The reagent delivery network 100 can further include a resistor(s) 140. In some examples a resistor can be operable to generate a pressure above a pressure threshold of a capillary retention meniscus in an amount sufficient to break the capillary retention meniscus or push the capillary retention meniscus into the reagent storage chamber thereby allowing a fluid to enter the reagent storage chamber when fluid is situated within the inlet microfluidic channel. The resistor can create a burst of pressure or a pressure pulse above the pressure threshold of the capillary retention meniscus. The pressure can expand and burst or push the capillary retention meniscus out of the constriction region.

The resistor(s) 140 can be sized and shaped to have a power sufficient to generate said pressure. In an example, a resistor can have a width ranging from 4 μm to 100 μm and can have an aspect ratio from 1:1 to 1:100. A power density of the resistor can range from 100 MW/m²to 1,000 MW/m², from about 100 MW/m²to 500 MW/m², from 250 MW/m²to 750 MW/m², or from 500 MW/m²to 1,000 MW/m². A resistor can be operable to generate a voltage ranging from 5 V to 400 V, from 100 V to 300 V, from 200 V to 4000 V from 5 V to 150 V, from 5 V to 40 V, or from 5 V to 75 V.

In other examples, resistor(s) 140 can be positioned to break or burst a capillary retention meniscus. In an example, a resistor can be located across from the microfluidic cross-channel along the inlet microfluidic channel, the outlet microfluidic channel, or a combination thereof. In an example, a resistor can be located along an inlet microfluidic channel across from the constriction region. A resistor in said location can push a gas bubble forming the capillary retention meniscus into the reagent storage chamber, burst a gas bubble forming the capillary retention meniscus, or push a fluid into and/or out of the reagent storage chamber. In some examples, a reagent delivery network can include a resistor along an inlet microfluidic channel and an outlet microfluidic channel. The inclusion of two resistors along opposing ends of the reagent storage chamber can generate back and forth pressure which can push and pull a fluid in and out of the reagent storage chamber thereby providing a mixing force. A resistor along the outlet microfluidic channel may be positioned to push or pull fluid through the reagent storage chamber. The resistors can also be positioned to function as pumps which can control fluid delivery into microfluidic channels.

Microfluidic Processing Systems

Microfluidic processing systems 200 are illustrated in FIGS. 7-11, and can include any of the reagent delivery networks 100 described herein and/or illustrated in FIGS. 1-6. The reagent delivery networks may not be referenced in as much detail as in FIGS. 1-6, but those details are incorporated into the microfluidic processing system examples herein.

As shown by way of example in FIG. 7, a microfluidic processing system can include a substrate 210, a sample port 220 fluidly coupled to an inlet microfluidic channel, and a reagent delivery network. In this example, the sample port is fluidly coupled to the inlet microfluidic channel via a sample receiving chamber 230, but could include the sample-receiving port coupled directly to the inlet microfluidic channel (now shown). In other examples, the reagent delivery network may include multiple reagent storage chambers such as that described and illustrated by way of example in FIG. 6. In this particularly example, the substrate may be a single layer or multi-layer substrate. A material of the substrate can include SU-8, glass, silicon, polydimethylsiloxane (PDMS), polystyrene, polycarbonate, polymethyl methacrylate, poly-ethylene glycol diacrylate, perflouroaloxy, fluorinated ethylenepropylene, polyfluoropolyether diol methacrylate, polyurethane, cyclic olefin polymer, Teflon, copolymers, and combinations thereof. In some examples, the microfluidic substrate can include a hydrogel, ceramic, thermoset polyester, thermoplastic polymer, or a combination thereof. In other examples, the microfluidic substrate can include silicon. In still other examples, the substrate can include a low-temperature co-fired ceramic. In a further example, the substrate can include SU-8. The substrate may include an optically transparent area. “Optically transparent” indicates that a material of the substrate (or a portion thereof) is of a material that permits at least 90% of a wavelength of light within an emission range of a light source or a detection range of an optical detector to pass through the optically transparent area.

The sample port 220 may be configured to allow for the introduction of a sample fluid into the microfluidic processing system 200. The sample port can be fluidly coupled to the inlet microfluidic channel 110. The fluidic coupling can be direct or indirect. For example, the sample port may be directly coupled to the inlet microfluidic channel without intervening structures in-between. In other examples, a sample port can be coupled to the sample receiving chamber 230 and the sample receiving chamber can be coupled to the inlet microfluidic channel, as illustrated by way of example in FIG. 7.

In still other examples, as shown in FIG. 8, the sample port 220 can be coupled to the sample receiving chamber 230, which can be coupled to a microfluidic processing channel 240. In this example, the microfluidic processing channel can be fluidly coupled to the reagent delivery network 100 at the outlet microfluidic channel 130. When fluidly coupled to the outlet microfluidic channel 130, a carrier fluid or buffer solution, for example, can be flowed into the reagent delivery network and into the inlet microfluidic channel to disperse or dissolve the reagent 126a, 126b, 126c still further downstream in one or more of the reagent storage chambers 124, resulting in a reconstituted reagent that is carried downstream via the outlet microfluidic channel and into the microfluidic processing channel. This configuration includes an inlet port 250, separate of the sample port.

In further detail, the microfluidic processing system 200 can include a heating element 260. The heating element may be integrated into the substrate or may be a separate component from the substrate. The types of heating elements that can be used include a resistive heating element, a field-effect transistor, a p-n junction diode, a thin film heater, a thermal diode, or a combination thereof. The heating element may be a resistive heating element. A resistive heating element may be coupled with a p-n junction diode. In other examples, the heating element can include a resistive heating element and a thermistor. A thermal resistor, if present, can apply heat to speed up a chemical reaction. The heating element can include a resistive heating element, field-effect transistor, p-n junction diode, thin film heater, thermal diode, or a combination thereof, and the heating element can include platinum, aluminum, copper, gold, silver, tantalum, titanium, nickel, tin, zinc, chromium, tungsten silicon nitride, tantalum aluminum, nichrome, tantalum nitride, chromium silicon oxide, poly-silicon, germanium, oxides, alloys, and combinations thereof. The heating element may be operable to heat fluid at a rate of 100° C./s to 50,000,000° C./s (e.g., pulses of heat ranging in duration from the order of hundreds of nanoseconds to milliseconds) or at a rate of 1,000° C./s to 10,000,000° C./s.

The heating element can permit consistent or pulsed heating. In some examples, the heating can be pulsed. Pulsed heating can provide suitable control in heating a fluid. In some examples, the heating element can be positioned to elevate a temperature of a fluid by 20° C. to 50° C. when pulsed on for 0.1 μs to 1 second. In an example, the heating element can be part of a microfluidic processing system that can be used for nucleic acid amplification. The heating element can allow for rapid thermal cycling and can be used to rapidly amplify a nucleic acid on time scale limits imposed by physical and chemical kinetics. Rapid thermal cycling can be used to amplify a nucleic acid within hold times from 0.05 seconds to 10 seconds, from 0.05 seconds to 1 second, from 0.5 seconds to 10 seconds, or from 0.5 seconds to 3 seconds for a denaturing, annealing, and extending phase during the amplification process. Rapid thermal cycling can be used for polymerase chain reaction, isothermal amplification, reverse transcription, forward transcription, or a combination thereof. In other examples, a continual heat can be applied for isothermal amplification. Other processes that may be carried out include loop mediated isothermal amplification (LAMP) or recombinase polymerase amplification (RPA), for example.

FIG. 9 and FIG. 10 depict other examples of microfluidic processing systems 200 where multiple reagent delivery networks 100A and 100B are connected in parallel or in series. As shown in FIG. 9, multiple reagent delivery networks are shown connected to downstream microfluidics, e.g., a microfluidic processing channel 240 or other fluidic processing architecture, in parallel. Thus, processing from these multiple reagent delivery networks can occur independently of one another and fed the microfluidic processing channel separately to work together or independent of one another.

On the other hand, as shown in FIG. 10, the microfluidic processing channels 100A and 100B may be fluidly coupled together by segments of microfluidic processing channel 240, so that fluid flowing through a first reagent delivery network 100A will ultimately also pass through a second microfluidic network 100B, and so forth. Thus, reagent 126a picked up and reconstituted in the reagent delivery network 100A will be sent passed through reagent delivery network 100B, including the reconstituted reagent formed in the reagent delivery network 100A. Notably, processing components are not shown in this example, but may be present along the various microfluidic mixing channels. Thus, there may be processing components used that are not shown positioned prior to reaching the first reagent delivery network, between reagent delivery networks, or after passing through the multiple reagent delivery networks. Various processing that may occur at any of these locations is shown in greater detail by example in FIG. 11, for example. Thus, as shown, it is notable that the microfluidic inlet channels are fed from below (though orientation is not particularly an issue on the microfluidic scale, but this is mentioned for clarity in understanding fluid flow as shown in FIG. 10).

In further detail, individual outlet microfluidic channels 130 receive fluid as it passes through its microfluidic cross-channel and passes that fluid along to the next microfluidic processing channel, and so forth. The microfluidic processing channels can independently feed other architecture, such as the next reagent delivery network or an outlet 150. A system that includes a plurality of microfluidic cross-channels can include a single inlet microfluidic channel and a single outlet microfluidic channel that can respectively feed or receive a fluid there through. Each of the reagent delivery networks may have different reagents therein. The reagent delivery networks may be accessible in series or randomly. In some examples, a single fluid can be fed through the different reagent chambers. In yet other examples, multiple fluids can be fed through the different reagent chambers. Selectivity can be based on fluid flow or can be based on actuating of resistors. The fluid may be fed from the inlet microfluidic channel to the microfluidic cross-channel.

Another microfluidic processing system 200 including a reagent delivery network 100 can be utilized for temporal multiplexing as shown as microfluidic processing system 200 at FIG. 11. This FIG. depicts several fluid ports, several fluid ejectors, surface-active microparticles shown at multiple locations, several magnets for moving magnetizing surface-active microparticles, multiple detectors, etc. A microfluidic processing system for use in accordance with the present location can include or use some or all of these components, or even additional components not shown. FIG. 11 is provided to describe various processing examples.

The microfluidic processing system 200 shown in FIG. 11 thus includes a reagent delivery network 100 similar to that described in FIGS. 1-10, but in this instance, the multiple microfluidic cross-channels 120 each contain a different reagent 126a, 126b, and 127c in their respective reagent storage chamber 124. As in the prior examples, there is an inlet microfluidic channel 110 (with an inlet port 250), an outlet microfluidic channel 130, and a plurality of resistors 140. In this example, the inlet port may be used to introduce fluids such as elution buffer, and the reagents can be used selectively to interact with the elution buffer based on how or when the individual resistors are energized. The reagent delivery network portion of the microfluidic processing system can operate similarly as that described previously.

The reagent delivery network 100 of the microfluidic processing system 200 can be fluidly coupled to other microfluidics, as shown by way of example in FIG. 11. For example, the microfluidic processing system can include a sample port 220 and a sample receiving chamber 230 for receiving sample fluid, which in this example includes surface-active microparticles which may be magnetizing microparticles. In this example, from the sample receiving chamber, a microfluidic processing channel 240 can be present that transports the sample fluid (with or without surface-active microparticles, depending on the application) in a direction toward an ejector 290 (E3) for dispensing of the fluid after processing within the microfluidic processing system. Other ejectors may also be included, shown at E1 and E2, for ejecting fluid into the microfluidic processing channel, for example. Also shown in this example are three additional secondary inlets 270 with secondary inlet microchannels 280, identified further as C1, C2, and C3. The secondary inlet microchannels located upstream of the reagent delivery network (C1 and C2) can permit loading of wash buffer, transport buffer, buffer containing reagents for downstream reactions, or the like. In the case of nucleic acid amplification, a post processing wash buffer may be introduced via another secondary inlet microchannel (C3), which can be used to wash the downstream portion of fluid after processing and/or mixing of fluids and/or dried reagents, e.g., a post amplification wash buffer. Washing can permit multiplexing of different nucleic acids in a sample fluid with different sets of primers introduced sequentially using the reagent delivery network 100 described previously. Following multiplexing of a nucleic acid, for example, the microfluidic processing channel can be washed with a wash buffer, a portion of sample fluid passed into the microfluidic processing channel, and another primer multiplexed with that portion of the sample fluid. In other examples, the microfluidic processing system can include integrated chambers prefilled with buffers in addition to or in place of additional fluid inlets.

In further detail, the microfluidic processing system 200 along the microfluidic processing channel 240 can include magnets 245. The magnets can be used for collecting and/or moving magnetizing microparticles 235 that are surface-activated along the microfluidic processing channel, for example. The magnets, identified individually as M1, M2, and M3, can be movable along the microfluidic processing channel and/or can be moved closer to or further from the microfluidic processing channel, or can be electromagnetically modified to be interactive with the magnetizing microparticles or not interactive with the magnetizing microparticles. In further detail, the magnet can be capable of generating a magnetic field, such as a magnetic field that can be turned on and off by introducing electrical current/voltage to the magnet. Alternatively, the magnet can be a permanent magnet that is placed in proximity to the microfluidic processing system to effect movement of surface-activated microparticles that are magnetic or magnetizing. The magnet can be permanently placed within this proximity, or can be movable along the microfluidic processing system, or movable in position and/or out of position to effect movement of the surface-activated microparticles. Magnetic surface-activated microparticles can be magnetized by the magnetic field generated by the magnet. In addition, the magnet can create a force capable of pulling the magnetic surface-activated microparticles through the microfluidic processing system. When the magnet is turned off or not in appropriate proximity, the magnetic surface-activated microparticles can reside in place or be passed through the microfluidic processing system using fluid flow. The use of magnetic surface-activated microparticles can permit portions of the sample fluid to be passed through a microfluidic processing channel, while other portions of the sample fluid remain in the sample receiving chamber.

One or multiple thermocycling heaters 260 can be included along the microfluidic processing channel adjacent to or operable in association with a thermocycling heater 260 to thermocycle fluidic or fluidic microparticle mixtures prepared using the microfluidic processing system. Two thermocyling heaters are shown by way of example at H1 and H2. In some examples, the fluids after processing can be thermocycled, for example, if the nucleic acid corresponding to the primer picked up from the reagent delivery network amplified, and amplification can be detected using a detector 260 (D1) outside the channel, for example, e.g., amplification detection with a fluorescence detector. After amplification, for example, post amplification wash buffer can be pumped through the thermocycling heater region to remove any amplified nucleic acid there as well as any primers, and the resulting fluid composition can be dispensed via an ejector 290 (E3). This wash buffer may contain reagents to degrade nucleic acids, e.g., nucleases, to prevent contamination of amplification for other runs with the same primer set.

Regarding the detector(s) 255, two are shown at D1 and D2. A detector can be used for purposes of detecting sample fluid along with fluids/microparticles admixed therewith that may be positioned along microfluidic processing channel 240 as well, either associated or located at or near a magnet 245 and/or a thermocycling heater 260, or elsewhere along the microfluidic processing channel or other location. The detector may be an optical detector, for example, with an illumination source (not shown) to illuminate the fluids within the microfluidic processing channel. The optical detector can receive data relating to the sample fluid. An illumination source can be operable to emit light towards the wall or portion of the wall of the substrate that is optically transparent. The detector can be operable to detect fluorescence emitted from a fluorescent molecule which can be loaded in the system and can become conjugated with a nucleic acid in the sample fluid during nucleic acid amplification. In other examples, the optical detector can be located with respect to the optically transparent area such that a light beam passes through the optically transparent area onto an optical detector or to an element capable of directing the light beam to the optical detector. In some examples, an optical detector can include a band pass filter and a p-n junction diode. A band pass filter passes frequencies in a certain range while attenuating frequencies outside that range. The band pass filter can permit a fluorescing wavelength emitted by the excited fluorescent molecule, allowing a p-n junction diode is a two-terminal semiconductor capable of converting light into an electrical current when photons are absorbed in the photodiode.

In further detail regarding the surface-activated microparticles, the surfaces can be adapted to bind with a biological component or can be bound to the biological component in the sample fluid. Surface activated microparticles can include surface groups that are interactive with a biological component of a biological sample or can include a covalently attached ligand attached to a surface of the microparticles to likewise bind with a biological component of a biological sample. In some examples, the ligand can include proteins, antibodies, antigens, nucleic acid primers, amino groups, carboxyl groups, epoxy groups, tosyl groups, sulphydryl groups, or the like. The ligand can be selected to correspond with and bind with the biological component and can vary based on the type of biological component being isolated from the biological sample. For example, the ligand can include a nucleic acid primer when isolating a biological component that includes a nucleic acid sequence. In other examples, the ligand can include an antibody when isolating a biological component that includes antigen.

In some examples, the surface-activated microparticles can have an average particle size that can range from about 0.1 μm to about 70 μm. The term “average particle size” describes a diameter or diameter of the volume of the particles if modified to a spherical size based on the particle volume, which may vary, depending upon the morphology of the individual particle. A shape of the surface-activated microparticles can be spherical, irregular spherical, rounded, semi-rounded, discoidal, angular, sub-angular, cubic, cylindrical, or any combination thereof. In some examples, the particles can include spherical particles, irregular spherical particles, or rounded particles. The shape of the surface-activated microparticles can be spherical and uniform, which can be defined herein as spherical or near-spherical, e.g., having a sphericity of >0.84. Thus, any individual particles having a sphericity of <0.84 are considered non-spherical (irregularly shaped). The particle size of the substantially spherical particle may be provided by its diameter, and the particle size of a non-spherical particle may be provided by its average diameter (e.g., the average of multiple dimensions across the particle) or by an effective diameter, e.g., the diameter of a sphere with the same mass and density as the non-spherical particle. In further examples, the average particle size of the surface-activated microparticles can range from about 1 μm to about 50 μm, from about 5 μm to about 25 μm, from about 0.1 μm to about 30 μm, from about 40 μm to about 60 μm, or from about 25 μm to about 50 μm.