MULTILAYER DISPOSABLE CARTRIDGE FOR FERROFLUID-BASED ASSAYS AND METHOD OF USE

Abstract

The disclosed embodiments relate to method, system and apparatus for assay testing. In an exemplary embodiment, the disclosure relates to a cartridge for testing an assay. The cartridge includes a sample reservoir to receive a mixture of a plurality of target particles and a ferrofluidic solution; a capture region formed on the cartridge; a fluidic channel to communicate the mixture between the sample reservoir and the capture region; a magnetic ferrofluidic solution positioned inside the fluidic channel; and at least one pneumatic valve to communicate a quantity of the mixture from the sample reservoir. The magnetic ferrofluidic solution is excitable in response to an externally applied electromagnetic field to affect the ferrofluidic solution in the mixture.

Description

BACKGROUND

The present application claims priority to U.S. Provisional Application Ser. No. 62/369,151, filed Jul. 31, 2016, and entitled “Systems, Devices and Methods for Cartridge Securement,” and U.S. Provisional Application Ser. No. 62/369,163, filed Jul. 31, 2016, and entitled “Multilayer Disposable Cartridge for Ferrofluid-Based Assays and Method of Use.” The disclosures of each of these applications are incorporated herein by reference in their entireties.

FIELD

The disclosure generally relates to a multilayered disposable cartridge for ferrofluid based assays and method for using same.

BACKGROUND

Conventional laboratory testing and measurement systems are comprised of at least two components: an instrument and a cartridge. The instrument provides power and excitation signals to perform a given assay and measures generated signals to ultimately quantify the result of the said assay. The cartridge can be inserted into the instrument and provides an interface between the instrument and the assay.

The cartridge may be replaced and/or disposed of at the end of each assay; a new cartridge may be inserted into the instrument at the beginning of the next assay. In most applications, the disposable cartridge is inserted into the instrument through an opening at the beginning of the new assay. This opening can be a door, a slot or a compartment built into the instrument to receive the cartridge. In some assays, reagents flow within channels inside the cartridge. The reagents transport biological and/or chemical moieties relevant to the assay from input reservoirs into different compartments within that cartridge. The fluid motion leads to pressure variations between different segments or channels of the cartridge. The fluid motion also leads to pressure differences between the inside of the cartridge and the ambient pressure. As such, cartridge walls are normally built so that they are thick enough to withstand and tolerate pressure differences between its channels and the ambient pressure.

Conventional fluidic devices make use of physical phenomena that apply controlled forces on a stationary collection or a stream of non-biological and biological particles, molecules, cells, or microbeads to manipulate them in the context of a given assay. Examples of such approaches include microfluidic devices that utilize dielectrophoresis or acoustophoresis for cell separation and capture, as well as immune-magnetic separation devices that use functionalized magnetic microbeads and externally applied magnetic fields to enrich cell populations. In the case of highly localized forces (e.g. electrostatic, dielectrophoretic, or acoustophoretic), the force transducer typically needs to be integrated within the body of the fluidic cartridge. Hence, the cartridge requires electrical ports in addition to fluidic and pneumatic ports. These requirements substantially increase the cost of the cartridge.

Conventional ferrofluid-based cellular and biological particle manipulation scheme rely on current-carrying electrodes on an industrial printed circuit board (PCB). The magnetic fields generated in such devices may be short-range and limited by electrode spacing on the PCB traces (e.g., 250 microns or less). In order to keep the design of the fluidic cartridge simple and low-cost, the excitation PCB resides outside the cartridge volume.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other embodiments of the disclosure will be discussed with reference to the following exemplary and non-limiting illustrations, in which like elements are numbered similarly, and where:

FIG. 1 is a perspective view of an exemplary embodiment of a cartridge;

FIG. 2 is a schematic illustration of a multi-layer cartridge according to one embodiment of the disclosure;

FIG. 3 is a top view of an exemplary cartridge showing components according to some embodiments of the disclosure;

FIG. 4 illustrates one or more layers having a pumping valve and degassing chambers;

FIG. 5A provides a simplified schematic of the fluidic network;

FIGS. 5B-5D illustrate an exemplary pumping sequence for a multilayer cartridge; and

FIG. 6 schematically illustrates an exemplary layer of the multi-layered cartridge containing the main assay channel.

DETAILED DESCRIPTION

In some embodiments, the design of a fluidic cartridge can be simplified and production cost can be reduced by, for example, placing an excitation printed circuit board (PCB) outside the cartridge volume. The excitation PCB may comprise one or more excitation electrodes to generate an electromagnetic field. A disposable fluidic cartridge for ferrofluid-based assays according to some embodiments is schematically illustrated in FIG. 1. The integrated cartridges allow ferrofluid and sample mixtures to run in self-contained fluidic networks for performing biological assays. The cartridge architecture provides main channels in which biological particle manipulation, concentration, separation, sorting and/or capture takes place. Such biological particles may include cells, microbeads, bacteria, fungi, algae, viruses, etc. The main channels may be in intimate proximity to the magnetic excitation fields that are generated off the cartridge. For example, the magnetic excitation fields may be generated by an instrument which receives the cartridge. Such instrument may be stand-alone instrument with a cavity or a receptacle configured to receive the fluidic cartridge.

The cartridge may be configured with a plurality of capture molecules that are configured for capturing capture particles. The capture particles may be one or more of a biological or a non-biological particle. The capture molecules (which may be, e.g., at least one receptor, antibody, lectin, etc.) may be configured downstream of the main channels and are configured to bind with the capture particles. The capture molecules may be affixed to a capture zone of the cartridge. As such, these main channels may reside just inside one of the faces of the disposable cartridge (typically the bottom face). In some embodiments, the main channels may be capped by a very thin (typically 25-50 microns) layer of film. The film may comprise PET, PMMA, or some other flexible material. As used herein, the term particle may include: latex beads; any bead ranging in size from 0.1 micron to 20 microns; cells; bacteria; algae; parasites; cysts; viruses; spores; macromolecular biological assemblies; cellular organelles; oocytes; sperm cells; and/or the like.

In some exemplary embodiments, the magnetic field excitation is generated by current-carrying electrode traces on a PCB, which can be a part of the instrument. The cartridge may be placed directly over the PCB with the main channels lined up directly (or approximately) over the electrodes.

Fluid flow within the cartridge creates a positive pressure (with respect to ambient pressure) inside the flow channels, which may cause bulging or inflation of the thin bottom channels during an assay. In order to account for this, the disclosed cartridge can be configured to work in conjunction with a lid that applies controlled pressure over the top surface of the cartridge in order to maintain dimensional integrity of the main channels. In one embodiment, the lid is a mechanical door or a clamp that closes over the cartridge and restrains it with sufficient pressure to prevent channel inflation. In another embodiment, an inflatable bladder may be used to provide a substantially even pressure to the channels, as discussed in Applicant's U.S. Provisional Patent Application No. 62/369,151, which has been incorporated by reference herein. For example, the lid may incorporate an air bladder that can be pneumatically and/or electronically actuated to inflate over and conformally cover the top side of the cartridge to apply a uniform, tunable pressure over the cartridge.

FIG. 1 is a perspective depiction of an exemplary embodiment of a cartridge that is configured to perform eleven independent, parallel assays. Other embodiments of a cartridge may be configured to perform a different number of parallel assays (such as 8 or 12), or they may be configured to run a single assay. The width of the cartridge 100 may change depending on the total number of assays supported.

Cartridge 100 may comprise multiple layers integrated into a unitary or an integrated cartridge. In an alternative embodiment, cartridge 100 may comprise a single construction with various features discussed below integrated therein. Cartridge 100 may include base layer 102, cartridge-instrument alignment features 118, a reagent spotting mask 114, pump valves 120 and a reservoir stack 108. Reservoir stack 108 may further include main reservoirs 112, return chimneys 122 and a plurality of secondary (and, in some implementations, tertiary, etc.) reservoirs 110. The cartridge may also comprise internal alignment features 104 and 116 that may be used to ensure proper registration between the internal layers during its construction.

Cartridge-instrument alignment features 118 enable aligning placement of cartridge 100 within an assay instrument (not shown). The alignment may ensure, in part, that the cartridge main channels can align directly (or approximately) over the electrodes of the excitation PCB. This may also ensure that any other interface to the cartridge (such as pneumatic input ports for pumping fluid reagents within the cartridge) are aligned with the corresponding output from the instrument. Cartridge 100 may be inserted into an instrument slot (not shown) or may be placed at a designated space (such as a dedicated receptacle) within the assay instrument (not shown).

A plurality of cartridge analysis windows (or viewing ports) 106 may correspond with each of a plurality of reaction channels (not shown). As described below, the reaction channels (not shown) may be embedded or formed over base 102. Cartridge analysis windows 106 provide optical viewing ports to each of the reaction channels.

The reagent spotting mask 114 may optionally be added to accommodate, for example, the precise positioning and spotting of assay reagents (e.g., capture reagents such as antibodies, aptamers, DNA fragments, other proteins or molecules used for surface modification or detection, etc.). The mask may consist of a matrix of patterned openings over an adhesive or a soft gasket (e.g., silicone rubber, PDMS, etc.) that is temporarily affixed over one of the bounding surfaces of the main assay channels. The assay reagents may thus be coated (or spotted) over that surface of the cartridge through the mask openings, either during the assembly of the cartridge or prior to running the assay by the end-user. Following an optional incubation period, the coated (or spotted) windows might be washed and/or dried, and the reagent spotting mask 114 may be removed (e.g., peeled off the cartridge surface) prior to capping the main assay channels with the final capping layer of the multi-stack assembly.

The internal alignment features 104 and 116 may optionally be used to assist in the assembly of the cartridge internal layers in order to ensure that each layer is properly aligned with and registered to its neighbors within a given positional tolerance. In some embodiments, the alignment features may be holes of a given shape (e.g., circular, square, hexagonal, diamond, etc.) that mate with alignment posts on an alignment jig.

In some embodiments, the cartridge may have pneumatic input ports 120. These ports may lead into pneumatic lines integrated into the cartridge. Together, they relay pressure and/or vacuum signals from the instrument to membrane valves (not shown) integrated into the body of the cartridge.

Reservoir stack 108, as described below, can retain the cartridge input fluids. For example, the reservoir stack 108 may receive and retain assay reagents which are then directed to the fluidic network (not shown in FIG. 1) of cartridge 100. Main reservoirs 112 typically receive ferrofluid and/or input sample reagents that are intended for the ferrofluidic assay. They may also be configured to receive additional reagents, as needed.

In some embodiments, reservoir stack 108 may support more than one set of reservoir wells per independent assay. Secondary reservoirs 110 may be configured to receive secondary reagents used for an assay under study. The secondary reagents may include labels, dyes, secondary antibodies, PCR reagents required for DNA amplification after cell capture, etc. In some implementations, the secondary reservoirs may be left blank or empty.

In certain embodiments, assay cartridge 100 may include multiple, patterned, alternating layers of double-sided adhesive tapes and plain plastic film that are laminated to each other in a specific sequence. One such embodiment is illustrated in FIG. 2. The multi-layered cartridge provides ease of manufacturing and reduced cost. In the multi-layered cartridge, each layer may be independently patterned by a subtractive process (such as laser or die cutting, etc.) and subsequently laminated to its neighbors through a manual and/or automated lamination procedure. When pressure-sensitive adhesive (PSA) layers are used, the lamination process may involve a roll-laminator or a hydraulic linear press. Other types of adhesives (such as heat-activated or UV-activated) may also be used, depending on compatibility with the assay. In some embodiments, neighboring layers are bonded using thermal compression or solvent-based bonding techniques in place of using adhesives.

FIG. 2 is a schematic illustration of a multi-layer cartridge according to some embodiments. Specifically, FIG. 2 is an exploded view of cartridge layers that may be combined to form cartridge 100 of FIG. 1. In FIG. 2, layer 1 may be a base layer (e.g., base layer 102, FIG. 1). Layers 2, 3-6 and 7-13 provide integral and optional components of the cartridge. Layer M may be an optional masking layer that may be used for coating reagents directly on Layer 3. Layer D may be used as the degasser component. In an exemplary manufacturing process, the layers with sequential numbers in FIG. 2 may be laminated to each other, with the largest number being on top and in descending order towards the bottom.

In some embodiments, certain neighboring functional layers of the cartridge may be combined into single, injection-molded segments that can be patterned on both sides with the desired features. In this hybrid approach, the total number of layers (and, hence, the assembly complexity of the cartridge) may be reduced, which lowers manufacturing costs. For example, layers 4-6 of FIG. 2 may be combined into a single injection-molded layer with the corresponding flow channels patterned on either side. The connecting holes of the middle layer may be patterned through the molded layer.

In certain embodiments, the cartridge architecture can be based on defining the outlines of flow channels and chambers within the adhesive layers. The plain plastic layers (interchangeably, ‘end layers’) may support flow connections and via holes between neighboring channel levels. Here, the channels can be capped from the top and bottom by the end layers and from the side by the cut boundaries of adhesive tape (not shown). By using different materials or coatings for the end layers, it is possible to exert substantial control on the wetting and filling properties of the channels in various levels. Another advantage of such designs is that channel depths can be controlled with relative precision, since each channel is defined by the tightly controlled thickness values of roll adhesive films to provide better than 5% variation in thickness.

Since fluid flow within the cartridge creates a positive pressure inside the flow channels, the thin bottom capping layer (i.e., Layer 1 in FIG. 2) may bulge or inflate during operation. To address this, in certain implementations, the cartridge may operate in concert with a lid that applies controlled pressure over the top surface of the cartridge to maintain dimensional integrity of the channels. The lid can be a mechanical door or a clamp that closes over the cartridge and restrains the cartridge with sufficient pressure to prevent channel inflation. In other embodiments, the lid may incorporate an inflatable bladder which can be pneumatically actuated to inflate over the cartridge. The inflatable bladder can conformally cover the top side of the cartridge to apply a substantially uniform tunable pressure over the cartridge.

FIG. 3 is a top view of an exemplary cartridge showing components according to some embodiments of the disclosure. Cartridge 300 of FIG. 3 shows cartridge alignment features 318, capture and analysis region 350, degasser component 330, integrated pneumatic pumping system 340, reservoir tank 308, secondary reservoir 310, main reservoir 312, return chimney 322, pump valve 320, pneumatic line 344, pneumatic port 342, degasser vent 332, capture/analysis windows 306 and optical viewing port 307.

It should be noted that FIG. 3 (as well as other Figures) are produced for illustrative purposes. The configuration and the layout of the various components shown in the cartridge 300 of FIG. 3 may be rearranged or removed without departing from the disclosed principles.

Reservoir stack 308 may be configured to receive sample-ferrofluid mixtures prior to the assay. The sample-ferrofluid mixtures may be introduced to main reservoir 312, for example, via pipetting by a technician or through an independent robotic system (not shown). While not shown, a filtering region may be included before or after reservoir section stack 308 to exclude particles above a certain size from the downstream fluidic components.

Integrated pneumatic pumping system 340 may be configured to introduce and circulate the sample and reagents through the fluidic networks of cartridge 300. The integrated-pneumatic pumping section may include pump valves 320, pneumatic line(s) 344 and pneumatic port 342. Pump valves 320 may be used to facilitate movement of sample and/or reagents from the reservoir stack into the fluidic network downstream, as described in more detail below.

Degasser component 330 occupies a section of cartridge 300 and is shown with degasser vent 332. Degasser component 330 may be optional and may be omitted when degassing is not required. In certain embodiments, the degassing section selectively removes or vents gas bubbles over a certain size from the recirculating fluid in the fluidic network.

While not shown, cartridge 300 includes a main channel section in which ferrofluid-mediated particle manipulation, concentration, sorting and capture takes place.

Reservoir stack 308 of FIG. 3 may be used as a holding vessel for the input liquid reagents that are introduced into the downstream fluidic network. The number of reservoir wells 313 may depend on the number of independent assays that cartridge 300 supports. In the exemplary embodiment of FIG. 3, eleven independent assays are supported by cartridge 300; this is denoted by the number of main reservoir wells 313. The number of assays supported by a cartridge may depend on the particular needs of a given application. Thus, for example, the cartridge may be constructed with a particular number assays based on the application for that cartridge. While not shown, each reservoir may communicate with a respective main fluidic channel. The main fluidic channels are illustrated, for example, in layer 2 of FIG. 2.

In some embodiments, the main reservoir wells may be configured to receive the ferrofluid-sample mixture. The sample to be placed in the main reservoir may be mixed with a corresponding amount of ferrofluid, either outside the cartridge prior to introduction into the reservoir well (e.g., in a micro-centrifuge tube) or inside the reservoir well itself. In the latter application, a given amount (an aliquot) of ferrofluid may be previously stored and sealed inside the main reservoir well for each assay on the cartridge. Alternatively, the ferrofluid may be added to the reservoir well before, after, or simultaneously with the sample.

In some implementations, a pipettor may be used to introduce the sample (or ferrofluid-sample mixture) into the main reservoirs. In certain embodiments, the reservoirs may be hermetically sealed (e.g., with plastic or foil cover) and may be punctured with the pipette tip during sample introduction. Thus, the inner volume of the reservoirs and the fluidic network downstream can be kept clean and sterile, evaporation of the reagents can be mitigated during the run, and the puncture can provide a visual indicator to the user that the well has been filled.

The reservoir stack 308 may be configured to support more than one set of reservoir wells per independent assay. For example, secondary reservoir 310 (including secondary wells 315) may be configured to receive secondary reagents for a given assay. The secondary reagents may include labels, dyes, secondary antibodies, PCR reagents to run DNA amplification following cell capture, etc. In certain applications, the secondary wells may be left blank or empty.

In some embodiments, at least one valve may be used to connect each reservoir well 313, 315 to the fluidic network downstream. The valve may be part of the pumping system 340 and may be integrated onto cartridge 300. In such embodiments, the appropriate reservoir (e.g., main reservoir 312 or secondary reservoir 310) to remove fluid is selected by actuating its corresponding valve while the other reservoir valves are kept closed.

In certain embodiments, the reservoir stack 308 may be located at the end of the cartridge on a side that is configured to be closest to the user (not shown).

In some embodiments, the stack may have return chimneys that are configured to return the circulated reagents back into the main reservoir. This way, the main reservoir reagents (i.e., ferrofluid-sample mixture) may be recirculated through the entirety of the fluidic network as many times as necessary. The reservoir stack may also be configured to receive a filter mesh directly underneath (e.g., layer 10, FIG. 2). The filter may retain particles larger than a certain size which can be tunable using externally applied magnetic fields as described below, as well as aggregates of particles, aggregates of extracellular matrix, fatty globules, and other debris.

In some embodiments, an active filter system may be incorporated into cartridge 300. The filter may be positioned at the bottom of the reservoir stack. One such architecture is shown in FIG. 2, where filter layer 10 is positioned below reservoir stack layer 12. In the depicted embodiment, Layers 9 and 11 are adhesive layers that affix the filter layer to its neighboring layers.

The filter may prevent particles larger than a predetermined threshold from leaving the input reservoirs and entering the flow channels positioned downstream of the reservoir. When used in the context of ferrofluid-mediated assays, the filter layer may be used both in passive or active filtration modes.

The openings (interchangeably, ‘pores’ or ‘apertures’) of the filter may be configured to be substantially larger than the biological and non-biological particles, including microbeads, of interest. In passive filtration mode, the filter mesh removes large particulate contaminants and debris present in the sample or in the sample-ferrofluid mixture. For example, the target particles of the assay may include bacteria (1-5 microns in length, typically less than 1 micron in width), and the filter pore size may be selected to be between 20 and 50 microns (i.e., much larger than the target bacteria). In this embodiment, the filter mesh removes large particulates, such as sand, small rocks, or large aggregates of extracellular matrices, as well as aggregates of particles. The filter pore size can be selected to be somewhat smaller than the smallest dimension present in the fluidic network downstream (such as the width or height of the smallest channel) to ensure that the fluidic networks and channels would not be physically clogged by particulate contaminants.

When used in the so-called active mode, the filter may be electromagnetically tuned to allow passage of particles of a desired size. In an exemplary implementation, when a ferrofluid-sample mixture flows from the input reservoir through the filter, application of an external magnetic field changes the threshold particle size that ends up being filtered by the active filter. Specifically, even if the applied magnetic field is uniform (or locally-uniform around the filter mesh), the field lines in the immediate vicinity of the filter go through the higher susceptibility ferrofluid medium within the pores, as opposed to going through the non-magnetic filter material. Thus, negative magnetic field gradient forms around each pore, leading to a very localized magnetic force acting on each non-magnetic particle that attempts to travel through the filter mesh.

The magnetic force follows the direction of the field gradient, so as to direct each particle away from the pore and towards the filter material between the pores. Since the ferrofluid-mediated magnetic force on a non-magnetic particle suspended in ferrofluid is proportional to the volume of that particle, larger particles will feel much larger diversion forces and will tend to land on the material between the pores, while smaller particles will succumb to hydrodynamic drag and travel through the pores. The separated particles stay away from the pores. Therefore, the filter does not clog in its active mode of operation.

As the amplitude of the externally applied magnetic field excitation is increased, the maximum particle size that ends up passing through the filter is proportionally reduced. This component is therefore an actively tunable filter in which the threshold particle size can be adjusted (i.e., tuned) by varying the applied magnetic fields. The magnetic fields may be adjusted either at the beginning of an assay or in real-time during an assay. In this manner, a filter that features a pore size of 30 microns can be utilized to effectively hold on to particles that are 5 microns and larger. The threshold size can be changed in real-time depending on the particular stage of a given assay. Particles that have been held back on the filter can also be released into the fluidic network downstream simply by lowering the magnetic field amplitude.

Referring once again to FIG. 3, the exemplary cartridge 300 may include an integrated pneumatic pumping system. The pumping system allows biological and/or non-biological particles that enter the main channels (see e.g., layer 2, FIG. 2) of the cartridge to be continually pushed towards the ceiling of those channels by an external magnetic field generated directly (or approximately) below the cartridge. The local flow rate near a channel wall can be much lower than the average fluid flow rate within that channel. The lower flow rate can be due to non-slip boundary conditions of the fluid. The biological and non-biological particles rolling over the channel ceiling travel much slower than the average linear flow rate. Consequently, the ferrofluid medium may be recirculated through the fluidic system to give the cells sufficient time to reach the analysis regions located near the downstream end of the main channel ceiling.

The cartridge may feature continuous, closed-loop recirculating flow of ferrofluid-sample mixture, in from the reservoir stack, through the internal components and fluidic network, and back to the reservoir stack again. In this approach, it may not be necessary to add any additional ferrofluid, sample or other reagents into the cartridge after the initial loading of the reservoir stack at the beginning of the assay. Thus, all reagents necessary for the assay are conveniently confined to the inside of the disposable cartridge, enabling easy disposal of potential biohazards at the end of each assay. Further, the instrument requires no reagent storage or dispense capabilities. This simplifies the design and operation of the cartridge, and is therefore more cost effective.

FIG. 4 illustrates layers 4-6 of FIG. 2, and more specifically, FIG. 4 is a schematic representation of layers 4-6 of FIG. 2, illustrating the pumping valves and the degassing chamber. Cartridge 400 of FIG. 4 includes degassing compartment 430 with multiple degassing chambers 432. Each degassing chamber 432 may correspond to a respective fluidic channel in the fluidic network. Cartridge 400 also shows valves 421, 422, 423 and 424. Valve 421 may correspond to the secondary reservoir control valve and valve 422 may correspond to the main reservoir control valve. In some embodiments, the recirculating flow may be set up by the peristaltic action of a series of integrated membrane valves downstream of the reservoir stack (308, FIG. 3) and the active filter mesh (layer 10, FIG. 2).

Valves 421-424 can be actuated by pneumatic input pulses (for example, by alternating between pressure and vacuum) generated by the instrument (not shown) and relayed to cartridge 400 through pneumatic ports (342, FIG. 3) located on a surface of the cartridge. When a relative negative pressure (i.e., vacuum) is applied to a specific valve, the valve membrane is pulled up into the open position to thereby fill the chamber with fluid. In contrast, when a relative positive pressure is applied (e.g., to about 20 psi or between 10-25 psi), the valve membrane is pushed down into the closed position to thereby evacuate the liquid within the volume of the valve chamber. Where the fluid is drawn from, or where it is evacuated to, can be determined by which fluidic path is available. This, in turn, can be determined by the position of the neighboring valves. By actuating the valves in a specific, repeating sequence, valves 421-424 can pump reagents from the reservoir stack (308, FIG. 3) to the fluidic network downstream (layer 2, FIG. 2) and eventually back into the reservoir stack (308, FIG. 3).

FIG. 5A provides a simplified schematic of the fluidic network depicting the main and secondary reservoirs (“S”), the pumping valves and the main channel. FIG. 5B, 5C and 5D illustrate an exemplary pumping sequence for an exemplary multilayer cartridge as shown in FIGS. 2-4. In FIGS. 5B-5D, the numeral 1 indicates that the valve is pressurized (i.e., closed). The numeral 0 means the valve is open. Other valve sequences are possible and equally applicable without departing from the disclosed principles. The valve sequence may be configured depending on the tolerable levels of flow pulsation versus back-flow that the pumping sequence generates. Typically, the faster the valves cycle through the predetermined set of switching states, the faster the recirculating flow will be. Thus, average flow rate may be controlled via changing the time period spent at each set of valve states (i.e., by changing the duration of the pressure and vacuum pulses sent to the pneumatic ports). It is also possible to implement variable valve timing (i.e., different set of valve states could have different active durations) in order to minimize the impact of flow pulsation and back-flow issues.

Referring to FIG. 5A, secondary reservoir (S) 502 is directed to valve 1 (V1) which is also in fluidic communication with valve 2 (V2). V2 is in fluid communication with main reservoir 503. The output of V2 is directed to valve 3 (V3), which is serially connected to valve 4 (V4) downstream. The output of valve 4 (V4) leads eventually to the fluid channel 504. As shown in FIG. 5A, channel 504 is also connected to the main reservoir 503.

The pneumatic valves (V1-V4) integrated into the cartridge can also act as stop valves. In the exemplary embodiments of FIGS. 4 and 5A, valve 2 (V2) connects directly to the main sample-ferrofluid reservoir, while valve 1 (V1) connects to the secondary (e.g., label, dye) reservoir 502. The main portion of the assay circulates the sample-ferrofluid mixture through the fluidic network. Hence, valves V2, V3, and V4 may be actuated in sequence while valve 1 remains closed (i.e., pressurized). After particle manipulation and/or capture is complete, V2 is closed, and V1, V3 and V4 are actuated in sequence to introduce the secondary reagent (e.g., for a label or dye) into the channels. This process of pumping from a different reservoir may be repeated as needed. The approach can be flexible and can easily accommodate additional input reservoirs (i.e. beyond the two exemplified here) as long as one additional valve is added to the pump subsystem for each new reservoir. The opening and closing sequences of valves V1-V4 of FIG. 5A are illustrated in the tables of FIGS. 5B, 5C and 5D.

As stated, the cartridge may also incorporate a degasser component downstream of the integrated pump. The degasser may remove gas bubbles that may be either initially present or subsequently generated within the input reagents (e.g., cavitation around the pump valves). Gas bubbles may be removed from fluids prior to the fluid introduction to the cartridge channels downstream. An exemplary degasser component was illustrated in FIGS. 3 (degasser 330) and 4 (degasser 430).

In some embodiments, the degassing functionality may be achieved by forming a flow chamber where at least one wall is comprised of a hydrophobic (or super-hydrophobic) porous membrane on one side and open to the atmosphere on the other side. As a gas bubble suspended in the fluid reagent flows through the degassing chamber, the bubble makes contact with the hydrophobic membrane and is pushed into the pores by the fluid pressure. As the residence time inside the volume of the degassing chamber is increased, the degassing becomes more effective and efficient. The smaller the pore size of the hydrophobic membrane, the stronger the capillary forces in the pores and the more fluid back pressure the membrane can withstand before the fluid leaks through the pores.

In some embodiments, the membrane may be made of a hydrophobic material. Such materials include poly-tetrafluoro-ethylene (PTFE). In an exemplary embodiment, the vent pore diameter may be in a range of about 100 nanometers (0.1 microns) or smaller. In such embodiments, the degasser can withstand at least several tens of psi of fluid pressure. Pores of about 0.1 microns wide or smaller may not let bacteria and larger cells through when there is a fluid leak. Thus, if a spill out of the cartridge occurs, the spill is sterile and/or cell-free. The membrane may be made of different materials including plastics and may be further coated to improve hydrophobic properties.

In an exemplary embodiment, the porous PTFE membrane may be bonded to a polyester or a polypropylene mesh backbone. The thin PTFE membrane may be fragile and difficult to handle without wrinkling. Thus, in some embodiments, a mesh backbone may be added to make it much easier to process, cut, and handle the degasser film. The mesh also permits gas bubbles to vent laterally from the top side of the PTFE membrane which allows proper operation of the degasser even when it is covered from the mesh side by other capping layers of the cartridge.

By way of illustration, FIG. 3 shows an exemplary embodiment where the degasser film is covered by the top layer (i.e., the molded plastic backbone of the cartridge) and vents to atmosphere from either side of the cartridge 332.

In some embodiments, the degasser may vent into channels that connect with overflow reservoirs that are part of the reservoir stack. In such applications, any accidental leak through the degasser may be contained without leaking outside the cartridge volume.

FIG. 6 schematically illustrates an exemplary layer of the multi-layered cartridge containing the main assay channel. Layer 600 may define a layer within the cartridge. For example, layer 600 may define layer 2 of FIG. 2. Layer 600 is shown with eleven independent channels 610. It should be noted that the illustrated number of channels is purely exemplary; more or fewer assay channels may be included without departing from the disclosed principles.

FIG. 6 also shows fluidic connector channels 612 and 614. These small channels carry the outputs of the main and secondary reservoirs (directly downstream of the reservoir stack and filter components) to valves V2 and V1, respectively.

In some embodiments, main channel layer 600 may be located downstream of the degasser component of the cartridge. The main channel layer may be where the ferrofluid-mediated particle sorting, separation, manipulation, concentration, enrichment, specific capture and/or eventual quantification takes place. Additional biochemical reactions may also take place in the channel layer.

An exemplary cartridge may be positioned in an instrument having excitation electrodes. The excitation electrodes generate fields that can manipulate ferrofluidic material in channels 610. Thus, the main channels may be configured to line up proximal to excitation electrodes of the instrument PCB (not shown). Further, the width of channels 610 may closely correlate to the width of each PCB electrode (not shown). In an exemplary implementation, the electrode set was about 4.00 millimeters wide and the main channel width of the corresponding cartridge was about 3.85 millimeters.

The length of the main channels may also correlate with the electrode length (not shown) of the PCB. This dimension may be determined as a function of the particles traveling within the main channels and the length needed to push up and/or sort the particles prior to capture/analysis regions which are downstream and at the end of the channels. By way of example, for a main channel depth of around 85 microns and width of 3.85 millimeters, a channel length on the order of about 5 cm is needed. This ensures that bacteria suspended in a moderate strength ferrofluid mixture (i.e., magnetic susceptibility on the order of 0.1-0.5) flowing at about 10-50 microliters/min can be focused between the two central electrode traces (i.e., within a narrow central band of about 200 microns) when the electrodes generate up to 10 mT of magnetic flux density within the channel volume.

In some embodiments, the main channel inner walls may be featureless and smooth. In another embodiment, the channel inner walls may include micro-scale patterns that interact with the ferrohydrodynamic flow to assist in particle sorting/separation and particle capture. Such surface features may include micro posts or chevron structures (patterns) to assist in hydrodynamic separation of particles based on size. These features may act independently of the magnetic fields applied to the cartridge or they may interact with the fields to enhance or augment the intended function. Some micro-structures within the main channels may interact with the applied fields to act as secondary active filters. Microposts functionalized with capture ligands (such as antibodies, aptamers, single-strand DNA, etc.) may also be utilized in the analysis/capture region.

Near the downstream end of the main channels 610, a capture/analysis region may be positioned. In one embodiment, the main channels feature antibody-coated capture windows. An exemplary window is shown in FIG. 3 as capture/analysis window 306. The cartridge layers directly above these windows are designed to be optically transparent. The optical transparency can be accomplished either by using layers that are transparent themselves or by simply cutting out viewing ports through the otherwise opaque layers.

In some embodiments, the capture/analysis region may feature integrated thin electrodes and quantification of the assay results may be based on measuring impedance changes over the window at various frequencies. In some implementations, the sensor integrated in this region is a piezoelectric mass balance or an electrochemical sensor. Such sensors can provide additional information based on observations made at the capture/analysis region. A piezoelectric mass balance, for example, can provide information about the captured particles. The electrochemical sensor can provide information about the captured particles' charge or pH. In such non-optical sensor approaches, the cartridge may not need additional optical viewing ports but may have other components such as thin electrodes integrated or printed on top of thin sheets of plastic film.

It should be noted that an exemplary cartridge may be configured to include a number of assays without departing from the disclosed principles. For example, the cartridge may be configured to include as few as one or more assays. In one exemplary embodiment, the cartridge includes up to twelve or more assays.

The following embodiments illustrate exemplary and non-limiting embodiments of the disclosure. Example 1 is directed to a biological particle capture device, comprising: a sample reservoir to receive a mixture of a plurality of target particles and a ferrofluidic solution; a capture region formed on the cartridge; a fluidic channel to communicate the mixture between the sample reservoir and the capture region; a magnetic ferrofluidic solution positioned inside the fluidic channel; and at least one pneumatic valve to communicate a quantity of the mixture from the sample reservoir; wherein the a magnetic ferrofluidic solution is excitable in response to an externally applied electromagnetic field to affect the ferrofluidic solution in the mixture.

Example 2 is directed to the device of example 1, wherein the a magnetic ferrofluidic solution is excitable in response to an externally applied electromagnetic field to attract the ferrofluidic solution to a proximal region of the fluidic channel.

Example 3 is directed to the device of example 1, wherein the pneumatic valve is responsive to an external pressure to communicate the mixture from the reservoir to the fluidic channel.

Example 4 is directed to the device of example 1, further comprising a dye reservoir to receive a dye solution.

Example 5 is directed to the device of example 1, wherein the reservoir further comprises a plurality of reservoir wells and wherein each well is configured to receive an independent assay.

Example 6 is directed to the device of example 1, further comprising a filter mesh positioned between the sample reservoir and the at least one fluidic channel.

Example 7 is directed to the device of example 6, further comprising a controller to cause application of a magnetic field to the filter mesh to dynamically change a threshold filter particle size.

Example 8 is directed to the device of example 1, further comprising a degasser region to remove gas from the one or more fluidic channels.

Example 9 is directed to the device of example 1, further comprising a plurality of capture molecules proximate to the capture region to capture at least some of the plurality of target particles through proximity with the capture molecules.

Example 10 is directed to a method to sort biological particle in a cartridge, the method comprising: communicating a mixture of a plurality of target particles and a ferrofluidic solution from a reservoir to a capture region through a fluidic channel; activating a magnetic ferrofluidic solution inside the fluidic channel by applying an electromagnetic field; substantially localizing a quantity of the ferrofluidic solution to a region influenced by the electromagnetic field while directing target particles toward the capture regions; and identifying target particles at the capture region; wherein the magnetic ferrofluidic solution is activated in response to an externally applied electromagnetic field to affect the ferrofluidic solution in the mixture.

Example 11 is directed to the method of example 10, further comprising communicating a quantity of the mixture from the reservoir to the fluidic channel using a pneumatic valve integrated into the fluidic channel.

Example 12 is directed to the method of example 10, wherein the step of directing target particles toward the capture regions further comprises pneumatically moving the particles toward the capture region.

Example 13 is directed to the method of example 12, wherein the pneumatic valve is responsive to an external pressure to communicate the mixture from the reservoir to the fluidic channel.

Example 14 is directed to the method of example 10, wherein activating the electrode further comprises applying an external electromagnetic field to attract the ferrofluidic solution to a proximal region of the fluidic channel.

Example 15 is directed to the method of example 10, further comprising introducing a dye to the reservoir.

Example 16 is directed to the method of example 10, filtering the mixture through a filter to capture a first particle before communicating the mixture from the reservoir to the fluidic channel.

Example 17 is directed to the method of example 16, further comprising electromagnetically tuning the filer to capture the first particle.

Example 18 is directed to the method of example 10, further comprising degassing the mixture.

Example 19 is directed to the method of example 10, further comprising positioning the cartridge proximal to an external excitation source to align an excitation source electrode with the fluidic channel to thereby provide an externally applied electromagnetic force to the magnetic ferrofluidic solution positioned inside the fluidic channel.

Example 20 is directed to an integrated cartridge to separate particles from a mixture, the cartridge comprising: a sample reservoir to receive a mixture of a plurality of target particles and a ferrofluidic solution; a capture region formed on the cartridge; a fluidic channel to communicate the mixture between the sample reservoir and the capture region; a filter positioned between the sample reservoir and the fluidic channel, the filter having at least one aperture configured to retain particles larger than a threshold size; and a fluidic pump to convey the mixture from the filter to the capture region.

Example 21 is directed to the cartridge of example 20, wherein the filter comprises an electromagnetic filter.

Example 22 is directed to the cartridge of example 21, wherein the electromagnetic filter communicates with an external source to dynamically tune the at least one aperture size.

Example 23 is directed to the cartridge of example 20, wherein the fluidic pump comprises a movable diaphragm responsive to an external pressure and wherein the diaphragm is integrated with the cartridge.

Example 24 is directed to the cartridge of example 20, wherein the fluidic channel comprises a smooth surface to communicate the mixture.

Example 25 is directed to the cartridge of example 20, wherein the fluidic channel comprises a pattern to communicate the mixture.

Example 26 is directed to the cartridge of example 20, wherein the capture region further comprises a piezoelectric sensor.

Example 27 is directed to the cartridge of example 20, wherein the capture region further comprises an integrated electrode.

Exemplary embodiments of the devices, systems and methods have been described herein. As noted elsewhere, these embodiments have been described for illustrative purposes only and are not limiting. Other embodiments are possible and are covered by the disclosure, which will be apparent from the teachings contained herein. Thus, the breadth and scope of the disclosure should not be limited by any of the above-described embodiments but should be defined only in accordance with claims supported by the present disclosure and their equivalents. Moreover, embodiments of the subject disclosure may include methods, systems and devices which may further include any and all elements from any other disclosed methods, systems, and devices, including any and all elements corresponding cartridges and systems thereof. In other words, elements from one or another disclosed embodiment may be interchangeable with elements from other disclosed embodiments. In addition, one or more features/elements of disclosed embodiments may be removed and still result in patentable subject matter (and thus, resulting in yet more embodiments of the subject disclosure). Correspondingly, some embodiments of the present disclosure may be patentably distinct from one and/or another reference by specifically lacking one or more elements/features. In other words, claims to certain embodiments may contain negative limitation to specifically exclude one or more elements/features resulting in embodiments which are patentably distinct from the prior art which include such features/elements.

While the principles of the disclosure have been illustrated in relation to the exemplary embodiments shown herein, the principles of the disclosure are not limited thereto and include any modification, variation or permutation thereof.

Claims

1. A biological particle capture device, comprising:

a sample reservoir to receive a mixture of a plurality of target particles and a ferrofluidic solution;

a capture region formed on the cartridge;

a fluidic channel to communicate the mixture between the sample reservoir and the capture region, the fluidic channel configured to receive a magnetic ferrofluidic solution; and

at least one pneumatic valve to communicate a quantity of the mixture from the sample reservoir;

wherein the magnetic ferrofluidic solution is excitable in response to an externally applied electromagnetic field to affect the ferrofluidic solution in the mixture.

2. The device of claim 1, wherein the magnetic ferrofluidic solution is excitable in response to an externally applied electromagnetic field to attract the ferrofluidic solution to a proximal region of the fluidic channel.

3. The device of claim 1, wherein the at least one pneumatic valve is responsive to an external pressure to communicate the mixture from the reservoir to the fluidic channel.

4. The device of claim 1, further comprising a secondary reservoir to receive a secondary solution.

5. The device of claim 1, wherein the sample reservoir further comprises a plurality of reservoir wells and wherein each well is configured to receive an independent mixture.

6. The device of claim 1, further comprising a filter mesh positioned between the sample reservoir and the fluidic channel.

7. The device of claim 6, further comprising a controller to cause application of a magnetic field to the filter mesh to dynamically change a threshold filter particle size.

8. The device of claim 1, further comprising a degasser region to remove gas from the one or more fluidic channels.

9. The device of claim 1, further comprising a plurality of capture molecules proximate to the capture region to capture at least some of the plurality of target particles through proximity with the capture molecules.

10. A method to sort biological particle in a cartridge, the method comprising:

communicating a mixture of a plurality of target particles and a ferrofluidic solution from a reservoir to a capture region through a fluidic channel;

activating the ferrofluidic solution inside the fluidic channel by applying an electromagnetic field;

substantially localizing a quantity of the ferrofluidic solution to a region influenced by the electromagnetic field while directing target particles toward the capture region; and

identifying target particles at the capture region;

wherein the ferrofluidic solution is activated in response to an externally applied electromagnetic field to affect the ferrofluidic solution in the mixture.

11. The method of claim 10, further comprising communicating a quantity of the mixture from the reservoir to the fluidic channel using a pneumatic valve integrated into the fluidic channel.

12. The method of claim 10, wherein the directing of target particles toward the capture regions comprises pneumatically moving the particles toward the capture region.

13. The method of claim 11, wherein the pneumatic valve is responsive to an external pressure to communicate the mixture from the reservoir to the fluidic channel.

14. The method of claim 10, wherein applying the electromagnetic field comprises applying an external electromagnetic field to attract the ferrofluidic solution to a proximal region of the fluidic channel.

15. The method of claim 10, further comprising introducing a dye to the reservoir.

16. The method of claim 10, further comprising filtering the mixture through a filter to capture at least a first particle before communicating the mixture from the reservoir to the fluidic channel.

17. The method of claim 16, further comprising electromagnetically tuning the filter to capture the at least first particle.

18. The method of claim 10, further comprising degassing the mixture.

19. The method of claim 10, further comprising positioning the cartridge proximal to an external excitation source to align an excitation source electrode with the fluidic channel to provide an externally applied electromagnetic force to the ferrofluidic solution positioned inside the fluidic channel.

20. An integrated cartridge to separate particles from a mixture, the cartridge comprising:

a sample reservoir to receive a mixture of a plurality of target particles and a ferrofluidic solution;

a capture region formed on the cartridge;

a fluidic channel to communicate the mixture between the sample reservoir and the capture region;

a filter positioned between the sample reservoir and the fluidic channel, the filter having at least one aperture configured to retain particles larger than a threshold size; and

a fluidic pump to convey the mixture from the filter to the capture region.

21. The cartridge of claim 20, wherein the filter comprises an electromagnetic filter.

22. The cartridge of claim 21, wherein the electromagnetic filter communicates with an external source to dynamically tune the at least one aperture size.

23. The cartridge of claim 20, wherein the fluidic pump comprises a movable diaphragm responsive to an external pressure and wherein the diaphragm is integrated with the cartridge.

24. The cartridge of claim 20, wherein the fluidic channel comprises a smooth surface to communicate the mixture.

25. The cartridge of claim 20, wherein the fluidic channel comprises a pattern to communicate the mixture.

26. The cartridge of claim 20, wherein the capture region further comprises a piezoelectric sensor.

27. The cartridge of claim 20, wherein the capture region further comprises an integrated electrode.