CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Application Serial No. 61/263,097, entitled "Method and Apparatus for Isolating Microvesicles" filed on November 20, 2009. The entire content of this application is incorporated herein by reference.

The entire contents of the following applications are also incorporated herein by reference: U.S. Application Serial No. 60/782,470, filed on March 15, 2006; PCT Application Serial No. PCT/US07/06791 (Published Application No. WO 2007/106598), entitled "Devices and Methods for Detecting Cells and Other Analytes," having an international filing date of March 15, 2007; and U.S. Application Serial No. 12/293,046 entitled "Devices and Methods for Detecting Cells and Other Analytes," filed on

September 15, 2008.

TECHNICAL FIELD

This specification relates to isolating microvesicles using microfluidic systems.

BACKGROUND

Many cell types actively shed small microvesicles (also known as exosomes) into the extracellular space including blood and other body fluids such as urine, milk, saliva, amniotic fluid, and malignant effusions. These membrane vesicles range in size from 30-

100 nm in diameter. Exosomes can arise by invagination of the limiting membrane of late endosomes, which leads to the formation of multivesicular bodies (MVBs). The contents of the MVBs are released into the extracellular milieu upon fusion of the MVBs with the plasma membrane. Exosomes can also bud directly from the plasma membrane. Hence, exosomes contain cellular components and expose the extracellular domain of receptors at their surface.

The molecular composition of exosomes is influenced by the type and activation state of the cell of origin. In addition to a set of membrane and cytosolic molecules common among donor cell types, exosomes can harbor unique and selected subsets of proteins, mR A, and microRNAs (miR As) associated with specific cell type - associated functions and genomic state.

Exosomes were first identified through their involvement in the elimination of excess proteins. Several recent findings indicate that exosomes constitute a potential mode of targeted intercellular transfer of molecules. Following interaction with the recipient cell by fusion, adhesion, or direct binding, exosomes can confer different functions on the recipient cell. The physiologic functions of exosomes vary with the cell of origin and include modulation of immune status and stimulation of angiogenesis, for example. Exosomes are increasingly recognized to play a role in inflammation, cancer, thrombosis, cell polarization, development, and neurological disorders.

The exosomal-acellular mode of communication can aid in the development of diagnostic and therapeutic strategies. Microvesicles contain sorted sets of molecules involved in many different cellular processes, have the capacity to transmit signaling and genetic information, and can be obtained non-invasively from body fluids. For example, some tumor cells are very active at shedding exosomes into the circulating vasculature.

These tumor exosomes and their constituent RNAs present unique genetic information about the tumor concerning its presence, cellular type, state of malignancy, and susceptibility to therapeutic treatment. Isolation of RNA from serum exosomes can yield up to sixty times greater concentrations of high integrity RNA as compared to that extracted directly from blood, serum or plasma. Therefore, exosomal RNA analysis provides an increase in the diagnostic sensitivity of transcriptome analysis and a tool to identify potential disease biomarkers.

Some protocols for isolation of microvesicles from blood involve high speed centrifugation and filtration. A method to purify exosomes from cell culture supematants or body fluids involves a series of centrifugations and filtration to remove dead cells, large debris, and other cellular contaminants resulting from cell lysis, followed by a final high-speed ultracentrifugation to pellet small membrane vesicles. This

ultracentrifugation technique can take 4-5 hours to yield a recovery of exosomes, ranging from 5-25% of the starting exosome MHC class II concentration. Contaminating material, such as protein aggregates, apoptotic vesicles, or nucleosomal fragments that are released by apoptotic cells, can be separated from exosomes by flotation on continuous sucrose gradient. Exosomes 'float' to a density close to 1.13 g/ml. This density may vary among different cells of origin depending on the content of the microvesicles.

Another method of exosome isolation based on ultrafiltration and centrifugation in sucrose deuterium oxide (D ₂ 0) cushions is capable of preparing clinical-grade purified exosomes. This other method can take 4-6 hours to yield an exosome recovery rate of

36-65% based on the starting exosome MHC class II concentration. Microvesicles of tumor origin can also be isolated utilizing adherence to magnetic beads coated with antibodies against tumor-associated markers, such as epithelial cell adhesion molecule (EpCAM). The purification involves ultracentrifugation and can take longer than 3 hours to recover exosomes diluted in the elution from magnetic beads.

Microfluidics and miniaturized lab-on-a-chip-type devices are applicable for medical diagnostics and blood analysis. The small dimensions of microfluidic devices, small sample sizes, and minimal amounts of reagents needed allow for faster reaction times, increased sensitivity, and reduced procedural costs.

SUMMARY

Microvesicles shed from cells provide a means whereby cells can communicate with cells nearby or at a distance and appear to play important roles in immunology and tumor development. This specification describes technologies relating to microfluidic systems for isolating microvesicles.

One innovative aspect of the subject matter described in this specification can be implemented as a method for isolating microvesicles in a serum sample. A serum sample that includes microvesicles and other particles is flowed through a microchannel formed in a microfluidic device at a first flow rate. The microfluidic device includes a binding agent selective for selectively binding substantially all of the microvesicles, but not the other particles, within the microchannel. An eluent solution is flowed through the microchannel at a second flow rate to release the microvesicles from the surface of the microchannel.

This, and other aspects, can include one or more of the following features. The eluent solution can include 1% BSA and 1 mM EDTA. The binding agent can be selected from a group consisting of anti-CD63, IgG, and anti-CD4. The binding agent can be adhered to a surface of the microchannel. The surface of the microchannel can be treated to bind the binding agent to the surface by pre-treating the microchannel with 4% (v/v) solution of 3-mercaptopropyl trimethoxysilane in ethanol for 30 minutes at room temperature, incubating the microchannel with Ο.ΟΙμιηοΙ/mL GMBS in ethanol for 15 minutes at room temperature, incubating the microchannel with 10 μg/mL NeutrAvidin solution in PBS for 1 h at 4 °C, and injecting 10 μg/mL of biotinylated anti-CD63, control IgG, or anti-CD4 solution in PBS containing 1% (w/v) BSA and 0.09% (w/v) sodium azide into the microchannel. Between each step of treating the surface of the

microchannel, the microchannel can be rinsed with one of ethanol or PBS. The surface of the microchannel can include multiple grooves. The microchannel can be 19 mm wide, 20 μιη deep, and 4.5 cm long. Each groove can be 50 μιη wide and 10 μιη deep. The microchannel can be 5 cm long, 4 mm wide, and 30 μιη deep. Multiple posts can be formed within the microchannel. The binding agent can be adhered to the multiple posts, such that the microvesicles, and not the other particles, bind to the multiple posts. The first flow rate can be one of substantially 16 μί/ηιίη or substantially 4 μΕ/ηιίη. Flowing the eluent solution through the microchannel at the second flow rate can include flowing 168 μΙ_, at the second flow rate of 30 μί/ηιίη or flowing 50 μΙ_, at the second flow rate of

Another innovative aspect of the subject matter described in this specification can be implemented as a method for extracting RNA from microvesicles in a microfluidic device. A serum sample that includes microvesicles and other particles is flowed through a microchannel formed in a microfluidic device at a first flow rate. The microfluidic device includes a binding agent selective for selectively binding substantially all of the microvesicles, but not the other particles, within the microchannel. A volume of lysis buffer and a volume of air equal to the volume of the lysis buffer are flowed through the microchannel at a second flow rate. When flowed through the microchannel at the second flow rate, the lysis buffer lyses the microvesicles bound to the microchannel resulting in lysate. The lysate is eluted from the microchannel. RNA is extracted from the lysate.

This, and other aspects, can include one or more of the following features. The binding agent can be adhered to a surface of the microchannel that includes multiple grooves. The multiple grooves can be formed in a ceiling of the microchannel. The microchannel can be 19 mm wide, 20 μιη deep, and 4.5 cm long. Each groove can be 50 μιη wide and 10 μιη deep. The binding agent can be adhered to multiple posts formed within the microchannel. 30 μΐ _^ of homogenate can be added to the lysate. RNA can be extracted from the lysate by phenol-chloroform separation. The extracted RNA can be precipitated with 100% ethanol. The microchannel can be 5 cm long, 4 mm wide, and 30 μιη deep.

Particular implementations of the subject matter described in this specification can be implemented so as to realize one or more of the following advantages. The microfluidic systems can be implemented as a rapid, point-of-care tool for the diagnosis of cancer. The immunoaffinity methods described here enable fast, simple, and specific isolation of microvesicles, including microvesicles of specific cell origin, from small volumes of both serum from blood samples and conditioned medium from cells in culture. Exosomes can be isolated from human sera or cell culture supernatants without requiring ultracentrifugation or resolution on sucrose gradients. With the micro vesicle microfluidic procedure, high quality RNA can be extracted from small volumes of serum to increase the sensitivity of tumor detection and mutational/quantitative evaluation of tumor-derived RNAs

Ribonucleic Acid (RNA) of high quality can be extracted from the isolated microvesicles, for example, within one hour from 100 - 400 serum samples. In particular, microvesicles shed by tumors can specifically be pulled out using tumor- specific cell surface markers, for example, mutant/variant cell surface protein like EGFRvIII32 and other tumor-enriched proteins. Further, organ-specific microvesicles when organ-specific surface markers are present. Also, the half-life of free RNA in serum is seconds to minutes, whereas RNA is protected from RNases in microvesicles. Thus, RNA extracted directly from serum includes a larger fraction of degraded RNA and more RNA from dead normal cells, as compared to living tumor cells. Extraction of microvesicles from serum can be scaled. The quality and quantity of RNA were sufficient for mutation-specific PCR or sequence analysis to potentially identify the point mutation of the IDH-1 transcript.

In addition, the microfluidic isolation of microvesicles can sort microvesicles directly from serum in a single step. This contrasts with magnetic-bead-based systems that require multiple open-system preparation steps (incubation, washing, centrifugation and ultracentrifugation), which can result in fusion and loss of a significant proportion of microvesicles, and are difficult to validate for clinical manufacturing. Microvesicles can be readily purified without any cellular contamination and are continuously shed by tumor cells into the circulation while the accumulation of microvesicles of non-tumor cell origins is rarely observed, which facilitates the genetic and proteomic analysis of tumor- derived microvesicles. Although microvesicles do not mirror the exact transcriptome of the tumor cells of origin, but rather a subset composition of these cells, tumor-derived microvesicles can have enhanced expression of tumor antigens and unique genetic information, which can present a distinctive opportunity for early diagnosis and monitoring of cancer.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.

DESCRIPTION OF DRAWINGS FIG. 1 is an example of a microfluidic system for isolating exosomes.

FIG. 2 is an illustration of an experimental setup of a microfluidic device for isolating exosomes.

FIG. 3 is an example of a microfluidic device having grooves. FIGS. 4A-D show particle flow paths in a microchannel having flat walls and another microchannel having grooves formed in a wall.

FIG. 5A-C show exemplary grooves.

FIGS. 6A-C show an exemplary method of forming the microfluidic device of

FIG. 3.

FIG. 7 is a flowchart of an example process for isolating exosomes using a microfluidic device.

FIGS. 8A-D are scanning electron microscopy (SEM) images of captured microvesicles.

FIG. 9A and 9B show distributions of the projected area diameters and Feret's diameters of microvesicles in the SEM images of FIG. 8A and FIG. 8B, respectively.

FIGS. 10A and 10B show intensities and size distributions of fluorescently- labeled total R A extracted from microvesicles.

FIG. 11 shows images of RNA extracted from micro fluidically isolated microvesicles.

Like reference symbols in the various drawings indicate like elements.

DETAILED DESCRIPTION FIG. 1 is an example of a microfluidic system 100 for isolating exosomes. The system 100 can be implemented to flow a serum that includes microvesicles and other particles that have been shed from a surface of cells through a microchannel in a microfluidic device such as microfluidic device 120. As described later, the serum can be extracted from blood samples obtained from patients and donors that are processed, for example, by centrifugation and ultracentrifugation to obtain pellets of microvesicles together with other particles. The micro fluidic device described with reference to the following figures and having a surface as described below can bind the microvesicles and not the other particles such that only microvesicles are isolated from the serum. In particular, implementing the immunoaffinity method described here reduces and nearly eliminates contamination of the microvesicles by the other particles that have been shed from the surface of cells.

In some implementations, the micro fluidic system 100 includes a syringe pump 105 to flow serum samples in a syringe 110 through a micro fluidic device 120. The micro fluidic device 120 includes a microchannel having an inlet and an outlet. The syringe pump 110 is coupled to the inlet through a tubing 115. The serum sample flows out of the outlet of the microchannel through another tubing 125. As described later, isolated exosomes or lysed exosomes can be collected through the other tubing 125. In some implementations, the microchannel is a straight channel having grooves formed on a surface (for example, the ceiling).

Alternatively, the microchannel can be curved (symmetrically or asymmetrically).

In some implementations, the microchannel can include combinations of one or more straight portions and one or more curved portions. All or some of the microchannel can include grooves on the surface while other portions need not include grooves. Further, the grooves can be formed in more than one surface of the microchannel. In some implementations, multiple posts can be formed within a microchannel. Binding agents, described below, can be bound to the multiple posts or the surface of the microchannel or both. FIG. 2 is an illustration of an experimental setup of a microfluidic device for isolating exosomes. An example of the microfluidic device that has grooves formed on a surface of the microchannel is described with reference to FIG. 3.

In another example in which a microfluidic device having a smaller footprint was used, the optimized flow rate was 4 μΙ7. The optimized flow rate was selected based on the dimensions of the microchannels and the estimated diffusivity of micro vesicles. In this example, 50 of 1% BSA and 1 mM EDTA were flowed at 10 μί/ηιίη.

FIG. 3 is an example of a microfluidic device 300 having grooves 335, 340 extending into one of the walls defining a channel 315 of the device 300. In some embodiments, microfluidic devices include protrusions extending outward from the wall

(for example, V-shaped protrusions) rather than grooves extending into a wall of the channel 315. In some implementations, a microfluidic device 300 can include an upper substrate 305 bonded to a lower substrate 310, each of which can be fabricated using an appropriate material. For example, the upper substrate 305 can be fabricated using an elastomer such as, for example, polydimethylsiloxane (PDMS), and the lower substrate can be fabricated using glass, PDMS, or another elastomer. Alternatively, or in addition, the substrates can be manufactured using plastics such as, for example,

polymethylmethacrylate (PMMA), polycarbonate, cyclic olefin copolymer (COC), and the like. In general, the materials selected to fabricate the upper and lower substrates can be easy to manufacture, for example, easy to etch, and can offer optical properties that facilitate ease of testing, for example, can be optically clear, and can be non-toxic so as to not negatively affect the cells attached to the substrate. In addition, the materials are preferred to exhibit no or limited autofluorescence. Further, the materials can be easy to functionalize so that analytes can be attached to the substrate. Furthermore, the materials can be mechanically strong to provide strength to the micro fluidic device 300. The upper substrate 305 can be securely fastened to the lower substrate 310, with a micro-channel formed between them, as described below.

In some implementations, the micro-channel 315 can have a rectangular cross- section including two side walls 320 and 325, and an upper wall 330 formed in the upper substrate 305. Terms of relative location such as, for example, "upper" and "lower" are used for ease of description and denote location in the figures rather than necessary relative positions of the features. For example, the device can be oriented such that the grooves are on a bottom surface of the channel or such that a central axis of the channel extends vertically. Alternatively, the cross-section of the micro-channel 315 can be one of several shapes including but not limited to triangle, trapezoid, half-moon, and the like. The lower substrate 310 can form the lower wall of the micro-channel 315 once bonded to the upper substrate 305. In some implementations, the micro-channel 315 includes multiple grooves 335 formed in the upper wall 330 of the micro-channel 315.

Alternatively, the grooves 335 can be formed in any of the walls, and/or can be formed in more than one wall of the micro-channel 315. The grooves 335 can span an entire length of a wall, or only a portion of the wall.

FIGS. 4A-D show particle flow paths in a microchannel having flat walls and another microchannel having grooves formed in a wall. The figures illustrate particle suspensions flowing through a micro-channel having flat walls and another micro- channel having grooves formed in a wall. FIG. 4A shows a microfluidic device 400 that includes a micro-channel 405 having a rectangular cross-section. The walls of the micro- channel 405 do not include grooves such as those described with respect to the microfiuidic device 300, i.e., surfaces of the walls are fiat. A particle suspension 420 including particles 425 suspended in a fluid is flowing through the micro-channel 405. In contrast, FIG. 4B shows a similar suspension flowing through the microfiuidic device 300.

As the fluid flows past a herringbone pattern formed by arranging grooves 335 in a column in the micro-channel 315, the grooves 335 in the path of the fluid disrupt fiuid fiow. In some embodiments, depending upon fiow velocity and the dimensions of the grooves, specifically, for example, a size of the grooves and an angle between the two arms of a groove, the disruption in the fiuid fiow leads to a generation of microvortices in the fluid. The microvortices are generated because the grooves induce fluid flow in a direction that is transverse to a principal direction of fiuid fiow, i.e., the axial direction. In some embodiments, although microvortices are not generated, the grooves 335, 340 induce sufficient disruption to alter the flow path of portions of the fluid to increase wall- particle interactions.

In an absence of the grooves, as shown in FIG. 4C, the particles 425 suspended in the fluid travel through the flat micro-channel 405 in a substantially linear fashion such that only those particles 425 near the edges of the fiow field (for example, immediately adjacent to the walls of the micro-channel 405) are likely to interact with the micro- channel 405 walls. In contrast, as shown in FIG. 4D, fiowpaths of the particles 425 traveling past the herringbone patterns experience can be disrupted by the microvortices in the fiuid, increasing the number of particle-micro-channel wall interactions. The microvortices are affected by the structural features of each groove 445 formed in the upper wall 330 of the micro fluidic device 300. Exemplary dimensions of a groove 445 are described with reference to FIGS. 3A-C.

FIG. 5A-C show exemplary grooves. In particular, FIGS. 5A and 5B illustrate a groove 335 formed on an upper wall 330 of a micro-channel 315. As shown in FIG. 5 A, a symmetric groove 335 includes two arms, each spanning a length between a first end 350 and the apex 345 (U), and a second end 355 and the apex 345 (1 ₂ ). In the illustrated embodiments, the angle a between the two arms is 90°. In some embodiments, the angle a between the arms ranges between 10° and 170°. FIG. 5B is a view of the micro-channel 315 including the groove 335 formed in the upper surface 315. As shown in FIG. 5B, the width of the groove is w, the height of the side walls 320 and 325 of the micro-channel 315 is h _c and the height of the groove 335 formed on the upper wall 315 is h _g . In some embodiments, U and 1 ₂ , each range between 250 μιη - 400 μιη, h _g ranges between 3 μιη and 70 μιη, h _c is 100 μιη. For example, when h _c is 100 μιη, h _g is 25 μιη.

FIG. 5C illustrates an asymmetric groove 340 including two arms, each spanning a length between a first end 370 and an apex 365 (1 ₃ ), and a second end 375 and the apex (I ₄ ), respectively. In the illustrated embodiment, the angle β between the two arms is 90°, and can range between 10° and 170°. In some implementations, the groove 340 can be manufactured such that a ratio between 1 ₃ and 1 ₄ is 0.5. For example, 1 ₃ is 141 μιη and 1 ₄ is 282 μιη. The groove 340 has a thickness of 35 μιη. An effect of the height of the groove, h _g , on particle capture is described with reference to FIG. 15.

A herringbone pattern can be created by forming a column of herringbones in which each groove is positioned adjacent to another groove. Further, all grooves in the column can face the same direction. In some embodiments, a distance between each groove is 50 μηι. Alternatively, the grooves can be positioned at any distance from each other. A column can include any number of grooves, for example, ten grooves. The herringbone pattern can further include multiple columns of grooves formed serially from an inlet to the outlet. In some embodiments, two adjacent columns of grooves can be separated by 100 μιη. In other words, a first groove of the second column can be positioned 100 μιη away from a last groove of the first column. This pattern can be repeated from an inlet to the micro-channel 315 to the outlet.

In some embodiments, grooves or groups of grooves in a column can be laterally offset from each other. For example, as can be see in FIG. 4B, the column of grooves in micro fluidic device 300 includes a first set of grooves with apexes set to the right (facing downstream) of the channel centerline and a second set of grooves with apexes set to the left of the channel centerline. Such offsets are thought to further increase wall-particle interactions.

The dimensions shown in FIGS. 5A-5C are exemplary. In general, the choice of groove heights can depend on factors including channel dimensions, particle properties including size, density, and the like, and particle suspension flow rates. Although deeper grooves offer more disruption, other factors can impose limits on groove heights. For example, up to a certain limit, the groove height can be increased in proportion with the channel height. The channel height, and consequently the groove height, can depend upon the particle to micro-channel 315 surface contact area. An increase in channel dimensions can cause a decrease in particle-micro-channel 315 interactions as surface contact area available for the particles to interact decreases relative to the cross-sectional flow area. Also, a lower limit on the channel height, and consequently the groove height, can be imposed to prevent clogging. In some implementations, a ratio between groove height and channel height can be less than one, for example, in a range between 0.1 to 0.6. In some implementations, the ratio can be equal to one (for example, the groove height can be equal to the channel height), or can be greater than one (for example, the groove height, for example, 60 μιη, can be greater than the channel height, for example, 50 μιη). Further, the shape of the groove can be different from a "V" shape, for example, "U" shape, "L" shape, and the like.

The micro-channel 315 can be formed in the upper substrate 105, for example, using soft lithography techniques. In some implementations, negative photoresist (SU-8, MicroChem, Newton, MA, USA) can be photolithographically patterned on silicon wafers to create masters with two-layer features. The masters thus formed can include SU-8 features that form the basis for the features of the micro-channel 315, for example, channel cross-section, channel size, and the like. The heights of SU-8 features (ranging from 3 μιη - 100 μιη) on the masters can be measured with a surface profilometer such as a Dektak ST System Profilometer, commercially available from Veeco Instruments Inc., Plainview NY. The masters can then be used as molds on which PDMS pre-polymer can be poured and allowed to cure in a conventional oven at 65 °C for 24 hours. The upper substrate 305, including the micro-channel 315, is formed when the poured PDMS pre- polymer is cured. The cured upper substrate 310 can be removed from the molds and bonded to the lower substrate 305, for example using oxygen plasma treatment, to form the micro fluidic device 300. Alternatively, other types of bonding, for example, using a reversible sealant, using physical clamping and holding under pressure, and the like, can be used. In some implementations, the substrates can be securely bonded together through chemical bonds, and can subsequently be separated by breaking the bonds under the application of mechanical forces.

FIGS. 6A-C show an exemplary method of forming the microfluidic device of FIG. 3. In particular, FIGS. 6A-6C illustrate the formation of a microfluidic device 600 including an upper substrate 605 manufactured using PDMS and a lower substrate 610 manufactured using glass. The upper substrate 605 including the upper and side walls of the microchannel 615 can be formed using previously described techniques.

Alternatively, or in addition, the upper wall can include multiple grooves 640, each formed in an asymmetric "V" shape. In some implementations, symmetric grooves 640 and asymmetric grooves 645 can be interspersed in the herringbone pattern. Each groove further includes an apex 645 and two ends 650 and 655. In addition, the micro-channel 615 includes two side walls 620 and 625.

To configure the microfluidic device 600 to capture the biological analyte of interest, an adherent 660 is disposed on the inner surfaces of the micro-channel 615. Specifically, surface modification is performed on the inner surfaces. In some implementations, as shown in FIG. 6B, the adherent 660 can be mixed in a solution and flowed through the micro-channel 615. As the solution flows through the micro-channel 615, the adherent 660 binds to, and is thereby disposed in the inner surfaces of the channel 615.

Techniques other than flowing the adherent through the micro-channel 615 can also be used to dispose the adherent. For example, in implementations in which plastic substrates are employed, the adherent can be disposed on the substrate, for example, by ultra-violet (UV) radiation treatment to alter the surface properties such that analytes bind to the altered surface prior to bonding the upper and lower substrates. In implementations in which the lower substrate is glass, the glass can be functionalized, for example, by sputtering, by gas phase deposition, by building up layers of nanoparticle monolayers, and the like prior to bonding the glass substrate to the upper substrate.

As shown in FIG. 6C, the adherent 660 can be disposed throughout the inner surfaces of the micro-channel 615. Alternatively, the adherent 660 can be disposed in one or more walls of the micro-channel 615, for example, in the wall in which the grooves 645 are formed. In some embodiments, the adherent 660 can be disposed only on a lower substrate 610 manufactured from glass. In such embodiments, the lower substrate 610 can be bonded to the upper substrate 605 after the adherent is disposed on the lower substrate. In such implementations, the flow rate of the fluid is selected such that the microvortices established by the grooves 640 drive the cells in the fluid toward the lower substrate 610 increasing a number of cell-lower substrate 610 interactions. Subsequently, the lower substrate 610 can be separated from the upper substrate 605 and the captured cells can be cultured.

In some implementations, the adherent 660 can be selected such that the micro- channel 615 can be used for affinity-based cell capture utilizing wet chemistry techniques. In such implementations, the adherent 660 can be an antibody, for example, antibody for EpCAM, or an aptamer, for example, aptamer for surface proteins, with which the inner surfaces of the micro-channel 615 are functionalized. Additional examples of adherent 660 include avidin coated surfaces to capture amplified target cells that express biotin through the biotin-avidin linkage. Further examples of adherents corresponding to cells that can be captured are shown in Table 1 below. Table 1

Once functionalized, the inner surfaces function as capture devices that can bind analytes of interest. As described with reference to the following figures, the inner surfaces of the microchannel can be functionalized to capture exosomes that are shed by healthy cells and/or by cancerous cells. By doing so, the microfluidic device 300 can be used to isolate the exosomes.

In some implementations, the microfluidic device 120 can include a straight flow microchannel, for example, of 19 mm width, 20 μιη depth, and 4.5 cm length. The microchannel can have a ceiling with herringbone groves, as described previously, that are, for example, 50 μιη wide, 10 μιη deep for processing samples of volumes of 400 μΐ,. In some implementations, the microfluidic device 120 can have dimensions of 5 cm (L) x 4 mm (W) x 30 μιη (H) for processing samples of volumes smaller than 100 μΐ,. Such a device can also be used for SEM imaging described later. The Herringbone or slanted grooves in microchannels can be utilized for mixing of fluids/particles, separation and separation of particles. These patterns can increase the capture efficiency, for example, due to the increased surface area and enhanced contact of particles with surfaces of the microchannel. The micro fiuidic device 120 can be fabricated using one of several materials, for example, PDMS. A device thus fabricated can be bonded permanently to plain PDMS slabs after treated with oxygen plasma.

The surfaces of the microchannel can be activated to capture exosomes in a serum sample that is flowed through the microchannel. For example, anti-human CD63 IgG can be adhered to the surfaces of the microchannel, as described previously. One of the most abundant protein families that are found in exosomes comprises the tetraspanins and several members of this family, including CD9, CD63, CD81 and CD82— are highly enriched in exosomes from virtually any cell type. Anti-human CD63 IgG was chosen for isolating microvesicles from all cell origins because of its high expression levels. However, CD63 antigens are also expressed on the surface membrane of platelets, granulocytes and monocytes at low levels. When serum samples were flowed into anti- CD63 IgG coated microchannels, very few platelets and nucleated cells were found to adhere to the microchannel surface. Certain implementations are described in the following examples, which do not limit the scope of the invention recited in the claims.

Example 1 :

Next, micro fiuidic capture of microvesicles was performed by injecting 10 μΐ,

PKH67-labeled microvesicle preparations obtained from differential centrifugations of the glioblastoma cell culture supernatant into microchannels coated with anti-CD63 antibodies or anti-CD4 antibodies as a negative control. Fluorescence intensity measurements inside the microchannel showed that microvesicle capture was higher in anti-CD63 IgG coated microchannels, as compared to anti-CD-4 coated channels and increased in the case of anti-CD63 with increasing concentration of micro vesicles.

Example 2:

The microchannel was pretreated with 4% (v/v) solution of 3-mercaptopropyl trimethoxysilane in ethanol for 30 minutes at room temperature. The pretreated microchannel was incubated with Ο.ΟΙμιηοΙ/mL GMBS in ethanol for 15 minutes at room temperature. Subsequently, the microchannel was incubated with 10 μg/mL NeutrAvidin solution in PBS for 1 h at 4 °C. Finally, 10 μg/mL biotinylated anti-CD63, control IgG, or anti-CD4 solution in PBS containing 1% (w/v) BSA and 0.09% (w/v) sodium azide was flowed through the microchannel to react with NeutrAvidin at room temperature for 15 min. After each step, the surfaces of the microchannel were rinsed with 8 device volumes of either ethanol or PBS, depending on the solvent used in the previous step, to flush away unreacted molecules. Serum samples, prepared using techniques described below, were flowed through the surface-treated microchannel to isolate exosomes.

Serum samples were obtained from healthy donors as well as from glioblastoma- confirmed subjects. In particular, the blood samples were obtained through the Cancer

Center Amsterdam, VU University Medical Center, Amsterdam, Netherlands under IRB approved protocols. Samples of 10 mL of peripheral blood were collected by

venipuncture in serum separation tubes (BD Biosciences, Franklin Lakes, NJ) at the time of surgery and processed according to the manufacturer's protocol (< 2 hours from time of collection until freezing of serum), followed by passage through a 0.8 μιη filter. These samples were kept at -80°C until use. For primary cell culture, brain tumor specimens from patients diagnosed by a neuropathologist as glioblastoma multiforme (GBM) were taken directly from surgery and placed in cold sterile Neurobasal media (Invitrogen, Carlsban, CA). The specimens were dissociated into single cells within 1 hour from the time of surgery using a neural Tissue Dissociation Kit (Miltenyi Biotech, Bergisch Gladbach, Germany) and plated in DMEM supplemented with 5% micro vesicle-depleted FBS (dFBS; prepared by ultracentrifugation at 110,000xg for 16 hour to remove bovine microvesicles), and penicillin-streptomycin (10 IU/mL and 10 mg/mL, respectively, Sigma-Aldrich, St. Louis, MO).

Primary glioblastoma cells were cultured in DMEM supplemented with 5% dFBS. The conditioned media was removed after 48 hours of cell growth. The microvesicles in the conditioned medium were purified by a series of centrifugations at 4°C. First, the medium was centrifuged at 700 ^x g for 10 minutes to eliminate any cell contamination. The medium was then centrifuged again at 16,500 ^x g for 20 min, and passed through a 0.22 um filter. Microvesicles were then pelleted by ultracentrifugation at 110,000 ^x g for 70 min. The microvesicle pellets were washed in 13 mL of PBS, re- pelleted, and re-suspended in PBS. The microvesicle pellets, re-suspended in PBS were flowed through the surface-treated microchannel to isolate the microvesicles in the microchannel. In one example, 10-400 serum samples were flowed into the microchannel at an optimized flow rate, for example, 16 μΕ/ηιίη. After rinsing with PBS containing 1% BSA and 1 mM EDTA (168 at a flow rate of 30 μΕ/ηιίη), microvesicles adherent to the surface were fixed for scanning electromicrograph (SEM) or lysed for R A extraction. FIG. 7 is a flowchart of an example process 700 for isolating exosomes using a microfluidic device. The process 700 is one implementation of flowing serum samples that include exosomes through a surface-treated microchannel of a microfluidic device included in a microfluidic system, for example, system 100 described with reference to FIG. 1. The process 700 includes injecting culture supernatant/fluid at 16 μΕ/ηώηιίεβ for 25 minutes (step 705). The exosomes in the injected culture adhere to the previously treated surface of the microchannel. The process 700 includes rinsing the microchannel with PBS at 30 μΕ/ηώηιίεβ for 5.6 minutes (step 710). The process 700 includes isolating the exosomes adhered to the microchannel surface (step 315), for example, by fixing for SEM or lysing for R A extraction.

FIGS. 8A-D are scanning electron microscopy (SEM) images of captured microvesicles. FIG. 8A is an SEM image showing microvesicles prepared by sequential centrifugation of cell culture supernatant bound to the microchannel surface. FIG. 8B is an SEM image showing microvesicles bound to the microchannel surface after 10 of GBM patient serum was passed through the previously-treated microchannel. FIG. 8C and 8D are the images of FIG. 8 A and 8B, respectively, at higher magnification. The SEM images show that surfaces of anti-CD63 antibodies coated microchannels were covered by microvesicles separated from 10 μΐ _^ οΐ microvesicle-containing medium obtained from primary glioblastoma cell cultures (FIG. 8A) or serum from a GBM patient (Figure 8B-D).

FIG. 9A and 9B show distributions of the projected area diameters and Feret's diameters of microvesicles in the SEM images of FIG. 8A and FIG. 8B, respectively. The size distributions of captured microvesicles in SEM images were analyzed using an image processing program (Image J, National Institutes of Health). Images were converted to binary images by background subtraction followed by setting thresholds. The projected area diameters were calculated from the projected area assuming circular geometry while Feret's diameters were determined as the greatest distance possible between any two points along the boundary of a region of interest. The size distributions of microvesicles captured from these two samples (shown in FIGS. 9A and 9B, respectively), were well within the range reported in the literature. Only 3% of the projected area diameters of microvesicles from serum were greater than 100 nm (FIG. 8C and 9B). However, more than 13% of microvesicles from medium had a projected area diameter greater than 100 nm (FIG. 8 A and 9A).

Total RNA was purified using the MirVana RNA isolation Kit (Ambion, Austin, TX) according to the manufacturer's protocol. In brief, 300 μΐ, of Lysis/Binding buffer followed by an equal amount of air was passed through the microchannel at a flow rate of 25 μΕ/ηώηιίεβ to lyse microvesicles captured on the chip. The lysate was collected in a collection tube via two inches of Teflon tubing (Small Parts, Inc., Miramar, FL). 30 homogenate provided in the isolation kit was added to the lysate. Total RNA was extracted by phenol-chloroform separation and precipitated with 100% ethanol and collected in the Elution solution. RNA was further purified and concentrated using Qiagen miniElute RNA cleanup kit (Qiagne Inc., Valencia, CA) according to the manufacture's protocol. The quality of RNA was examined on a Eukaryote Total RNA Pico chip with a 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA).

FIG. 11 shows images of RNA extracted from microfluidically isolated microvesicles. Total RNA purified was then converted into cDNA using Sensiscript RT kit (Qiagen Inc, Valencia, CA) with a mix of oligo dT and random nonamers according to the manufacture's protocol. The mRNA for GAPDH (glyceraldehyde 3-phosphate dehydrogenase) was selected as a control because it is usually stably and constitutively expressed at high levels in most tissues, cells and micro vesicles 10. IDH-1 (isocitrate dehydrogenase 1) mRNA was analyzed as being of particular interest, as 12% GBM patients have a specific point mutation in this gene, which favors a better prognosis28, 29. The sequences of primers used in the PCR were: GAPDH primers - Forward 5'-CAG CCT CAA GAT CAT CAG CA-3', Reverse 5'-TGT GGT CAT GAG TCC TTC CA-3'; IDH-1 primers - Forward 5'-CGG TCT TCA GAG AAG CCA TT -3', Reverse 5'-TAT TGA TCC CCA TAA GCA TGA T -3'. PCR protocol: 95 °C 3 min; 95 °C 20 s, 59 °C 20 s, 72 °C 30 s 35 cycles; 72 °C 7 min.

RNAs from microvesicles from serum captured on microchannels as well as from the corresponding serum samples were extracted and analyzed with a Bioanalyzer, showing that the microvesicles contained a broad range of RNA sizes consistent with a variety of mRNAs and miRNAs (Fig. 3), but lacked the ribosomal peaks characteristic of cellular RNA. There appeared to be fewer microvesicles and less RNA in the normal serum compared to the GBM patient serum as indicated by RNA concentrations measured with a Bioanalyzer (Fig. 3 and Table 1). In addition, more RNAs were extracted from lysates from anti-CD63-coated chips than from both GBM patient/normal control sera directly. RT-PCR analysis showed that the mRNAs for GAPDH (106 bp product) and IDH-1 (100 bp product) were present in the microvesicles captured from both GBM patient/normal control sera on anti-CD63 -coated microchannels (Fig. 4, lane 1 and 3), but little were present in lysates from control IgG-coated microchannels (Fig. 4, lane 2 and 4).

The percentage recovery of microvesicles ranged from 42-94% (n=4) based on the total RNA amounts extracted on chip and from the effluent. The capture yield of microvesicle-chip can be increased, for example, by increasing microvesicle-surface interactions within the chip, such as modifying the dimensions of the microchannel, incorporating structures inside the microchannel, augmenting transverse flow, and increasing the coverage of active antibodies on the surface.

In summary, exosomes shed from both normal and cancerous cells provide a means of intercellular communication allowing for exchange of charged molecules, including RNA and non-secreted proteins between cells. The easy and rapid micro fluidic immunoaffinity method can be implemented to isolate exosomes from both serum and cell culture medium. High quality RNA can be extracted in a single wash-through procedure with RT-PCR of mRNA and miRNA providing biomarkers for mutational status and expression profiles for diagnosis and prognosis of tumors.

While this specification contains many specific implementation details, these should not be construed as limitations on the scope of any inventions or of what may be claimed, but rather as descriptions of features specific to particular implementations of particular inventions. Certain features that are described in this specification in the context of separate implementations can also be implemented in combination in a single implementation. Conversely, various features that are described in the context of a single implementation can also be implemented in multiple implementations separately or in any suitable subcombination. Moreover, although features may be described above as acting in certain combinations and even initially claimed as such, one or more features from a claimed combination can in some cases be excised from the combination, and the claimed combination may be directed to a subcombination or variation of a

subcombination.

Similarly, while operations are depicted in the drawings in a particular order, this should not be understood as requiring that such operations be performed in the particular order shown or in sequential order, or that all illustrated operations be performed, to achieve desirable results. Thus, particular implementations of the subject matter have been described. Other implementations are within the scope of the following claims. In some cases, the actions recited in the claims can be performed in a different order and still achieve desirable results. In addition, the processes depicted in the accompanying figures do not necessarily require the particular order shown, or sequential order, to achieve desirable results.