 milliliters per hour.
  • the microfluidic device of the invention has a variable sample volume of 0.25 milliliters to 50 milliliters.
  • the microfluidic device of the invention has one or more of the following properties: reduces optical interference, enhances visualization, and improves rare cell identification.
  • the microfluidic device of the invention includes a bottom surface having capture ligands attached thereto.
  • the whole biological cells bind to the capture ligands so as to isolate cells present on the top of the surface, to facilitate automated scanning and rare cell detection, analysis and characterization.
  • the surface profile comprises microValley features, and the micro Valleys have dimensional ranges of 10 to 1000 micrometers wide, 20 to 1000 micrometers long, and 20 to 1000 micrometers high.
  • the surface can include an array of microValley features having regular relative positions with gap size between 5 and 500 micrometers.
  • the microValley has a regularly shaped profile.
  • the microValley is in an array of micro Valleys, and the array is arranged irregularly.
  • ⁇ Valley is a micro concave structure that can be simulated with a smooth functions such as Gaussian curves. This micro structure can promote high flow rate, increasing fluid mixing and reduce share stress to the cells.
  • the surface profile comprises an array of microFunnel features and microSp litter features, the array having a dimensional range of 10 to 100 micrometers wide, 20 to 200 micrometers long, and 20 to 200 micrometers high.
  • the array can be arranged in a layout of optimized externally tangent circles.
  • the cell capture module is sufficiently large to process 7.5 mL of blood between 1 hour to 24 hours.
  • the cell capture chambers are characterized by matching surface profiles.
  • the devices further comprise a non-cell biomarker module, wherein the non-cell biomarker module is in fluid communication between the cell capture module and the outlet.
  • the non-cell biomarker module can include capture ligands for binding to analytes in a sample fluid, the analytes selected from the group consisting of a DNA marker such as ctDNA or cfDNA, an RNA marker, a peptide marker, and a protein, a biomarker-carrying microparticle such as microvesicles, exosomes, and apoptoic bodies.
  • the invention includes a method for enriching a fluid sample for a rare whole cell, comprising flowing a fluid sample comprising a rare amount of the whole cells through any of the micro fluidic devices disclosed herein.
  • the rare whole cells are circulating tumor cells.
  • the method further includes releasing the whole cells from the cell capture module, the releasing via a combination of mechanical, chemical and biological mechanisms.
  • the invention further includes a method a for enriching a fluid sample for a rare whole cell, comprising flowing a fluid sample comprising a rare amount of the whole cells through any of the microfluidic devices disclosed herein.
  • the non- cell biomarker detection module comprises more than one type of capture ligand.
  • the method can comprise collecting fluid from the outlet, wherein the fluid is enriched for analytes released from capture ligands immobilized to the non-cell biomarker detection module.
  • analyte is meant a molecule or component in a fluid which is the target of a method of, as the case may be, detection, isolation, concentration, or extraction.
  • exemplary analytes include cells, viruses, nucleic acids, proteins, carbohydrates, and small organic molecules.
  • blood component any component of whole blood, including host red blood cells, white blood cells, and platelets. Blood components also include the components of plasma, e.g., proteins, lipids, nucleic acids, and carbohydrates, and any other cells that may be present in blood, e.g., because of current or past pregnancy, organ transplant, or infection.
  • Bio fluid is meant to include naturally occurring fluids (e.g., blood, lymph, cerebrospinal fluid, urine, cervical lavage, saliva, and water samples), portions of such fluids, and fluids into which cells have been introduced (e.g., culture media and liquefied tissue samples).
  • the term also includes a lysate.
  • capture moiety or “capture ligand” is meant a chemical species to which, as the case may be, an analyte binds or a whole cell binds by virtue of its surface components.
  • a capture moiety may be a compound coupled to a surface or the material making up the surface.
  • Exemplary capture moieties include antibodies, oligo- or polypeptides, nucleic acids, other proteins, synthetic polymers, and carbohydrates.
  • channel is meant a gap through which fluid may flow.
  • a channel may be a capillary, a conduit, or a strip of hydrophilic pattern on an otherwise hydrophobic surface wherein aqueous fluids are confined.
  • CTC circulating tumor cell
  • component of a cell is meant any component of a cell that may be at least partially isolated upon lysis of the cell.
  • Cellular components may be organelles (e.g., nuclei, peri-nuclear compartments, nuclear membranes, mitochondria, chloroplasts, or cell membranes), polymers or molecular complexes (e.g., lipids, polysaccharides, proteins (membrane, trans-membrane, or cytosolic), nucleic acids (native, therapeutic, or pathogenic), viral particles, or ribosomes), or other molecules (e.g., hormones, ions, cofactors, or drugs).
  • component of a cellular sample is meant a subset of cells contained within the sample.
  • enriched sample is meant a sample containing an analyte that has been processed to increase the relative amount of the analyte relative to other analytes typically present in a sample.
  • samples may be enriched by increasing the amount of the analyte of interest by at least 10%, 25%, 50%, 75%, 100% or by a factor of at least 1000, 10,000, 100,000, or 1,000,000.
  • sample a sample containing an analyte that has been processed to decrease the amount of the analyte relative to other analytes typically present in a sample.
  • samples may be depleted by decreasing the amount of the analyte of interest by at least 5%, 10%, 25%, 50%, 75%, 90%, 95%, 97%, 98%, 99%, or even 100%.
  • gap is meant an open passage through which a fluid may flow.
  • micro- is meant having at least one dimension of less than 1 millimeter.
  • naturally-occurring water body is meant sea, ocean, river, or stream.
  • profile is meant a contour side view image.
  • a "rare amount" of cells refers to less than 100 cells in a milliliter of fluid, less than 10 cells per milliliter fluid, or even less than 1 cell per milliliter fluid.
  • “regular” is meant a pattern which is recurring, orderly, methodical or occurs at fixed, uniform or normal intervals; “irregular” means not regular.
  • surface feature is meant a perturbation of the surface above or below the plane of the surface which is co-planar with the bottom of the cell capture chamber.
  • micro-mountain micro-valley
  • micro-funnel micro-funnel
  • micro-splinter describe the contour of a surface feature by analogy to the expected contour of the a conventional mountain, valley, funnel, or splinter, respectively.
  • FIG. 1 illustrates a top view layout of a micro fluidic device having a two chamber cell capture module 1 and a two meander non-cell capture module 2.
  • FIG. 2 is a pictorial illustration of the topography of a seafloor (A, B, and C) and a river bed (D). The direction of flow goes from left to right.
  • Fig. 3 is a three-dimensional representation of a Gaussian curve having a two-dimensional domain.
  • Fig. 4 is a side view of two chip assemblies: (A) topographic chamber floor with flat ceiling; and (B) topographic floor and topographic ceiling.
  • Fig. 5 is a (A) topographic view; and (B) side view of the bottom surface of a microValley array used to isolate rare whole cells from blood samples.
  • FIG. 6A is an illustration of a topographic feature on the bottom surface of a microValley having design control parameters a, b, c, o("sigma"), and A.
  • Fig. 6B is an illustration of array design parameters to consider positioning of micro Valleys relative to each other.
  • FIG. 6C-6H is each an illustration of a different microValley arrangement, where 6C: rectangular positioning; 6D. A-B-A; 6E. short gap-long gap along flow direction; 6F. short gap-long gap perpendicular to flow direction; 6G. short gap
  • Fig. 7 is a schematic illustration of different ways of assembling microValley chips, where: Fig. 7A is an illustration of a regular microValley chip with a flat top; Fig. 7B is an illustration of two matching microValley chips assembled together; Fig. 7C is an illustration of two mismatched microValley chips assembled together; Fig. 7D, Fig. 7E, and Fig. 7F are illustrations of variations from Fig. 7A- Fig. 7C, respectively, where the microValley chips are irregular.
  • FIG. 8 is a process diagram illustrating the progress of rare whole cells in a blood sample on micro Valley-surfaced microfluidic device of the invention, where Fig. 8A: Flow; Fig. 8B: capture; Fig. 8C: wash; and Fig. 8D: release.
  • FIG. 9 is a top-down schematic illustration of a pattern of micro Funnels and microSp litter surface features on the bottom surface of a microfluidic device of the invention. Arrows between the surface features illustrate the direction of flow of a biological fluid between the surface features.
  • FIGs. 10A and 10B are illustrations of the top-down view (Fig. 10A) and profile (Fig. 10B) of a single microFunnel.
  • Figs. IOC and 10D are illustrations of the top-down view (Fig. IOC) and elevated (Fig. 10D) of a single micro Splitter.
  • Fig. 1 1 is a layout optimization of microFunnels and micro Splitters.
  • Fig. 12 is an example of microFunnels and micro Splitters layout and CTC capture from whole blood.
  • Fig. 13 is a side view illustration of four surface chemistries for use to capture whole sales and biomarkers from a biological fluid, where each capture ligand is attached to a substrate through a spacer, the four capture ligands being a complete antibody (Fig. 13A); F(ab')2 (Fig. 13B); rlgG (Fig. 13C); and Fab' (Fig. 13D).
  • An improved microfluidic device for processing rare whole cells, such as circulating tumor cells.
  • the improvements include 1) a smooth surface design in the cell module; 2) cell and biomarker combination capture mechanism, and 3) the "multiplexing" capturing of different biomarkers in the separated bands in the biomarker module.
  • the microfluidic device has two integrated capture modules.
  • the first module contains a capture zone having internal surfaces.
  • the bottom of the internal surface of the capture module contains seafloor or river bed mimicking smooth features at the micrometer ( ⁇ ) to millimeter (mm) level that promote cell-device surface interactions.
  • the smooth surface features are simulated using Gaussian or other smooth mathematical functions.
  • the smooth features create a gentle environment to enhance sample mixing, continuous cell-antibody contact, and rare cell capture.
  • the smooth surface design also reduces shear-force applied to the cells to prevent cell damage during the enrichment processing.
  • the layout of such features is optimized with computer simulation based on Gaussian or other smooth functions. The result is improved cell-surface contact efficiency, reduced shear stress, and enhanced throughput.
  • the first module is coated with ligands that bind to target cells. When biological fluids flow through the device, the target cells are captured on these seafloor or river bed mimicking features via ligand-cell binding.
  • the second module contains chemically separated areas that are coated with capture molecules of non-cell biomarkers. This module takes advantages of the large volume of blood sample used for rare cell isolation to enrich the non-cell biomarkers by at least 100- 1000 fold before further analysis.
  • the detection of capture rare cells and non-cell biomarkers are detected via immuno-staining with detection tags.
  • the captured rare cells can be released and collected by reverse the flow direction and wash with a combination of mechanical, chemical, and biological releasing mechanism.
  • Smooth curves or structures with different sizes can be randomly placed in a microfluidic chamber to form smooth capture surfaces (simulating blood vessels, or simulating the bottom of a naturally-occurring water body) that 1) promote high flow rate; 2) increase fluid mixing and 3) reduce share stress to the cells.
  • the smooth surface is designed to avoid disadvantages of prior art devices which relied on a "post", or on a prior art “micro structure”, or on a prior art “obstacle” (hereafter prior art micro structure).
  • the prior art micro structure has major disadvantages due to short cell-post contact length, high shear stress, and low capture rate.
  • the smooth surface design disclosed herein is optimized to achieve a gentle cell-surface contact without sacrificing the larger overall capture surface area of the entire module chamber. In the microstructure "post” design, the larger surface area is achieved by placing a huge number of small structures (posts).
  • the micro fluidic device 100 is equipped with two modules: cell capture module 1 and non-cell biomarker detection module 2.
  • a fluid sample enters microfluidic device 100 at input 10.
  • the fluid sample can be a sample of a biological fluid, e.g., whole blood. Fluid is pushed or pulled through microfluidic device 100 by a flow force exerted by a pump or by a vacuum (not shown).
  • Cell capture module 1 is composed of one or more cell capture chamber 5. Each cell capture chamber 5 is connected to inlet 10 by an equal length of inlet channel 11. Each channel 1 1 is of the same depth and width. Thus, the sample fluid is evenly split in volume between the one or more chamber 5. Each portion of the blood sample moves through each cell capture chamber 5 simultaneously.
  • the cell capture chambers 5 are filled with computer simulation optimized features and layout which facilitate and prolong cell- surface contact while minimizing shear stress experienced by the cells.
  • the surface 6 of cell capture chamber 5 is coated with specific ligand or ligand combinations (not shown), such as antibody or antibody combos that bind to cell surface antigen or antigens, and as a result, capture cells via Ab-Ag interactions.
  • Target rare cells such as CTCs are captured in this cell capture module 1. Non-target cells flow through the cell capture chamber 5 and exit the cell capture chamber 5 at capture module exit port 3. The captured rare cells are identified and quantified by immuno-staining.
  • the sample fluid After the sample fluid leaves the cell capture chamber 5, the sample fluid enters non-cell biomarker detection module 2.
  • This detection module 2 is composed of micro-channel meanders 7.
  • the inside walls 8 of the channel meanders 7 are coated with bands of specific receptors 12, 13, 14.
  • these receptors capture corresponding biomarkers from the sample fluid.
  • the captured biomarkers are identified and quantified by immuno-staining.
  • cell-capture module 1 and non-cell biomarker detection module 2 can be on one single chip or two chips connected via a connector.
  • Fig. 2 shows the typical topographies of seafloor ( Figure 2A and 2B) and river bed (Figure 2C).
  • Fig. 3 shows a Gaussian curve with two-dimensional domain.
  • suitable mathematical functions for use in simulating the topography of a naturally-occurring water body include sine waves, polynomials of degree 2 or higher, and cubic spline curves.
  • the internal surfaces of the cell capture module 1 of micro fluidic device 100 are designed to mimic the topographies, at a micro-meter level, of the bottom surface of a natural waterway, e.g., a seafloor, ocean floor, or river bed
  • surface designs of the invention are used on the floor and ceiling surfaces of cell- isolation chamber 5.
  • the surfaces can be made smooth with Gaussian-function based computer simulation to optimize the surface in the following aspects :
  • the surface designs of the invention promote mixing of the sample fluid, e.g., blood mixing, preventing constituents in the sample, e.g., CTCs in whole blood, from settling or fractioning. Mixing is optimized by varying the heights and slopes of the Gaussian "mountains,” and depth of the "valleys" in between.
  • Additional benefits of such design include ease of release of captured rare cells due to the capture mechanism that relies mainly on affinity capture enhanced by cell-rolling on the surface. Little physical hindrance exists in releasing cell as experience by existing cell capture devices.
  • the surface designs of the invention also ease visualization and detection by optical methods such as fluorescent imaging due to the fact that angled surface topography greatly reduce the optical interferences.
  • Chip Assembly There are two different ways that cell-capture module 1 can be assembled.
  • the embodiment shown in Fig. 4A uses a flat chamber cover 21, which can be a piece of tape applied via adhesives, or a thin slab of plastic applied via thermal or chemical bonding.
  • the height of chamber walls can be varied to adjust the size of chamber gap 23 between the chamber bottom 22 and the chamber ceiling (the underside of flat chamber cover 21) for best balance between capture efficiency and throughput.
  • Fig. 4B one chip can be flipped over a second chip and held together via an assembly gig. Again, this can be done with variable sizes of chamber gap 33.
  • the benefits are twofold. One benefit is the doubled capture area, because capture ligands can be affixed to the underside (inner side) of top chip 31, as well as to the upper (inner) side of bottom chip 32.
  • the other benefit is the ease of post-isolation processing such as molecular analysis as the two-chip assembly can be reversed and opened for CTC collection or DNA/RNA analysis.
  • FIG. 5 shows the top (Fig. 5A) and side views (Fig. 5B) of a rare cell-isolation chamber.
  • Fig. 5A is the bottom surface 6 of a cell capture chamber 5.
  • the inset displays the micro Valleys array of bottom surface 6.
  • the array design is performed in three aspects: depth and surface profile on individual micro Valleys, relative positioning of micro Valleys in the array, and the optimized combination of the above two aspects.
  • Figure 6A and 6B show the parameters of microValley array optimizations.
  • Figure 6C-H are six examples of many feasible array designs.
  • the parameters a, b, c, ⁇ , A, XI , X2, Yl , Y2, D, are identified as follows:
  • a, b, c, ⁇ , A the parameters that define a two-dimensional elliptical Gaussian function, which is an typical smooth micro structure.
  • the parameters a, b, c define the shape of the elliptical Gaussian function; furthermore, the parameters a, b, c can be derived from the standard deviation ( ⁇ ) of the elliptical Gaussian.
  • XI, X2, Yl , Y2, and D are the parameters that define the arrangement pattern of the micro structures (micro Valley):
  • XI and X2 - defines the column alignment width of the micro structures Yl and Y2 - defines the row alignment width of the micro structures
  • Chip assembly can follow a few routes as shown in Fig. 7.
  • the micro Valley array chip can be assembled with a flat top or another microValley chip. When two micro Valley chips are assembled, different alignment is another parameter that can be optimized for most efficient rare cell isolation.
  • microValley chips are functionalized with rare cell capture ligands, and assembled.
  • the rare cell isolation can be carried out as shown in Fig. 8.
  • surface immobilized capture ligands such as antibodies against rare cell surface antigens.
  • a small amount of blood cells stick to the chip surface through weak physical adsorption. Washing with proper buffer will remove the vast majority of the blood cells and leave behind highly purified rare cells.
  • These rare cells can be immuno-stained for enumeration or released and recollected for molecular analysis.
  • Fig. 9 shows the design scheme for a simplified version of the Gaussian simulation optimized seafloor design.
  • the design consists of many modules of microFunnels and micro Splitters of smooth structure that benefits smooth flow, promotes cell capture by enhancing cell-surface contact, while reduces shear stress to cells.
  • Fig. 10A shows the optimization of each half of the microFunnel's top view profile. This profile has a backbone made of a series of externally tangent circles with various radiuses. Along the flow direction, the radiuses increase gradually to narrow the width of the microFunnel, then decrease for a wider opening.
  • Fig. 10B shows the side view cross section profile of a microSp litter or one half of a microFunnel.
  • Fig. 10D is a 3-D rendering of a microSplitter or one half of a microFunnel.
  • Fig. IOC is top view of a microSplitter.
  • the opening angle (2a) of the microFunnel is optimized to enhance cell- funnel wall interaction while maintaining a flow rate high enough to sustain desired throughput.
  • the outlet opening (do) of the microFunnel is optimized relative to the size of the target cell.
  • the distance between two microFunnels (di) is adjusted according to ratio of (between microFunnel volume/2X in microFunnel volume).
  • the relative position of microSp litters relative to surrounding microFunnels (LI, L2, Ul and U2) are adjusted to split and distribute upstream blood flow towards the inside walls of the microFunnels.
  • Fig. 1 IB shows the elevation map of such a chip layout.
  • FIG. 12 shows the scheme of CTC isolation from whole blood.
  • the chip surfaces are coated with capture molecules that bind to targets on the cell surface of CTCs.
  • the design facilitates smooth rolling of CTCs on the chip surface.
  • CTCs are captured via capture-target molecule bindings.
  • the modules do not include capture ligands which recognize targets on other types of cells in the sample, e.g., blood cells. As blood cells lack the target binding modules, they flow through the chip without being captured.
  • the plastic device surface is treated with oxygen (O 2 ) plasma or ultraviolet (UV) to generate active binding sites. Either treatment also changes the plastic surface from hydrophobic to hydrophilic, which is beneficial for antibody or other capture coating on plastic surface.
  • the activated surface is coated with a carbohydrate based substrate to minimize nonspecific of blood cells, proteins and nucleotides.
  • Cell binding ligands e.g., antibodies to cell surface antigens, are then immobilized to the carbohydrate substrate via a hydrophilic spacer such as polyethylene glycol (PEG) with functional groups on both ends.
  • Monoclonal antibodies can be immobilized in different forms. Whole antibodies can be immobilized to the surface (Fig. 13 A).
  • Fragments of antibodies can be immobilized for better orientation of binding sites or for lower nonspecific binding to blood cells (Fig. 13B-D).
  • the antibody coating is preserved with stabilizers for storage before use.
  • Release mechanism built into surface chemistry The release and collection of capture CTCs are realized by a combination of mechanical, chemical, and biological forces. Mechanically, the design of the chip would not trap CTCs by physical force (size trap and physical adsorption) alone. There are a few chemical and biological mechanisms at work when CTCs are released after capture. PH and chemical adjustment of the releasing buffer significantly weakens the binding between capture molecules on chips surface and target molecules on cell surface.
  • the carbohydrate substrate coating on the plastic surface can be dissolved in an enzyme solution that digests the substrate and releases the captured CTCs.
  • the cross linker between substrate and capture molecules can be enzymatically or chemically cleaved, so as to release the CTCs.
  • a cell capture module can include one or more cell capture chambers running simultaneously. With a single-chamber format, a whole sample, e.g., an entire blood sample, is processed under one single condition. In embodiments having more than one cell capture chamber (“multi-chamber format”), the sample is, optimally, processed in all chambers in equal portions
  • the multiple chambers have identical layouts and surface coating.
  • the benefits are twofold.
  • One benefit is reduced failure, because one chamber's failure is just a portion (1/n) of the total sample.
  • the other benefit is the ability to gain additional heterogeneous information of captured CTCs, by using different capture and detection technologies in each of the cell capture chambers. This can yield valuable information when the target cell in the sample fluid exhibits a variety of analytes and/or surface ligands.
  • the embodiments of a multi- chamber format can yield valuable information given the notoriously heterogamous nature of CTCs.
  • the second way is to have different layouts or different surface coatings or different layouts and surface chemistry in each chamber.
  • each chamber is coated with a different capture antibody.
  • the two chambers will capture two sub-populations of CTCs of different surface markers.
  • the combined information of two types of CTCs can be of greater clinical value than each sub-population individually or two subpopulations in one chamber.
  • the two chambers capture 2 subpopulations of CTCs of different physical properties.
  • CTCs can be separated into many small subpopulations as well as isolated.
  • each chamber can be processed separately post capture. For example, when two identical chambers are used to isolate CTCs, CTCs from one chamber can be enumerated for CTC count, while CTCs from the other cell capture chamber can be processed for molecular analysis.
  • Non-cell biomarker detection module is
  • a regular CTC enrichment blood sample volume is 100-1000 times higher than a regular sample for non-cell markers.
  • One advantage of the micro fluidic device 100 is that the large blood volume leads to an enhancement of detection for non-cell biomarkers (e.g., DNA, RNA, peptides, proteins and etc.), exhibiting an increase in sensitivity gain of 100-1000 times.
  • Non-cell biomarker detection module 2 is immediately downstream of cell capture module 1.
  • Non-cell biomarker detection module 2 is composed of micro channel meanders. The channel dimension and density is optimized for efficient non-cell biomarker capture via not affect module one flow rate.
  • the non-cell biomarker module 2 can be part of the whole chip that includes the rare -cell capture module 1, or, alternatively, non-cell biomarker module 2 can be in a separate chip connected to a separate microfluidic device which includes rare-cell capture module 1. The two chips would be connected sequentially, with the sample (e.g., sample of blood) flowing through the rare-cell capture module in its chip, then through the non-cell biomarker module in the second chip.
  • sample e.g., sample of blood
  • the channel surface is coated with bands of receptors of target non-cell biomarkers and corresponding positive and negative controls.
  • the gap between bands is long enough to prevent cross-contamination.
  • the coating can be physical adsorption or covalent binding of receptor molecules.
  • the plastic surface will be treated with oxygen ((3 ⁇ 4) plasma or ultraviolet (UV) to generate binding sites.
  • Receptor molecules can then be immobilized.
  • a spacer may be needed in some cases.
  • Substrate coating may also be needed due to the fact that the large amount of blood processed may cause high background signals.
  • Multiplexing is easy to implement by increasing the number of receptor bands in the non-cell biomarker module 2.
  • the several regions, or segments (bands), of the non-cell biomarker detection module can be coated with different capture molecules to capture different bio markers (multiplexing).
  • Capture ligands for each of the various target biomarkers are immobilized inside of the channel meanders, which are curved micro-channels in the non-cell biomarker detection module.
  • band 12, band 13, and band 14 each indicate different bands (regions) and the "gaps" between the "bands" of the "multiplexing" capturing of different biomarkers.

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Abstract

Microfluidic devices and methods of enrichment and detection of rare cells, e.g. circulating tumor cells (CTC), and other biomarkers including protein and DNA/RNA, from biological fluid samples are disclosed. The device is an improvement over prior micro fluidics devices by having in its capture chamber smooth surfaces which are designed to mimic the floor of a naturally occurring water body, such as a seafloor or river bed, at the micrometer level. The smooth surface features create a gentle environment, and enhance sample mixing, continuous cell-antibody contact, and rare cell capture. Shear- force and cell damage are reduced. The cell capture module is coated with cell-capture ligands. A second optional module binds non-cell biomarkers with high sensitivity due to the large volume of sample used for rare cell isolation to enrich the non-cell biomarkers by at least 100-1000 times.

Description

MICROFLUIDIC DEVICE WITH SMOOTH SURFACE FOR ENRICHMENT OF RARE CELLS AND BIOMARKERS FROM A BIOLOGICAL
FLUID
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of the filing date of U.S. Provisional Patent Application Serial No. 62/031 ,004, filed July 30, 2014, which application is hereby incorporated by reference.
FIELD OF THE INVENTION
[0002] The invention relates to the fields of micro fluidic devices, cell detection, cell separation, and molecular biomarkers.
BACKGROUND
[0003] Circulating tumor cells (CTCs) originate from primary tumors and perhaps from metastatic sites. Cancer cells disseminate from a primary tumor into the circulatory system as a first step toward metastasis in distant organs. An average tumor may release an estimated million cells per day into the bloodstream. Butler et al., "The Physical and functional behavior of capture antibodies adsorbed on polystyrene. Butler et al., "" L
Immunol. Meth., 150:77-90 (1992). Most CTCs do not survive, but those that do survive pose a risk of further cancer to the host organism. Metastasis is the leading cause of death in cancer patients. For solid tumors, the presence of CTCs is evident late in disease and is most apparent when metastatic disease sites are already established. Identification and characterization of CTCs offers an opportunity to study, monitor, and, ultimately, alter the metastatic process.
[0004] CTCs are exceedingly rare cells, being present at 1-10 CTCs per milliliter (mL) of whole blood. Ordinary blood cell types are present in tremendous numbers by comparison, and patients with cancer may have altered levels of other blood cell types to consider. These other cell types may comprise leukocytes (about 7 million/mL blood) and red blood cells (about 5 billion/mL blood).
[0005] Considerable evidence exists that subpopulations of CTCs may be destined for selected sites in tumor progression and metastasis. (Norton et al, "Is Cancer a Disease of Self-Seeding?, Nat. Med., 12(8):875-878 (2006); Attard et al., "Characterization of ERG, AR and PTEN gene status in circulating tumor cells from patients with castration-resistant prostate cancer." Cancer Res.. 69(7):2912-2918 (2009)).
[0006] The number of circulating tumor cells before treatment is an independent predictor of progression-free survival and overall survival in patients with metastatic breast cancer. Cristofanilli et al, "Circulating tumor cells, disease progression, and survival in metastatic breast cancer," N Engl J Med, 2004; 351(8):781 -91.
[0007] Further reports on the characterization of blood constituents indicate that larger numbers of cellular fragments are present in the circulatory system of patients with cancer along with undamaged CTCs. Enumeration of tumor cell fragments and intact CTCs can be used to accurately predict the survival of patients with cancer. (Ann Oncol. 2010; 21 : 1851-1857)
[0008] One prior CTC-chip is a silicon chamber etched with 78,000 microposts in staggered arrangement with 50-μιη spacing. The posts are coated with anti-EpCAM antibodies. The CTC capture yield is limited at about 60% (Nagrath et al., Isolation of rare circulating tumour cells in cancer patients by microchip technology," Nature, 450: 1235- 1239 (2007). The "post" like structures have major disadvantages due to short cell-post contact length, high shear stress, and low capture rate.
[0009] Identification and characterization CTCs would offer an opportunity to study, monitor, and, ultimately, alter the metastatic process. However, isolating CTCs from blood without breakage or other damage is difficult. The main limitation in most existing microfluidic systems is that the device efficiency is limited by the shear stress the devices exert on the blood cells and CTCs. Too large a shear stress will break the cells. When CTCs break, their internal constituents (the analytes of the microfluidic system) are lost to the fluid. When blood cells break, you create interference. Some prior devices have attempted to avoid breaking cells, including CTCs, by reducing shear stress using a microfluidic device in which the ceiling of the channel is low. However, the low channel causes reduced volume of fluid passing through the channel, which leads in turn to a very low flow rate. Such devices cannot process a large amount of blood quick enough to be practical and useful. Processing a large amount (7-3 OmL) of blood quickly (i.e., in a 24hr window [Stott et al., "Isolation and Characterization of Circulating Tumor Cells from Patients with Localized and Metastatic Prostate Cancer," Sci. Transl. Med.. 2:25ra23 (2010); Meng et al., "Circulating tumor cells in patients with breast cancer dormancy," Clin Cancer Res., 10:8152-8162 (2004)]) is a prerequisite of high yield isolation of intact CTCs. [00010] Besides CTCs, circulating cell-free DNA (cfDNA) and circulating tumor D A (ctDNA) in blood have emerged as important biomarkers in cancer diagnostics and detection of various clinical conditions. Studies have shown that cfDNA may be used for translational genomic research and monitoring personalized therapies based on specific tumor profiles. In order to extract valuable information, it is critical to be able to detect the low levels of cfDNA from whole blood samples. Investigations are now focusing on blood based assays that detect and characterize both CTCs and cfDNA. Study has shown that the ctDNA was detected more frequently than CTCs in blood and ctDNA might be more useful in identifying mutations that could provide therapeutic targets than as a prognostic biomarker like CTCs (Madic et al., Int. J. Cancer, 136:2158-2165 (2015)). In combination, these minimally invasive, real-time 'liquid biopsies' can be performed at multiple intervals with complementary information to monitor disease and tailor cancer treatment. (Pantel, et al., Cancer Res., 73(21): l-5 (2013); Haber et al., Cancer Discov; 4(6):650-61 (2014); Kidess et al., Genome Medicine, 5:70 (2013)).
SUMMARY
[00011] In one aspect of the invention, a microfluidic device includes an inlet; an outlet; an enclosed fluid channel and, in operation, a biological fluid flowing within the channel, the biological fluid containing a rare amount of whole biological cell analyte; and a cell capture module in fluid communication between the inlet and the outlet via the channel, the cell capture module comprising at least one cell capture chamber comprising a smooth- featured surface on the bottom of the chamber, the smooth- featured surface characterized by a smooth topographic surface profile that simulates the topographic surface profile of a naturally-occurring water body.
[00012] In an embodiment of the microfluidic device of the prior paragraph, the cell capture module comprises greater than one cell capture chamber having a smooth- featured surface, each capture chamber in fluid communication with the inlet through the channel, and the smooth-featured surface characterized by a smooth topographic surface profile that simulates the topographic surface profile of a naturally-occurring water body. Optionally, each of the cell capture chambers is of equivalent dimensions. In addition, or in the alternative, the cell capture modules have equal flow rates. Alternatively, the microfluidic device has at least one of the cell capture chambers having dimensions which differ from dimensions of another cell capture chamber. In another embodiment, the microfluidic device of the invention can include a cell capture chamber which further comprises a smooth- featured surface on the top of the chamber.
[00013] All of the micro fluidic devices disclosed herein are improved from prior microfluidic devices by having surfaces within one, some, or all of the one or more cell capture chambers which are smooth featured surfaces. The smooth featured surfaces are designed to simulate, or to mimic, the topographic features of the bottom surface of a naturally-occurring water body, i.e., the naturally-occurring topographic features of a sea floor, an ocean floor, or a river bed or stream. In most embodiments, the smooth-featured surface includes an array of smooth features, each of the smooth features characterized by a profile. The smooth features can be designed by simulation can be based on a
mathematically smooth function. Suitable mathematical functions for use in such simulation include Gaussians, Sine waves, Polynomials of degree 2 or higher, and cubic spline curves. In one embodiment, the surface profile includes surface features with a half peak width between 10 and 5000 micrometers.
[00014] The surface profile which is characteristic of the surfaces of microfluidic devices of the invention enhance cell rolling on the surface. This surface profile prolongs cell-surface interactions, promotes affinity based rare-cell isolation, and reduces shear stress.
[00015] The surface profile which is characteristic of the surfaces of microfluidic devices of the invention also can include surface features of optimal peak height
("mountain"), optimal trough depth (depth of "valley"), and optimal slope and curvature between peak and trough. Such surfaces promote local blood mixing in the cell capture chamber, and facilitate easy release of captured rare cells while maintaining cell viability and integrity.
[00016] In another aspect, the microfluidic device of the invention has a sample throughput of 1 to 20 milliliters per hour.
[00017] In another aspect, the microfluidic device of the invention has a variable sample volume of 0.25 milliliters to 50 milliliters.
[00018] In another aspect, the microfluidic device of the invention has one or more of the following properties: reduces optical interference, enhances visualization, and improves rare cell identification.
[00019] In another aspect, the microfluidic device of the invention includes a bottom surface having capture ligands attached thereto. [00020] It is another aspect of the micro fluidic device of the invention that, in operation, the whole biological cells bind to the capture ligands so as to isolate cells present on the top of the surface, to facilitate automated scanning and rare cell detection, analysis and characterization.
[00021] It is another aspect of the micro fluidic device of the invention that the surface profile comprises microValley features, and the micro Valleys have dimensional ranges of 10 to 1000 micrometers wide, 20 to 1000 micrometers long, and 20 to 1000 micrometers high. The surface can include an array of microValley features having regular relative positions with gap size between 5 and 500 micrometers. In one embodiment, the microValley has a regularly shaped profile. Alternatively, the microfluidic device of claim 27, wherein the microValley has an irregularly shaped profile. In another embodiment, the microValley is in an array of micro Valleys, and the array is arranged irregularly. μValley is a micro concave structure that can be simulated with a smooth functions such as Gaussian curves. This micro structure can promote high flow rate, increasing fluid mixing and reduce share stress to the cells.
[00022] It is another aspect of the microfluidic device of the invention that the surface profile comprises an array of microFunnel features and microSp litter features, the array having a dimensional range of 10 to 100 micrometers wide, 20 to 200 micrometers long, and 20 to 200 micrometers high. The array can be arranged in a layout of optimized externally tangent circles.
[00023] It is an objective in the microfluidic devices of the invention that the cell capture module is sufficiently large to process 7.5 mL of blood between 1 hour to 24 hours.
[00024] In embodiments in which the cell capture module includes more than one cell capture chamber, the cell capture chambers are characterized by matching surface profiles.
[00025] In another aspect of the microfluidic device of the invention, the devices further comprise a non-cell biomarker module, wherein the non-cell biomarker module is in fluid communication between the cell capture module and the outlet. The non-cell biomarker module can include capture ligands for binding to analytes in a sample fluid, the analytes selected from the group consisting of a DNA marker such as ctDNA or cfDNA, an RNA marker, a peptide marker, and a protein, a biomarker-carrying microparticle such as microvesicles, exosomes, and apoptoic bodies. [00026] It yet another aspect, the invention includes a method for enriching a fluid sample for a rare whole cell, comprising flowing a fluid sample comprising a rare amount of the whole cells through any of the micro fluidic devices disclosed herein. In an
embodiment, the rare whole cells are circulating tumor cells. In this or a separate embodiment, the method further includes releasing the whole cells from the cell capture module, the releasing via a combination of mechanical, chemical and biological mechanisms.
[00027] The invention further includes a method a for enriching a fluid sample for a rare whole cell, comprising flowing a fluid sample comprising a rare amount of the whole cells through any of the microfluidic devices disclosed herein. In one embodiment, the non- cell biomarker detection module comprises more than one type of capture ligand. The method can comprise collecting fluid from the outlet, wherein the fluid is enriched for analytes released from capture ligands immobilized to the non-cell biomarker detection module.
[00028] By "analyte" is meant a molecule or component in a fluid which is the target of a method of, as the case may be, detection, isolation, concentration, or extraction. Exemplary analytes include cells, viruses, nucleic acids, proteins, carbohydrates, and small organic molecules.
[00029] By "blood component" is meant any component of whole blood, including host red blood cells, white blood cells, and platelets. Blood components also include the components of plasma, e.g., proteins, lipids, nucleic acids, and carbohydrates, and any other cells that may be present in blood, e.g., because of current or past pregnancy, organ transplant, or infection.
[00030] "Biological fluid" is meant to include naturally occurring fluids (e.g., blood, lymph, cerebrospinal fluid, urine, cervical lavage, saliva, and water samples), portions of such fluids, and fluids into which cells have been introduced (e.g., culture media and liquefied tissue samples). The term also includes a lysate.
[00031] By "capture moiety" or "capture ligand" is meant a chemical species to which, as the case may be, an analyte binds or a whole cell binds by virtue of its surface components. A capture moiety may be a compound coupled to a surface or the material making up the surface. Exemplary capture moieties include antibodies, oligo- or polypeptides, nucleic acids, other proteins, synthetic polymers, and carbohydrates. [00032] By "channel" is meant a gap through which fluid may flow. A channel may be a capillary, a conduit, or a strip of hydrophilic pattern on an otherwise hydrophobic surface wherein aqueous fluids are confined.
[00033] By "circulating tumor cell" (CTC) is meant a cancer cell that is exfoliated from a solid tumor of a subject and is found in the subject's circulating blood.
[00034] By "component" of a cell is meant any component of a cell that may be at least partially isolated upon lysis of the cell. Cellular components may be organelles (e.g., nuclei, peri-nuclear compartments, nuclear membranes, mitochondria, chloroplasts, or cell membranes), polymers or molecular complexes (e.g., lipids, polysaccharides, proteins (membrane, trans-membrane, or cytosolic), nucleic acids (native, therapeutic, or pathogenic), viral particles, or ribosomes), or other molecules (e.g., hormones, ions, cofactors, or drugs). By "component" of a cellular sample is meant a subset of cells contained within the sample.
[00035] By "enriched sample" is meant a sample containing an analyte that has been processed to increase the relative amount of the analyte relative to other analytes typically present in a sample. For example, samples may be enriched by increasing the amount of the analyte of interest by at least 10%, 25%, 50%, 75%, 100% or by a factor of at least 1000, 10,000, 100,000, or 1,000,000.
[00036] By "depleted sample" is meant a sample containing an analyte that has been processed to decrease the amount of the analyte relative to other analytes typically present in a sample. For example, samples may be depleted by decreasing the amount of the analyte of interest by at least 5%, 10%, 25%, 50%, 75%, 90%, 95%, 97%, 98%, 99%, or even 100%.
[00037] By "gap" is meant an open passage through which a fluid may flow.
[00038] By "micro-" is meant having at least one dimension of less than 1 millimeter.
[00039] By "naturally-occurring water body" is meant sea, ocean, river, or stream.
[00040] By "profile" is meant a contour side view image.
[00041] A "rare amount" of cells refers to less than 100 cells in a milliliter of fluid, less than 10 cells per milliliter fluid, or even less than 1 cell per milliliter fluid.
[00042] By "regular" is meant a pattern which is recurring, orderly, methodical or occurs at fixed, uniform or normal intervals; "irregular" means not regular. [00043] By "surface feature" is meant a perturbation of the surface above or below the plane of the surface which is co-planar with the bottom of the cell capture chamber. The terms "micro-mountain", "micro-valley", "micro-funnel", and micro-splinter" describe the contour of a surface feature by analogy to the expected contour of the a conventional mountain, valley, funnel, or splinter, respectively.
[00044] Other features and advantages will be apparent from the following description and the claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[00045] A preferred embodiment of the present invention is shown in the accompanying drawings which, together with the description thereof, will serve to exemplify the invention. The particular structure illustrated can be modified by those skilled in the art without departing from the broad scope of the invention.
[00046] Fig. 1 illustrates a top view layout of a micro fluidic device having a two chamber cell capture module 1 and a two meander non-cell capture module 2.
[00047] Fig. 2 is a pictorial illustration of the topography of a seafloor (A, B, and C) and a river bed (D). The direction of flow goes from left to right.
[00048] Fig. 3 is a three-dimensional representation of a Gaussian curve having a two-dimensional domain.
[00049] Fig. 4 is a side view of two chip assemblies: (A) topographic chamber floor with flat ceiling; and (B) topographic floor and topographic ceiling.
[00050] Fig. 5 is a (A) topographic view; and (B) side view of the bottom surface of a microValley array used to isolate rare whole cells from blood samples.
[00051] Fig. 6A is an illustration of a topographic feature on the bottom surface of a microValley having design control parameters a, b, c, o("sigma"), and A.
[00052] Fig. 6B is an illustration of array design parameters to consider positioning of micro Valleys relative to each other.
[00053] Fig. 6C-6H is each an illustration of a different microValley arrangement, where 6C: rectangular positioning; 6D. A-B-A; 6E. short gap-long gap along flow direction; 6F. short gap-long gap perpendicular to flow direction; 6G. short gap
perpendicular to flow direction; 6H. short gap along and perpendicular to flow direction.
[00054] Fig. 7 is a schematic illustration of different ways of assembling microValley chips, where: Fig. 7A is an illustration of a regular microValley chip with a flat top; Fig. 7B is an illustration of two matching microValley chips assembled together; Fig. 7C is an illustration of two mismatched microValley chips assembled together; Fig. 7D, Fig. 7E, and Fig. 7F are illustrations of variations from Fig. 7A- Fig. 7C, respectively, where the microValley chips are irregular.
[00055] Fig. 8 is a process diagram illustrating the progress of rare whole cells in a blood sample on micro Valley-surfaced microfluidic device of the invention, where Fig. 8A: Flow; Fig. 8B: capture; Fig. 8C: wash; and Fig. 8D: release.
[00056] Fig. 9 is a top-down schematic illustration of a pattern of micro Funnels and microSp litter surface features on the bottom surface of a microfluidic device of the invention. Arrows between the surface features illustrate the direction of flow of a biological fluid between the surface features.
[00057] Figs. 10A and 10B are illustrations of the top-down view (Fig. 10A) and profile (Fig. 10B) of a single microFunnel.
[00058] Figs. IOC and 10D are illustrations of the top-down view (Fig. IOC) and elevated (Fig. 10D) of a single micro Splitter.
[00059] Fig. 1 1 is a layout optimization of microFunnels and micro Splitters.
[00060] Fig. 12 is an example of microFunnels and micro Splitters layout and CTC capture from whole blood.
[00061] Fig. 13 is a side view illustration of four surface chemistries for use to capture whole sales and biomarkers from a biological fluid, where each capture ligand is attached to a substrate through a spacer, the four capture ligands being a complete antibody (Fig. 13A); F(ab')2 (Fig. 13B); rlgG (Fig. 13C); and Fab' (Fig. 13D).
DETAILED DESCRIPTION
[0001] An improved microfluidic device is disclosed for processing rare whole cells, such as circulating tumor cells. The improvements include 1) a smooth surface design in the cell module; 2) cell and biomarker combination capture mechanism, and 3) the "multiplexing" capturing of different biomarkers in the separated bands in the biomarker module.
[0002] In overview, the microfluidic device has two integrated capture modules. The first module contains a capture zone having internal surfaces. The bottom of the internal surface of the capture module contains seafloor or river bed mimicking smooth features at the micrometer (μιη) to millimeter (mm) level that promote cell-device surface interactions. The smooth surface features are simulated using Gaussian or other smooth mathematical functions. The smooth features create a gentle environment to enhance sample mixing, continuous cell-antibody contact, and rare cell capture. The smooth surface design also reduces shear-force applied to the cells to prevent cell damage during the enrichment processing. The layout of such features is optimized with computer simulation based on Gaussian or other smooth functions. The result is improved cell-surface contact efficiency, reduced shear stress, and enhanced throughput. The first module is coated with ligands that bind to target cells. When biological fluids flow through the device, the target cells are captured on these seafloor or river bed mimicking features via ligand-cell binding. The second module contains chemically separated areas that are coated with capture molecules of non-cell biomarkers. This module takes advantages of the large volume of blood sample used for rare cell isolation to enrich the non-cell biomarkers by at least 100- 1000 fold before further analysis. The detection of capture rare cells and non-cell biomarkers are detected via immuno-staining with detection tags. The captured rare cells can be released and collected by reverse the flow direction and wash with a combination of mechanical, chemical, and biological releasing mechanism.
[0003] Smooth curves or structures with different sizes can be randomly placed in a microfluidic chamber to form smooth capture surfaces (simulating blood vessels, or simulating the bottom of a naturally-occurring water body) that 1) promote high flow rate; 2) increase fluid mixing and 3) reduce share stress to the cells. The smooth surface is designed to avoid disadvantages of prior art devices which relied on a "post", or on a prior art "micro structure", or on a prior art "obstacle" (hereafter prior art micro structure). The prior art micro structure has major disadvantages due to short cell-post contact length, high shear stress, and low capture rate. In contrast, the smooth surface design disclosed herein is optimized to achieve a gentle cell-surface contact without sacrificing the larger overall capture surface area of the entire module chamber. In the microstructure "post" design, the larger surface area is achieved by placing a huge number of small structures (posts).
However, cells may contact many posts but still may not be captured due to a very short cell-post contact distance (rolling length) at each post. In contrast, the smooth surface design of the present invention provides a continuous cell-antibody contact for extended rolling distance (e.g. several cell perimeters). In addition, the smooth surface simulates blood flow in the blood vessel so that reduces shear stress to the cell and prevents cell damage. [0004] Referring to the embodiment shown in Fig. 1, the micro fluidic device 100 is equipped with two modules: cell capture module 1 and non-cell biomarker detection module 2. A fluid sample enters microfluidic device 100 at input 10. The fluid sample can be a sample of a biological fluid, e.g., whole blood. Fluid is pushed or pulled through microfluidic device 100 by a flow force exerted by a pump or by a vacuum (not shown).
[0005] Cell capture module 1 is composed of one or more cell capture chamber 5. Each cell capture chamber 5 is connected to inlet 10 by an equal length of inlet channel 11. Each channel 1 1 is of the same depth and width. Thus, the sample fluid is evenly split in volume between the one or more chamber 5. Each portion of the blood sample moves through each cell capture chamber 5 simultaneously. The cell capture chambers 5 are filled with computer simulation optimized features and layout which facilitate and prolong cell- surface contact while minimizing shear stress experienced by the cells. The surface 6 of cell capture chamber 5 is coated with specific ligand or ligand combinations (not shown), such as antibody or antibody combos that bind to cell surface antigen or antigens, and as a result, capture cells via Ab-Ag interactions. Target rare cells such as CTCs are captured in this cell capture module 1. Non-target cells flow through the cell capture chamber 5 and exit the cell capture chamber 5 at capture module exit port 3. The captured rare cells are identified and quantified by immuno-staining.
[0006] After the sample fluid leaves the cell capture chamber 5, the sample fluid enters non-cell biomarker detection module 2. This detection module 2 is composed of micro-channel meanders 7. The inside walls 8 of the channel meanders 7 are coated with bands of specific receptors 12, 13, 14. As the sample flows through the channel meanders 7, these receptors capture corresponding biomarkers from the sample fluid. The captured biomarkers are identified and quantified by immuno-staining.
[0007] A variation on the embodiment above is that cell-capture module 1 and non-cell biomarker detection module 2 can be on one single chip or two chips connected via a connector.
Cell capture module
[0008] "Seqfloor" and "River bed" smooth surface designs of the CTC isolation chip. Fig. 2 shows the typical topographies of seafloor (Figure 2A and 2B) and river bed (Figure 2C). Fig. 3 shows a Gaussian curve with two-dimensional domain. In addition to a Gaussian curve, suitable mathematical functions for use in simulating the topography of a naturally-occurring water body include sine waves, polynomials of degree 2 or higher, and cubic spline curves.
[0009] In all embodiments of the set forth herein, the internal surfaces of the cell capture module 1 of micro fluidic device 100, or at a minimum at least the bottom surface of the cell capture module 1 , are designed to mimic the topographies, at a micro-meter level, of the bottom surface of a natural waterway, e.g., a seafloor, ocean floor, or river bed
(hereafter "surface designs of the invention". These topographical surface designs of the invention are used on the floor and ceiling surfaces of cell- isolation chamber 5. The surfaces can be made smooth with Gaussian-function based computer simulation to optimize the surface in the following aspects :
[00010] 1) The total surface area in the cell capture chamber 5 should be maximized in order to enhance contact between rare cells and the surface 6.
[00011] 2) The surface designs of the invention enhance cell-rolling on the chip surface and affinity-based rare cell isolation.
[00012] 3) The surface designs of the invention minimize shear stress on cells and reduce damage to captured rare cells.
[00013] 4) The surface designs of the invention promote mixing of the sample fluid, e.g., blood mixing, preventing constituents in the sample, e.g., CTCs in whole blood, from settling or fractioning. Mixing is optimized by varying the heights and slopes of the Gaussian "mountains," and depth of the "valleys" in between.
[00014] 5) The surface designs of the invention balance sample processing throughput and capture efficiency.
[00015] 6) Additional benefits of such design include ease of release of captured rare cells due to the capture mechanism that relies mainly on affinity capture enhanced by cell-rolling on the surface. Little physical hindrance exists in releasing cell as experience by existing cell capture devices. The surface designs of the invention also ease visualization and detection by optical methods such as fluorescent imaging due to the fact that angled surface topography greatly reduce the optical interferences.
[00016] Chip Assembly. There are two different ways that cell-capture module 1 can be assembled. The embodiment shown in Fig. 4A uses a flat chamber cover 21, which can be a piece of tape applied via adhesives, or a thin slab of plastic applied via thermal or chemical bonding. The height of chamber walls can be varied to adjust the size of chamber gap 23 between the chamber bottom 22 and the chamber ceiling (the underside of flat chamber cover 21) for best balance between capture efficiency and throughput.
[00017] In Fig. 4B, one chip can be flipped over a second chip and held together via an assembly gig. Again, this can be done with variable sizes of chamber gap 33. The benefits are twofold. One benefit is the doubled capture area, because capture ligands can be affixed to the underside (inner side) of top chip 31, as well as to the upper (inner) side of bottom chip 32. The other benefit is the ease of post-isolation processing such as molecular analysis as the two-chip assembly can be reversed and opened for CTC collection or DNA/RNA analysis.
[00018] Special case 1 of Gaussian simulation optimized seafloor design:
microValley arrays for rare cell isolation. A simplified version of the Gaussian simulation optimized seafloor design consists of arrays of micro Valleys as seen on a typical seafloor. These micro Valleys promote smooth flow, low mixing of blood sample, cell rolling on surface and results in high efficiency capture of rare cells such as CTCs. Fig. 5 shows the top (Fig. 5A) and side views (Fig. 5B) of a rare cell-isolation chamber. Fig. 5A is the bottom surface 6 of a cell capture chamber 5. The inset displays the micro Valleys array of bottom surface 6.
[00019] Generally as to the surface designs of the invention, the array design is performed in three aspects: depth and surface profile on individual micro Valleys, relative positioning of micro Valleys in the array, and the optimized combination of the above two aspects. Figure 6A and 6B show the parameters of microValley array optimizations. Figure 6C-H are six examples of many feasible array designs. The parameters a, b, c, σ, A, XI , X2, Yl , Y2, D, are identified as follows:
a, b, c, σ, A: the parameters that define a two-dimensional elliptical Gaussian function, which is an typical smooth micro structure.
In general, a two-dimensional elliptical Gaussian function is expressed as:
Figure imgf000015_0001
where A is the height of the Gaussian function, the parameters a, b, c define the shape of the elliptical Gaussian function; furthermore, the parameters a, b, c can be derived from the standard deviation (σ) of the elliptical Gaussian. XI, X2, Yl , Y2, and D are the parameters that define the arrangement pattern of the micro structures (micro Valley):
XI and X2 - defines the column alignment width of the micro structures Yl and Y2 - defines the row alignment width of the micro structures
D - the center column shifting distance
[00020] Chip assembly can follow a few routes as shown in Fig. 7. The micro Valley array chip can be assembled with a flat top or another microValley chip. When two micro Valley chips are assembled, different alignment is another parameter that can be optimized for most efficient rare cell isolation.
[00021] Once the microValley chips are functionalized with rare cell capture ligands, and assembled. The rare cell isolation can be carried out as shown in Fig. 8. When blood sample flows through the microValley chip, rare cells are captured through surface immobilized capture ligands such as antibodies against rare cell surface antigens. Along with rare cells, a small amount of blood cells stick to the chip surface through weak physical adsorption. Washing with proper buffer will remove the vast majority of the blood cells and leave behind highly purified rare cells. These rare cells can be immuno-stained for enumeration or released and recollected for molecular analysis.
[00022] Special case 2 of Gaussian simulation optimized seafloor design:
microFunnels and micro Splitters. Fig. 9 shows the design scheme for a simplified version of the Gaussian simulation optimized seafloor design. The design consists of many modules of microFunnels and micro Splitters of smooth structure that benefits smooth flow, promotes cell capture by enhancing cell-surface contact, while reduces shear stress to cells.
[00023] Optimization of microFunnels and microSplitters. Referring to Fig. 10, the shapes, curvatures and sizes of both microFunnels and microSplitters are computer simulation optimized for enhanced cell rolling along the microFunnel's and micro Splitter's walls. Fig. 10A shows the optimization of each half of the microFunnel's top view profile. This profile has a backbone made of a series of externally tangent circles with various radiuses. Along the flow direction, the radiuses increase gradually to narrow the width of the microFunnel, then decrease for a wider opening. The neighboring circles' radius (e.g., rl, r2) and alignment are optimized with typical size of relevant cell types (e.g., Rl, R2). Fig. 10B shows the side view cross section profile of a microSp litter or one half of a microFunnel. Correspondingly, Fig. 10D is a 3-D rendering of a microSplitter or one half of a microFunnel. Fig. IOC is top view of a microSplitter.
[00024] Optimization of relative positions of microFunnels and microSp litters. As shown in Figure 11 A, the opening angle (2a) of the microFunnel is optimized to enhance cell- funnel wall interaction while maintaining a flow rate high enough to sustain desired throughput. The outlet opening (do) of the microFunnel is optimized relative to the size of the target cell. The distance between two microFunnels (di) is adjusted according to ratio of (between microFunnel volume/2X in microFunnel volume). The relative position of microSp litters relative to surrounding microFunnels (LI, L2, Ul and U2) are adjusted to split and distribute upstream blood flow towards the inside walls of the microFunnels.
[00025] The three dimensional (3-D) profile of individual microFunnel and microSplitter also play a role in enhancing cell-surface interaction, and thus is included in optimizing the relative positions of microFunnels and micro Splitters. Fig. 1 IB shows the elevation map of such a chip layout.
[00026] CTC isolation scheme. Fig. 12 shows the scheme of CTC isolation from whole blood. The chip surfaces are coated with capture molecules that bind to targets on the cell surface of CTCs. The design facilitates smooth rolling of CTCs on the chip surface. CTCs are captured via capture-target molecule bindings. The modules do not include capture ligands which recognize targets on other types of cells in the sample, e.g., blood cells. As blood cells lack the target binding modules, they flow through the chip without being captured.
[00027] Surface chemistry. The plastic device surface is treated with oxygen (O2) plasma or ultraviolet (UV) to generate active binding sites. Either treatment also changes the plastic surface from hydrophobic to hydrophilic, which is beneficial for antibody or other capture coating on plastic surface. The activated surface is coated with a carbohydrate based substrate to minimize nonspecific of blood cells, proteins and nucleotides. Cell binding ligands, e.g., antibodies to cell surface antigens, are then immobilized to the carbohydrate substrate via a hydrophilic spacer such as polyethylene glycol (PEG) with functional groups on both ends. Monoclonal antibodies can be immobilized in different forms. Whole antibodies can be immobilized to the surface (Fig. 13 A). Fragments of antibodies can be immobilized for better orientation of binding sites or for lower nonspecific binding to blood cells (Fig. 13B-D). The antibody coating is preserved with stabilizers for storage before use. [00028] Release mechanism built into surface chemistry. The release and collection of capture CTCs are realized by a combination of mechanical, chemical, and biological forces. Mechanically, the design of the chip would not trap CTCs by physical force (size trap and physical adsorption) alone. There are a few chemical and biological mechanisms at work when CTCs are released after capture. PH and chemical adjustment of the releasing buffer significantly weakens the binding between capture molecules on chips surface and target molecules on cell surface. The carbohydrate substrate coating on the plastic surface can be dissolved in an enzyme solution that digests the substrate and releases the captured CTCs. In certain embodiments, where a cross linker is used between substrate (surface) and capture molecules, the cross linker between substrate and capture molecules can be enzymatically or chemically cleaved, so as to release the CTCs.
[00029] This can be achieved by reverse flow direction and wash with releasing buffer that combines optimized chemical and biological factors.
[00030] Multi-chamber format. In various embodiments, a cell capture module can include one or more cell capture chambers running simultaneously. With a single-chamber format, a whole sample, e.g., an entire blood sample, is processed under one single condition. In embodiments having more than one cell capture chamber ("multi-chamber format"), the sample is, optimally, processed in all chambers in equal portions
simultaneously.
[00031] This can happen in two ways. First, the multiple chambers (n chambers) have identical layouts and surface coating. The benefits are twofold. One benefit is reduced failure, because one chamber's failure is just a portion (1/n) of the total sample. The other benefit is the ability to gain additional heterogeneous information of captured CTCs, by using different capture and detection technologies in each of the cell capture chambers. This can yield valuable information when the target cell in the sample fluid exhibits a variety of analytes and/or surface ligands. By way of example, the embodiments of a multi- chamber format can yield valuable information given the notoriously heterogamous nature of CTCs.
[00032] The second way is to have different layouts or different surface coatings or different layouts and surface chemistry in each chamber. For example, in a two-chamber format, each chamber is coated with a different capture antibody. The two chambers will capture two sub-populations of CTCs of different surface markers. The combined information of two types of CTCs can be of greater clinical value than each sub-population individually or two subpopulations in one chamber. When the two chambers have the same capture molecules and different layouts, the two chambers capture 2 subpopulations of CTCs of different physical properties. When multiple layouts and multiple capture molecules are combined, CTCs can be separated into many small subpopulations as well as isolated.
[00033] Additionally, each chamber can be processed separately post capture. For example, when two identical chambers are used to isolate CTCs, CTCs from one chamber can be enumerated for CTC count, while CTCs from the other cell capture chamber can be processed for molecular analysis.
Non-cell biomarker detection module.
[00034] A regular CTC enrichment blood sample volume is 100-1000 times higher than a regular sample for non-cell markers. One advantage of the micro fluidic device 100 is that the large blood volume leads to an enhancement of detection for non-cell biomarkers (e.g., DNA, RNA, peptides, proteins and etc.), exhibiting an increase in sensitivity gain of 100-1000 times.
[00035] Chip Design. Referring back to Fig. 1, the non-cell biomarker detection module 2 is immediately downstream of cell capture module 1. Non-cell biomarker detection module 2 is composed of micro channel meanders. The channel dimension and density is optimized for efficient non-cell biomarker capture via not affect module one flow rate.
[00036] In other embodiments, the non-cell biomarker module 2 can be part of the whole chip that includes the rare -cell capture module 1, or, alternatively, non-cell biomarker module 2 can be in a separate chip connected to a separate microfluidic device which includes rare-cell capture module 1. The two chips would be connected sequentially, with the sample (e.g., sample of blood) flowing through the rare-cell capture module in its chip, then through the non-cell biomarker module in the second chip.
[00037] Surface chemistry. In any of the various embodiments disclosed herein, the channel surface is coated with bands of receptors of target non-cell biomarkers and corresponding positive and negative controls. The gap between bands is long enough to prevent cross-contamination. The coating can be physical adsorption or covalent binding of receptor molecules. In the case of covalent binding, the plastic surface will be treated with oxygen ((¾) plasma or ultraviolet (UV) to generate binding sites. Receptor molecules can then be immobilized. A spacer may be needed in some cases. Substrate coating may also be needed due to the fact that the large amount of blood processed may cause high background signals.
[00038] Multiplexing. Multiplexing is easy to implement by increasing the number of receptor bands in the non-cell biomarker module 2. The several regions, or segments (bands), of the non-cell biomarker detection module can be coated with different capture molecules to capture different bio markers (multiplexing). Capture ligands for each of the various target biomarkers are immobilized inside of the channel meanders, which are curved micro-channels in the non-cell biomarker detection module. In Fig. 1, for example, band 12, band 13, and band 14 each indicate different bands (regions) and the "gaps" between the "bands" of the "multiplexing" capturing of different biomarkers.

Claims

1. A micro fluidic device comprising:
a. an inlet;
b. an outlet;
c. an enclosed fluid channel and, in operation, a biological fluid flowing within the channel, the biological fluid containing a rare amount of whole biological cell analyte; and
d. a cell capture module in fluid communication between the inlet and the outlet via the channel, the cell capture module comprising at least one cell capture chamber comprising a smooth- featured surface on the bottom of the chamber, the smooth- featured surface characterized by a smooth topographic surface profile that simulates the topographic surface profile of a naturally-occurring water body.
2. The micro fluidic device of claim 1, wherein the cell capture module comprises greater than one cell capture chamber having a smooth-featured surface, each capture chamber in fluid communication with the inlet through the channel, and the smooth- featured surface characterized by a smooth topographic surface profile that simulates the topographic surface profile of a naturally-occurring water body.
3. The micro fluidic device of claim 2, wherein each the cell capture chamber is of
equivalent dimensions.
4. The micro fluidic device of claim 2, wherein all of the cell capture modules have equal flow rates.
5. The microfluidic device of claim 2, wherein at least one of the cell capture chambers has dimensions which differ from dimensions of another cell capture chamber.
6. The microfluidic device of any one of claims 1-5, wherein the cell capture chamber further comprises a smooth-featured surface on the top of the chamber.
7. The micro fluidic device of any one of claims 1-6, wherein the topographic surface of a naturally-occurring water body is selected from the group consisting of a sea floor, an ocean floor, and a river bed.
8. The micro fluidic device of claim 7, wherein the smooth- featured surface comprises an array of smooth features, each of the smooth features characterized by a profile.
9. The micro fluidic device of claim 8, wherein the simulation is based on a
mathematically smooth function.
10. The micro fluidic device of claim 9, wherein the simulation is based on a Gaussian function.
11. The micro fluidic device of claim 10, wherein the surface profile comprises surface features with a half peak width between 10 and 5000 micrometers.
12. The micro fluidic device of claim 8, wherein the surface profile enhances cell rolling on the surface.
13. The micro fluidic device of any one of claims 1-6, wherein the surface profile prolongs cell-surface interactions.
14. The micro fluidic device of any one of claims 1-6, wherein the surface profile
promotes affinity based rare-cell isolation.
15. The micro fluidic device of any one of claims 1-6, wherein the surface profile reduces shear stress.
16. The micro fluidic device of any one of claims 1-6, wherein the surface profile
comprises surface features of optimal peak height ("mountain"), optimal trough depth (depth of "valley"), and optimal slope and curvature between peak and trough.
17. The microfluidic device of any one of claims 1 -6, wherein the surface profile
promotes local blood mixing in the cell capture chamber.
18. The microfluidic device of any one of claims 1-6, wherein the surface profile facilitates easy release of captured rare cells while maintaining cell viability and integrity.
19. The microfluidic device of any one of claims 1-6, wherein the microfluidic device has a sample throughput of 1 to 20 milliliters per hour.
20. The microfluidic device of any one of claims 1-6, wherein the microfluidic device has a variable sample volume of 0.25 milliliters to 50 milliliters.
21. The microfluidic device of any one of claims 1-6, wherein the surface profile reduces optical interference.
22. The microfluidic device of any one of claims 1-6, wherein the surface profile
enhances visualization.
23. The microfluidic device of any one of claims 1 -6, wherein the bottom surface profile improves rare cell identification.
24. The microfluidic device of any one of claims 1 -6, wherein the bottom surface
comprises capture ligands attached thereto.
25. The microfluidic device of any one of claims 1 -6, wherein, in operation, the whole biological cells bind to the capture ligands so as to isolated cells present the cells on the top of the surface.
26. The microfluidic device of any one of claims 1-6, wherein the surface profile
comprises microValley features, and the micro Valleys have dimensional ranges of 10 to 1000 micrometers wide, 20 to 1000 micrometers long, and 20 to 1000 micrometers high.
27. The microfluidic device of any one of claims 26, wherein the surface comprises an array of microValley features having regular relative positions with gap size between 5 and 500 micrometers.
28. The micro fluidic device of any one of claims 27, wherein the micro Valley has a regularly shaped profile.
29. The micro fluidic device of claim 27, wherein the microValley has an irregularly shaped profile.
30. The micro fluidic device of claim 27, wherein the microValley is in an array of
micro Valleys, and the array is arranged irregularly.
31. The micro fluidic device of any one of claims 1-6, wherein the surface profile
comprises an array of microFunnel features and microSp litter features, the array having a dimensional range of 10 to 100 micrometers wide, 20 to 200 micrometers long, and 20 to 200 micrometers high.
32. The micro fluidic device of claim 31 , wherein the array is arranged in a layout of optimized externally tangent circles.
33. The micro fluidic device of any one of claims 1-6, wherein the cell capture module is sufficiently large to process 7.5 mL of blood between 1 hour to 24 hours.
34. The micro fluidic device any one of claims 2-6, wherein the cell capture chambers are characterized by matching surface profiles.
35. The microfluidic device of any one of claims 1 -6, further comprising a non-cell biomarker module, wherein the non-cell biomarker module is in fluid communication between the cell capture module and the outlet.
36. The microfluidic device of claim 35, wherein the non-cell biomarker module
comprises capture ligands for binding to analytes in a sample fluid, the analytes selected from the group consisting of a DNA marker such as ctDNA or cfDNA, an RNA marker, a peptide marker, and a protein.
37. A method for enriching a fluid sample for a rare whole cell, comprising flowing a fluid sample comprising a rare amount of the whole cells through a micro fluidic device of any one of the above claims.
38. The method of claim 37, wherein the rare whole cells are circulating tumor cells.
39. The method of claim 37, further comprising releasing the whole cells from the cell capture module, the releasing via a combination of mechanical, chemical and biological mechanisms.
40. A method for enriching a fluid sample for a rare whole cell, comprising flowing a fluid sample comprising a rare amount of the whole cells through the micro fluidic device of any one of the claim 35 and claim 36.
41. The method of claim 40, wherein the non-cell biomarker detection module comprises more than one type of capture ligand.
42. The method of claim 40, wherein the method comprises collecting fluid from the outlet, wherein the fluid is enriched for analytes released from capture ligands immobilized to the non-cell biomarker detection module.
PCT/US2015/053337 2014-07-30 2015-09-30 Microfluidic device with smooth surface for enrichment of rare cells and biomarkers from a biological fluid WO2016019401A1 (en)

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