Patent Grant
11504681
The present invention relates generally to microfluidic devices for mixing fluidized biological samples and reagents for preparation, processing and/or analysis of the samples.
Biological analytes of relevance to clinical, biological, or environmental testing frequently are found at low concentrations in complex fluid mixtures. It is important to capture, concentrate, and enrich the specific analyte away from background inhibitory or interfering matrix components that can limit the sensitivity and/or specificity of analyte detection assays. Specific analytes include but are not limited to nucleic acids, proteins, including for example antigens or antibodies, prokaryotic or eukaryotic cells, and viruses, and small molecules such as drugs and metabolites. Conventional sample preparation methods include centrifugation, solid phase capture, selective precipitation, filtration, and extraction. These methods are not generally amenable to efficient automation and integration with subsequent assay steps, especially in a manner compatible with the development of point of care testing.
Microfluidic devices have become popular in recent years for performing analytical testing. Using tools developed by the semiconductor industry to miniaturize electronics, it has become possible to fabricate intricate fluid systems that can be inexpensively mass-produced. Systems have been developed to perform a variety of analytical techniques for the acquisition and processing of information.
The ability to perform analyses microfluidically provides substantial advantages of throughput, reagent consumption, and automatability. Another advantage of microfluidic systems is the ability to integrate a plurality of different operations in a single “lap-on-a-chip” device for performing processing of reactants for analysis and/or synthesis. Microfluidic devices may be constructed in a multi-layer laminated structure wherein each layer has channels and structures fabricated from a laminate material to form microscale voids or channels where fluids flow. A microscale or microfluidic channel is conventionally defined as a fluid passage, which has at least one internal cross-sectional dimension that is less than 500 μm, and typically between about 0.1 μm and about 500 μm.
U.S. Pat. No. 5,716,852, hereby incorporated by reference in its entirety, is an example of a microfluidic device. The '852 patent teaches a microfluidic system for detecting the presence of analyte particles in a sample stream using a laminar flow channel having at least two input channels which provide an indicator stream and a sample stream, where the laminar flow channel has a depth sufficiently small to allow laminar flow of the streams and length sufficient to allow diffusion of particles of the analyte into the indicator stream to form a detection area, and having an outlet out of the channel to form a single mixed stream. This device, which is known as a T-Sensor, allows the movement of different fluidic layers next to each other within a channel without mixing other than by diffusion. A sample stream, such as whole blood, a receptor stream, such as an indicator solution, and a reference stream, which may be a known analyte standard, is introduced into a common microfluidic channel within the T-Sensor, and the streams flow next to each other until they exit the channel. Smaller particles, such as ions or small proteins, diffuse rapidly across the fluid boundaries, whereas larger molecules diffuse more slowly. Large particles, such as blood cells, show no significant diffusion within the time the two flow streams are in contact.
There is general agreement that, in the laminar flow regime characteristic of microfluidic channels, mixing is limited to diffusion. Because of the dimensions involved, wherein diffusional free path lengths are roughly equal the device dimensions, diffusional mixing can be very effective for solutes. This condition enables ribbon flow, T-sensor, and other useful microfluidic phenomena. However, for larger analytes such as cells, bacteria, viral particles, and for macromolecular complexes and linear polymers, diffusional mixing is slow and processes for capture or depletion of these species require prolonged incubation. Diffusional limits on mixing thus present a problem in microfluidic devices where bulk mixing or combination of a sample and reagents or beads is required. This problem has not been fully solved and methods, devices and apparatuses for improving the mixing arts are being actively sought.
In brief, the present invention relates to microfluidic devices, apparatuses, and methods involving manipulating and mixing fluidized biological samples with reagents of different physical and chemical properties. In particular, disclosed microfluidic mixers utilize a plurality of microfluidic channels, vias, valves, pumps and other elements arranged in various configurations to manipulate the flow and mixing of fluid samples and reagents to prepare samples for subsequent analysis.
A preferred embodiment disclosed herein is a microfluidic mixing device, including a first bellows pump with a chamber bisected in coronal plane by a first elastomeric membrane, a second bellows pump with a chamber bisected in coronal plane by a second elastomeric membrane, a first microchannel fluidly interconnecting the first bellows pump with a sample inlet and a reagent reservoir, wherein the first microchannel comprises a valve interposed between the pump and the inlet and a valve interposed between the pump and the reservoir, a second microchannel fluidly interconnecting the first bellows pump with the second bellows pump, wherein the second micro channel comprises a valve interposed between the first and second pump, a third microchannel fluidly interconnecting the first bellows pump with the second bellows pump, wherein the third micro channel comprises a valve interposed between the first and second pump, a first and second pneumatic members pneumatically connected to the first and second bellows pumps; wherein, the volume of the second bellows pump is great than the volume of the first bellows pump. In certain embodiments, the first, second, and third microchannels intersect to form a web in fluid communication with the first bellows pump. In yet other embodiments, each of the channels of the microweb is in fluid communication with a liquid via. In yet another embodiment, each of the channels of the microweb is in fluid communication with a liquid via. In yet another embodiment, the microweb is configured to enable both laminar and turbulent fluid flow. In another embodiment, the second and third microfluidic channels comprise perpendicular extensions in fluid communication with the second bellows pump. In yet another embodiment, each of the extensions is in fluid communication with more than one via. In yet another embodiment, each of the extensions is in fluid communication with three vias. In another embodiment, the vias are configured to enable dispersed flow of liquid over substantially the entire surface area of the second bellows pump.
In another aspect, the invention provides a microfluidic cartridge including any of the mixing devices described herein.
In another aspect, the invention provides a method of processing serial aliquots of a test sample using any of the cartridges described herein.
As an aid in better explaining the invention, the following definitions are provided. If any definition provided herein is inconsistent with a dictionary meaning, meaning as commonly understood in the art, or meaning as incorporated by reference to a patent or literature citation, the definition presented here shall prevail.
Microfluidic cartridge: a “device”, “card”, or “chip” with fluidic structures and internal channels having microfluidic dimensions. These fluidic structures may include chambers, valves, vents, vias, pumps, inlets, nipples, and detection means, for example. Generally, microfluidic channels are fluid passages having at least one internal cross-sectional dimension that is less than about 500 μm and typically between about 0.1 μm and about 500 μm. Therefore, as defined herein, microfluidic channels are fluid passages having at least one internal cross-sectional dimension that is less than 500 μm. The microfluidic flow regime is characterized by Poiseuille or “laminar” flow.
Bellows Pump: is a device formed as a cavity, often cylindrical in shape, bisected in coronal section by an elastomeric diaphragm to form a first and a second half-chamber which are not fluidically connected. The diaphragm is controlled by a pneumatic pulse generator connected to the first half-chamber. Positive pressure above the diaphragm distends it, displacing the contents of the second half-chamber, negative gauge pressure (suction) retracts it, expanding the second half chamber and drawing fluid in. By half-chamber, it should be understood that the effective area of the diaphragm is the lesser of the volume displacement under positive pressure and the volume displacement under suction pressure, and it thus optimal when the first and second half chambers are roughly symmetrical or equal in volume above and below the diaphragm. The second half-chamber is connected to a fluid in-port and out-port. The fluid in-port and out-port may be separate ports or a single port, but in either case, are under valve control. As described above, a pneumatic pulse generator is pneumatically connected to the first half-chamber, generally by a microchannel, which is also valved. In the complete apparatus, pneumatic actuation is programmable. Thus, programmable pneumatic pressure logic used by the pulse generator has two functions, to actuate the diaphragm on signal, and to open and close valves on signal. When the pulse generator is off-cartridge, nipples or inlets, a pneumatic manifold and solenoid valves are provided.
In use, fluid enters the second half-chamber of a bellows pump through the inlet valve when negative pressure is applied to the diaphragm (or passively, when fluid is pushed in by a second bellows pump). Then, when positive pressure is applied to the diaphragm, the fluid contents of the chamber are displaced out through the outlet valve. Similarly, positive and negative pressure signals control valve opening and closing. By supplying a train of positive and negative pressure pulses to a diaphragm, fluid can be moved in and out of a bellows pump chamber. This fluid motion becomes directional by the application of synchronized valve logic, thus the pumping action.
As disclosed here, pairs of bellows pumps, i.e., “dual bellows pumps”, can mix suspensions of biological samples and reagents for sample preparation and/or analysis when configured with a first diaphragm pressure-actuated and a second diaphragm passive so as to force reciprocating flow between the two bellows chambers after the inlet and outlet valves are closed. Reciprocating flow can also be obtained by synchronously actuating both diaphragms with alternating or inverted pneumatic pulses. Similarly, a multiplicity of bellows pumps can be fluidly connected in series to perform a mixing function.
Test samples: Representative biological samples include, for example: blood, serum, plasma, buffy coat, saliva, wound exudates, pus, lung and other respiratory aspirates, nasal aspirates and washes, sinus drainage, bronchial lavage fluids, sputum, medial and inner ear aspirates, cyst aspirates, cerebral spinal fluid, stool, diarrhoeal fluid, urine, tears, mammary secretions, ovarian contents, ascites fluid, mucous, gastric fluid, gastrointestinal contents, urethral discharge, synovial fluid, peritoneal fluid, meconium, vaginal fluid or discharge, amniotic fluid, semen, penile discharge, or the like may be tested. Assay from swabs or lavages representative of mucosal secretions and epithelia are acceptable, for example mucosal swabs of the throat, tonsils, gingival, nasal passages, vagina, urethra, rectum, lower colon, and eyes, as are homogenates, lysates and digests of tissue specimens of all sorts. Mammalian cells are acceptable samples. Besides physiological fluids, samples of water, industrial discharges, food products, milk, air filtrates, and so forth are also test specimens. These include food, environmental and industrial samples. In some embodiments, test samples are placed directly in the device; in other embodiments, pre-analytical processing is contemplated. For example, fluidization of a generally solid sample is a process that can readily be accomplished off-cartridge.
Reagent: refers broadly to any chemical or biochemical agent used in a reaction, including enzymes. A reagent can include a single agent which itself can be monitored (e.g., a substance that is monitored as it is heated) or a mixture of two or more agents. A reagent may be living (e.g., a cell) or non-living. Exemplary reagents for a nucleic acid amplification reaction include, but are not limited to, buffer, metal ion (for example magnesium salt), chelator, polymerase, primer, template, nucleotide triphosphate, label, dye, nuclease inhibitor, and the like. Reagents for enzyme reactions include, for example, substrates, chromogens, cofactors, coupling enzymes, buffer, metal ions, inhibitors and activators. Not all reagents are reactants, tags, or ligands, and no reagents are target analytes.
Via: A step in a microfluidic channel that provides a fluid pathway from one substrate layer to another substrate layer above or below, characteristic of laminated devices built from layers.
Air ports: refer to the arms of a pneumatic manifold under programmable control of external servomechanisms. The pneumatic manifold may be charged with positive or negative gauge pressure. Operating pressures of +/−5 to 10 psig have been found to be satisfactory. Air and other gasses may be used.
“Conventional” is a term designating that which is known in the prior art to which this invention relates, particularly that which relates to microfluidic mixing devices.
“About”, “around”, “generally”, and “roughly” are broadening expressions of inexactitude, describing a condition of being “more or less”, approximately, or almost, where variations would be obvious, insignificant, or of lesser or equivalent utility or function, and further indicating the existence of obvious exceptions to a norm, rule or limit.
As noted previously, embodiments of the present invention relate to microfluidic mixing devices, apparatuses, and methods utilizing a plurality of microfluidic channels, inlets, valves, membranes, pumps, liquid barriers and other elements arranged in various configurations to manipulate the flow of a fluid sample in order to prepare such sample for analysis and to analyze the fluid sample. In the following description, certain specific embodiments of the present devices and methods are set forth, however, persons skilled in the art will understand that the various embodiments and elements described below may be combined or modified without deviating from the spirit and scope of the invention.
Following introduction of the sample into the device, in the integrated devices of the invention, the remaining assay steps are automated or semi-automated.
Lysis buffer in the lysis buffer pouch contains, e.g., a chaotrope in combination with a detergent to effect cellular lysis and reduce associations between nucleic acids and adherent molecules, and optionally contains a nuclease inhibitor and chelator, such as EDTA to reduce nucleic acid degradation prior to wash.
We have found that guanidinium thiocyanate (GSCN), for example 4.5M GSCN, in combination with detergents such as sarcosine and Triton X-100, with weakly acidic buffer, successfully extract nucleic acids from stool that are suitable for PCR. This lysis buffer is also sufficient to remove hemoglobin from whole blood and lyse Gram negative bacteria.
However, mixing of the sample and the lysis buffer at the microscale requires ingenuity. Adaptation of biochemistry to microscale fluid assay devices has required novel engineering. In our experience, for example, a preferred mixing mechanism in the microfluidic devices of the present invention is to alternate fluid dynamics between laminar and turbulent flow. Motion in the laminar regime is characterized by parallel particle trajectories, and turbulent motion in transitional “puffs” represents strong mixing in the radial direction. Flow in conventional microfluidic structures is generally laminar and allows mixing by diffusion along boundary layers and interfaces. However, such phenomena present a problem in microfluidic devices in which bulk mixing, e.g. of solutions of different viscosities is required.
Embodiments of the present invention solve the problem of mixing solutions of different viscosities at the microscale by providing laminated or molded mixing devices including a pair of bellows pumps separated and connected by a circuit of flow-restricting channels. In this system, solutions moving through the channels experience laminar, focused flow. Upon exit from the channels into the chambers of the bellows pumps, the solutions form fluid “jets” and disperse as vortices in the bulk fluid of the chamber. These vortices, or “turbulent puffs” are characteristic of transition to turbulent flow. Turbulent mixing increases the surface area over which the solutions of different viscosities can interact and thus promotes and accelerates mixing of the two solutions. The increased surface area of the chambers relative to the channels also provides a platform enabling the faster moving, less viscous solution to contact the slower moving, more viscous solution. In the device, pneumatic actuators are provided so as to permit reciprocating flow of the two solutions between the two bellows pump chambers. Elastomeric membranes ensure forward and reverse isolation.
Operation of the microfluidic mixing subcircuit of
In a second step, valve V2 is closed, valve V10 is opened, bellows pump B1 pumps the sample into bellows pump B2 and valve V10 is closed. In an optional third step, sample is again introduced into the sample inlet, valve V2 is opened, and bellows pump B1 draws the sample into the bellows. In an optional forth step, valve V2 is closed, valve V10 is opened bellows pump B1 draws the sample into bellows pump B2 and valve V10 is closed.
In a fifth step, valve V1 is opened, valve V11 is opened and lysis buffer is introduced into bellows pump B2 after traversing bellows B1.
In a sixth step, valve V1 is closed, valve V10 is closed, bellows pump B2 pushes lysis buffer and sample through channels and valve V11 to bellows pump B1; valve V11 is closed, valve V10 is opened, bellows pump B1 pushes the mixture through channels and valve V10 to bellows pump B2. Step six is repeated multiple times to effectively mix the two samples as they flow through the circuit formed by the channels and bellows pumps. While in the channels, fluid flow is laminar; however, upon entry into the bellows chambers, fluid flow is turbulent. This repeated cycling of laminar flow in microchannels and turbulent flow in bellows chambers is surprisingly effective in mixing solutions of different viscosities, e.g., a biological sample and a lysis buffer based on chaotropes, such as guanidinium.
One advantageous feature of the microfluidic mixing subcircuits of the present invention is that they enable serial aliquots of a sample to be introduced into the mixing device, as discussed above. This functionality is achieved by designing the two pumps such that bellows pump B2 is larger in size, and thus accommodates a greater volume, than bellows pump B1. The ability of this mixing device to process serial aliquots of a single sample as well as to optionally bypass either of the pumps during operation provides advantageous flexibility to the user of the system, e.g. to customize a particular assay as required.
Turning to large bellows pump 115, in this embodiment, each of the channels connected to the pump is extended in the perpendicular direction so that multiple vias can spread the flow of liquid entering the chamber of the pump. In the exemplary configuration depicted in
In
The features of the bellows pumps of the microfluidic mixers of the present invention are shown in greater detail in
The two bellows pumps are fluidly connected by the network of microchannels, as described with reference to
Embodiments of the invention include, but are not limited to, the following:
Embodiment 1. A microfluidic mixing device, comprising:
The various embodiments described above can be combined to provide further embodiments. U.S. Provisional Application 62/297,497, filed Feb. 19, 2016 is incorporated herein by reference, in its entirety. These and other changes can be made to the embodiments in light of the above-detailed description. In general, in the following claims, the terms used should not be construed to limit the claims to the specific embodiments disclosed in the specification and the claims, but should be construed to include all possible embodiments along with the full scope of equivalents to which such claims are entitled. Accordingly, the claims are not limited by the disclosure.
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| PCT/US2017/018268 | 2/17/2017 | WO |
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| WO2017/143120 | 8/24/2017 | WO | A |
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