Biomarker detection using integrated purification-detection devices

Techniques regarding integrated purification-detection devices for detecting one or more biomarkers are provided. For example, one or more embodiments described herein are directed to an apparatus, comprising a housing and a microfluidic chip contained within the housing. The microfluidic chip comprises a separation unit that separates, using one or more nano deterministic lateral displacement (nanoDLD) arrays, target biological entities having a defined size range from other biological entities included in a biological fluid sample. The microfluidic chip further comprises a detection unit that facilitates detecting presence of one or more biomarkers associated with the target biological entities using one or more detection molecules or macromolecules that chemically reacts with the one or more biomarkers.

BACKGROUND

The subject disclosure relates to integrated purification-detection devices for detecting one or more biomarkers, and more specifically, to integrating lateral deterministic displacement arrays for particle purification and one or more sensor arrays for biomarker detection onto a single microfluidic chip.

Technologies capable of detecting the presence of biomarkers are ubiquitous in biochemistry and a necessary element of diagnostic devices in healthcare. Common methods of detection, such as enzyme-linked immunosorbent assays (ELISAs), utilize high-affinity interactions between antibodies and their target epitope to achieve chemical specificity in detecting a particular analyte. One exemplary application of this method of revealing chemical specificity is targeting the epitopes of exosomes. Exosomes are extracellular vesicles (EVs) ranging in size from 30-150 nanometers (nm) found in minimally invasive and completely non-invasive biological fluids, or liquid biopsies, such as blood, urine, saliva, etc. Exosomes have emerged as a promising class of biomarkers for studying and identifying various disease conditions. These EVs contain a rich set of genetic information, including tumor-specific proteins, micro ribonucleic acid (microRNA), messenger RNA (mRNA), and deoxy ribonucleic acid (DNA), that can individually and/or collectively provide a glimpse into the health state of an individual at the sub-cellular level. To extract meaningful information from these nanoscale prognosticators first requires the ability to isolate them from a complex biological fluid. Once they are extracted, some form of biochemical analysis or genetic sequencing is needed to detect presence of biomarkers. Both the extraction and detection processes at present are cumbersome, costly, and impractical for frequently running a diagnosis to catch a disease at an early stage or for monitoring a patient's response to a particular treatment.

Focusing on the first requirement, the extraction piece, many standard biochemistry methods have been applied to isolate exosomes, each with its own set of drawbacks, and, in general, the community is actively seeking for better solutions to the sample preparation problem of EVs. The most common methods currently employed for the task include ultracentrifugation (UC), filtration, precipitation, immunoaffinity-based capture, nano deterministic lateral displacement (nanoDLD), Exodisc, viscoelastic flows, and exoTIC.

Ultracentrifugation (UC) exploits size differences between cells, EVs, and proteins to isolate these materials from each other using progressively higher spin speeds with intermediate extraction protocol. Major drawbacks are high spin speeds that can impact EV quality and long run times (around 5 hours). UC is also a manual, batch process often resulting in lower exosome recovery and less than optimal EV quality. Filtration isolation techniques employ membrane filters, such as polyvinylidene difluoride (PVDF) or polycarbonate filters, to sieve cells and large EVs from biological samples. Filtration is sometimes coupled with ultracentrifugation to further separate exosomes from proteins. These types of multistep arrangements require a bulky centrifuge or vacuum system, use large sample volumes (30-100 milliliters (mL)), require batch processing, and typically result in poor yields due to clogging.

Several precipitation kit-based solutions have emerged to circumvent the need for UC, including EXOEASY®, EXO-SPIN®, EXOQUICK® exosome precipitation, TOTAL EXOSOME ISOLATION REAGENT®, and/or PUREEXO®, to name a few. These products use special reagents to induce precipitation of exosomes, such as polyethylene glycol (PEG) based additives. These kits typically suffer from unacceptable purity due to polymer contamination, making downstream analysis difficult. These precipitation kits are also often limited to small, batched sample volumes.

The immunoaffinity-based capture isolation method specifically targets exosomes from a complex biological fluid using, for example, tetraspanin proteins such as CD81 found on the surface of exosomes or markers specific to the exosome's cell of origin to isolate them. A common technique utilizes antibody coated magnetic beads to capture exosomes that contain specific markers from bodily fluids. These methods are expensive, relying on specific antibodies that can vary batch to batch and suffer from stability issues. Thus, while these methods allow specific subpopulations of exosomes to be isolated, the cost of antibodies makes them generally unsuitable for isolating exosomes from large quantities of biological samples.

In light of the inherent drawbacks surrounding the above-mentioned isolation standards, exploration of new solutions that can provide a route toward a simple, inexpensive, automated, and rapid EV isolation techniques have been reported in literature, including for example, lab-on-a-chip based approaches. Exemplary techniques within this realm include nanoDLD, Exodisc, viscoelastic flows, and ExoTIC. NanoDLD refers to a technique wherein deterministic lateral displacement (DLD) technology is shrunk to the nanoscale, demonstrating the ability to subfractionate exosome populations with tens of nanometers resolution in a continuous flow system (no batch processing) with a theory of operation. However, current nanoDLD techniques can only process very low sample volumes at low throughput rates (e.g., about 0.2 microliters (μLs) per hour (hr)). Exodisc is a lab-on-a-disc separation technique presented in H.-K. Woo, et al., ACS Nano, vol. 11, pp. 1360, 2017. The Exodisc technique integrates two on-disc nanofilters that allow fully automated and label-free enrichment of EVs in the size range of 20-600 nanometers (nm) within 30 minutes using a tabletop-sized centrifugal microfluidic system. Although the Exodisc technique have reportedly demonstrated high yields (e.g., greater than 95% recovery of EVs from cell culture and greater than a 100-fold higher concentration of mRNA as compared with UC), the discs employed are large and costly. In addition, sample processing is batched rather than continuous flow, and subfractionation of exosomes is not demonstrated or straightforwardly applicable.

Viscoelastic flow techniques have been used to isolate exosomes from cell culture media and serum in a continuous flow, field-free, and label-free manner using an additive polymer (poly-oxyethylene or PEO) to control the viscoelastic forces exerted on nanoscale EVs. As reported in C. Liu, et al., ACS Nano, vol. 11, pp. 6968, 2017, viscoelastic flow techniques have demonstrated a separation purity greater than 90% with a recovery of greater than 80% and a throughput of 200 μL/hr. However, these techniques also suffer from disadvantages. In particular, viscoelastic flow devices are large (and thus more cumbersome and costly), requiring channels of 32 millimeters (mm) in length to achieve lateral resolution of particle streams (plus space for input/outports), and although isolation of 100 nm and 500 nm particles sizes have been shown, this size selectivity does not lend itself to exosome fractionation.

The exosome total isolation chip (ExoTIC) filtration technique is another exosome isolation technique reported in F. Liu, et al., ACS Nano, vol. 11, pp. 10712-10723, 2017. ExoTIC employs a filtration arrangement to achieve EV yields from 4 to 1000 fold higher than UC using a low protein binding filter membrane from track-etched polycarbonate and a syringe pump driver at flowrates up to 30 mL/hr shown on 6 parallel syringes. A buffer wash step allows for EV purification from smaller contaminates. Subfractionation is also demonstrated by staging filters down to the nanoscale and exosomes from specific cell lines are analyzed in terms of their size distribution. However, since filtration and purification are inherently sequential processes, Exotic is a batch process requiring over 2 hours to perform a sample preparation. In addition, nanoparticle tracking analysis (NTA) performed on ExoTIC subfractionated EV populations does not indicate strong control of fractionated sizes, which calls into question run-to-run reliability.

Exosome detection and molecular profiling of exosomes presents an added challenge for exosome-based cancer diagnostics. Few technologies have arisen that attempt to tackle this problem. One technique described in H. Im, et al.,Nat. Biotechnol., vol. 32, pp. 490-495, 2014, includes a nano-plasmonic exosome (nPLEX) assay, which uses transmission surface plasmon resonance through periodic nanohole arrays functionalized with antibodies to profile the surface proteins of exosomes as well as proteins present in exosome lysates. This technique was successful at identifying exosomes derived from ovarian cancer cells by their expression of CD24 and EpCAM with 100 times the sensitivity of an ELISA, and the exosomal and cellular protein profiles showed excellent correlation. However, upfront sample preparation was required for the nPLEX device to obtain a clean signal using standard UC or filtration. Another technique has been developed that uses an integrated magneto-electrochemical sensor for exosome (iMEX) analysis. (See S. Jeong, et al.,ACS Nano, vol. 10, pp. 1802-1809, 2016). The iMEX technique involves enriching exosomes directly from blood and profiling them for molecular information. The platform uses magnetic selection and electrochemical enrichment to isolate cell-specific exosomes from complex media and achieved high sensitivity through magnetic enrichment and enzymatic amplification to detect these markers electrically. This technique however requires magnetic beads bearing horseradish peroxidase (HRP) labels to isolate the exosomes and produce a signal. In addition, off-platform sample preparation is required for each biomarker along with manual loading of the prepared sample onto each electrode.

SUMMARY

In accordance with various embodiments, an apparatus is provided that comprises a housing and a microfluidic chip contained within the housing. The microfluidic chip comprises a separation unit that separates, using one or more nanoDLD arrays, target biological entities having a defined size range from other biological entities included in a biological fluid sample. The microfluidic chip further comprises a detection unit that facilitates detecting presence of one or more biomarkers associated with the target biological entities using one or more detection molecules or macromolecules that chemically reacts with the one or more biomarkers. In some implementations, the biological entities comprise exosomes. The biological entities can also include other biological molecules and macromolecules ranging in size from 10.0 nm to 200 nm, viruses, DNA sequences, RNA sequences and the like. In some implementations, the one or more detection molecules or macromolecules comprise an antibody or aptamer that binds with a target epitope of the one or more biomarkers.

In various implementations, the detection unit can comprise a sensing element, wherein a surface of the sensing element is coated with the one or more detection molecules or macromolecules. With these implementations, the detection molecules or macromolecules can chemically react with the one or more biomarkers by binding to the one or more detection molecules or macromolecules. In some implementations, based on the binding, the one or more detection molecules or macromolecules generate a visual signal, such as a florescent signal. The sensing element can also comprise a signal enhancing structure selected from a group consisting of a photonic grating structure, a photonic pillar array structure, an optoelectrical structure, and a plasmonic structure. In one or more implementations, a portion of the housing formed adjacent to the sensing element is transparent or partially transparent and enables visual observation of the fluorescent signal.

The microfluidic chip can further comprise at least one conduit from the separation unit to the detection unit that facilitates passage of buffer fluid comprising the target biological entities, as separated from the other biological entities, from the separation unit to the detection unit. At least one inlet can be included on the microfluidic chip through which the buffer fluid passes from the conduit to the surface of the sensing element. In some implementations, the detection unit further comprises a blocking element formed at an interface between the surface of the sensing element and the at least one inlet. The blocking element can inhibit reverse flow of one or more reacted or unreacted molecular complexes (e.g., antibody/exosome complexes) from the surface of the sensing element through the at least one inlet, wherein the one or more reacted molecular complexes are formed as a result of a chemical reaction between the one or more detection molecules or macromolecules and the one or more biomarkers (e.g., an epitope on the surface of the exosomes). The microfluidic chip can further comprise at least one outlet from which the buffer fluid and unreacted portions of the target biological entities that fail to chemically react with the one or more detection molecules or macromolecules, are excreted from the detection unit.

In some implementations, the microfluidic chip further comprises at least one inlet via which solution comprising the one or more detection molecules or macromolecules are injected into the detection unit to coat the surface of the sensing element. In addition, in order to facilitate simultaneous detection of a plurality of biomarkers, the detection unit can comprise two or more separate detection chambers, wherein respective chambers of the two or more separate detection chambers comprise different types of detection molecules or macromolecules of the one or more detection molecules or macromolecules. In this regard, the different types of detection molecules or macromolecules can chemically react with different types of biomarkers.

In another embodiment, a method is provided that comprises isolating target biological entities (e.g., exosomes) having a defined size range from other biological entities included in a biological fluid sample using a separation unit comprising one or more nanoDLD arrays formed on a microfluidic chip, thereby resulting in isolated target biological entities. The method further includes driving flow of a buffer fluid comprising the isolated target biological entities through a conduit of the microfluidic chip from the separation unit to a sensing element formed on the microfluidic chip, and facilitating detection of presence of one or more biomarkers associated with the isolated target biological entities based on whether a detectable signal is generated by the sensing element in response to the driving.

For example, the sensing element can comprise one or more detection molecules or macromolecules, and the detectable signal can comprise a reaction signal that is indicative of a chemical interaction between the one or more detection molecules or macromolecules and the one or more biomarkers. For instance, the chemical reaction can include a reaction selected from a group consisting of a covalent bonding reaction, an electrostatic interaction, a hydrophobic interaction, an antibody-epitope interaction, an aptamer-epitope reaction, a protein-protein interaction, a protein-small molecule interaction, a polymerization reaction, a complementarity reaction, a complementary DNA strand hybridization interaction, and a complementary RNA strand hybridization interaction. In some implementations, prior to the driving, the method can comprise. Functionalizing a surface of the sensing element with the one or more detection molecules or macromolecules, wherein the functionalizing comprises injecting a solution comprising the one or more detection molecules or macromolecules into a chamber enclosing the surface of sensing element via at least one injection inlet of the microfluidic chip.

In some implementations of the subject method, the detectable signal comprises a visual signal. With these implementations, the method can further comprise determining whether the detectable signal is generated using a microscope positioned adjacent the sensing element. The method can also include capturing, by a device operatively coupled to a processor, image data of the sensing element in association with the driving, and determining, by the device, whether the visual signal is generated based on the image data.

In another embodiment, an apparatus is provided comprising a housing and a microfluidic chip contained within the housing. The microfluidic chip comprises a separation unit that separates, using one or more nanoDLD arrays, exosomes from other biological entities included in a biological fluid sample, resulting in isolated exomes. The microfluidic chip further comprises a detection unit that facilitates detecting presence of different biomarkers located on or within with the exosomes using different detection entities that respectively chemically react with the different biomarkers, wherein the different detection entities are selected from a group consisting of molecules and macromolecules, and at least one channel from the separation unit to the detection unit that facilitates flow of a buffer solution comprising the isolated exomes to the detection unit. In some implementations, the detection unit comprises different chambers that respectively detect presence of a different type of biomarker of the different types of biomarkers, and wherein the different chambers are respectively coated with a different detection entity of the different detection entities.

In one or more additional embodiments, a system is provided comprising a microfluidic chip contained within a housing, wherein the microfluidic chip comprises a separation unit that separates, using one or more nanoDLD arrays, target biological entities having a defined size range from other biological entities included in a biological fluid sample, resulting in isolated target biological entities. The microfluidic chip further comprises a detection unit that facilitates detecting presence of one or more biomarkers associated with the isolated target biological entities using one or more detection molecules or macromolecules that chemically react with the one or more biomarkers, and at least one channel from the separation unit to the detection unit that facilitates flow of a buffer solution comprising the isolated target biological entities to the detection unit. The system further comprises an imaging device (e.g., a microscope, a camera, etc.) that captures image data in association with flow of the buffer solution to the detection unit and contact of the buffer solution with the one or more detection molecules or macromolecules. In some implementations, the system further comprises a memory that stores computer executable components, and a processor that executes the computer executable components stored in the memory. The computer executable components can comprise an analysis component that evaluates the image data to determine biomarker information regarding the presence of the one or more biomarkers. The computer executable components can also comprise a diagnosis component that determines diagnostic information regarding a medical condition of a patient from which the biological fluid is sampled from based on the biomarker information.

In yet another embodiment, a method is provided that comprises isolating target biological entities having a defined size range from other biological entities included in a biological fluid sample using a separation unit comprising one or more nanoDLD arrays, thereby resulting in isolated target biological entities, wherein the separation unit is formed on a microfluidic chip contained within a housing. The method further comprises, driving flow of a buffer fluid comprising the isolated target biological entities through a conduit of the microfluidic chip from the separation unit to a sensing element formed on the microfluidic chip, wherein the sensing element generates a visual signal in response to detection of presence of one or more defined biomarkers associated with the target biological entities. The method further comprises capturing image data of the detection unit in association with the driving.

DETAILED DESCRIPTION

Various embodiments described herein are directed to microfluidic chip devices and systems that integrate biomarker detection together with upstream isolation and purification of biological entities, all on a single chip, providing a powerful self-contained, and portable solution to biochemical identification of disease-related biomarkers. The integrated purification-detection devices can be tailored to isolate and detect biomarkers associated with various types of biological particles, including exosomes, as well as viruses and other biological entities. In one or more embodiments, the microfluidic chip comprises as sensing element that provides for real-time detection of one or many biomarkers located downstream of a continuous flow isolation and purification separation element that is also located on the microfluid chip. By integrating an upstream separation element with the sensing element, the noise floor of the sample is minimized to enhance sensitivity by removing background contaminates and larger unwanted material, such as cellular debris and multi-vesicular bodies (MBVs).

The separation element can employ an arrangement of multiplexed lateral deterministic displacement (DLD) arrays, (e.g., nanoDLD arrays) for a buffer exchange of target biomolecules (e.g., exosomes) from an input sample with smaller contaminants, such as small molecules, proteins, and salts, exiting a common set of waste outlets. The nanoDLD arrays can be configured to bump or otherwise direct purified target biological entities into the portion of the buffer medium that flows into a common bus toward the downstream sensing element. In various embodiments, the sensing element can provide for detecting presence of one or more biomarkers present on the surface of the purified target biomolecules via chemical specificity between the one or more biomarkers and another chemical coated on the surface of the sensing element. For example, the sensing element can be coated with antibodies having a chemical specificity for a known epitope that may be present on the surface of isolated exosomes. In some implementations, the sensing element can incorporate a plurality of different antibodies that provide for simultaneous detection of two or more biomarkers. Simultaneous detection of multiple markers allows for fast, effective diagnosis of disease, such as certain forms of cancer. However, the sensing element biomarker detection methods may be more broadly extended to any specific chemical or biochemical interaction between two molecules or macromolecules, can be naturally occurring or synthetic and can be a permanent covalent linkage or a reversible bond (e.g., electrostatic interactions, hydrophobic interactions, complementarity, etc.).

In various exemplary embodiments, the sensing element can be located at or near the center of the chip and provide for optical readout of chemical reactions indicative of biomarker presence. For example, the purified sample can flow from the common bus mover the sensing element, which can include a signal-enhancing element such as a photonic grating, an optoelectrical element or plasmonic structure, coated with antibodies or aptamers known to bind with target epitopes or surface markers. For instance, the sensing element can be configured to detected chemical reactions that produce a fluorescent signal that is observable with fluorescence microscopy, and therefore manually detectable by eye or through software to automate the process. Accordingly, the sensing element can provide for real-time monitoring and diagnosis of a particular disease condition.

In one or more embodiments, the disclosed microfluidic chip can be coupled to a housing to facilitate a real-time exosome separation and biomarker detection process. For example, one exemplary process can include loading (e.g., pipetting) several fluids into various reservoirs onto the housing containing a microfluidic chip. The fluids include a biological sample (e.g., urine, blood, saliva, etc.), a buffer, and one or more fluids containing antibody or aptamer chemistries for surface functionalization of all or dedicated parts of the on-chip sensing element. A pressure-driven can be used to first drives the antibody or aptamer containing fluids onto the sensing element to functionalize the surface for immunocapture of exosomes containing certain target surface markers. Next, the biological sample and buffer can be pressure driven onto the chip where exosomes are harvested and purified using the nano-DLD arrays of the detection unit, and then captured on the downstream sensing element for detection and analysis using fluorescence microscopy, either manually by an operator or using a software analysis program.

In this regard, the subject integrated purification-detection devices and systems provide an all-in-one solution for sample preparation from complex patient fluids together with detection of multiple surface markers all on a single chip. The disclosed exosome isolation and biomarker detection devices provide a uniquely powerful, self-contained, and portable solution to biochemical identification of disease-related exosomal cohorts for biomarker discovery and diagnostic applications. Thus, the technology provides a means of semi-automating the biomarker discovery process as well as aids in rapid sample screening that can potentially be performed at the clinic.

As used herein, the term lab-on-a-chip (LOC) can refer to one or more devices that can integrate one or more laboratory functions onto an integrated circuit (e.g., a semiconductor substrate structure) to achieve autonomous screening of one or more samples. LOCs can utilize microelectromechanical systems and/or microfluidic systems to facilitate screening the one or more samples. One of ordinary skill in the art will recognize that a LOC devices can range in size from, for example, one or more square millimeters to one or more square centimeters. One or more embodiments can utilize microfluidics in a LOC device to detect one or more target biomarkers, wherein the biomarkers can be indicative of various traits (e.g., physical properties) and/or health conditions (e.g., diseases). Thus, in some embodiments, the one or more integrated purification-detection devices described herein can be considered LOC devices that can facilitate biomarker detection, wherein the one or more LOC devices can be operated quickly (e.g., near instantaneously), in a variety of locations (e.g., at an entity's home), and without the typical need for specialized laboratory equipment.

As used herein the term deterministic lateral displacement (DLD) can refer to one or more microfluidic techniques that can size fractionate a polydisperse suspension of molecules through the use of one or more arrays of obstacles. For example, DLD arrays can laterally displace target molecules within a sample stream based on size. Further, DLD arrays can comprise a plurality of pillars arranged in a lattice structure. Rows of pillars comprising the lattice structure can be positioned offset of each other at a defined angle, and pillars can be separated from each other by a defined gap size. The defined angle and/or gap size can facilitate displacement of one or more molecules of a target size range comprised within a stream flowing through the DLD array.

As used herein the term nanoDLD array can refer to a DLD array that can be characterized by one or more dimensions ranging from greater than or equal to 1 nanometer (nm) and less than or equal to 999 nm. For example, a nanoDLD array can be a DLD array characterized by a gap size (e.g., a distance between adjacent pillars comprised within the lattice structure) of greater than or equal to 1 nm and less than or equal to 999 nm (e.g., greater than or equal to 25 nm and less than or equal to 235 nm). In one or more embodiments, a nanoDLD array can facilitate displacement of exosomes, viruses, and other biomolecules or micromodules of various sizes (e.g., from 1 nm to 999 nm). In some implementations, the nanoDLD arrays described herein can also isolate genetic code sequences that can be characterized as having an exemplary length ranging from, but not limited to, greater than or equal to 25 base pairs (bp) and less than or equal to 200 bp.

As used herein, unless otherwise specified, terms such as on, overlying, atop, on top, positioned on, or positioned atop mean that a first element is present on a second element, wherein intervening elements may be present between the first element and the second element. As used herein, unless otherwise specified, the term directly used in connection with the terms on, overlying, atop, on top, positioned, positioned atop, contacting, directly contacting, or the term direct contact, mean that a first element and a second element are connected without any intervening elements, such as, for example, intermediary conducting, insulating or semiconductor layers, present between the first element and the second element. As used herein, terms such as upper, lower, above, below, directly above, directly below, aligned with, adjacent to, right, left, vertical, horizontal, top, bottom, and derivatives thereof shall relate to the disclosed structures as oriented in the drawing figures.

One or more embodiments are now described with reference to the drawings, wherein like referenced numerals are used to refer to like elements throughout. In the following description, for purposes of explanation, numerous specific details are set forth in order to provide a more thorough understanding of the one or more embodiments. It is evident, however, in various cases, that the one or more embodiments can be practiced without these specific details. Further, it is to be understood that common cross-hatching and/or shading depicted across the drawings can represent common features, compositions, and/or conditions described herein in accordance with one or more embodiments.

FIGS. 1A and 1Billustrates a diagram of an example, non-limiting, separation-purification apparatus100that integrates on-chip particle purification and biomarker detection functionality in accordance with one or more embodiments described herein. As shown inFIG. 1A, the separation-purification apparatus100comprises a microfluidic chip106provided within a housing102. The housing is composed of a top plate102A and a bottom plate102B.FIG. 2Bdepicts the separation-purification apparatus100with the top plate102A removed. In various embodiments, the housing102can be or correspond to a flow cell or other form of packaging that houses the microfluidic chip106. For example, in some embodiments, the top plate102A and the bottom plate102B can be physically coupled to one another (e.g., via one or more screws or another suitable attachment mechanism) with the microfluidic chip106sandwiched therebetween. In the embodiment shown, the top plate102A is transparent or semitransparent. For example, the top plate102A can be formed of a clear acrylic plastic, glass, or another suitable material. The bottom plate102B can also be formed with a transparent or semitransparent material, such as clear acrylic plastic, glass or another suitable material. In other embodiments, the bottom plate102B can be formed with a non-transparent material, such as silicon or another material in which microchannels, reservoirs, vias, etc., can be fabricated thereon and/or therein.

The microfluidic chip106comprises a substrate material with a plurality of elements formed on or within the substrate material that facilitate on-chip particle filtration and biomarker detection. For example, in some embodiments, the microfluidic chip106can comprise a silicon substrate with elements formed therein and/or thereon using various semiconductor fabrication techniques. Other suitable materials for the microfluidic chip106can include glass, plastic, or a combination thereof. The elements formed on and/or within the microfluidic chip106can include a separation unit that includes one or more DLD arrays and/or nanoDLD configured to separate particles of interest (e.g., exosomes) from other particles included in a biological fluid sample. The biological fluid sample can include for example (but is not limited to), a blood sample, a urine sample, a tissue sample, a saliva sample, a plasma sample, a cell culture medium, an in vitro sample, a plant sample, a food samples, a combination thereof, and/or the like. The microfluidic chip106further includes a detection unit that facilitates detecting one or more biomarkers located on or within the particles of interest using a sensing element108. For example, in one or more embodiments, the sensing element108can be coated with one or more detection molecules or macromolecules configured to chemically react with the one or more biomarkers. In accordance with theses embodiments, the detection unit can facilitate flowing solution comprising the isolated particles of interest over the sensing element108. If the one or more biomarkers are present, the one or more detection molecules or macromolecules will chemically react with the biomarkers and produce some form of detectable signal (e.g., a visual signal) that can be read from the sensing element108. The microfluidic chip106further includes a microfluidic busing network consisting of a plurality of microchannels, busses, vias and/or reservoirs formed on or within the microfluidic chip. The microfluidic bussing network facilitates transporting fluid streams between the separation unit, the detection unit, and other elements present on or within the microfluidic chip106.

As shown inFIG. 1A, the top plate102A can include a window region124formed on or within the top surface of the top plate102A. This window region124can comprise glass or another transparent material (e.g., in implementations in which the material employed for the top plat102is semitransparent) that facilitates clearly visualizing the sensing element108of the microfluidic chip106. For example, in some implementations, the window region124can be formed with transparent glass and the remainder of the top plate102A can be formed with transparent or semitransparent acrylic plastic. The window region can be formed in an area of the top plate that is aligned with the sensing element108when the top plate102A is attached to the bottom plate102B. In some embodiments, (as shown inFIG. 1Aand more clearly shown inFIG. 1B), a capping layer110can be formed on the top surface of the microfluidic chip110. The capping layer110can comprise a transparent material (e.g., glass, acrylic plastic, etc.) that provides for fluidically sealing the microfluidic elements formed on the top surface of the microfluidic chip110.

FIG. 2Apresents an example 3D view of microfluidic chip106as separated from separation-purification apparatus housing in accordance with one or more embodiments described herein. In the embodiment shown, the capping layer110is also removed.FIG. 2Bpresents an orthogonal, 2D, perspective view of microfluidic chip106taken along axis B-B′ shown inFIG. 2A, in accordance with one or more embodiments described herein. Repetitive description of like elements employed in respective embodiments is omitted for sake of brevity.

As shown inFIG. 2B, the microfluidic chip106can include a circular architecture with several elements formed around the sensing element108provided at or near the center of the chip. In particular, (shown in light grey), the microfluidic chip106can include an inlet bus204formed around an outer perimeter area of the chip and fluidically coupled to a global inlet via202. For example, the inlet bus204can be etched or otherwise formed within a portion of the thickness of the chip. In some implementations, the inlet bus204can be etched deeper than other fluidic channels and/or elements formed within the thickness of the chip. For example, in some implementations, the base of the inlet bus204can be located 100 μm (or greater) from the bottom surface of the microfluidic chip (e.g., the surface opposite the capping layer110), without penetrating the bottom surface of the microfluidic chip. The global inlet via202can however penetrate through the bottom surface of the microfluidic chip to facilitate receiving and transporting fluid therethrough and into the inlet bus204. For example, in various embodiments, the inlet bus204can be configured to receive biological sample fluid introduced through the global inlet via202, and distribute the biological sample fluid evenly, (or substantially evenly) throughout the inlet bus204(e.g., in the direction shown via the dashed arrows extending from the global inlet via202).

The microfluidic chip106further includes a separation unit206formed around the sensing element108and within the perimeter of the inlet bus204. For example, in the embodiment shown, the separation unit206is divided into four segments respectively arranged in ring shape (or more accurately, a rectangular shape) within the perimeter of the inlet bus. However, it should be appreciated that the specific shape or geometrical configuration of the separation unit206can vary. In the embodiment shown, the separation unit206encompasses the alternating black and white checkered lines formed parallel to one another, as well as the dark grey region formed in between them. As discussed in greater detail, the dark grey region of the separation unit206can comprise a plurality of DLD or nanoDLD arrays configured to separate target particles of a particular size range from other particles included in the biological sample fluid, and the respective checked lines can correspond to inlet and outlet vias through which fluid passes into and out of the DLD or nanoDLD array.

For example, in some implementations, the first or outermost checkered line provided at the interface between the inlet bus204and the DLD or nanoDLD region (e.g., the dark grey region), can include a plurality of openings through which the biological fluid sample can flow from the inlet bus204and into the DLD or nanoDLD array, (e.g., in the direction shown by the dashed arrows). In some implementations, the first checkered line can also include a plurality of second inlet vias through which another fluid, such as a buffer fluid, can be introduced. For example, as discussed in greater detail infra, as the biological fluid and the buffer fluid can simultaneously flow through the DLD or nanoDLD arrays, the particles of interest can be bumped into or otherwise captured in first streams of the buffer fluid. Other undesired particles included in the biological fluid sample can be captured in second streams of the biological fluid sample that generally flow in a straight trajectory through the DLD or nanoDLD arrays. For example, in implementations in which the target particles include exosomes, the undesired particles removed by the separation unit can include potentially contaminating small molecules such as salts, proteins, lipids and the like. The second or innermost checked line (provided adjacent to outlet bus208), can further include a plurality of openings through which the first streams can exit the DLD or nanoDLD array (e.g., the dark grey region of the separation unit206) and enter into outlet bus208(e.g., in the direction shown by the dashed arrows). The second checked line can also include a plurality of outlet vias through which the respective second streams can be collected and expelled from the microfluidic chip106(e.g., as waste fluid).

The outlet bus208comprises an etched channel formed within the thickness of the microfluidic chip106. The outlet bus208can receive the filtered stream of the buffer fluid including the target particles from the separation unit206and transport the filtered target particle stream to the downstream, sensing element108(e.g., in the direction shown by the dashed arrows. In one or more embodiments, the interface (or interfaces) between the outlet bus208and the sensing element108can include one or more openings (not shown) through which the target particle buffer stream can enter and flow onto and over the sensing element (e.g., in the direction shown by the dashed grey arrows). The sensing element108can further include a global outlet via210through which the buffer stream can exit the microfluidic chip106, along with any unreacted and/or unbound particles included in the filtered, target particle buffer stream. In various embodiments, the outlet bus208and the inlet bus204bus can be etched deeper than both the separation unit206and the sensing element108. The purpose for this is to ensure that fluidic resistance is dropped or decreased across the separation unit206and the sensing element108.

The microfluidic chip106also include one or more third inlet vias212that are fluidically coupled to the sensing element108. In the embodiment shown, four third inlet vias212are shown, however the number of third inlet vias212can vary. The one or more third inlet vias212can facilitate introducing a detection fluid onto the sensing element108for coating and functionalizing the sensing element108. For example, the detection fluid can include one or more types of detection molecules or macromolecules (e.g., antibodies, aptamers, etc.), known to chemically react with one or more biomarkers of interest that may be present on or within the separated particles of interest. In some implementations, prior to injecting the biological sample fluid into the microfluidic chip, the detection fluid can be injected through the one or more third inlet vias212and flowed onto the sensing element108to coat and functionalize the sensing element108. Excess detection fluid or otherwise portions of the detection fluid that do not coat the surface of the sensing element108can also flow through the global outlet via210.

In the embodiment shown, a circular, distribution bus214can be formed around the perimeter of the sensing element108(depicted by the thin grey line formed around the sensing element108) to facilitate evenly distributing the detection fluid and the biological fluid over the surface of the sensing element108. For example, the distribution bus214can be formed around the perimeter of the sensing element108and minimize fluidic resistance to induce uniform fluid flow from the perimeter injection sites to the center of the sensing element108, thereby enabling uniform coverage of coating chemistry and sample over the sensing element during device operation.

In various embodiments, the sensing element108, the portion of the outlet bus208that connects to the sensing element108, the global outlet via210, the one or more third inlet vias212, and the distribution bus214, can constitute the detection unit of the subject microfluidic chips (e.g., microfluidic chip106).

In this regard,FIGS. 2C and 2Dpresent a 3D, perspective view of an example detection unit200of a microfluidic chip (e.g., microfluidic chip106) that integrates on-chip particle purification and biomarker detection functionality in accordance with one or more embodiments described herein. Repetitive description of like elements employed in respective embodiments is omitted for sake of brevity.

As shown inFIG. 2Cwith respect to the dashed arrow lines, detection fluid can be introduced at the respective third inlet vias212and flowed over the sensing element108and out the global outlet via210to coat and/or functionalize the surface of the sensing element108. As shown inFIGS. 2C and 2D, the outlet bus208can comprise a plurality of deeply etched channels that connect to the sensing element108. As shown inFIG. 2Dwith reference to the dashed arrow lines, after the sensing element has been functionalized, a stream of buffer fluid comprising purified target particles can flow from the separation unit206, up through the outlet bus208channels and onto the sensing element108. Excess detection fluid and target particle buffer stream can further flow into the global outlet bus210to be removed from the microfluidic chip106.

With reference again toFIGS. 1A and 1Bin connection with reference toFIGS. 2A-2D, in various embodiments, the microfluidic bussing network (e.g., including the global inlet via202, the inlet bus204, the second inlet vias (not shown) for introducing the buffer fluid into the separation unit206, the plurality of outlet vias (not shown) for removing waste fluid from the separation unit206, the outlet bus208, the global outlet via210, and/or the one or more third inlet vias212) can be fluidically coupled to one or more fluid inlets and outlets provided within the bottom plate102B of the housing102via which the microfluidic chip receives and excretes fluids. For example, in one or more embodiments, the housing102can be or include a flow cell or another form of packaging that facilitates flowing or otherwise injecting fluid into one or more input vias connected to the bussing network of the microfluidic chip106and removing fluid from the microfluidic chip106. The housing102can be formed with various materials, including silicon, glass, plastic, or a combination thereof. In this regard, the bottom plate102B of the housing102can include one or more inlet ports through which fluid is injected (e.g., using a syringe, pipette, or the like) into one or more flow cell channels (not shown) and/or reservoirs (not shown) provided on or within the bottom plate102B. The one or more flow cell channels/and reservoirs can be fluidically coupled to the microfluidic bussing network of the microfluidic chip106. The bottom plate102B can further include one or more output ports through which fluid is exported or otherwise removed from the microfluidic chip106and/or one or more reservoirs of the housing102. In this regard, one or more fluids can flow into the bottom plate102B of separation-purification apparatus100, through the microfluidic chip106, and then out of the microfluidic chip via the housing102.

For example,FIG. 3Apresents a 3D view of the bottom plate102B of the housing102as separated from the microfluidic chip106and the top plate102A. Repetitive description of like elements employed in respective embodiments is omitted for sake of brevity.

With reference toFIG. 3A, in conjunction with reference toFIGS. 1A-1B and 2A-2D, in the embodiments shown, the bottom plate102B of the housing can include a chip pocket312that can receive the microfluidic chip106. In this regard, the microfluidic chip106can be inserted into the chip pocket312such that a bottom surface (e.g., the surface opposite the sensing element108), is opposed to the upper surface of the bottom plate102B. The bottom surface of the microfluidic chip106can further include openings or vias which can correspond to one or more inlet vias and outlet vias of the microfluidic chip (e.g., the global inlet via202, the global outlet via210, and other vias described below). In some embodiments, these openings or vias in/through the bottom surface of the microfluid chip can align with and fluidically couple to corresponding fluid inlets/outlets provided by the bottom plate102B of the housing.

The bottom plate102B includes three inlet ports or capillaries, including inlet port112, inlet port114and inlet port116. These inlet ports can respectively be used to inject fluid (e.g., the biological fluid sample, the buffer fluid, and the detection fluid), into the microfluidic chip. The bottom plate102B also includes two outlet ports, outlet port118and outlet port120. These outlet ports can respectively be used to remove fluid (e.g., waste fluid, excesses detection fluid, and purified sample fluid as it flows over the sensing element108and out through the global outlet via210), from the microfluidic chip106and the bottom plate102B. In some embodiments, a single outlet port can be used. In other embodiments, more than two output ports can be used. In the embodiment shown, the inlet ports and outlet ports are depicted as tubes that extend from sides of the bottom plate102B. However, it should be appreciated that the location of the respective inlet and outlet ports can vary. In addition, although the inlet ports are shown as tubes, it should be appreciated that these tubes connect to corresponding openings/microfluidic channels (not shown) formed within the body of the bottom plate. In this regard, the tubes can be removably attached/detached from corresponding openings/microfluidic channels in the bottom plate102B.

For example, in various embodiments, an upper surface region of the bottom plate102B can include a plurality of fluidic connections and fluid reservoirs which can receive fluid from the one or more inlet ports (e.g., inlet port112, inlet port114and/or inlet port114) for introducing into the microfluid chip, and/or receive fluid as it is excreted from the microfluid chip. For example, in the embodiment shown, these fluidic connections/reservoirs respectively include fluidic connection302′, buffer fluid reservoir304, waste fluid reservoir306, detection fluid reservoir308, and fluidic connection310′. The respective reservoirs, including the buffer fluid reservoir304, the waste fluid reservoir306, and the detection fluid reservoir308, can be fluidic pools that can contain a fluid within. Specifically, in one or more embodiments, the buffer fluid reservoir304can receive and contain buffer fluid for injection into the separation unit of the microfluidic chip, the waste fluid reservoir306can receive and contain waste fluid (comprising unwanted particles) removed by the separation unit, and the detection fluid reservoir308can receive and contain detection fluid comprising the surface chemistry molecules/macromolecules for coating the sensing element108. Each of these reservoirs can include one or more openings (not shown) through which the corresponding fluid can be injected into the reservoir and one or more openings (not shown) through which fluid can removed from the reservoir.

In the embodiment shown, the buffer fluid reservoir304, waste fluid reservoir306, detection fluid reservoir308, are formed on/within an upper surface region of the bottom plate102B. For example, in some implementations, the respective reservoirs can be exposed on the top surface of the bottom plate on the housing. With these embodiments, the reservoirs can become enclosed by the bottom surface of the microfluidic chip when the microfluidic chip is inserted into the chip pocket312. In this regard, when the microfluidic chip is inserted into the chip pocket, the bottom surface of the microfluidic chip can cover and enclose the reservoirs. In other implementations, a top surface of the bottom plate102B can enclose the reservoirs. In another embodiment, one or more of these reservoirs can be formed within the microchip106and/or an intermediary layer (not shown) between the microchip106and the bottom plate102B. In various embodiments, these three reservoirs are collectively referred to herein as the reservoir region.

In one or more embodiments, fluidic connection302′ can correspond to an opening in an upper surface of the bottom plate102B that can align with and connect to the global inlet via202of the microfluid chip. In accordance with this example embodiment, the inlet port112can connect to the fluidic connection302′ and the global inlet via202of the microfluidic chip106when the microfluid chip106is inserted into the chip pocket102. In this regard, inlet port112can be configured to receive a biological fluid sample and facilitate flowing the biological fluid sample through a channel (not shown) formed within the bottom plate102B that connects to the fluidic connection302′ and which is further aligned with and connects to the global inlet via202of the microfluidic chip106. In one or more embodiments, the interface between the fluidic connection302′ and the global inlet via202can employ an o-ring seal or gasket to maintain fluidic isolation between the reservoirs (via compressive pressure applied to the o-ring seal or gasket) and controlling passage of the biological fluid from the bottom plate102B, through global inlet via202and into the inlet bus204. The inlet bus204can further receive the biological fluid and pass the fluid through the microfluidic chip106for processing by the separation unit and the detection unit of the microfluidic chip106, as herein. In some implementations, the biological fluid sample can be injected into the inlet port112via a pipette, via a syringe, or via from another off-chip biological sample reservoir connected to the inlet port112via a suitable tube or capillary. In various embodiments, the biological fluid sample can be injected through the inlet port112and into the microfluidic chip106(e.g., via the first global inlet via) using a pressure driving system or device (not shown) that is external to the separation-purification apparatus100.

Similarly, in some embodiments, the fluidic connection310′ can correspond to an opening in an upper surface of the bottom plate102B that can align with and connect to the global outlet via210of the microfluid chip. In accordance with this example embodiment, the outlet port120, can be fluidically connected to the fluidic connection310′ via a microfluidic channel (not shown) formed within the bottom plate102B (and through the center of the detection fluid reservoir308). The fluidic connection310′ can further be fluidically connected to the global outlet via210of the microfluidic chip106when inserted into the chip pocket312. In this regard, the outlet port120can be configured to export fluid passed over the sensing element108and flowed into the global outlet via120. For example, in some implementations, this fluid can initially include excess detection fluid that flows from inlet port116through separation-purification apparatus100, over the sensing element108of the microfluidic chip106and exits the separation-purification apparatus100via outlet port120. In this regard, outlet port120can provide for removing excesses reagent chemistry (e.g., antibodies, aptamers, etc.) from the sensing element108in association with the coating process used to functionalize the sensing element108. Outlet port120can also be employed to remove the stream of buffer fluid including separated particles of interest as the stream is passed over the sensing element108to detect presence of biomarkers on or within the particles of interest. In this regard, a stream of buffer fluid including separated particles of interest can flow from the separation unit206of the microfluidic chip106to the downstream detection unit and over the sensing element108in a steady manner, allowing for biomarkers to contact and react with the sensing element108, while unreacted or unbound particles in the buffer stream are excreted through the outlet port120. In some implementations, exit of fluid through the global outlet via210can be contained via an o-ring or another suitable gasket formed around and//or within the global outlet via210and/or the fluidic connection310′.

The introduction of buffer fluid and detection fluid into the microfluidic chip106via the corresponding buffer fluid reservoir304and detection fluid reservoir308(when the microfluidic chip is inserted into the chip pocket312), and the removal of waste fluid from the microfluidic chip106via the corresponding waste fluid reservoir306, is discussed with reference toFIGS. 3B and 3Cin connection withFIGS. 1A-1B, 2A-2D and 3A.

In this regard,FIGS. 3B and 3Cpresent orthogonal, 2D, top-down views of an example reservoir region that couples with a microfluidic chip to facilitate on-chip particle purification and biomarker detection functionality in accordance with one or more embodiments described herein. The reservoir region can comprise three reservoirs including the buffer fluid reservoir304, waste fluid reservoir306, and detection fluid reservoir308. Each of the reservoirs can be enclosed fluidic pools that can contain a fluid within. In this regard, each of these fluid reservoirs can be defined by an upper surface, a bottom surface, and a fluidic space between the bottom surface and the upper surface.FIG. 3Bdepicts the bottom surface300of the reservoir region.FIG. 3Cdepicts the upper surface301of the reservoir region. In the embodiment shown inFIG. 3A, the reservoir region is formed within an upper portion of the bottom plate102B. However, the specific location of the respective reservoirs can vary so long as they are located between the active features of the microfluidic chip (e.g., the separation unit and the detection unit) and the corresponding fluidic inlets/outlets of the bottom plate102B. In this regard, in some embodiments, the bottom surface of the300of the reservoir region can be part defined within the bottom plate102B and the upper surface300of the reservoir region can also be defined by/within the bottom plate (e.g., the upper surface of the bottom plate102B). In other embodiments, the upper surface301of the reservoir region can be defined by the bottom surface of the microfluidic chip106. For example, the reservoirs can be exposed and formed on the top surface of the bottom plate102B. The exposed reservoirs can further become enclosed and covered when the microfluidic chip106is inserted into the chip pocket312. With this implementation, the bottom surface of the microfluidic chip can correspond to the top surface301of the reservoir region. Other configurations are envisioned.

Each of these reservoirs (e.g., the buffer fluid reservoir304, the waste fluid reservoir306, and the detection fluid reservoir308) can include one or more openings through which the corresponding fluid can injected into the reservoir and one or more openings through which fluid is removed from the reservoir. For example, as shown inFIG. 3B, each of the reservoir regions can include a single fluidic connection or opening through which fluid is injected from the bottom plate102B and into the reservoir, or from which fluid is removed from the reservoir and ejected through the bottom plate102B. In particular, the buffer fluid reservoir304can include fluidic connection304′ through which buffer fluid can be injected into the buffer fluid reservoir304. Waste fluid reservoir306can include fluidic connection306′ through which waste fluid can be extracted from the waste fluid reservoir306. Detection fluid reservoir308can include fluidic connection308′ through which detection fluid can be inserted into the detection fluid reservoir308. For example, with reference toFIGS. 3A and 3B, in some embodiments, inlet port114can be configured to receive and facilitate injection of the buffer fluid into the buffer fluid reservoir304via a microfluidic channel (not shown) formed within the bottom plate102B that connects the inlet port114to the microfluidic connection304′. In some implementations, an o-ring or another suitable gasket material can form a fluid seal at the interface of the fluidic connection304′ and the fluidic inlet connected thereto. Similarly, inlet port116can be configured to receive and facilitate injection of the detection fluid into the detection fluid reservoir308via a microfluidic channel (not shown) formed within the bottom plate102B that connects the inlet port116to the microfluidic connection308′. In some embodiments, the solid lines318that separate the reservoirs can be or correspond to o-rings. With these embodiments the o-rings do not seal the fluid at the interface of the fluidic connections308′ and304′. Rather, they corral the fluid within the respective reservoirs. In addition, the outlet port118can be configured to receive and facilitate removal of waste fluid that is collected in the waste fluid reservoir306(e.g., as injected from the separation unit of the microfluidic chip into the waste fluid reservoir306) via a microfluidic channel (not shown) formed within the bottom plate102B that connects the oulet port118to the microfluidic connection306′. In some implementations, an o-ring or another suitable gasket material can form a fluid seal at the interface of the fluidic connection308′ and the fluidic inlet connected thereto.

With reference toFIG. 3Cin connection with reference toFIG. 2B, in one or more embodiments, the upper surface of the buffer fluid reservoir304can align with and be fluidically connected to the plurality of input vias of the separation unit of the microfluidic chip106. For example, the respective dashes of the dashed line314′ depicted within the buffer fluid reservoir304can respectively correspond projected inlet via locations of the separation unit through which the buffer fluid included in the buffer fluid reservoir304can be pushed to enter through aligned input vias (e.g., the second input vias described above) of the separation unit. With these implementations, the buffer fluid, can be injected into the buffer fluid reservoir304of the housing102via inlet port114and fluidic connection304′. As the buffer fluid reservoir304fills with buffer fluid, the buffer fluid can be pressure driven through the aligned and fluidically connected second inlet vias of the separation unit206. For example, in some embodiments, a pressure driving device or system coupled to the separation-purification apparatus100can be used to pressure drive the second fluid or buffer fluid through the inlet port114and/or fluid the buffer fluid reservoir304and onto the microfluidic chip106. In other implementations, the second fluid can be injected into inlet port114via a pipette, a syringe, or another suitable injection means. Similarly, the upper surface of the waste fluid reservoir306can align with and be fluidically connected to the plurality of outlet vias of the separation unit of the microfluidic chip106through which waste fluid is excreted. In this regard, the respective dashes of the dashed line316′ depicted within the waste fluid reservoir306can respectively correspond to projected outlet via locations of the separation unit through which waste fluid can be ejected into the waste fluid reservoir106. In addition, a plurality of fluidic connections212′ can be included in the upper surface of the detection fluid reservoir308which can be fluidically connected to the one or more third inlet vias212. With these implementations, the detection fluid reservoir308can be fluidically coupled with the one or more third inlet vias212of the microfluidic chip106through which the detection fluid can be injected and flowed onto the sensing element108to facilitate coating the sensing108prior to flow of biological sample fluid through the microfluidic chip106. In this regard, as the detection fluid is injected through inlet port116and fills the detection fluid reservoir308via fluidic connection308′, the detection fluid can evenly enter the one or more third inlet vias212and evenly coat the sensing element108. For example, in some embodiments, the pressure driving device or system coupled to the separation-purification apparatus100can also be used to pressure drive the detection fluid through inlet port116and/or the additional fluid reservoir and onto the microfluidic chip106. In other implementations, the third fluid can be injected into inlet port116via a pipette, a syringe, or another suitable injection means.

With reference again toFIGS. 1A and 1B, the separation-purification apparatus100can further include a sealing layer104formed between the microfluidic chip106and the housing102. For example, in the embodiment shown, the sealing layer104is provided between an upper surface of the bottom plate102B and a bottom surface or backside of the microfluidic chip106. In other implementations, the sealing layer104can be formed at an interface between the reservoir region and the upper surface of the bottom plate102B. In yet another embodiment, the sealing layer104can be formed at an interface between the reservoir region and the bottom surface of the microfluidic chip.

The sealing layer104can comprise one or more materials that facilitate creating a fluidic seal between one or more vias or openings in the backside of the microfluidic chip106, and one or more ports, capillaries, channels, and/or reservoirs of the bottom plate102B and/or the reservoir region. For example, in some implementations, the sealing layer104can comprise a gasketing material that creates a fluidic seal at the interface between one or more openings or vias on the backside of the microfluidic chip106, and one or more adjacent/aligned openings (e.g., a capillary, a channel, a via, a reservoir, etc.) in the upper surface of the bottom plate102B. For example, in some embodiments, the gasket material can be constructed from o-rings or a polymer, such as an elastomer, a thermoset, a thermoplast, and the like. In some implementations, the gasket material can be formed by 3D printing, stamped, embossed, injection molded, laser cut, or ablated from the polymeric starting material. For example, as can be observed by comparingFIGS. 2B and 3B, the placement of the global and local via inlet and outlets on the chip surface are superimposed or projected onto common reservoirs, whose borders can be defined by sets of o-rings on the backside of the chip, one means of providing a fluidic seal at the interface between the chip and flow-cell/packing.

The sealing layer104can thus comprise one or more or more o-rings or gasket arrangement that ensure each reservoir of the housing and/or microfluidic chip is continuous, allowing one fluid type to be universally introduced or extracted from one associated via or set of vias on the microfluidic chip106. In this regard, although the sealing layer104is depicted a continuous layer of material, it should be appreciated that this depiction is merely for exemplary purposes. For example, in implementations, the sealing layer104can consist of a plurality of o-rings formed in an arrangement only between portions of the interface between the microfluidic chip106and the housing102where adjacent openings are located. This arrangement allows the simultaneous fluid loading of all vias associated with a particular fluid group. Importantly, the fluid to/from each reservoir in the gasket must have an inlet port or outlet port (e.g., one or more of ports112-120) that can be accessed from the housing102. In the embodiment shown, only one access port to each reservoir is used to minimize the design complexity of the housing102.

FIG. 4illustrates an example cross-sectional view the detection region400of separation-purification apparatus100taken along axis A-A′ shown inFIG. 1in accordance with one or more embodiments described herein. Repetitive description of like elements employed in respective embodiments is omitted for sake of brevity.

With reference toFIG. 4in conjunction withFIGS. 1, 2A-2D and 3A-3C, in the embodiment shown, three vias or channels are formed within the thickness of the substrate (e.g., silicon) of the microfluidic chip106, respectively corresponding two of the third inlet vias212, and the global outlet via210. In this regard, the inlet vias212respectively connect to and/or correspond to respective third inlet vias212through which detection fluid can be introduced onto the sensing element108. Likewise, outlet via210connects to and/or corresponds to outlet via210through which excess detection fluid and biological fluid sample can be removed from the microfluidic chip. These channels are respectively fluidically coupled to and aligned with corresponding fluidic connections212′ and310′ in the reservoir layer. For example, inlet vias212are respectively connected to fluidic connections212′ in the detection fluid reservoir308, and outlet via210is connected to the fluidic connection310′ which connects to the outlet port120. In accordance with this embodiment, the sealing layer104consists or o-rings respectively formed between the bottom surface of the microfluidic chip106and the upper surface of the reservoir layer (e.g., included in the bottom plate102B) around the aligned channels vias212and210and their corresponding fluidic connections in the reservoir layer.

With reference again toFIG. 1, in various embodiments, although not shown, separation-purification apparatus100can be coupled to a pressure-driving system to control flow of the various fluids (e.g., the biological sample fluid, the buffer fluid, the detection fluid, and other potential cleaning/preparation fluids) into, out of, and through the housing102and the microfluidic chip106. The pressure-driving system can be a fully automated pressure driving machine, a manually operated pressure driving machine, or a combination thereof. The pressure driving system can form a pressure seal between the inlet ports (e.g., inlet port112, inlet port114, and inlet port116) and the respective reservoirs, channels, inlets, etc., of the housing102, and/or the respective outlet ports (e.g., outlet port118and outlet port120). In this regard, the pressure driving system can apply pressure to initiate fluid flow of one or more fluids within the different fluid reservoirs located within the housing and a reservoir comprising the biological sample fluid, referred to herein as the sample fluid reservoir. Although not explicitly shown inFIG. 3B, in some implementations, the housing102can include the sample fluid reservoir formed therein in which the biological sample fluid can be pre-loaded prior to running through the microfluidic chip106. In other implementations, the sample fluid reservoir can be provided external to separation-purification apparatus100. The sample fluid reservoir can be fluidically coupled to the global inlet via202by way of the (gasketed) opening302of the housing102aligned therewith.

The separation-purification apparatus100can further include a capping layer110formed on or over the microfluidic chip106. In various embodiments, the capping layer110can include a transparent or semi-transparent material (e.g., glass, plastic, etc.) that provides for hermetically sealing one or more fluidic elements (e.g., busses, channels, vias, reservoirs, sensing element108chambers, etc.), provided on or within the microfluidic chip106. In addition, by employing a transparent capping layer110, the upper surface of the microfluidic chip including the sensing element108can be visually observed (e.g., via the naked eye, a microscope, or another suitable imaging device).

In some embodiments, the microfluidic chip106can be permanently sealed within the housing102. With these embodiments, formation of separation-purification apparatus100can include a bonding procedure wherein the backside of the microfluidic chip106is bonded to or otherwise affixed to the upper surface of the bottom plate102B of the housing. The capping layer110can further be bound to the microfluidic chip106and/or the housing102to permanently seal the microfluidic chip106within the housing102. In other embodiments, the microfluidic chip106can be removably attached to the housing102. With these embodiments, the housing102can be re-used with new microfluidic chips inserted therein (or cleaned microfluidic chips reinserted therein). For example, in one implementation, the housing102can be configured to receive and snap-in, screw in, lock-in, etc., the microfluidic chip106in a manner that allows for the chip to be easily removed after use. The capping layer110can further be configured to removably attach or open and close to cover and seal the microfluidic chip106within the housing102during use. Alternatively, the capping layer110can be permanently affixed to the surface or the microfluidic chip106. With this implementation, the microfluidic chip106with the capping layer110bound thereto can for a single unit that can be inserted into the housing102. With these implementations, the top plate102A of the housing can be attached to the bottom plate102B of the housing (e.g., via one or more screws or another mechanism) to sandwich and seal the microfluidic chip106/capping layer110unit therein.

In accordance with an example usage scenario in which separation-purification apparatus100is used for biomarker discover and/or liquid biopsy screening, separation-purification apparatus100can be operated as follows. Optionally, the microfluidic chip106can be pre-wet with antifouling chemical agents including, but not limited to buffers of varying pH and ionic strength levels, surfactants, and biological coating agents such as bovine serum albumin (BSA). Next, a sample (including urine, blood, plasma, saliva, cell culture media, etc.), buffer, and antibody or aptamer containing chemistries can be loaded (e.g., via pipetting or another suitable mechanism) into separate reservoirs of the housing102(e.g., located on or within the bottom plate102B) with the microfluidic chip106sealed inside. The housing can further be being placed into or otherwise coupled to a pressure driven system to initiate purification and detection. Initially, the pressure-driven system can apply pressure to force the flow of the antibody or aptamer containing fluid onto the sensing element108to surface functionalize the sensing element108. The pressure-driven system can then stop the flow of antibody or aptamer chemistry and initiate the flow of sample and buffer simultaneously to initiate purification and downstream immunocapture of target macromolecules that flow over the sensing element108. The presence of one or more exosomes or other biomarkers bound to the antibodies or aptamers coated on the surface of the sensing element108can be observed either directly by an operator (e.g., via the naked eye, through a microscope lens placed over the sensing element108, etc.) or computer-based reception and analysis of image data captured of the sensing element108.

FIG. 5illustrates an enlarged view of an example separation unit206of the microfluidic chip106in accordance with one or more embodiments described herein. For example, call-out box501presents and enlarged or zoomed-in view of the area of the microfluidic chip106encircled by box500. Repetitive description of like elements employed in respective embodiments is omitted for sake of brevity.

With reference to call-out box501, the separation unit206can comprise a plurality of DLD on nanoDLD arrays formed between inlet bus204and outlet bus208. In this regard, the size and spacing of the respective pillars used for DLD array can be adapted to a particular target particle size or size range, which can include nanoparticles, such as exomes, viruses, DNA sequences, RNA sequences, etc., as well as larger particles such as cells. Thus, in implementations in which the microfluidic chip106is used for exosome filtration and biomarker analysis, the separation unit206can employ nanoDLD arrays. For larger particles, the separation unit206can employ DLD arrays with pillars and/or pillar spacing adapted to filter particles greater than 999 nm. For ease of explanation, the DLD array portion of the separation unit206is referred to as a nanoDLD array506.

The separation unit206can employ a multiplexed arrangement of a plurality of conjoined nanoDLD array pillars or units to facilitate enhanced throughput of target particle isolation from sample biological fluid. For example, with reference toFIG. 2BandFIG. 5, the separation unit206can employ four rows of densely packed nanoDLD array pairs arranged inside of the circular, inlet bus204feed that distributes the sample fluid from the global inlet via202. For example, the number of nanoDLD array pairs that can be integrated into the microfluidic chip can range from the thousands to the hundreds of thousands depending on the size of the chip. Usage of this massive, multiplexed parallelization framework can substantially increase the throughput rate of the separation unit206(e.g., to around 1.0 mL of sample fluid per hour or greater).

The nanoDLD array506can include alternating pillar array units with different pillar angles for outward deflection of an incoming sample fluid, and inward deflection for incoming buffer fluid. For example, in the embodiment shown, the separation unit206can include a plurality of second inlet vias502through which buffer fluid can be introduced into the separation unit206and flowed downstream (e.g., in the direction of arrow D) in association with simultaneous flow of biological sample fluid through the separation unit206from inlet bus204(e.g., in the direction of arrow D). As described with reference toFIGS. 2B and 3C, for example, the second inlet vias502can correspond to the respective dark boxes of the first checkered line drawn at the interface between the inlet bus204and the dark grey region of the separation unit206. The second inlet vias502can further connect to the buffer fluid reservoir304.

A zoomed-in view of the nanoDLD array pairs is shown in call out-boxes503and505. As shown in call-out box501and call-out box503, the nanoDLD array506can include outward deflection units506apositioned adjacent to the entry region of the separation unit206with the biological fluid sample is passed from the inlet bus204. As shown in call-out box501and call-out box505, the nanoDLD array506can further include inward deflection units506bpositioned adjacent to the second inlet vias502through which the buffer fluid is injected. As a result, larger, target particles of interest included in the biological fluid sample can be directed in an inward flow path direction that causes a first stream of buffer fluid including the particles of interest to flow toward and into the outlet bus208where they are collected. Smaller, unwanted particles (e.g., salts, proteins, lipids, and other small biomolecules/macromolecules), collectively referred to herein as waste particles, can be directed in an outward flow path direction that causes a second stream of buffer fluid to flow toward and into a plurality of outlet vias508. For example, as described with reference toFIGS. 2B and 3C, the outlet vias508can correspond to the respective dark boxes of the second checkered line drawn at the interface between the dark grey region of the separation unit206and the outlet bus208. The outlet vias508can further align with and connect to the waste fluid reservoir306. In this regard, the portion of the buffer stream including the waste particles can flow into the waste fluid reservoir306via the outlet vias508where the waste fluid can be collected and further exported off the chip and the housing102(e.g., through outlet port118).

In the embodiment shown, the separation unit206can also include a filtration element504(depicted by the plurality of speckles or dots) provided at or near the initial entry points of the buffer fluid and the sample fluid into the separation unit206. For example, the filtration element504can be formed upstream of the nanoDLD array506. The filtration element504can (optionally) be used to filter out or remove certain large particles of size greater than a defined threshold size, such as a size greater than the target particles (e.g., greater than exosomes). In one or more embodiments, the filtration element can include but is not limited to, a cross-flow of serpentine filters, traps, sieves, bladed loading features or a number of other microfluidic filter arrangements to capture cells, larger cellular debris, and/or larger multivesicular bodies (MVBs) while letting desired colloids pass into the downstream, nanoDLD array506.

FIG. 6illustrates another enlarged view of the portion of the microfluidic chip106included in call-out box401.FIG. 6further includes call-out box600depicting a nano-scale view of one of the outward deflection units506ain association with simultaneous flow of sample biological fluid and buffer fluid through the nanoDLD arrays in accordance with one or more embodiments described herein. Repetitive description of like elements employed in respective embodiments is omitted for sake of brevity.

FIG. 6highlights an example process of operation for the separation unit206of example separation-purification apparatus100. In the embodiment shown, the separation process of the separation-purification apparatus100involves inlet bus204for introduction of sample fluid, second inlet vias502through which buffer fluid is injected, filtration element504, nanoDLD array506, outlet vias508for excreting waste fluid, and outlet bus208for collecting purified or extracted particles of interest (e.g., exosomes) and carrying the particles of interest to the downstream detection unit. In this regard, with reference toFIGS. 2B, 3BandFIG. 6, a sample fluid, such as plasma, cultured medium, urine, etc., can be introduced into the microfluidic chip106(e.g., at global via202), and fed through the (circular) inlet bus204, which in turn, injects the sample through openings at the interface of the inlet bus204and the separation unit206. For example, these openings are located adjacent to the outward deflection units506aof the nanoDLD array506. Buffer fluid is further simultaneously injected into the separation unit206through the second inlet vias502adjacent to the inward deflection areas of the nanoDLD arrays. The buffer fluid can provide a fresh purification medium. Prior to flow of the sample fluid through the nanoDLD array506, the filtration element504can remove larger particles of material from the incoming sample fluid that might otherwise clog at the interface of nanoDLD arrays, reducing longevity.

As shown inFIG. 6, as the fluid sample and the buffer fluid simultaneously flow through the nanoDLD array506, the large target particles of a defined size range (e.g., exosomes having a defined size range), are deflected or bumped into a portion of the buffer fluid which gets focused at the inward deflection unit/outward deflection unit junction. As a result, the large target particles are directed into the outlet bus208. Smaller unwanted particles, such as salts, small molecules, lipids, proteins, etc. maintain their general trajectory in the direction of the sample fluid flow toward the outlet vias508, also referred to herein as the waste outlets. Through this process, a particular purified size range of target particles (e.g., exosomes) can be selectively loaded into the outlet bus208. This purification process, in effect, acts as a bandpass filter to remove the background contamination below and larger material above structurally defined cutoffs, making it much more straightforward to detect target particles bearing a particular surface marker or biomarker on the downstream, sensing element108.

FIG. 7illustrates an enlarged view of an example detection unit700of an example microfluidic chip (e.g., microfluidic chip106) that integrates on-chip particle purification and biomarker detection functionality in accordance with one or more embodiments described herein. Repetitive description of like elements employed in respective embodiments is omitted for sake of brevity.

The detection unit700can include the sensing element108. In the embodiment shown, the detection unit700can also include the portion of the outlet bus208that connects to the sensing element108and provides for injecting a stream of buffer fluid comprising the purified or separated target particles onto and over the surface of the sensing element108coated with a surface chemistry (e.g., one or more specific molecules or macromolecules) that provides chemical specificity for one or more known biomarkers that may be present on or within the target particles. In this regard, the as discussed above, the sensing element108can facilitate detecting presence of one or more biomarkers by coating or otherwise providing one or more reactive agents that have a known chemical specificity for the one or more biomarkers, such as antibodies and aptamers known to bind with one or more surface markers. In one or more example implementations, the sensing element108can include one or more antibodies or aptamers known to bind with exosomes bearing a particular oncogenic surface marker (e.g. CD81, PSMA, etc.). The chemical specificity however is not limited to antibody/epitope interactions but can be extended to any specific chemical or biochemical interaction between two molecules or macromolecules, including naturally occurring and synthetic molecules/macromolecules. For example, the chemical interactions can include permanent covalent linkage as well as reversible bond interactions, such as electrostatic interactions, hydrophobic interactions, complementarity interactions and the like. In this regard, the sensing element108can provide for detecting biomarkers are result of interactions including but not limited to, antibody-epitope interactions, complementary DNA or RNA strand hybridization interactions, DNA binding proteins and DNA consensus sequence interactions, protein-protein interactions, protein-small molecule interactions, polymerization reactions, biotin-streptavidin interactions, and others.

The sensing element itself108can take on a variety of structures and materials to enhance the signal generated as a result of a chemical reaction between the surface chemistry of the sensing element and the one or more biomarkers. For example, the sensing element108can include an optical element that enhances a visual signal generated or detected in association with binding of a antibody provided on the surfaced of the sensing element with an epitope of a target particle, such as fluorescence signal of immuno-bound exosomes, thereby enhancing the sensitivity of the sensing element, In this regard, the sensing element108can include but is not limited to, a photonic grating or pillar array, an optoelectrical element, or a plasmonic structure.

The detection unit700can further include one or more third inlet vias212through which detection fluid including the reactive analyte substances can be introduced and applied to coat the surface of the sensing element108. The detection unit700can also include a distribution bus214to facilitate evenly coating the sensing element with the introduced detection fluid. For example, in one or more embodiments, prior to introducing purified sample to the sensing element108, the sensing element108can be coated with appropriate detection molecules/macromolecules (e.g., antibodies, aptamers, etc.) in accordance with a coating process (noted inFIG. 7as coating step1). With reference toFIGS. 2C-2D,FIG. 3CandFIG. 7, in accordance with the coating process, detection fluid (e.g., antibody-containing fluid) can be introduced through the one or more third inlet vias212by applying a positive pressure at these inlets, or more directly to the detection fluid reservoir308to which the one or more third inlet vias212are fluidically connected (e.g., via fluidic connections212′). At the same time, a comparatively negative pressure can be applied at the global outlet via210. The binding chemistry of the detection fluid can thus be forced to flow from one or more third inlet vias212to the global outlet via210, thereby coating the sensing element108. In some implementations, the sensing element can be pre-wetted prior to the coating step to facilitate the coating process. Once the sensing element108has been coated the separation-purification apparatus100is ready to use particle purification and separation. In this regard, in accordance with step2, the upstream separation can then be performed in accordance with the techniques described herein, bringing the purified particles (e.g., exosomes) to the sensing element108by way of the outlet bus208, where they can potentially bind to the sensing element108(if a particular target exosome and/or biomarker is present) and generate a reactionary signal that can be detected. For example, in some implementations, a target particle can be detected through immunocapture, producing a fluorescent signal that can be observed with fluorescence microscopy, and therefore manually detectable by eye or through software to automate the process.

FIG. 8illustrates an enlarged view of another example detection unit800of an example microfluidic chip (e.g., microfluidic chip106) that integrates on-chip particle purification and biomarker detection functionality in accordance with one or more embodiments described herein. Repetitive description of like elements employed in respective embodiments is omitted for sake of brevity.

Detection unit800can include same or similar features and functionality as detection unit700with the addition of a blocking element802at the inlet interface between the one or more third inlet vias212and the sensing element108, (e.g., including openings where the detection fluid can flow from the one or more second inlet vias onto the surface of the sensing element108. The primary function of the blocking element802is to block free floating, unbound or unreacted target particles (e.g., exosomes), from reversely flowing away from the sensing element108and the global outlet via210toward the one or more third inlet vias212. In some implementations, in which detection molecules are not tethered to or become separated from the surface of the sensing element, the blocking element802can also prevent reverse flow of free floating reacted molecular complexes (e.g., antibody-exosome complexes) through the third inlet vias212. In this regard, the blocking element802can include a physical structure that prevents essentially any particles other than free floating detection molecules/macromolecules (which are generally very small) therethrough. The blocking element802can thus corral isolated target biological entities (e.g. exosomes, viruses, etc.) with biochemically specific markers within the field of view of an optical detector. For example, as shown in call out boxes801and803, in one embodiment, the blocking element802can comprise integrated pillars, a sieve, or the like, with gaps in between them at the perimeter of the sensing element108and adjacent to the detection fluid chemistry inlets. The gaps can be too small for free-flowing isolated target particles (e.g., exosomes) to fit through, but large enough for the coating chemistry to flow through (e.g., antibodies in the example shown). In this way, the blocking element can prevent loss of exosomes bearing the target surface proteins, or epitopes, keeping them contained completely within the field of view and surface of the sensing element108.

FIG. 9Aillustrates an enlarged view of another example detection unit900that can be employed in a microfluidic chip (e.g., microfluidic chip106) that integrates on-chip particle purification and biomarker detection functionality in accordance with one or more embodiments described herein. Detection unit900can include same or similar features and functionality as detection unit800, with the addition of a plurality of different detection chambers to the sensing element108. Repetitive description of like elements employed in respective embodiments is omitted for sake of brevity.

In the embodiment shown, the sensing element108can be subdivided into a plurality of separate detection chambers, respectively identified as detection chambers901,902,903and904. Each of the different detection chambers can provide for detecting a presence of different biomarker and/or target particle. For example, each (or in some implementations one or more) of the detection chambers901,902,903and904can be coated with different detection molecules/macromolecules known to react with different biomarkers. For instance, the respective detection chambers can be coated with different biomarker-specific antibodies or aptamers. With these embodiments, the sensing element108can provide for simultaneous detection of a plurality of potential biomarkers from a single input fluid sample and with a single purification-detection procedure. Simultaneous detection of multiple markers allows for fast, and effective diagnosis of various diseases, such as certain forms of cancer. In order to ensure isolation between the different detection chambers, the detection chambers can respectively be separated from one another via partition wall906.

In accordance with this embodiment, a separate detection fluid should be injected into each of the respective detection chambers in isolation. In this regard, each of the detection chambers can employ a different inlet via (of the one or more third inlet vias212) that can respectively be fluidically coupled to different detection fluid reservoirs (e.g., as opposed to the single, communal, detection fluid reservoir308, shown inFIGS. 3A-3C). For example, as shown inFIG. 9B, rather than a communal, detection fluid reservoir308, reservoir layer (included in the bottom plate102B or between the bottom plate102B and the microfluidic chip106) can include a plurality of isolated detection fluid reservoirs or channels910through which the different detection fluids can be injected to coat the different detection chambers.

FIGS. 9C and 9Dpresent a 3D, perspective view of example detection unit900in accordance with one or more embodiments described herein. With reference toFIGS. 2C and 2Din conjunction with reference toFIGS. 9C and 9D, detection unit900can include same or similar features and functionalities as detection unit200with the addition of separate detection chambers901,902,903and904to the sensing element108. The respective detection chambers are separated by separation walls906. In addition, a blocking element802is formed at the inlet region between the respective third inlet vias212and the sensing element108. As shown inFIG. 9Dwith reference to the dashed arrow lines, after the different detection chambers of the sensing element108have been functionalized with a different surface chemistry, a stream of buffer fluid comprising purified target particles can flow from the separation unit206, up through the outlet bus208channels and onto the sensing element108. In the embodiment shown, a single outlet bus208channel can feed two detection chambers at a time (e.g., detection chambers903and904in the demonstrated example).

FIG. 10illustrates an enlarged view of another example detection unit1000that can be employed in a microfluidic chip (e.g., microfluidic chip106) that integrates on-chip particle purification and biomarker detection functionality in accordance with one or more embodiments described herein. Detection unit1000can include same or similar features and functionalities as detection unit900with the addition of a greater number of detection chambers, including detection chambers1001-1008. In this regard, the number of detection chambers in which the sensing element is subdivided into can vary and is not limited to one, four or eight (as in the embodiments shown). However, as the number of detection units increases, the number of third inlet vias212will also increase, as each detection unit can comprise a separate injection via (and corresponding channel and/or reservoir) through which each different type of detection fluid chemistry can be provided. Repetitive description of like elements employed in respective embodiments is omitted for sake of brevity.

FIG. 11illustrates an example system1100that facilitates integrating real-time particle purification and biomarker detection in accordance with one or more embodiments described herein. Embodiments of systems (e.g., system1100and pressure driving system1102), imaging devices (e.g., imaging device1104) and computing devices (e.g., computing device1108) described herein can include one or more machine-executable components embodied within one or more machines (e.g., embodied in one or more computer readable storage media associated with one or more machines). Such components, when executed by the one or more machines (e.g., processors, computers, computing devices, virtual machines, etc.) can cause the one or more machines to perform the operations described. Repetitive description of like elements employed in respective embodiments is omitted for sake of brevity.

System1100includes separation-purification apparatus100, a pressure driving system1102an imaging device1104and a computing device1108. The computing device1108can be communicatively coupled to the imaging device1104and/or the pressure driving system1102via one or more wires and/or one or more wireless networks (e.g., a local area network (LAN), a wide area network (WAN), such as the Internet, and the like).

The pressure driving system1102can be operatively coupled to one or more inlet ports and outlet ports of the housing (e.g., which can be or correspond to a microfluidic flow cell or the like) to control flow of the various fluids (e.g., the biological sample fluid, the buffer fluid, the detection fluid, and other potential cleaning/preparation fluids) into, out of, and through the housing102and the microfluidic chip106. For example, in some implementations, after the above noted fluids are introduced into the designated reservoirs of the housing102(e.g., via pipetting or another suitable technique), the apparatus can be operatively coupled to the pressure driving system1102and while also being aligned with the imaging device1104. In some implementations, the imaging device1104and the pressure driving system1102can be a combined system. In other implementation, the pressure driving system1102can facilitate injecting the various fluids into the corresponding reservoirs of the housing102prior to and/or at runtime of the apparatus. The pressure-driving system can be a fully automated pressure driving machine, a manually operated pressure driving machine, or a combination thereof. The pressure driving system can form a pressure seal between the inlet ports (e.g., inlet port112, inlet port114, and inlet port116) and the respective reservoirs, channels, inlets, etc., of the housing102, and/or the respective outlet ports (e.g., outlet port118and outlet port120). In this regard, the pressure driving system can apply pressure to initiate fluid flow of one or more fluids within the different fluid reservoirs located within the housing and a reservoir comprising the biological sample fluid, referred to herein as the sample fluid reservoir.

The imaging device1104can comprise a lens1006or capture region that is aligned with the sensing element108. In this regard, the location of the sensing element108at or near the center of the microfluidic chip106(with a transparent window formed thereover as part of the capping layer110), can enable efficient optical readout of biochemically specific information that developed or captured by the sensing element (e.g., immunocaptured exosomes detected using for example fluorescence microscopy). In the embodiment shown, the imaging device1104is a microscope. In some implementations in which the imaging device1104comprises a microscope, the microscope can be or include a fluorescence microscope configured to capture immunofluorescent signals generated by fluorescent labeled molecules (e.g., antibodies or aptamers, target particles, binding proteins, etc.) captured by the sensing element108. The features and functionalities of the microscope can however vary. The imaging device1104can alternatively or additionally include other types of imagining devices or cameras configured to captures still images, 2D images, high dynamic range images, video, etc. of the sensing element108at various stages of operation of separation-purification apparatus100in accordance with the techniques described herein (e.g., in real-time during separation and detection flow or after completion of running of the biological fluid sample therethrough). In some implementations, these images can be sent to a computing device1108for rendering via a display1110of the computing device1108. In this regard, in some implementations, biomarker detection and/or biochemical analysis of target particles with specific surface markers (e.g., exosomes with specific carcinogenic surface markers) can be manually performed by examining the sensing element108through the microscope and/or via image data presented via the display1110in real-time during separation and detection flow, or after completion of running of the biological fluid through the apparatus. In other implementations, described infra, the computing device1108can include software configured to perform automated biomarker detection and analysis based on image data captured of the sensing element108before, during, and/or after running of biological fluid sample through separation-purification apparatus100.

FIG. 12illustrates an example computing device1108that facilitates real-time biomarker detection and analysis in accordance with one or more embodiments described herein. Repetitive description of like elements employed in respective embodiments is omitted for sake of brevity.

As presented in system1100, the computing device1108can include or be operatively coupled to a display1110via which image data captured of the sensing element108can be rendered. The computing device1108further includes or can be operatively coupled to at least one memory1212and at least one processor1210. In various embodiments, the at least one memory1212can store executable instructions that when executed by the at least one processor1210, facilitate performance of operations defined by the executable instruction. For example, in the embodiment shown, the computing device1108further include microfluidic sensor processing module1202which includes sensor data reception component1204, biomarker detection component1206, and diagnosis component1208. In one or more embodiments, these components (e.g., the microfluidic sensor processing module1202and additional components of the microfluidic sensor processing module1202) can be stored in memory1212and executed by the at least one processor1210. The computing device1108can further include a device bus1214that communicatively couples the various components of the computing device1108(e.g., the microfluidic sensor processing module1202, the processor1210, the memory1212and the display1110). Examples of said processor1210and memory1212, as well as other suitable computer or computing-based elements, can be found with reference toFIG. 16, and can be used in connection with implementing one or more of the systems or components shown and described in connection withFIGS. 11 and 12or other figures disclosed herein.

The microfluidic sensor processing module1202can facilitate various processing functionalities associated with evaluating chemical reactions that occur (or do not occur) at the sensing element108of the disclosed microfluidic chips. In this regard, the sensor data reception component1204can receive data regarding the chemical reaction that occur (or do not occur) at a particular sensing element in association with flow of purified target biological entities over the surface of a functionalized sensing element. In various embodiments, this data can include image data captured of the sensing unit before, during, and/or after flow process. For example, the image data can include still images of the sensing element108captured by an imaging device (e.g., imaging device1104) positioned in line-of-sight of the sensing element at one or more points before, during and/or after the flow process. In other implementations, the image data can include video captured during the flow process by such an imaging device. With these implementations, the microfluidic sensor processing module1202can provide for real-time or live biomarker detection and analysis. In some embodiments, the chemical reactions that occur at the sensing element108between a target particle or target biomarker and the one or more detection molecules/macromolecules with which the sensing element108is functionalized can result in other forms of detectable sensory data, other than visual signals. For example, in some implementations, a chemical reaction can be detected by generation of a detectable electrochemical signal, generation a heat signal, or another form of sensory data. With these implementations, the sensor data reception component1204can receive information regarding generation of such other types of sensory signals to facilitate biochemical analysis.

The biomarker detection component1206can analyze received sensory data (e.g., image data, or another form of sensory data) captured of and/or generated at the sensing element regarding occurrence, (or non-occurrence), of one or more chemical reactions between one or more particles in the purified biological sample stream and one or more detection molecules/macromolecules of the sensing element108, and determine whether a particular biomarker is present. In some implementations, the biomarker detection component1206can also determine a quantitative measure of the amount of detected biomarker. In this regard, the biomarker detection component1206can access and/or employ biomarker identification information (e.g., stored in memory) that correlates potential chemical reaction-based image signals that can be generated at a sensing element (e.g., based on the type of detection molecules/macromolecules with which the sensing element is functionalized), with known biomarkers and/or known particles. The biomarker detection component1206can further be configured to identify or otherwise recognize an image signal that correlates with a known biomarker or particle to determine whether the biomarker is present. For example, in some implementations, the image signals can include image data such, as fluorescent image data, that visually tags a chemical bond between a target biomarker or surface marker and a detection molecule/macromolecule. In other implementation, the image signal data can include a particular coloration or change in coloration, a particular brightness or change in brightness, a particular image pattern, and the like. In various embodiments, based on the analysis of the received sensory data (e.g., image data) the biomarker detection component1206can generate biomarker information (for rendering via the display or for otherwise providing to an entity via a suitable output device) identifying detected biomarkers and/or particles, and in some implementations, an amount of the detected biomarkers and/or particles.

The diagnosis component1208can further analyze biomarker information to determine diagnosis information regarding a disease state or medical condition of the entity (e.g., a patient) from which the biological fluid sample was taken. In this regard, the diagnosis component1208can access and employ information that correlates known biomarkers, known biomarker amounts, and/or known biomarker combinations (in implementations in which two or more biomarkers can be detected at a time, such as with respect to detection unit200, detection unit800, detection unit900, detection unit1000and the like), with particular diseases, disease states, and/or medical conditions, to determine whether a disease, disease state, and/or medical condition has been detected.

FIG. 13illustrates a flow diagram of an example, non-limiting method1300for performing particle purification and biomarker detection using an integrated microfluidic device in accordance with one or more embodiments described herein. Repetitive description of like elements employed in respective embodiments is omitted for sake of brevity.

At1302, target biological entities having a defined size range are isolated from other biological entities included in a biological fluid sample using a separation unit (e.g., separation unit206) comprising one or more nanoDLD arrays formed on a microfluidic chip (e.g., microfluidic chip106), thereby resulting in isolated target biological entities. At1304buffer fluid comprising the isolated target biological entities is driven through a conduit (e.g., outlet bus208) of the microfluidic chip from the separation unit to a sensing element (e.g., sensing element108) formed on the microfluidic chip. At1306, detection of the presence of one or more biomarkers associated with the isolated target biological entities is facilitated based on whether a detectable signal is generated at the sensing element in response to the driving.

FIG. 14illustrates a flow diagram of an example, non-limiting method1400for functionalizing a sensing element of an integrated microfluidic device and thereafter, employing the integrated microfluidic device to isolate exosomes and detect presence of exosomal surface markers based on reaction with the functionalized sensing element, in accordance with one or more embodiments described herein. Repetitive description of like elements employed in respective embodiments is omitted for sake of brevity.

At1402, functionalizing a surface of a sensing element (e.g., sensing element108) of a microfluidic chip (e.g., microfluidic chip106) with one or more detection molecules or macromolecules (e.g., antibodies, aptamers, etc.) that chemically react with one or more exosomal surface markers, wherein the functionalizing comprises injecting a solution comprising the one or more detection molecules or macromolecules into a chamber enclosing the surface of sensing element via at least one injection inlet (e.g., the one or more third inlet vias212) of the microfluidic chip. At1404, exosomes are isolated from other biological entities included in a biological fluid sample in response to driving flow of the biological fluid sample and a buffer fluid through a multiplexed nanoDLD array (e.g., nanoDLD array506) of the microfluidic chip. At1406, a stream of the buffer fluid comprising the exosomes is driven from the multiplexed nanoDLD array (e.g., via outlet bus208) over the surface of the sensing element and through an outlet via (e.g., global outlet via210) of the microfluidic chip located downstream of the sensing element.

FIG. 15illustrates a flow diagram of an example, non-limiting method1500that facilitates integrating real-time particle purification and biomarker detection in accordance with one or more embodiments described herein. Repetitive description of like elements employed in respective embodiments is omitted for sake of brevity.

At1502, target biological entities having a defined size range from other biological entities included in a biological fluid sample are isolated using a separation unit comprising one or more nanoDLD arrays (e.g., separation unit206), thereby resulting in isolated target biological entities, wherein the separation unit is formed on a microfluidic chip contained within a housing (e.g., housing102). At1504, a buffer fluid comprising the isolated target biological entities is driven through a conduit of the microfluidic chip (e.g., outlet bus208) from the separation unit to a sensing element (e.g., sensing element108) formed on the microfluidic chip, wherein the sensing element generates a visual signal in response to detection of presence of one or more defined biomarkers associated with the target biological entities. At1506, image data of the detection unit is captured in association with the driving (e.g., via imaging device1104). At1508, a computing device comprising a processor (e.g., computing device1108) determines a diagnosis regarding a medical condition or a disease state associated with the biological fluid sample based on analysis of the image data (e.g., using biomarker detection component1206and/or diagnosis component1208).

In order to provide a context for the various aspects of the disclosed subject matter,FIG. 16as well as the following discussion are intended to provide a general description of a suitable environment in which the various aspects of the disclosed subject matter can be implemented.FIG. 16illustrates a block diagram of an example, non-limiting operating environment1600in which one or more embodiments described herein can be facilitated. For example, the operating environment1600can comprise and/or otherwise facilitate one or more features of the pressure driving system1102, the imaging device1104, and/or the computing device1108described herein in accordance with one or more embodiments. Repetitive description of like elements employed in other embodiments described herein is omitted for sake of brevity.

With reference toFIG. 16, a suitable operating environment1600for implementing various aspects of this disclosure can include a computer1612. The computer1612can also include a processing unit1614, a system memory1616, and a system bus1618. The system bus1618can operably couple system components including, but not limited to, the system memory1616to the processing unit1614. The processing unit1614can be any of various available processors. Dual microprocessors and other multiprocessor architectures also can be employed as the processing unit1614. The system bus1618can be any of several types of bus structures including the memory bus or memory controller, a peripheral bus or external bus, and/or a local bus using any variety of available bus architectures including, but not limited to, Industrial Standard Architecture (ISA), Micro-Channel Architecture (MSA), Extended ISA (EISA), Intelligent Drive Electronics (IDE), VESA Local Bus (VLB), Peripheral Component Interconnect (PCI), Card Bus, Universal Serial Bus (USB), Advanced Graphics Port (AGP), Firewire, and Small Computer Systems Interface (SCSI). The system memory1616can also include volatile memory1620and nonvolatile memory1622. The basic input/output system (BIOS), containing the basic routines to transfer information between elements within the computer1612, such as during start-up, can be stored in nonvolatile memory1622. By way of illustration, and not limitation, nonvolatile memory1622can include read only memory (ROM), programmable ROM (PROM), electrically programmable ROM (EPROM), electrically erasable programmable ROM (EEPROM), flash memory, or nonvolatile random access memory (RAM) (e.g., ferroelectric RAM (FeRAM). Volatile memory1620can also include random access memory (RAM), which acts as external cache memory. By way of illustration and not limitation, RAM is available in many forms such as static RAM (SRAM), dynamic RAM (DRAM), synchronous DRAM (SDRAM), double data rate SDRAM (DDR SDRAM), enhanced SDRAM (ESDRAM), Synchlink DRAM (SLDRAM), direct Rambus RAM (DRRAM), direct Rambus dynamic RAM (DRDRAM), and Rambus dynamic RAM.

Computer1612can also include removable/non-removable, volatile/non-volatile computer storage media.FIG. 16illustrates, for example, a disk storage1624. Disk storage1624can also include, but is not limited to, devices like a magnetic disk drive, floppy disk drive, tape drive, Jaz drive, Zip drive, LS-100 drive, flash memory card, or memory stick. The disk storage1624also can include storage media separately or in combination with other storage media including, but not limited to, an optical disk drive such as a compact disk ROM device (CD-ROM), CD recordable drive (CD-R Drive), CD rewritable drive (CD-RW Drive) or a digital versatile disk ROM drive (DVD-ROM). To facilitate connection of the disk storage1624to the system bus1618, a removable or non-removable interface can be used, such as interface1626.FIG. 16also depicts software that can act as an intermediary between users and the basic computer resources described in the suitable operating environment1600. Such software can also include, for example, an operating system1628. Operating system1628, which can be stored on disk storage1624, acts to control and allocate resources of the computer1612. System applications1630can take advantage of the management of resources by operating system1628through program modules1632and program data1634, e.g., stored either in system memory1616or on disk storage1624. It is to be appreciated that this disclosure can be implemented with various operating systems or combinations of operating systems. A user enters commands or information into the computer1612through one or more input devices1636. Input devices1636can include, but are not limited to, a pointing device such as a mouse, trackball, stylus, touch pad, keyboard, microphone, joystick, game pad, satellite dish, scanner, TV tuner card, digital camera, digital video camera, web camera, and the like. These and other input devices can connect to the processing unit1614through the system bus1618via one or more interface ports1638. The one or more Interface ports1638can include, for example, a serial port, a parallel port, a game port, and a universal serial bus (USB). One or more output devices1640can use some of the same type of ports as input device1636. Thus, for example, a USB port can be used to provide input to computer1612, and to output information from computer1612to an output device1640. Output adapter1642can be provided to illustrate that there are some output devices1640like monitors, speakers, and printers, among other output devices1640, which require special adapters. The output adapters1642can include, by way of illustration and not limitation, video and sound cards that provide a means of connection between the output device1640and the system bus1618. It should be noted that other devices and/or systems of devices provide both input and output capabilities such as one or more remote computers1644.

Computer1612can operate in a networked environment using logical connections to one or more remote computers, such as remote computer1644. The remote computer1644can be a computer, a server, a router, a network PC, a workstation, a microprocessor based appliance, a peer device or other common network node and the like, and typically can also include many or all of the elements described relative to computer1612. For purposes of brevity, only a memory storage device1646is illustrated with remote computer1644. Remote computer1644can be logically connected to computer1612through a network interface1648and then physically connected via communication connection1650. Further, operation can be distributed across multiple (local and remote) systems. Network interface1648can encompass wire and/or wireless communication networks such as local-area networks (LAN), wide-area networks (WAN), cellular networks, etc. LAN technologies include Fiber Distributed Data Interface (I-DDI), Copper Distributed Data Interface (CDDI), Ethernet, Token Ring and the like. WAN technologies include, but are not limited to, point-to-point links, circuit switching networks like Integrated Services Digital Networks (ISDN) and variations thereon, packet switching networks, and Digital Subscriber Lines (DSL). One or more communication connections1650refers to the hardware/software employed to connect the network interface1648to the system bus1618. While communication connection1650is shown for illustrative clarity inside computer1612, it can also be external to computer1612. The hardware/software for connection to the network interface1648can also include, for exemplary purposes only, internal and external technologies such as, modems including regular telephone grade modems, cable modems and DSL modems, ISDN adapters, and Ethernet cards.