Patent Application
20180024134
The present invention relates to methods for the immobilization of cells or virus particles of interest expressing one or more predetermined surface marker(s) on defined spots on a solid support, comprising the steps of providing a biological fluid sample suspected of containing cells or virus particles of interest; labeling the cells or virus particles of interest with antibodies, antibody fragments or antibody mimetics directed against the one or more predetermined surface marker(s) and carrying a first binding agent; and contacting the labeled cells or virus particles of interest with a solid support (biochip), said solid support comprising an array of defined isolated spots of a solid support-bound second binding agent, wherein the first and second binding agents can bind to each other. The present invention further relates to devices for the isolation of cells or virus particles of interest expressing one or more predetermined surface marker(s), comprising a microfluidic chamber comprising a solid support comprising an array of defined isolated spots of a solid support-bound binding agent, and a surface opposing the solid support, said surface having transverse structures that facilitate chaotic mixing of the fluid containing the cells or virus particles of interest when said fluid flows along said surface; and a capillary micropipette.
The origin of metastases in cancer are circulating tumor cells (CTCs). These cells separate from the solid primary tumor and enter the blood stream. While circulating in their new environment, CTCs can settle in organs and undergo mitosis. Avoiding and controlling metastases is crucial for cancer treatment, since these metastases are most of the times responsible for the patient's ultimate fate, while the primary tumor seldom leads to life threatening conditions on itself. However, CTCs offer new perspectives in the observation and treatment of cancer. Known as liquid biopsy, frequent quantification and molecular analysis of CTCs before and during treatment supply a real-time status of the patient, rather than the fixed one-time histopathologic result of the primary tumor.
The identification and hence enrichment and localization of cells of interest, such as CTCs, but also other cells including bacterial cells, or virus particles of interest in biological fluids is still a challenge, even after decades of research. This is particularly true for cells or virus particles that are present in the biological fluid in very low numbers, possibly on a background of high numbers of other cells or virus particles. For example, in blood the ratio between CTCs and blood cells is approximately 1:109 (assuming <200 CTC/ml, 5×109 red blood cells/ml, 7×106 white blood cells/ml).
Various strategies for the enrichment and identification of cells in biological fluids have been described. Physical or biological characteristics of cells can be exploited to enrich their concentration in a given biological fluid. Filtration concerning cell size or stiffness can be performed as well as a density centrifugation, where particular cells can be isolated. As another target for enrichment and separation, specific surface markers can be facilitated in several ways for capturing. Binding agents binding to such surface markers conjugated with magnetic nanoparticles mark cells that separate from non-labeled cells in a magnetic field. The same principle can be performed by negative enrichment or depletion. Further, most microfluidic approaches are based on the immobilization of cells surfaces labeled with specific binding agents.
After enrichment according to methods known in the art, cells still need to be identified in an additional step. Cytospinning is a technique that centrifuges cells from suspension onto a surface. Immunostaining approaches can then be performed to mark proteins that are either specific to the cells of interest or identify said cells by exclusion of other cell types. Systems like CellSearch perform enrichment and identification in a fully automated way. Furthermore, automated imaging systems based on fluorescence microscopy observe cytospinned cell samples and supply micrographs of single cells. Genomic analyses of cell suspensions or even single cells allow the precise characterization and absolute identification of cells of interest, e.g. via procedures like the polymerase chain reaction and methods like whole genome sequencing. Besides exploitation of biological properties for enrichment, chip technologies also facilitate physical parameters for cell enrichment. Even both the biological and physical approach can be conducted in one chip serially.
Since the discovery of the epithelial cell adhesion molecule (EpCAM) as a marker for CTCs, various microfluidic approaches were developed showing promising results. Anti-EpCAM coated surfaces interact with EpCAM molecules in the cell membrane immobilizing the CTCs whereas blood cells transmit the system. Verification and further analysis is carried out by immunostaining. However, recent studies have shown that EpCAM is not always a reliable marker. Some patients develop metastases although EpCAM positive CTCs are not detectable in their blood. Further, CTCs were discovered that were negative for anti-EpCAM staining. On this account, the development of CTC-capturing devices that can easily target different surface epitomes besides EpCAM, are able to handle high blood volumes and allow single cell analysis is still challenging, but highly demanded.
Respective methods known in the art all require the use of labeled, e.g. fluorescently labeled, specific binding agents for identification of the isolated cells after immobilization. Further, said methods often achieve only inadequate binding efficiency, specificity and recovery of cells.
Accordingly, the technical problem underlying the present invention is to provide means for the immobilization, identification and isolation of cells or virus particles of interest from biological fluids, in particular cells or virus particles that are present in the biological fluid in very low numbers, possibly on a background of high numbers of other cells or virus particles. Such means should display a high binding efficiency, high specificity and high recovery, and enable the direct identification of the cells or virus particles of interest without the use of labeled specific binding agents. Further, such means should allow for an easy access to immobilized cells for subsequent single cell analysis.
The solution to the above technical problem is achieved by the embodiments characterized in the claims.
In particular, in a first aspect, the present invention relates to a method for the immobilization of cells or virus particles of interest expressing one or more predetermined surface marker(s) on defined spots on a solid support, comprising the steps:
In the present application, the term “comprising” is expressly intended to encompass the terms “consisting of” and “consisting essentially of”. Thus, said terms are interchangeable throughout the present application.
The method of the present invention can be used for the immobilization of particular cells of interest, or of particular virus particles of interest, provided that said cells or virus particles express a known surface marker, and provided that antibodies, antibody fragments or antibody mimetics that bind to said known surface marker in a specific manner are available.
Preferably, the method of the present invention is used for the immobilization of cells, wherein cells that are present in the biological fluid in very low numbers, possibly on a background of high numbers of other cells, are particularly preferred. In a specific embodiment, the cells are circulating tumor cells (CTCs).
Surface markers that can function as a labeling target in the context of the method of the present invention are not particularly limited, provided that antibodies, antibody fragments or antibody mimetics that bind to said known surface marker in a specific manner are available. In a specific embodiment, the surface marker is Epithelial Cell Adhesion Molecule (EpCAM) which is an important marker of CTCs. Further particular surface markers that can be of interest include human epidermal growth factor receptor 2 (HER-2) which is a marker of breast cancer cells, CD44, CD133, aldehyde dehydrogenase isoform 1 (ALDH1), c-Met, CD47, nerve-cell adhesion molecule (N-CAM), CD340, epidermal growth factor receptor (EGFR), CD318, Muc-1, Trop-2, Mpv-18, and musculin (MSC).
The biological fluid provided in the method of the present invention is a biological fluid that is suspected of containing cells or virus particles of interest. Respective biological fluids are not particularly limited. They include for example biological fluids selected from the group consisting of blood, blood components, amniotic fluid, cerebrospinal fluid, lymphatic fluid, peritoneal fluid, saliva, sputum, urine, and partitions or fractions thereof. In this context, the method of the present invention may, in a preferred embodiment, comprise after step (a) and prior to step (b), or after step (b) and prior to step (c), the step of enriching cells or virus particles of interest from the biological fluid. As an example, in case the biological fluid is blood, mononuclear cells may be enriched by way of a density gradient centrifugation as known in the art. Further means for enriching cells or virus particles of interest from a biological fluid are not particularly limited and are known in the art.
In step (b) of the method of the present invention, cells or virus particles of interest are labeled with antibodies, antibody fragments or antibody mimetics as defined above which are directed against the one or more predetermined surface marker(s), i.e., which bind in a specific manner to said surface markers, wherein said antibodies, antibody fragments or antibody mimetics carry a first binding agent. In this context, the term “carrying a first binding agent” as used herein indicates that the antibodies, antibody fragments or antibody mimetics are non-covalently or, preferably, covalently coupled to said first binding agent. Respective binding agents and binding agent pairings of first and second binding agents that can bind to each other are not particularly limited and are known in the art. In a preferred embodiment, the first binding agent is biotin. In another preferred embodiment, the first binding agent is a His-Tag.
In step (c) of the method of the present invention, the labeled cells or virus particles of interest are contacted with a solid support, said solid support comprising an array of defined isolated spots of a solid support-bound second binding agent, wherein the first and second binding agents can bind to each other.
The second binding agent that is bound to the solid support is not particularly limited, provided that it can bind to the first binding agent carried by the antibodies, antibody fragments or antibody mimetics. As indicated above, respective pairings of first and second binding agents that can bind to each other are not particularly limited and are known in the art. In a preferred embodiment, the first binding agent is biotin and the second binding agent is streptavidin or an analog thereof such as avidin and neutravidin. In another preferred embodiment, the first binding agent is a His-Tag and the second binding agent is a metal chelating group, e.g. nitrilotriacetic acid (NTA). Further, in a preferred embodiment, the second binding agent is fluorescently labeled, wherein suitable fluorescent dyes are not particularly limited and can be chosen from fluorescent dyes known in the art. In a particular example, said fluorescent dye is the cyanine dye Cy3.
The solid support that the cells or virus particles are contacted with is a solid substrate, preferably a transparent solid substrate. Suitable materials are not particularly limited and are known in the art. In a preferred embodiment, the solid support is a glass substrate, e.g. a microscopy slide. In other embodiments, the solid support is a substrate made of a transparent polymer material such as e.g. polystyrene and poly(methyl methacrylate). Said solid support is in some instances designated as “biochip” throughout the present application. Preferably, the solid support provides a plane, smooth surface.
The solid support used in the method of the present invention comprises an array of defined isolated spots of solid support-bound second binding agent. This array is preferably a regular array of spots, the array covering an area of 1 to 2500 mm2, preferably 25 to 1000 mm2, more preferably of 100 to 600 mm2, most preferably of 300 to 500 mm2, e.g. of 400 mm2, wherein the dots are spaced at a distance of at least 10 μm, preferably at least 15 μm, more preferably at least 20 μm, e.g. 20 to 100 μm, 20 to 80 μm, 20 to 60 μm, 20 to 40 μm, 20 to 30 μm, or 25 μm. The spot diameter is preferably 2 to 20 μm, more preferably 5 to 15 μm, more preferably 8 to 12 μm, e.g. 10 μm. Respective arrays are shown in
Methods for generating arrays of defined isolated spots of binding agents according to the present invention on a solid support are not particularly limited and are known in the art. They include methods involving the direct application of the binding agents or the application of anchoring molecules for subsequent binding of the binding agents to said anchoring molecules by dip-pen nanolithography (DPN), polymer pen lithography (PPL), spotting with capillaries, microchannel cantilevers or ink jet printing to the solid support. This may involve prior activation of the solid support by means known in the art. Specific exemplary and preferred methods include the methods described hereinafter. In a first example, the solid support is passivated with bovine serum albumin (BSA) by immersing the solid support in a solution of BSA. Subsequently, an array of biotin-4-fluorescein is printed on the solid support by PPL. Irradiation of the solid support with UV light covalently couples the biotin-4-fluorescein to the BSA on the solid support (cf. Examples, Experimental procedures). After washing, the solid support is incubated with streptavidin which binds to the immobilized biotin, thus generating an array of solid support-bound streptavidin. In a second example, the strong affinity of thiol groups (—SH) to gold is used. The solid support is coated with gold and subsequently an array of Thiol-biotin is printed by PPL or DPN on the solid support, followed by incubation with streptavidin (cf. Example 4). In a third example, cycloaddition of azide groups to alkyne groups is used. The solid support is e.g. immersed in a solution of (3-glycidyloxypropyl)trimethoxysilane (GPTMS) and an epoxy ring-opening is performed by immersing the solid support in a solution of propargylamine. Subsequently, an array of biotin-azide is printed on the solid support by DPN, followed by incubation with streptavidin (cf. Example 4). In related embodiments, biotin-alkyne is printed on a solid support having immobilized azide groups.
In preferred embodiments, the areas on the solid support surrounding the defined isolated spots of solid support-bound second binding agent are passivated. Suitable methods for the passivation of surfaces are not particularly limited and are known in the art. They include for examples the incubation of the surface with solutions of one or more suitable passivation agents. Respective passivation agents are not particularly limited and are known in the art. They include for example BSA, milk powder, casein, and polyethylene glycol (PEG). In this context, the term “passivation” as used herein indicates the process of rendering a surface unable to unspecifically bind or adhere proteins, cells, or virus particles.
In preferred embodiments, the solid support is part of a microfluidic chamber, said microfluidic chamber comprising said solid support, preferably as the bottom surface of the chamber, and a surface opposing the solid support, preferably as the ceiling surface of the chamber, said surface opposing the solid support having transverse structures that facilitate chaotic mixing of the fluid containing the cells or virus particles of interest when said fluid flows along said surface. The microfluidic chamber can further comprise inlet and outlet ports for supplying and discharging the biological fluid to and from the chamber, respectively. Preferably, all surfaces of the chamber are passivated in addition to the solid support, as specified above.
Transverse structures that facilitate chaotic mixing of the biological fluid when said fluid flows along said surface are not particularly limited and are known in the art. Preferably, said structures are surface ridges that are substantially perpendicular to the flow direction of the biological fluid, i.e., that form an angle with said flow direction of 45 to 135°. Preferably, said transverse structures are surface ridges having a herringbone-structure as known in the art and as depicted in
In preferred embodiments, the microfluidic chamber has a channel height of 10 to 150 μm, preferably 20 to 100 μm, more preferably 30 to 70 μm, or 40 to 60 μm, e.g. 50 μm. as measured from the solid support to the surface ridges. The surface ridges have preferably a height of 20 to 70 μm, preferably, 30 to 60 μm, more preferably 40 to 50 μm, e.g. 45 μm. Accordingly, the height from the solid support to the surface opposing the solid support in-between two surface ridges can be from 30 to 220 μm, e.g. from 80 to 110 μm, or from 85 to 95 μm.
Further, regarding the preferred herringbone-structure, two consecutive ridges of said structure can have a distance from each other as measured in the flow direction of 20 to 80 μm, preferably 30 to 70 μm, or 40 to 60 μm, e.g. 50 μm. A single ridge can be composed of alternating segments that are arranged to alternately form an angle with the flow direction of 45°, then 135°, then 45° and so on, i.e., said segments form a 90° angle with each other. Two consecutive segments can have a shorter length, e.g. of 120 to 160 μm, 130 to 150 μm, or 140 μm, followed by two consecutive segments having a longer length, e.g. the double of the shorter length, such as e.g. 260 to 300 μm, 270 to 290 μm, or 280 μm. Thus, a structure as depicted in
Means and methods for producing a microfluidic chamber as described above are not particularly limited and are known in the art. In particular, the surface opposing the solid support can be made of polydimethylsiloxane (PDMS) by molding in a prefabricated photolithographically patterned mold. The finished product can then be bonded to the solid support. In a specific and preferred example, the microfluidic chamber is produced as described in Examples, Experimental procedures.
In preferred embodiments, the method of the present invention further comprises, after step (c), the step of identifying solid support-bound cells or virus particles of interest microscopically. Suitable microscopy techniques are not particularly limited and are known in the art. They include, for example conventional light microscopy and/or fluorescence microscopy techniques.
In a further preferred embodiment, the method of the present invention further comprises, after step (c) and after microscopic identification as described above, the step of isolating one or more individual immobilized cells or virus particles from the chip. This can be achieved by means known in the art, e.g. by aspiration of individual cells with the help of a capillary micropipette, preferably a micropipette that is mounted on a micromanipulator. In the context of this embodiment, the surface opposing the solid support, e.g. as provided in the microfluidic chamber described above, forms a removable lid to allow for access to the immobilized cells or virus particles.
In preferred embodiments, the above additional methods steps of identifying and isolating solid support-bound cells or virus particles is performed in an automated manner. Respective means for automatic identification and isolation are not particularly limited and are known in the art. They include for example the CellaVista system (Roche, Switzerland) or the CellSelector system (ALS Germany) (
In a preferred embodiment, step (c) of the method of the present invention is performed at elevated temperatures, i.e., temperatures higher than room temperature, e.g. at 28 to 40° C., preferably at 32 to 38° C., more preferably at 36 to 38° C., e.g. at 37° C., in order to promote binding of cells or virus particles of interest to the second binding agent. In particular, it has been found that binding of cells or virus particles is unexpectedly improved at elevated temperatures. This can be realized e.g. by mounting the solid support to a heating device such as a heating plate. Alternatively, a heating device could be integrated directly into the solid support.
In preferred embodiments, the method of the present invention is performed using microfluidic chambers or devices as described above or as described in further aspects of the present invention hereinafter.
In a second aspect, the present invention relates to a microfluidic chamber, comprising:
In this aspect of the present invention, all definitions and embodiments described above for the method of the present invention apply in an analogous manner.
In particular, the microfluidic chamber itself, methods for producing the same, the solid support, the array of defined isolated spots of a solid support-bound binding agent, said binding agent, the surface opposing the sold support, the transverse structures that facilitate chaotic mixing of the biological fluid, and the herringbone-structure are preferably as defined above for the method of the present invention.
In specific examples, the transverse structures that facilitate chaotic mixing of the fluid containing the cells or virus particles of interest when said fluid flows along said surface are preferably surface ridges as defined above for the method of the present invention, preferably having a herringbone-structure as defined above for the method of the present invention. Further, the surface opposing the solid support preferably forms a removable lid. Furthermore, it is preferred that the areas on the solid support surrounding the defined isolated spots of solid support-bound second binding agent, as well as all additional surfaces of the chamber, are passivated as described above for the method of the present invention. Furthermore, the binding agent, which is preferably streptavidin, is preferably fluorescently labeled.
In a third aspect, the present invention relates to a device for the isolation of cells or virus particles of interest expressing one or more predetermined surface marker(s), comprising:
(a) a microfluidic chamber comprising:
(b) a capillary micropipette.
The microfluidic chamber of the device of the present invention is the microfluidic chamber as defined in the second aspect of the present invention. A schematic setup of the device of the present invention is shown in
According to this aspect of the present invention, cells or virus particles are isolated using a capillary micropipette, i.e., by aspiration with said capillary micropipette. Respective micropipettes are not particularly limited and are known in the art. They include e.g. glass capillary micropipettes having an inner diameter of from 10 to 100 μm, preferably from 20 to 80 μm, from 30 to 50 μm, e.g. 40 μm. Preferably, the micropipette is mounted to a micromanipulator as known in the art.
In preferred embodiments, the device of the present invention further comprises means for the automatic identification of solid support-bound cells or virus particles of interest, and/or means for the automatic isolation of immobilized cells or virus particles from the solid support.
In another preferred embodiment, the device of the present invention further comprises means for heating the solid support, e.g. a heating plate.
Here, a capture strategy is presented based on micropatterns that offers high specificity, large throughput and easy access to captured cells for single cell analysis (
One advantage of pre-labeling target cells with an antibody cocktail instead of anchoring one type of antibody onto the surface of the capture device is that the fabricated chip has no limitation concerning the type of cell (or virus particle) that is to be captured at the same time. In fact, any cell (or other target) that can be sensitized by biotinylated antibodies in a prior incubation step can be captured with one chip, allowing for great flexibility in this approach: E.g. only the general biotin conjugate will bind to the streptavidin. The binding mechanism is based solely on the interaction between e.g. biotin and streptavidin. Even if the incubation protocol varies and different antibodies are used, the fluidic parameters do not change. A further advantage of pre-labeling the target cells is an increased antibody stability and increased interaction of the antibodies with the solid support, thus facilitating cell capture on a plane smooth surface provided by the solid support.
Using a plane smooth surface provided by the solid support according to the present invention for cell capturing provides the additional advantage of an easy isolation of cells after capture for further analysis without excessively disturbing the cells. When using the solid support in combination with an opposing surface having transverse structures that facilitate chaotic mixing, the efficiency of cell capture is comparable to the use of structured capture surfaces lacking this advantage.
Capture approaches based on microfluidic chips aim primarily at cell quantification, however, immunocytochemistry and fluorescence in situ hybridization (FISH) can be conducted on captured cells inside these closed systems. Due to the three-dimensional inner architecture that is homogeneously coated with functional molecules captured CTCs cannot be extracted and individually investigated any further. Immobilizing CTCs on a plane surface according to the present invention has some advantages if further investigation needs to be performed. The approach taken in the present invention immobilizes cells exclusively on the functional micropattern; other surfaces like the herringbone-structured ceiling and the channel walls are passivated with e.g. BSA preventing cells to bind there. Having the captured CTCs on a flat surface allows picking single cells e.g. by a micromanipulator and capillary micropipette.
In contrast to other cell capture chips that have a homogeneous antibody coating on the inner walls, the capturing strategy according to the present invention is based on spot microarrays, offering intrinsic specificity enhancement by co-localization detection (
The genomic analysis and quantification of circulating tumor cells in the blood of cancer patients will play an important role for a precise tumor diagnosis and monitoring during and after treatment. Introduced as liquid biopsy of cancer, the analysis of CTCs as metastatic cells offers additional information of the primary tumor that are not accessible by other means. Furthermore, the patient is aggravated from first surgical interventions. During cancer treatment, e.g. chemo- or radiotherapy, physicians are able to evaluate its impact by the quantification of CTCs.
The strategy in cell capture according to the present invention immobilizes and aligns cells on a microarray for intrinsic detection validation. Streptavidin spots of 10 μm in diameter patterned over an area 10×40 mm2 function as capturing platform for cells pre-labeled with biotinylated antibodies. To maximize the chance of contact between cells and the microarray, the microarray is encapsulated within a microfluidic PDMS chip carrying a herringbone structure in its channel top wall that causes a chaotic stirring flow. In contrast to other cell capture chips, this approach allows identification of cells without secondary labeling due to a high specificity and intrinsic additional check against unspecific adhesion by the co-localization with the fluorescent capture spots and provides the ability to pick single cells with a micromanipulator to perform further single cell analysis. Furthermore, the pre-incubation allows for free choice of used antibody in our approach, without need for adjustments. For example, besides EpCAM, other biotinylated or otherwise tagged CTC-specific surface proteins can be targeted. This idea will lead to the application of an antibody cocktail to include EpCAM negative CTCs for capture and could even be expanded to any other antibody target desired to be captured. This proof-of-principle study shows the value of this approach for the ongoing development of microfluidic cell capture chips.
The present invention will be further illustrated in the following examples without being limited thereto.
Experimental Procedures:
Micropattern Fabrication
Biotin microarrays were generated by Polymer Pen Lithography. Briefly, the polydimethylsiloxane (ABCR, Karlsruhe, Germany) stamp is molded from a silicon master that was initially generated by photolithography. Oxygen plasma treatment renders the stamp's surface hydrophilic which is needed to homogeneously coat the pyramided tips when approaching the stamp into a stamp pad filled with ink. The stamp pad is a piece of silicon covered with biotin-4-fluorescein ink (Sigma Aldrich, Germany). The stamp is attached to the holder of an NLP2000 system (Nanolnk, Skokie, Ill., US) that offers a stage able to perform movements over some centimeters with sub-micrometer precision. Depending on the pen's spacing, either several approaches are carried out to realize the pattern with the desired parameters. The stamp itself has an area of 1×1 cm2. Pen inking is carried out at 70% relative humidity (RH), lithography at 50% RH.
A microscopy slide (Menzel Glaser, Germany) acts as substrate for the microarray. Prior cleaning is performed with sonication in chloroform, isopropanol (Merck, Germany) and deionized water (18.2 MΩ cm) for 10 min each. The slides were then immersed in a solution of bovine serum albumin (BSA, Sigma Aldrich, Germany) and phosphate buffered saline (PBS, Sigma Aldrich, Germany) of a concentration of 3% w/V. After 45 min, the slides were dipped in PBS 10 times to remove the excess of BSA from the surface and dried with nitrogen. Inking of the PPL stamps was carried out with the stamp pad method. Cleaned pieces of a silicon wafer were coated with the respective ink mixture by depositing 10-15 μL of ink onto the bare surface by a pipette and allowing the droplet to spread. Then the polymer pens were approached onto this silicon wafer (either by making 10×10 dot patterns with dwell time 1s or by making arbitrary lines) for inking at RH 70%. Immobilization of the biotin-4-fluorescein micropattern is carried out with a UV lamp at 350 nm for 45 min. The slides are again dipped into PBS 10 times to remove excess and dried with nitrogen.
Fabrication of the Microfluidic Device
Device fabrication was accomplished by a rapid, low-cost micromolding technique. A 4″ glass wafer was used as substrate material for the mold. The inverse of the microfluidic structures were formed within a biocompatible dry film resist (Ordyl SY330, Elga Europe, Italy) with a thickness of 55 μm. For the mold fabrication, the first resist layer was laminated onto the glass substrate at a temperature of 95° C., and photolithographically patterned. To achieve the desired herringbone structure in the microfluidic channel top wall, a second layer of dry film resist was laminated onto the structure and patterned, resulting in a total height of 110 μm. After resist development, liquid, degassed PDMS was poured over the finished mold. Once solidified, PDMS was peeled off and holes for fluid connections were punched. In a next step, an oxygen plasma treatment of the bottom side of the PDMS was applied. The PDMS was bonded to the micropatterned microscope glass slide. Finally, tubings with syringe needles were fitted into the punched fluid connector holes to finish the microfluidic chip.
Microfluidics and Chip Functionalization
The microfluidic chip is connected with 1/32 inch tubes that are inserted into provided inlet and outlet ports. The ingoing tube is connected to a 3-way-cock that is again connected to a syringe. A microfluidic syringe pump (NE-1002X, FisherScientific) is used to reproducibly pump the liquids through the chip. To allow a smooth wetting of the whole chamber and to block unspecific protein binding at the channel walls, 100 μL of bovine serum albumin (1% w/V) and Tween20 (1% V/V) are pumped into the chip and incubated for 30 min. Then, 200 μL streptavidin/cy3 in PBS (0.5% VN) replaces the prior solution to bind streptavidin to the biotin pattern. The chip is ready-to-use after flushing with 500 μL PBS.
Blood Sampling
4 mL whole blood from a healthy donor was stored in an EDTA tube. A general density gradient protocol was applied to separate red blood cells and mononuclear cells: Blood was given in a falcon tube and filled with 30 mL HBSS (Biochrom) and centrifuged at 400 g and 4° C. for 10 min. The cell pellet was resuspended in 30 mL PBS and 20 mL Ficoll (GE Healthcare) were added. The mixture was centrifuged at 400 g at 4° C. for 30 minutes. The interface and supernatant, containing the mononuclear cells (i.e. leukocytes), were transferred to a new 50 ml falcon tube. The tube was filled with PBS and centrifuged at 400 g at 4° C. for 10 min. Supernatant was discarded and cell pellet was resuspended in 1 mL 1×H-Lysis buffer (R&D Systems) and incubated for 3 minutes with gentle shaking at room temperature. 30 mL PBS was added and sample was centrifuged again at 400 g for 10 minutes at 4° C. Supernatant was discarded and pellet was resuspended in warm PBS to spike the biotinylated MCF-7 cells into the suspension.
Cancer Cell Sensitization
Cells from the cancer cell line MFC-7 (SKBR-3) were cultured in Dulbecco's modified eagle medium (DMEM) including 10% (5%) fetal calf serum (FCS) at 10% (5%) CO2 in a 25 cm2 flask. Cells were washed with warm PBS and detached with trypsin after 2 min incubation and rinsed with warm DMEM. After centrifugation, the cells were re-suspended in warm BSA/PBS 0.1% wN and counted. 100,000 cells in warm PBS/BSA were then incubated with either 0.5 μg biotinylated Anti-EpCAM (VU-1D9, Abcam, UK) or biotinylated Anti-HER-2 (3B5, Abcam, UK) in a shaker (Eppendorf Thermomixer Comfort, Hamburg, Germany) revolving at 300 rpm at 37° C. for 40 min. The cells are then centrifuged and suspended in warm PBS. The ratio of dead cells is counted before spiking. To achieve a precise number of spiked cancer cells, the suspension is diluted to 10,000 cells/mL; 5 drops of 10 μL suspension are then pipetted on a glass slide and the cells are counted.
Capturing Experiments
Positive and negative cells are mixed in 1 mL PBS and pipetted into a 3 mL syringe (BD Bioscience) that is covered with aluminum foil to hold a temperature of 37° C. To prevent air bubbles to enter the system and to allow the cells to enter the syringe's exit directly, the microfluidic pump is placed vertically (
Design of a Microfluidic Chip and an Integrated Microarray
The capturing strategy presented here is based on a functional dot pattern combined with a microfluidic chaotic mixing system. A standard microscopy slide is coated with bovine serum albumin (BSA) to create both a passive and integrative interface: passive, as BSA prevents proteins and cells from unspecific binding; integrative, as fluorescein can bind covalently to BSA molecules induced by photobleaching. The latter is exploited to generate a chemisorbed dot pattern on the microscopy slide that is stable under streaming liquid condition. Lithography is carried out by polymer pen lithography (PPL), a high throughput technique with full pattern flexibility.
The microfluidic PDMS chip is casted from a prefabricated mold and covalently bonded to the microscopy slide carrying the microarray after oxygen plasma treatment. The array matches the microfluidic channel design with its herringbone topography in the top wall. The microdevice can be stored for months before application since the biotin pattern is stable in air and not prone to degradation as compared to antibody patterns. By connecting the microfluidic chip to a syringe, the chamber is flushed with a solution of streptavidin/cy3, which binds to the biotin-array and turns it into the active capture array for biotin-labeled targets. After flushing with PBS to remove excess streptavidin, the device is ready for CTC capture. A photo of the microfluidic device is given in
Identification and Single Cell Extraction
Capture approaches based on microfluidic chips aim primarily at CTC quantification, however, immunocytochemistry and fluorescence in situ hybridization (FISH) can be conducted on captured cells inside these closed systems. Due to the three-dimensional inner architecture that is homogeneously coated with functional molecules captured CTCs cannot be extracted and individually investigated any further. Immobilizing CTCs on a plane surface has some advantages if further investigation needs to be performed. The approach taken in the present invention immobilizes cells exclusively on the functional micropattern; other surfaces like the herringbone-structured ceiling and the channel walls are passivated with BSA preventing cells to bind there. Having the captured CTCs on a flat surface allows picking single cells by a micromanipulator.
The advantage of facilitating dot patterns as capturing platform becomes clear when referring to
Performance
The low quantity of CTCs in blood is a challenge for all capturing strategies. A relatively large blood volume of several mL needs to be processed to isolate usually some hundred CTCs. When facilitating specific surface proteins as CTC marker, all blood cells need to be brought in contact with an interacting surface. The first experiments to proof the stability and binding dynamics of the micropatterns have been performed with 100 μL cell suspension on cover slips. EpCAM micropatterns have been created by direct protein deposition and it was tried to incubate biotinylated EpCAM on a streptavidin array. However, both approaches did not show sufficient binding results during a short-term incubation of 15 minutes. By sensitizing targeting cells with biotinylated EpCAM one can take advantage of the strong affinity between streptavidin and biotin. Live-imaging experiments show that 80% of biotinylated cells are trapped on a streptavidin dot immediately after touching. However, using cover slips alone will not solve the volume problem, as most of the blood cells will not have a chance to touch the functionalized surface. By designing a microfluidic chip with a herringbone structure enveloping the micropattern the cell-surface interaction and therefore the binding probability is enhanced.
Table 1 presents a set of experiments from a statistical point of view. MCF-7 cells have been sensitized with biotinylated EpCAM and spiked into lysed blood carrying 2×106 leukocytes (#1) or into an untreated cell suspension of MCF-7 cells (#2-4). These suspensions were then pumped through the microfluidic chip keeping the temperature at 37° C. Binding effects increase when the cells have a longer contact time to a dot, i.e. less velocity. Therefore, a herringbone-structured chip was facilitated to mix the cell suspension to allow any cell to get in touch with a streptavidin dot. By interrupting the flow the cell velocity in the chamber decreases which results into a larger reaction time between cells and dots. Following this protocol, we were able to achieve average recovery rates of about 50% could be achieved. All experiments presented in Table 1 show a high specificity between 86% and 96%.
As can be taken from Table 1, the recovery varies between 40 and 60%, the specificity is higher than 85%. All experiments were performed with cells of the cell line MCF-7 that have been labeled with biotinylated anti-EpCAM before. Experiment 1 was carried out with 1 mL lysed blood and 2050 spiked cells. To allow cell binding, the flow of 20 μL/min was interrupted in periods of 2 minutes for 3 minutes each. 99% of leukocytes passed the chip, whereas 68% of the positive cells remained in the chamber. The other experiments were performed with non-sensitized cells as negative sample.
Additional Methods for Micropattern Fabrication
Gold/Thiol-Biotin
The gold/thiol substrate/ink system is the most widely employed platform in DPN and PPL. For application in PPL, the PPL stamp is generally plasma cleaned, then spin coated with or completely immersed into the respected thiol solution in ethanol. After drying of the stamp, printing takes place in the same way as described for the fluorescein-biotin patterning. Prior to incubation of the substrate with streptavidin, the substrate is treated with a blocking agent such as BSA.
Glass/Alkyne-Group/Azide-Biotin
Functionalization of Silica Substrates:
Glass slides were cleaned in piranha acid (3:1 v/v solution of H2SO4 and H2O2) for 30 min, rinsed with copious amounts of MilliQ water and dried with pressurized air. Caution: piranha acid is a strong oxidizer and a strong acid. It should be handled with extreme care, as it reacts violently with most organic materials. The samples were then immersed in a solution of (3-glycidyloxypropyl)trimethoxysilane (GPTMS) in toluene (1% v/v) for 8 h at room temperature, afterwards rinsed with acetone, and dried with pressurized air. Immediately afterwards, the epoxy ring-opening was performed by immersing the substrates in a solution of propargylamine in acetonitrile (2% v/v) for 8 h at 45° C. The samples were taken out of the solution, sonicated in ethanol for 5 min, rinsed thoroughly with ethanol and MilliQ water and dried with pressurized air. If the samples were not used directly, they were stored at 4° C. under argon atmosphere.
Patterning of Alexa Fluor 555 Azide and Biotin-Azide by DPN:
Dip-pen nanolithography was done on a DPN 5000 system (Nanoink, Skokie, USA). A 1D cantilever array containing 26 Si3N4 tips was cleaned with oxygen plasma, 10 sccm at 100 mTorr with 30 W for 5 min. The cantilevers were subsequently immersed for 10 min in a solution of Alexa Fluor 555 azide (150 μg mL−1) or biotin azide (PEG4 carboxamide-6-azidohexanyl biotin, 2 mg mL−1), each ink containing 20 mM sodium ascorbate and 10 mM CuSO4. After inking the cantilever arrays were blow-dried with pressurized nitrogen. Writing of the Alexa Fluor 555 patterns was performed in feedback mode with a velocity of 1 μm s−1 at 74.8% relative humidity and 26.4° C. The biotin azide dot patterns were generated with a dwell time of 2 s per dot at 74.0% relative humidity and 26.4° C. After the DPN process for the biotin pattern the sample was blocked with a PBS solution containing 0.5% BSA for 15 min, then washed with PBS three times and incubated for another 15 min with PBS containing 1 vol % Streptavidin-Cy3 (Sigma-Aldrich GmbH, Germany). After that, the sample was washed again three times with PBS, rinsed with MilliQ DI water and blow-dried with nitrogen before inspection with fluorescent microscopy. Prior to incubation of the substrate with streptavidin, the substrate is treated with a blocking agent such as BSA.
In a related method, an azide can be immobilized on the substrate, e.g. via substrate-bound silanes, and a biotin-alkyne bound thereto.
CVD-Coating with Alkyne Functionalization and Biotin-Azide
CVD Polymerization of poly(p-xylylenes):
Poly(4-ethynyl-p-xylylene-co-p-xylylene) was synthesized via CVD (chemical vapor deposition) polymerization in a custom-made CVD polymerization system, and were prepared on substrates including PDMS, gold, PMMA, PS, PTFE, and stainless steel. The synthesis of ethynyl[2.2]paracyclophane followed routes known in the art. Ethynyl[2.2]paracyclophane was used as starting material and was sublimed under vacuum and converted by pyrolysis into the corresponding quinodimethanes, which spontaneously polymerized upon condensation to the cooled substrate surface at 15° C. Throughout CVD polymerization, a constant argon flow of 20 sccm and a working pressure of 0.3 mbar were maintained. The pyrolysis temperature was set to be 670° C. and sublimation temperatures were between 90 to 110° C. under these conditions. CVD polymerization spontaneously occurred on samples placed on a rotating, cooled sample holder. The resulting poly(4-ethynyl-p-xylylene-co-p-xylylene) film thickness was controlled at 100 nm on all substrates studied. Film thickness was measured using a single wavelength (532 nm) EP3-SW imaging ellipsometry (Nanofilm Technologie GmbH, Germany).
Dip-Pen Nanolithography Process:
For writing on poly(4-ethynyl-p-xylylene-co-p-xylylene) surfaces, all dip-pen nanolithography was done on a DPN 5000 system (Nanolnk, Skokie, USA). A 1-D cantilever array containing 26 Si3N4 tips was first treated with oxygen plasma, 10 sccm, at 100 mTorr and with 30 W for 5 min. The tips were then dipped either in Alexa Fluor® 555 azide, triethylammonium salt (150 μg/ml, Invitrogen GmbH, Germany) or biotin azide (PEG4 carboxamide-6-azidohexanyl biotin) (100 μg/ml, Invitrogen GmbH, Germany) solution containing sodium ascorbate (20 mM) and CuSO4 (10 mM) for 15 min, and then were dried using a nitrogen stream. For writing on poly(4-formyl-p-xylylene-co-p-xylylene) surfaces, a 1-D cantilever array containing 26 Si3N4 tips was also treated with oxygen plasma with the same process parameters. The tips were then dipped in biotin-LC-hydrazide (100 μg/ml, Thermo Scientific GmbH, Germany) solution at a pH of 5-6 for 15 min, and then were dried using a nitrogen stream. After DPN writing, the resulting samples were rinsed with DI-water in order to remove loosely bound azide molecules. Finally, the samples were examined using a fluorescence microscope (Eclipse 80i upright microscope, Nikon Instruments, Japan). Prior to incubation of the substrate with streptavidin, the substrate is treated with a blocking agent such as BSA.
In a related method, an azide can be immobilized on the substrate via CVD and a biotin-alkyne bound thereto.
Direct Printing of Streptavidin
Direct printing of streptavidin on the substrate can be achieved using streptavidin in a protein carrier matrix, e.g. PEG or glycerol.
Discussion:
The experiments presented here were carried out with the model system of the cancer cell line MCF-7 and biotinylated anti-EpCAM. Streptavidin spots on the bottom surface function as binding sites. To ensure a saturated binding of anti-EpCAM, 100,000 cells were incubated in 1 mL PBS carrying 0.5 μg biotinylated EpCAM representing approximately 8×1012 molecules (assuming 45 kDa). Prior incubation experiments have shown that antibody labeling should be carried out in a shaker set to 37° C. for 40 minutes.
Anti-EpCAM is the standard marker for CTC capturing and is therefore used in this study. CTCs derived from a solid tumor express EpCAM as epithelial cells, but they may undergo a transition to a mesenchymal phenotype. This process is known as the epithelial to mesenchymal transition (EMT). Until then, CTCs can be identified and labeled with anti-EpCAM. To the present knowledge, all surface-marker-based CTC capturing approaches focus on EpCAM. This is a severe drawback as EpCAM negative CTCs are invisible for these approaches. By labeling CTCs with other specific surface antibodies, like the human epidermal growth factor receptor 2 (HER-2) in the case of breast cancer, or even with an antibody cocktail, the probability to capture EpCAM negative cells increases. The capturing methods and devices of the present invention are compatible for the application of antibody cocktails without any further adaptation, as shown by no need for adjustment on the microfluidic parameters when switching from EpCAM to other antibodies.
| Number | Date | Country | Kind |
|---|---|---|---|
| 15000479.4 | Feb 2015 | EP | regional |
This application is a U.S. National Phase patent application of International Application Serial Number PCT/EP2016/000191, filed on Feb. 5, 2016, which is hereby incorporated by reference in its entirety, and which claims the benefit of European Patent Application Number 15000479.4, filed on Feb. 13, 2015, which is hereby incorporated by reference in its entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2016/000191 | 2/5/2016 | WO | 00 |
