Patent Application
20220380709
The present invention relates, in general terms, to an apparatus for forming one or more compartments. The present invention also relates to methods for forming one or more compartments. The present invention is applicable for use in crystallisation, bioassays and chemical microreactors, for example.
Traditionally, droplets are produced in large stirred batches of immiscible fluids. In their simplest form, droplets can be formed in an emulsion of two immiscible liquids (for example, oil and water) in which one liquid (the dispersed phase) is in the form of macroscopic or microscopic droplets dispersed in the other (continuous) phase. Emulsions can also contain emulsifiers to lower the interfacial tension and hence reduce the energy required to break the dispersed phase into droplets, and also preventing them from coalescing by generating a repulsive force or a physical barrier between them. However, droplets formed using this method have sizes which are usually spread over a Gaussian distribution. This is undesirable if precise control over the chemistry within the droplets is required.
Fluidic manipulation of micro- and milliscale droplets has become core to many fields and applications, including particle synthesis, chemistry, and biology. Microfluidic techniques improve on these batch methods and offer precise control over droplet composition and size by mixing fluids during flow within small diameter fixtures and tubing, or custom-fabricated devices. However, as these droplets are required to travel through fixtures and tubings or devices, the droplets are subjected to surface forces and fluid dynamics which may influence the chemistry occurring within the droplets. Further, persistent challenges and limitations remain such as the complexity of device manufacturing, a lack of flexibility with discrete channels and fixed geometries, and difficulty in selectively processing and extracting individual droplets. Efforts to address some of these challenges include the development of dynamic flow patterning, patterned substrates with “fluid walls”, and open microfluidic capillary systems, yet inherent limitations are still present due to the primary reliance of these and other microfluidic methods on phenomena and fluids that are purely Newtonian.
It would be desirable to overcome or ameliorate at least one of the above-described problems, or at least to provide a useful alternative.
In accordance with the present disclosure, there is provided a method for forming one or more compartments in a yield-stress fluid, including: a) introducing one or more volumes from an outlet of a nozzle into a yield-stress fluid, the outlet of the nozzle being in contact with the yield-stress fluid; and b) displacing the nozzle and/or the yield-stress fluid relative to each other to thereby form one or more compartments in the yield-stress fluid, the one or more compartments being formed by, or being formable from, the one or more volumes.
Advantageously, the volumes may be selectively introduced at pre-determined locations within the immiscible or partially miscible yield-stress fluid. Such volumes are embedded within the yield-stress fluid, such that the volumes are spatially isolated from each other and not influenced by solid boundaries (e.g. solid containing walls) that may influence induced internal processes.
In certain embodiments, the method further includes a step of selectively perturbing a flow of an input fluid for forming one or more volumes.
In certain embodiments, the selective perturbation of the flow of the input fluid may be synchronised to the displacement of the nozzle and/or the yield-stress fluid relative to each other.
In certain embodiments, the selective perturbation of the flow of the input fluid may be a periodic variation of the flow of the input fluid.
In certain embodiments, the one or more compartments are one or more droplets, and the one or more droplets may be suspended in the yield-stress fluid.
The method may further include a microfluidic system in communication with an inlet of the nozzle for supplying the input fluid.
The method may further include a flow system in communication with the yield-stress fluid for supplying the yield-stress fluid as a continuous flow to the outlet of the nozzle.
In certain embodiments, the one or more compartments is, or are, non-miscible with the yield-stress fluid.
In certain embodiments, the yield-stress fluid is at rest.
In certain embodiments, the yield-stress fluid is contained in a vessel.
In certain embodiments, the vessel is displaceable relative to the nozzle.
In certain embodiments, the displacement is in Cartesian coordinates.
In certain embodiments, the yield-stress fluid has a yield-stress value of about 0.1 Pa to about 10 Pa.
In certain embodiments, the yield-stress fluid has a surface tension of about 5 mN/m to about 75 mN/m.
In certain embodiments, the yield-stress fluid has a critical shear rate e,dot γc of about 0.01 1/s to 1000 1/s, and a flow index n of about 0.25 to about 1.
In certain embodiments, the yield-stress fluid has a characteristic thixotropic timescale of about 0 seconds to about 10 seconds.
In certain embodiments, the yield-stress fluid is selected from polydimethylsiloxane, silicone oil, colloidal or granular particles in water or oil, diblock or triblock copolymers in water or oil, microfibrillar cellulose, xanthan gum and a combination thereof.
In some embodiments, the yield-stress fluid is semi-transparent or transparent to allow for direct observation of the compartments.
In certain embodiments, the one or more compartments is, or are, suspended within the yield-stress fluid at least 1 mm below a surface of the yield-stress fluid.
Advantageously, this ensures that the compartments are always enclosed within the yield-stress fluid and perturbations will not cause the compartments to be displaced and escape the yield-stress fluid.
In certain embodiments, the input fluid is a liquid.
In certain embodiments, the one or more volumes in the one or more compartments is, or are, diffusible out from the one or more compartments for use in crystallisation.
In certain embodiments, each of the one or more compartments has a different composition for following progress of a chemical reaction.
In certain embodiments, each of the one or more compartments includes a microorganism for use in bioassays.
There is also provided an apparatus for forming one or more compartments, including: a) a nozzle including an outlet, the outlet for introducing one or more volumes; b) a yield-stress fluid, the yield-stress fluid in contact with the outlet of the nozzle; and c) a controller configured to displace the nozzle and/or the yield-stress fluid relative to each other to introduce one or more volumes into the yield-stress fluid to thereby form one or more compartments from the one or more volumes.
In certain embodiments, the apparatus further includes a controller configured to selectively perturb a flow of an input fluid for forming one or more volumes.
In certain embodiments, the selective perturbation of the flow of the input fluid is synchronised to the displacement of the nozzle and/or the yield-stress fluid relative to each other.
In certain embodiments, the selective perturbation of the flow of the input fluid is a periodic variation of the flow of the input fluid.
The apparatus may further include a microfluidic system in communication with an inlet of the nozzle.
In certain embodiments, the yield-stress fluid is contained in a vessel.
Embodiments of the present invention will now be described, by way of non-limiting example, with reference to the drawings in which:
As used herein, ‘yield-stress fluid’ is a material that reversibly transitions from effectively solid to effectively fluid and back again as a consequence of applied mechanical stress. It is a material that is solid-like below a critical stress but undergoes a dramatic drop in viscosity and flows above its yield stress. Yield-stress fluids are generally considered non-equilibrium systems both in their solid-like state and their tendency to age and flow over long timescales. Yielding may be gradual across a range of stress magnitudes and may occur as localized brittle cracking, shear-banding, or system spanning diffuse failure. An important distinction is that of the ‘static’ yield stress (for the transition from solid to liquid, e.g. start-up shear tests) versus the ‘dynamic’ yield stress (for the transition from liquid to solid, e.g. steady flow tests of decreasing shear rate). These can be very different; when they are, the static yield stress is typically larger. Furthermore, the forward and reverse transitions between solid-like and liquid-like are not instantaneous; the microstructural units require time to return to an arrested state. This reversible time-dependence is known as thixotropy and it is intimately linked with yielding in these materials. For practical purposes, ‘simple yield-stress fluids’ may be defined as those for which thixotropic timescales are too short to observe with available techniques while ‘thixotropic yield-stress fluids’ are those with a measurable time scale.
At least some embodiments of the invention have advantages over batch techniques. These advantages include precise control of small sample volumes and high frequency of automated generation. Furthermore, the bath material (yield-stress fluid) provides an intrinsic barrier against atmospheric contamination or evaporation of the fluid samples. At least some embodiments of the invention can also overcome some of the disadvantages of traditional microfluidic technologies by removing the aspects of solid boundaries (e.g. tubing and fixtures) and continuous flow. Microfluidic device designs exist for trapping many droplets in a large reservoir or in arrays of chambers but droplet mobility may make indexing difficult and the presence of shared boundaries may lead to fouling or affect processes like nucleation. By embedding compartments within a yield-stress fluid, compartments can be processed in spatial isolation for essentially an indefinite period of time with no risk of coalescence or collapse, barring some degradation of the bath. This is not possible with traditional microfluidics since droplets are generally constrained by flow through the length of a given microfluidic device. For example, in some microfluidic cases, the flow may be paused but there is a significant risk of continued droplet mobility and droplet coalescence. It has been suggested that droplets in microfluidic devices may be circulated in very long tubing to develop. However, this was found to be challenging to execute because droplets will become unstable and collapse or coalesce due to the non-negligible hydraulic resistance.
To this end, embodiments of the present invention are suitable for the generation, trapping, and processing of fluid droplets within yield-stress fluids. By printing discrete droplets that are embedded within a bath of yield-stress fluid, the droplets are not limited to fixed geometric boundaries, and can freely manipulate and process selected droplets for extended periods of time, and there is no complex device manufacturing. Furthermore, droplets embedded in yield-stress fluids are not affected by any solid boundaries or convective effects which are often inherent to other forms of microfluidics. This results in droplets with no mobility or risk of coalescence whatsoever, even without utilizing surfactants.
Rayleigh instability is the phenomenon where a falling stream of fluid breaks up into smaller packets with the same volume but less surface area. The driving force of Rayleigh instability is that liquids, by virtue of their surface tensions, tend to minimize their surface area. In the past, a considerable amount of work has been done to reduce or avoid this fluid instability in, for example, 3D printing in sacrificial yield-stress fluid baths as it introduces inconsistencies in a technique which strives for precision and control. Advantageously, the inventors have found that by embracing Rayleigh instability, which would otherwise be problematic in many contexts, droplets of controllable size and volume can be formed in a yield-stress fluid. The use of a yield-stress fluid to support and maintain the droplets further removes aspects of solid boundaries and continuous flow, thus providing a reproducible environment substantially removed from external forces and influences.
At step 110, an outlet of a nozzle is positioned in contact with a yield-stress fluid. The nozzle is suitable for supplying one or more volumes of input fluid to the yield-stress fluid. At step 120, the one or more volumes exit from the outlet of the nozzle and are introduced into the yield-stress fluid. At step 130, the nozzle and yield-stress fluid are displaced relative to each other. In this regard, the nozzle can be moved while the yield-stress fluid is stationary, or the yield-stress fluid can be moved while the nozzle is stationary, or both the nozzle and yield-stress fluid can be moved. The displacement allows for the formation of one or more compartments in the yield-stress fluid, the one or more compartments being formed from the one or more volumes.
The nozzle is optionally in communication with a fluid reservoir, and with a pump that can be used to introduce one or more volumes of fluid from the nozzle into the yield-stress fluid. For example, optional step 102 can include providing a microfluidic system in communication with an inlet of the nozzle. The microfluidic system can be modulated to provide the one or more volumes. Alternatively, optional step 104 can include selectively perturbing a flow of the input fluid for forming one or more volumes.
The yield-stress fluid is optionally in communication with a fluid reservoir. The fluid reservoir is for supplying a flow of yield-stress fluid to the nozzle. In this regard, the yield-stress fluid is moved/translated while the nozzle is stationary. For example, optional step 106 can be a microfluidic system provided in communication with the yield-stress fluid for displacing the yield-stress fluid relative to the nozzle. Preferably, the yield-stress fluid is provided as constant flow to the nozzle.
As used herein, ‘compartment’ refers to an enclosed pocket of a substance. This substance can be a liquid and/or a gas. When the compartment is, or contains, a liquid or both a liquid and a gas, the compartment may be referred to as a droplet. When the compartment only contains a gas, the compartment may be referred to as a bubble. As will be shown herein, the compartment can contain chemical compounds, biological materials such as cells, genetic materials and viruses, crystals, nanoparticles precursors and/or nanoparticles.
The perturbation of the input fluid to allow for the formation of the volumes and compartments can be by stop-flow setup, which can be manually operated or programmed to release a volume of input fluid periodically. Alternatively, the volumes and compartments can be formed by relying on Rayleigh instability.
The rheology of the yield-stress fluid bath determines the theoretical operating space for the present invention. The compartments are generated by yielding the yield-stress fluid to an inserted nozzle that injects fluid. As the nozzle and yield-stress fluid are displaced relative to each other, the localised displacement applies a localised mechanical stress to the yield-stress fluid, thus converting the localised volume to transit into a fluid. When the volume exits the nozzle, the fluid state yield-stress fluid allows for the formation of a compartment. As the yield-stress fluid reverts back to the solid state, the compartment is held in place, embedded at a pre-determined location within the yield-stress fluid. Compartments are formed when the surface tension between the injected fluid and the yield-stress bath obeys the relation (γ/r) σ_y (surface tension, γ, divided by the radius of the compartment, r, is greater than or equal to the bath yield stress, σ_y).
Without wanting to be bound by theory, comparing the yield stress of the bath, σγ, to the stress exerted on the bath by a spherical droplet of diameter, d, due to buoyancy from a density difference, Δρ, the equation obtained is
where g is the gravitational acceleration constant, and Ycrit is a dimensionless parameter defined as the ratio of the fluid's yield stress to the stress exerted on the fluid by the droplet when motion occurs due to a yielding transition. The definition of Ycrit was rearranged to describe the operating space of interest. One might naively expect Ycrit to be unity for yielding to occur, but due to the droplet needing only to yield a finite volume to move, theoretical studies have found a value of Ycrit=0.14 for an idealized fluid; experiments have shown Ycrit to be material specific, ranging between 0.05 and 0.6. Equation 1 is the condition for a formed droplet to be statically suspended within the yield-stress fluid bath rather than sedimenting or floating due to buoyancy. Comparing the yield stress to the stress exerted due to surface tension γ, for a curved interface with diameter d, we obtain
This inequality is the condition for whether droplet formation will occur. If the yield stress of the bath is too great, a continuous thread of the injected phase will form rather than discrete droplets. With these conditions, an operating space for embedded droplet printing for a pairing of a model yield-stress fluid and an immiscible phase can be determined.
In some embodiments, the yield-stress fluid has a Ycrit of about 0.05 to about 1, or preferably about 0.05 to about 0.6. In other embodiments, Ycrit is about 0.1 to about 0.6, about 0.1 to about 0.5, about 0.1 to about 0.4, about 0.1 to about 0.3, or about 0.1 to about 0.2.
The present invention allows for the manipulation of droplets under conditions that are simply unattainable with conventional microfluidic methods, namely the elimination of exterior influences including convection and solid boundaries. Advantageously, the compartments are held for an effectively indefinite period of time in an “absolutely quiescent” state with a complete absence of exterior convective forces (including buoyancy-driven flows), solid boundaries that may influence induced internal processes (e.g. solid containing walls) and continuous flow. Further, there is no possibility for cross-contamination between compartments since they are always spatially isolated, and there are no shared solid boundaries that can lead to fouling or uncontrolled nucleation. The present invention also removes some troublesome aspects of microfluidics including the use of surfactants and the complexity of device manufacturing.
Additionally, as Rayleigh instability generates the volumes as a periodic function and amplitude, the compartments formed are consistent in size and shape. The compartments are also maintainable without the use of emulsifiers and surfactants.
In contrast,
However, the disadvantage with this method is that the continuous phase is constantly being added to the yield-stress fluid. This can cause the yield-stress fluid to gradually lose its thixotropic property (for example). The inability to maintain a consistent environment in the yield-stress fluid can result in a gradual change in compartment morphology and/or content. In some scenarios, the compartments can collapse on entering the yield-stress fluid. Further, the dilution of the yield-stress fluid by the continuous phase can cause the yield-stress fluid to lose its solid-like state such that the compartments cannot be selective positioned. In contrast, the present invention allows for the maintenance yield-stress fluid such that the compartments are homogenous throughout the deposition method. The present invention also allows for selective placement of the compartments.
The selective perturbation of the flow of the input fluid may be synchronised to the displacement of the nozzle and the yield-stress fluid relative to each other. This synchronisation enables compartments of a consistent size and shape to be formed in the yield-stress fluid. Further, the synchronisation also reduces and/or eliminates the formation of satellite compartments; i.e. smaller sized compartments formed at the tail end of the primary compartment. Additionally, by timing the relative movement of the nozzle and the yield-stress fluid, due to surface tension, any formed satellite compartments are able to coalesce with the primary compartment, thus eliminating the presence of the satellite compartments which may be undesirable. Such synchronisation would be dependent on several factors, for example the properties of the yield-stress fluid and the rheology of the input fluid.
In certain embodiments, the selective perturbation of the flow of the input fluid may be a periodic variation of the flow of the input fluid.
As shown in
In certain embodiments, the one or more compartments are one or more droplets. The one or more compartments may be suspended in the yield-stress fluid. Advantageously, and in addition to the removal of external forces, suspending the compartments allows them to be visualised in a 3D environment. In this regard, changes in the internal chemistry within the compartment can be tracked and sampled at any time point.
The velocity of the nozzle relative to the bath was systematically varied for different nozzle diameters and a fixed injection flow rate of 50 μL/min. The nozzle materials were also varied between stainless steel and glass that has been plasma cleaned shortly before droplet generation to be hydrophilic and thus more readily wet by the bath phase. The droplet size obtained as a function of velocity for droplets that range in diameter from about 300 μm to about 1.5 mm. The generated droplets are highly uniform in size, as depicted in
The analysis of the conventional, purely Newtonian case of droplet formation from coflowing streams balances the viscous Stokes' drag with the interfacial tension. This results in a normalized droplet size, d/di, where di is the inner diameter of the nozzle; and a capillary number, 3Vη/y, where V is the velocity of the nozzle relative to the bath; η is the non-Newtonian viscosity of the yield-stress fluid bath at a shear rate of V/do, where do is the outer diameter of the nozzle (see
In some embodiments, a diameter of the compartment is related to the displacement rate of the nozzle and/or the yield-stress fluid relative to each other by a power-law scaling of about −1.
The compartments can have a diameter of about 50 μm to about 1.5 mm, or about 100 μm to about 1.5 mm, about 150 μm to about 1.5 mm, about 200 μm to about 1.5 mm, about 250 μm to about 1.5 mm, about 300 μm to about 1.5 mm, about 400 μm to about 1.5 mm, about 500 μm to about 1.5 mm, about 600 μm to about 1.5 mm, about 700 μm to about 1.5 mm, about 800 μm to about 1.5 mm, about 900 μm to about 1.5 mm, or about 1 mm to about 1.5 mm.
The compartments can be printed with a linear density of about 0.1 droplets per millimeter to about 1 droplets per millimeter. The compartments can be printed with a volumetric density of about 1 droplets per milliliter of yield-stress fluid to about 70 droplets per milliliter of yield-stress fluid.
The method may further include providing a microfluidic system in communication with an inlet of the nozzle for supplying the input fluid. The method may also include providing/placing a microfluidic system in communication with the yield-stress fluid. As shown in
The method may further include providing a system in communication with the yield-stress fluid for supplying the yield-stress fluid as a continuous stream to the outlet of the nozzle. The system can be a microfluidic system. In this regard, the yield-stress fluid is displaced with respect to the nozzle and the nozzle is stationary. As shown in
For the compartment to be suspended in the yield-stress fluid, some degree of immiscibility is advantageous. Immiscibility is the property where two substances are not capable of combining to form a homogeneous mixture; i.e. the two substances (oil and water) will separate from each other. In certain embodiments, the one or more compartments is non-miscible with the yield-stress fluid. As discussed above, by accounting for the surface tension of the compartments and the density difference, the compartments can be maintained as an enclosed reaction environment which is suitable for use as a chemical microreactor. When the compartments are arranged in, for example an array, the reaction condition or reaction progress can be tracked over time.
In other embodiments, the one or more compartments is, or are, partially miscible with the yield-stress fluid. In this regard, partially miscible substances can be substances that do not mix in all proportions at all temperatures; i.e. they are miscible only in a limited range of concentrations. A partially miscible substance (or compartment) can be made up of one or more components, one component of which can be miscible and the other component immiscible (hydrophobic compound in ethyl acetate). In this way, when the two substances are combined (compartment in yield-stress fluid), the miscible component of a first substance (ethyl acetate) is able to diffuse and mix with a second substance (Carbopol in water), leaving the immiscible component (hydrophobic compound) behind. This allows for the use of the compartments as a crystallisation platform, for example via an anti-solvent process.
In certain embodiments, the yield-stress fluid is at rest. In certain embodiments, the yield-stress fluid is contained in a vessel. In certain embodiments, the nozzle is displaceable relative to the vessel. Alternatively, the vessel is displaceable relative to the nozzle. For example, as shown in
In certain embodiments, the displacement is in Cartesian coordinates. In some embodiments, the yield-stress fluid is mounted on an X, Y-axis motion-controlled stage and one or more nozzles mounted on a Z-axis motion controller. The motion controllers can be programmed or operated to move in a determined pattern at a pre-determined and controlled movement rate.
The yield-stress fluid may be water-based or oil-based depending on the input fluid and/or compartment formed. For example, the yield-stress fluid can be water-based if the input fluid is oil-based and vice versa. It is further advantageous if the yield-stress fluid has low opacity (and/or good clarity) for being able to visualise changes in the compartments in applications such as microreactors. To improve the consistency of the yield-stress fluid and to minimize false readings, the yield-stress fluid can be subjected to a vacuum prior to introducing the one or more volumes for removing gases/air bubbles.
In certain embodiments, the yield-stress fluid is selected from polydimethylsiloxane, silicone oil, colloidal particles in water or oil, diblock or triblock copolymers in water or oil, microfibrillar cellulose, xanthan gum and a combination thereof. For example, water-based yield-stress fluids can be made up of appropriate amounts of xanthan gum, micro-fibrillated cellulose and/or microgels of polyacrylic acid. Examples of oil-based yield-stress fluid systems include mixtures of polydimethylsiloxane, silicone oil, and hydrophilically modified fumed silica nanoparticles; and mixtures of diblock and triblock copolymers in mineral oil.
Additives can also be added to the yield-stress fluid to alter some of the properties of the yield-stress fluid. In some embodiments, the rheological additives do not interact with the compartments. In this regard, the additives do not affect with the interior processes within the compartments. In some embodiments, additives are added at less than 5% wt/wt, or less than 4% wt/wt, or less than 3% wt/wt, or less than 2% wt/wt, or less than 1% wt/wt.
In some embodiments, the yield-stress fluid is composed of additives that are generally regarded as safe by the United States Food and Drug Administration. In some embodiments, the yield-stress fluid is selected from Carbopol in water, fumed silica in silicone oil and polydimethylsiloxane (PDMS). Carbopol is a polyacrylic acid microgel sold by Lubrizol in powdered form. This would be considered a system of swollen granular particles (˜10 μm diameter) in water. This system is transparent and can be triggered to lose its yield stress through the shrinkage of the granular particles. Fumed silica can be for example Aerosil 200 (pharmaceutical grade sold by Evonik). Silicone oil can for example be obtained from Sigma Aldrich and PDMS purchased from Dow Chemical Company. Fumed silica in silicone oil and polydimethylsiloxane would be considered a system of colloidal particles in oil. This system is semi-transparent. Further advantageously, when PDMS is caused to be crosslinked by addition of a crosslinking agent, this system can be triggered to permanently gel. This allows the compartments to be entrapped within for ease of transportation. In some embodiments, the yield-stress fluid is about 0.07 wt % to about 1 wt % Carbopol.
For example, the yield-stress fluid can be selected from:
a) 0.1 wt % Carbopol (polyacrylic acid microgels) in water;
b) 4 wt % fumed silica, 48 % silicone oil. 48 % PDMS;
c) 0.1 wt % xanthan gum in water;
d) 0.4 wt % microfibrillar cellulose in water;
e) 2.25 wt % diblock, triblock copolymer, 95.5 % mineral oil;
f) 1-2wt % Laponite in water;
g) 15-20 wt % Pluronic F127 in water; and
h) 60-70 wt % silicone oil in water emulsion.
Other examples of yield-stress fluids are shown in the table below (but not limited to):
To control the compartments size and placement in the yield-stress fluid, the following properties of yield-stress fluid and other engineering properties can be modulated (but not limited to):
a) yield stress (Pa);
b) elastic modulus pre-yield, G (Pa);
c) yield strain (%);
d) thixotropic restructuring time (s);
e) post-yield viscosity (Pa s) or viscous effects e.g. Herschel-Bulkley parameters:
critical shear rate, flow index;
f) uniaxial strain at break (%);
g) density (kg/m3);
h) homogeneity of the material (particle, building block, or aggregate size) (pm);
i) surface energy and wetting;
j) optical properties, e.g. maintain light transmittance for photo-chemical responsiveness; and
k) transformation requirements, e.g. chemical or thermal.
Some of the engineering properties of yield-stress fluids considered in the present invention include the yield-stress value, the post-yield viscous effects [i.e., Herschel-Bulkley parameters], optical properties, surface energy and wetting, and the ability to trigger mechanical changes in the yield-stress fluid.
In some embodiments, the yield-stress fluid can be characterised by the Herschel-Bulkley model. A Herschel-Bulkley fluid is a generalized model of a non-Newtonian fluid, in which the strain experienced by the fluid is related to the stress in a complicated, non-linear way. Three parameters characterize this relationship: the critical shear rate, {dot over (γ)}c, the flow index n, and the yield shear stress To. The critical shear rate is the rate of deformation at which the flow stress is twice the yield stress, while the flow index measures the degree to which the fluid is shear-thinning or shear-thickening. Finally, the yield stress quantifies the amount of stress that the fluid may experience before it yields and begins to flow. Alternatively, a proportionality constant, k, may be used in place of {dot over (γ)}c, where k =τ0/({dot over (γ)}c)n.
In some embodiments, the critical shear rate {dot over (γ)}c is about 0.01 1/s to 1000 1/s. In other embodiments, the critical shear rate {dot over (γ)}c is about 0.1 1/s to 1000 1/s, about 1 1/s to 1000 1/s, about 10 1/s to 1000 1/s, about 50 1/s to 1000 1/s, about 100 1/s to 1000 1/s, or about 500 1/s to 1000 1/s.
In some embodiments, the flow index n is about 0.25 to about 1. In other embodiments, the flow index n is about 0.3 to about 1, about 0.4 to about 1, about 0.5 to about 1, about 0.6 to about 1, about 0.7 to about 1, or about 0.8 to about 1.
In some embodiments, the yield shear stress To is about 0.1 Pa to 100 Pa. In other embodiments, the yield shear stress To is about 0.1 Pa to 100 Pa, about 1 Pa to 100 Pa, about 10 Pa to 100 Pa, about 50 Pa to 100 Pa, or about 80 Pa to 100 Pa.
The yield stress of the yield-stress fluid is selected to be large enough to prevent the compartments from settling to the bottom or floating to the top. If the yield stress is too high or if the surface tension is too low, a continuous thread of fluid will form rather than discrete compartments. If the yield stress is too high or if the nozzle does not have sufficient affinity (wettability) for the bath, the injected fluid may wet the nozzle and flow back upwards. If the yield stress is too high or the printing depth is too shallow, a static crevice will be created by the motion of the nozzle through the bath. Such are not desirable.
In certain embodiments, the yield-stress fluid has a yield-stress value of about 0.1 Pa to about 10 Pa. In other embodiments, the yield-stress fluid is about 0.2 Pa to about 10 Pa, about 0.3 Pa to about 10 Pa, about 0.5 Pa to about 10 Pa, about 1 Pa to about 10Pa, about 2 Pa to about 10 Pa, about 3 Pa to about 10 Pa, about 4 Pa to about 10 Pa, about 5 Pa to about 10 Pa, about 6 Pa to about 10 Pa, about 1 Pa to about 9 Pa, about 1 Pa to about 8 Pa, about 1 Pa to about 7 Pa, about 1 Pa to about 6 Pa, about 2 Pa to about 6 Pa, or about 2 Pa to about 5 Pa.
In certain embodiments, the yield-stress fluid has a surface tension of about 5 mN/m to about 75 mN/m. Some examples of fluids with surface tensions within this range are water in ethyl acetate (6.8 mN/m), water in mineral oil (50 mN/m) and air in water (73 mN/m).
In certain embodiments, the yield-stress fluid has density of about 800 kg/m3 to about 1000 kg/m3. Alternatively, the density can be about 800 kg/m3 to about 950 kg/m3, 800 kg/m3 to about 900 kg/m3 or 800 kg/m3 to about 850 kg/m3.
In certain embodiments, the yield-stress fluid has a characteristic thixotropic timescale of about 0 seconds to about 10 seconds. Alternatively, the characteristic thixotropic timescale can be about 1 s to about 10 s, about 2 s to about 10 s, about 3 s to about 10 s, about 3 s to about 9 s, about 3 s to about 8 s or about 3 s to about 7 s.
In some embodiments, the yield-stress fluid is semi-transparent or transparent to allow for direct observation of the compartments. In this regard, the yield-stress fluid allows light to pass through so that objects within can be partially or distinctly seen.
The one or more compartments is, or are, suspended at a distance below the surface of the yield-stress fluid. In certain embodiments, the one or more compartments is, or are, suspended within the yield-stress fluid at least 1 mm below a surface of the yield-stress fluid. In other embodiments, the distance is at least a diameter of the one or more compartments. In other embodiments, the distance is at least twice the diameter of the one or more compartments. In this regard, the compartments are printed sufficiently far from the boundaries of the container or from the surface of the fluid to avoid contact with the air-liquid interface or solid-liquid interface. Advantageously, this ensures that the compartments are totally embedded within the yield-stress fluid and are less likely to be perturbed by external forces. This also provides a buffer distance and ensures that the compartment is maintained within the yield-stress fluid when subjected to sudden and unintentional movement.
As the output of the nozzle is at the interface of the volume and the yield-stress fluid, the chemical and physical properties of the nozzle can also influence the formation of the compartments. For example, factors such as the geometry, surface energy and wettability of the nozzle, movement rate of the nozzle, and injected flowrate of the input fluid can affect the compartment size.
For modifying the wettability of the nozzle, the nozzles can be coated with polydimethyl siloxane (hydrophobic), coated with poly vinyl alcohol (hydrophilic), and/or plasma cleaned (hydrophilic).
In some embodiments, the water surface energy is about 5 mN/m to about 100 mN/m. In other embodiments, the surface energy is about 5 mN/m to about 800 mN/m, or about 5 mN/m to about 50 mN/m.
In some embodiments, the nozzle has an output diameter of less than 2 mm, or less than 3 mm, less than 4 mm, less than 5 mm, less than 10 mm or less than 20 mm. In other embodiments, the nozzle has a movement rate of about 50 mm/min to about 4000 mm/min, about 50 mm/min to about 3800 mm/min, about 50 mm/min to about 3500 mm/min, about 50 mm/min to about 3200 mm/min, about 50 mm/min to about 3000 mm/min, about 60 mm/min to about 3000 mm/min, about 70 mm/min to about 3000 mm/min, about 100 mm/min to about 3000 mm/min, about 200 mm/min to about 3000 mm/min, about 500 mm/min to about 3000 mm/min, about 750 mm/min to about 3000 mm/min, about 1000 mm/min to about 3000 mm/min or about 1500 mm/min to about 3000 mm/min. In other embodiments, the flowrate of the input fluid is about 1 μL/min to about 3000 μL/min, about 2 μL/min to about 3000 μL/min, about 5 μL/min to about 3000 μpL/min, about 10 μL/min to about 3000 μL/min, about 20 μL/min to about 3000 μL/min, about 50 μL/min to about 3000 μL/min, about 70 μL/min to about 3000 μL/min, about 100 μL/min to about 3000 μL/min, about 150 μL/min to about 3000 μL/min, about 200 μL/min to about 3000 μL/min, about 500 μL/min to about 3000 μL/min, about 750 μL/min to about 3000 μL/min, about 1000 μL/min to about 3000 μL/min, about 1000 μL/min to about 2500 μL/min, or about 1500 μL/min to about 2000 μL/min.
In certain embodiments, the input fluid is a liquid. In other embodiments, the input fluid is a gas.
The compartments formed using this presently disclosed method can function as “microreactors” for many different processes and applications related to crystallization, biology, or chemical reactions.
The present invention can be used in crystallisation of spherical drug particles. In the manufacturing of pharmaceuticals, common secondary manufacturing processes are drug crystallization, followed by milling, and granulation. The goal of these processes is to obtain a flowable drug powder for further processing into a final tablet. The present invention can be used to combine the three aforementioned processes into one step through spherical crystallization with a high degree of control over key attributes including size, shape, and structure by use of evaporative means or the careful choice of partially miscible solvents (anti-solvent crystallization). In certain embodiments, the input fluid in the one or more compartments is diffusible out from the one or more compartments for use in crystallisation.
In the production of pharmaceutical tablets, it is often necessary to have a flowable powder of crystallized active pharmaceutical ingredient (API) and excipient. Granules of agglomerated crystals often have poor flowability in large part due to irregular shapes and wide size distributions, with many recent batch methods for crystallizing particles often still resulting in only what may be described as “approximately spherical” at best. Microfluidic approaches to spherical crystallization offer significant improvements in this regard, but crystals are often still marred by the deforming effects of viscous drag and interactions with other particles or solid surfaces, leading to noticeable deviations from sphericity (see
Embodiments of the present invention allows for crystallization to occur under absolutely quiescent conditions, by utilizing an antisolvent crystallization approach to produce uniform spherical particles inside aqueous Carbopol yield-stress fluid as depicted in
As shown in
Accordingly, in some embodiments, the yield-stress fluid is capable of a triggered change in mechanical properties such as the loss of its yield stress properties.
In certain embodiments, the one or more compartments each has a different composition for following a progress of a chemical reaction.
The present invention allows for the ability to perform chemical reactions in high throughputs and with small and precise volumes of potentially expensive reagents. By further removing interactions with solid boundaries and external flow, considerably more freedom in arraying droplets is obtained. Embedded droplet printing eliminates reported challenges in droplet microfluidics such as droplet coalescence and collapse, reduces the risk of fouling in fixed geometries, and easily allows droplets to develop for extended timescales and be selectively addressed or directly sampled.
Embodiments of the present invention can provide a versatile platform for performing micro-batch chemical reactions. Precisely controlled quantities of multiple reagents may be simultaneously injected and allowed to react. After the reaction has progressed, fluid may either be directly extracted, optically characterized or both in sequence.
In many conventional microfluidic systems, procedures that require significant reaction times often have significant drawbacks. For example, flow and droplet generation might be paused as a reaction proceeds, limiting throughput and risking the merging of still-mobile droplets; droplets might also be circulated through very long or looping tubing, but this reportedly risks coalescence and collapse. Alternatively, droplets could be deposited into a large reservoir or a trapping device that may be complex to manufacture; these techniques typically randomize the droplet placement inside sealed devices, making indexing and extraction difficult. Using embedded droplet printing, such limitations are removed or at least minimized, thus greatly facilitating time-lapse studies.
Further advantageously, products can be directly extracted from droplets of interest for characterization such as with transmission electron microscopy (TEM) as shown in
Accordingly, in some embodiments, the yield-stress fluid is capable of a triggered change in mechanical properties such as permanently gelling.
In this regard, the method can further include a step of collapsing or gelling the yield-stress fluid for collecting or handling the compartments.
The present invention can also be used to investigate gaseous chemical reactions. In this regard, the compartments contain gases and the yield-stress fluid are impervious to the gases being tested. Advantageously, this allows for small volumes of gases to be used, which can be beneficial if the gaseous reagents are expensive, flammable and/or explosive.
Embedded droplet printing allows for small volume bio-assays with directly accessible droplets. In certain embodiments, the one or more compartments each includes a microorganism for use in bioassays.
Alternatively,
Similar to chemical applications, biological experiments are often extremely limited in terms of sample quantities and availability of expensive reagents. With embedded droplet printing, droplets containing biological samples are able to incubate for long periods of time but are continually directly addressable and recoverable; the droplets are stabilized without the use of molecular surfactants and are protected from exterior contaminants by the bath material. The lack of added surfactant molecules eliminates micelle formation as a mechanism for cross-droplet material transfer, an issue which has been reported to occur with droplet-based microfluidics systems. Additionally, compared to traditional droplet microfluidics assays containing microorganisms, the long term stability of the droplets of the present invention is not affected by mechanical agitation, wetting of the droplets with sidewalls, or the synthesis of new biomolecules by the microorganisms.
At low citric acid concentrations, nearly the entire bacterial population is alive. The bath material is permeable to oxygen and thus the bacteria are able to survive as long as there are sufficient nutrients and low concentrations of waste within the growth media droplets. At a critical concentration above 2 mg/mL of citric acid, the bacteria rapidly die. As with many conventional microfluidic techniques, in general, embedded droplet printing would allow for faster drug screening compared to traditional batch methods. If desired, the droplet contents could be transferred onto an agar plate to detect the presence of any colony-forming units.
The present invention also allows for one to easily extract fluid and samples from a given droplet for further culturing outside the bath if desired or for proteomic analysis. By combining extraction with secondary injection, one could remove waste and supply new growth media for extended periods of culturing inside the bath. In embedded droplet printing, as long as the buoyant stress does not exceed the yield stress (Equation 1), the droplets act as chambers that would be bound in size only by the overall dimensions of the yield-stress fluid bath; this means that biological samples could potentially grow inside a droplet that continually scales up in size to accommodate them via the injection of additional fluid. A unique attribute of this is that it is possible to reconfigure and merge two droplets at some selected time point by pushing them into contact using a nonwetting nozzle and then bridging them, which would not be possible with well-plate experiments. This feature could be exploited to simulate an infection in which one host grows for some time before encountering another species or multispecies studies to better understand the microbiome.
The one or more volumes can be provided by a microfluidic system 1740. The microfluidic system can be provided in communication with an inlet of the nozzle. The microfluidic system can be modulated to provide the one or more volumes. Alternatively, the flow of the input fluid can be selectively perturbed to form one or more volumes via a pump 1730.
The yield-stress bath 1710 can optionally be located on a translation stage 1750. The translation stage 1750 allows the displacement of the yield-stress fluid relative to the nozzle.
Accordingly, there is also provided an apparatus for forming one or more compartments, including: a) a nozzle including an outlet, the outlet for introducing one or more volumes; b) a yield-stress fluid, the yield-stress fluid in contact with the outlet of the nozzle; and c) a controller configured to displace the nozzle and/or the yield-stress fluid relative to each other to introduce one or more volumes into the yield-stress fluid to thereby form one or more compartments from the one or more volumes.
The apparatus can further include a controller configured to selectively perturb a flow of an input fluid for forming one or more volumes.
In certain embodiments, the selective perturbation of the flow of the input fluid is synchronised to the displacement the nozzle and/or the yield-stress fluid relative to each other. In certain embodiments, the selective perturbation of the flow of the input fluid is a periodic variation of the flow of the input fluid. In other embodiments, the controller configured to selectively perturb the flow is a compartment/volume generator. The compartment generator can rely on Rayleigh instability, or can be based on a stop-flow modulator.
The apparatus may further include a microfluidic system in communication with an inlet of the nozzle. Alternatively, the apparatus may further include a pumping system in communication with the inlet of the nozzle. The same or a separate microfluidic system and/or pumping system can also be in communication with the yield-stress fluid.
In certain embodiments, the yield-stress fluid is contained in a vessel.
In certain embodiments, nozzle is displaceable relative to the vessel. Alternatively, the vessel is displaceable relative to the nozzle. In certain embodiments, the displacement is in Cartesian coordinates. In some embodiments, the yield-stress fluid is mounted on an X, Y-axis motion-controlled stage and one or more nozzles mounted on a Z-axis motion controller. The motion controllers can be programmed or operated to move in a determined pattern at a pre-determined and controlled movement rate.
High degrees of particle uniformity and sphericity are key manufacturing targets for enhancing powder flowability and processability. As an example for further improving suitability for manufacturing applications, a platform that enables the continuous production of embedded droplets and for use to produce flowable pharmaceutical particles is exemplified. The setup in
In particular, the above example was setup using a robot arm and conveyor belt from Dobot and attaching a glass capillary nozzle with a nominal tip diameter of 30 μm to the end of the arm. Elastic bands are placed around the conveyor belt to create side walls of a nominal height of 6 mm and a center channel with a nominal width of 50 mm. The length of the belt is approximately 700 mm. Nozzles are connected to glass syringes mounted in a Harvard Apparatus syringe pump. The yield-stress fluid is Carbopol 980 in water, prepared to a concentration of 0.08 wt %. This fluid is deposited on the conveyor belt via a BT100S peristaltic pump through a flexible 1 cm silicone tube. Antisolvent crystallization using a solution of 50 mg/mL Naproxen (a model hydrophobic active pharmaceutical ingredient), 20 mg/mL ethyl cellulose (a model excipient) in ethyl acetate was performed.
To prevent the droplets from interacting, it requires δ to be greater than or equal to two times the radius of a droplet. Combined, these two conditions set the following boundaries on the belt speed in terms of the printing parameters, droplet size, and geometry of the belt setup:
The robot arm 1830 was programmed to print at a speed of 2000 mm/min and the syringe pump to inject at a volumetric flowrate of 200 μL/min, producing droplets approximately 800 microns in diameter at a total mass throughput of approximately 840 mg/h. For ethyl acetate diffusing in water, this results in a belt speed range of 0.8 to 1 mm/s to satisfy Equation 3. Choosing 1 mm/s, we produce the several mL of powder needed for rheological characterization in approximately 1 hour, significantly less time than would have been required with the semi-batch method. Particles were recovered from the yield-stress fluid bath via salt addition, and dried overnight before characterizing. The pharmaceutical powder is shown in the inset of
The platform can help pave the way for applying the unique functionality of embedded droplet printing to many continuous processes, including particle manufacturing. For pharmaceuticals, the use of similar platforms to enable precise, rapid, customized, and distributed drug manufacturing is envisioned.
Images of various phases of performance of another example method according to the present invention are shown in
Materials. Carbopol 980 (cross-linked polyacrylic acid particles) was obtained from Lubrizol. Ultrapure water (18.2 MΩ) was obtained from a Sartorius H2OPRO-DI-T Arium
Pro purifier. Sodium hydroxide (221465) and silicone oil (317667) were purchased from Sigma-Aldrich. Hydrophilic silica nanoparticles (AEROSIL 200) were obtained from Evonik Industries. PDMS and curing agent (Sylgard 184) were obtained from Dow Corning.
Water-based yield-stress fluid. Aqueous jammed suspensions of polymer microgels were prepared by mixing Carbopol 980 powder in ultrapure water at a concentration of 0.1 wt %. This solution was mixed for 30 min before being neutralized to pH 7 using a 1 M sodium hydroxide solution. This solution was placed in a vacuum chamber to remove air bubbles.
Oil-based yield-stress fluid. Silica nanoparticle oleogels were prepared by adding PDMS and then silicone oil to hydrophilic silica powder to achieve a final composition of 4 wt % silica, 48 wt % PDMS, 48 wt % silicone oil. This solution was first mixed vigorously by hand and then placed on an IKA Eurostar 40 digital stand mixer with a four-bladed propeller stirrer (R1342) to mix for 15 min at 2,000 rpm. If the bath was to be cross-linked, immediately prior to droplet printing, the yield-stress fluid and curing agent were mixed in a 10:1 ratio (wt/wt) and placed in a vacuum chamber to remove air bubbles. For consistent shear history and printing conditions, even if no cross-linker was to be added, immediately prior to droplet printing, this bath material would be vigorously mixed and the bubbles removed.
Motorized Platform System. For computer control of our droplet generation, we use the motorized platform of a 3Drag open-source 3D printer purchased from Open Electronics with an Arduino running Marlin firmware that accepts G-code commands through the freely available software Repetier. To the stationary frame of this platform, we mount nozzles that are either flattipped needles or surface-treated glass capillaries. Nozzle surfaces were modified to be preferentially wet by either the bath or the droplet phase, depending on the situation as discussed in Toolbox of Embedded Droplet Printing. For hydrophobic modification, nozzles were plasma cleaned and then treated with the commercial product Rain-X according to the manufacturer instructions. For hydrophilic modification, nozzles were plasma cleaned and submerged in ultrapure water for storage before printing. For secondary injection and extraction, prepulled glass micropipettes with a nominal tip diameter of 20 μm from FIVEphoton Biochemicals were used. Nozzles are connected via poly(tetrafluoroethylene) tubes (inner diameter 1 mm) purchased from Cole Parmer to 2.5-mL glass syringes (Hamilton GASTIGHT) purchased from Sigma-Aldrich mounted in Harvard Apparatus syringe pumps (PHD ULTRA 70-3007). We use custom-made 3D printed mounts to hold containers of yield-stress fluid in place on the motorized stage.
Materials. Light mineral oil (330779) was purchased from Sigma-Aldrich. For
Single-phase droplet printing and characterization. Nozzles were mounted to the stationary frame of the motorized platform described above and connected to a gas-tight glass syringe filled with mineral oil that was placed in a Harvard syringe pump. Petri dishes were filled with Carbopol solution to a thickness of ˜1 cm and mounted on the motorized stage. The nozzle was positioned at a nominal height of 5 mm above the bottom of the dish. The platform was programmed to move in a serpentine pattern at progressively faster speeds starting at 100 mm/min and ending at 3,000 mm/min, with 2.5 mm between rows. Row spacing of 2.5 mm and layer spacing of 5 mm were used to calculate the volumetric density of droplets. Just after nozzle translation initiated, the mineral oil was injected at a volumetric flow rate of 50 or 100 μL/min via the syringe pump. Droplets were imaged on an inverted Olympus IX71 microscope with a 4x or 10x objective. Droplet size and linear density measurements were made using ImageJ .
Rheological characterization of bath material. Rheological characterization of steady flow properties was performed on an AR-G2 combined motor/transducer rotational rheometer from TA Instruments, using parallel-plate geometry with a diameter of 40 mm and a Peltier temperature controller set to 25° C. A range of shear rates was applied from high to low and the apparent steady stress was recorded and fitted to a Herschel-Bulkley model using Origin 2019 and used to calculate the corresponding viscosity as a function of the shear rate.
Spherical Crystallization of Pharmaceutical Particles. Materials. Naproxen (N8280), ethyl cellulose (viscosity 10 cP 200689), and sodium chloride (S5886) were purchased from Sigma-Aldrich. Ethyl acetate (HiPerSolv
CHROMANOFORM for HPLC, ≥99.8%) was purchased from VWR International, LLC, and used as received. Ultrapure water (18.2 MΩ at 25° C.) was obtained from a Sartorius H2OPRO-DI-T Arium Pro purifier. Cell strainers with a mesh size of 100 μm were purchased from Fisher Scientific. Carbopol solution (0.1 wt %) was prepared as detailed above.
Production of crystallized particles. The drug-loaded injected phase was prepared by dissolving 50 mg of naproxen and 20 mg of ethyl cellulose per milliliter of ethyl acetate. This solution was delivered at a volumetric flow rate of 50 μL/min through a flat-tipped needle of nominal inner diameter 0.44 mm and nominal outer diameter 0.8 mm, submerged in the Carbopol bath material contained by a rectangular plastic box. The motorized platform was programmed to move at a constant linear speed of 1,000 mm/min in a multilevel serpentine pattern. After suspended droplets were generated, the bath and droplets were left undisturbed for at least 20 min (double the characteristic diffusion time estimated in the main text) for crystallization to proceed. To collapse the
Carbopol material, a volume of 1 M NaCI solution approximately equal to 5% of the volume of the bath was added followed by agitation of the bath. Particles were collected on a cell strainer, rinsed three times with ultrapure water, and vacuum dried at room temperature for at least 12 h prior to structural and polymorphic characterization.
Characterization of pharmaceutical particles. All samples were prepared for scanning electron microscopy on conventional stubs with a silicon wafer surface and were coated with ˜10 nm of platinum by sputter coating. A field-emission scanning electron microscope (JEOL JSM-6700F) at 5 kV accelerating voltage was used to image the particles. Polymorphic characterization of particles was analyzed using differential scanning calorimetry (DSC) and powder X-ray diffraction (PXRD) to examine their crystallinity. An X-ray diffractometer (Bruker; D8 Advance) was operated at 40 kV, 30 mA, and at a scanning rate of 1.06°/min over a range of 2θfrom 2.5-30° , using a Cu radiation wavelength of 1.54 Å. For DSC, a Mettler Toledo DSC 882 apparatus was used. Approximately 5 mg of sample was crimped in a sealed aluminum pan and heated at 5 ° C/min in the range of −20 to 180 ° C. using an empty sealed pan as a reference.
Embedded Chemical Reaction Chambers. Materials. Silver nitrate (99.9%) from Strem Chemicals and hydrazine hydrate (50 to 60%), sodium borohydride (98%), poly(vinyl alcohol) (molecular weight 67,000), and sodium citrate tribasic dihydrate (99%) from Sigma-Aldrich Co. Ltd. were used as received without any further purification. Ultrapure water (18.2 MQ at 25 ° C.) was obtained from a Milli-Q purifier. Petri dishes were purchased from Thermo Scientific.
Synthesis of silver seed solution. A total of 2 mL of 1 mM silver nitrate solution and 2 mL of 1 wt % aqueous poly(vinyl alcohol) solution are pipetted into a 20-mL glass bottle with a magnetic stirrer. The solution is stirred at 1,400 rpm tomix the precursor solution. After that, 2 mL of 2.5mMsodium borohydride solution is added rapidly into the glass bottle. The product is left to stir for 15 min.
Synthesis of silver nanoparticles. Aqueous growth solution consists of 2 mL of seed solution, 3 mL 100 mM sodium citrate solution, 5 mL of 1 wt % poly(vinyl alcohol) solution, and 5 mL of 40 mM hydrazine solution. A total of 10 mL of 1 mM silver nitrate solution is prepared as the precursor solution. These two solutions are filled into separate gas-tight Hamilton syringes. The solutions are delivered by using two syringe pumps (Harvard PHD ULTRA) into a coaxial glass capillary microfluidic device with the outermost surface (1.5 mm outer diameter) treated with Rain-X according to the manufacturer instructions to be hydrophobic and more readily wet by the bath material. The growth solution flows through the inner channel while the fluid in the outer channel is the precursor solution. Near the tip of the device, the two solutions meet and are printed as a single droplet in the oil-based yield-stress bath that contains cross-linking agent as described above. Before printing, the oil-based yield-stress bath was conditioned as described above in a Petri dish at a thickness of ˜1 cm. To produce silver nanoparticles of various sizes in each droplet, a ramp program is used, where the growth solution flow rate is decreased from 100 to 55 μL/min and the precursor solution flow rate is increased from 5 to 50 μL/min.
Characterization of silver nanoparticles. To prepare for TEM imaging, the silver nanoparticles solution in the printed droplet is extracted from the bath via a pipette and diluted with ultrapure water. A drop of this diluted sample is placed onto a 200-mesh copper grid, which is dried overnight and analyzed using TEM (JOEL 2010; accelerating voltage 200 K). In addition, the optical properties of these silver nanoparticles are identified via Resonon Hyperspectral Imaging Systems (Pika L with Backlight stage). Before characterization, the Petri dish containing the bath and droplets was placed on a hot plate at 35° C. for several hours to cross-link the bath. Droplets were characterized within 24 h to avoid any observed evaporation through the now-porous PDMS material. With this hyperspectral system, the intensity of the light passing through each silver nanoparticle-containing droplet is recorded and used to compute the absorbance of the silver nanoparticle solution. Droplets of the same size containing only ultrapure water were used to obtain a reference absorbance.
Biofilm formation in microarrays. P. aeruginosa mucA strains, which were fluorescently tagged with green fluorescent protein (eGFP), were used to make the microprinted droplet arrays of biofilms. Overnight cultures of P. aeruginosa strains were grown in
Luria—Bertani broth (5 g/L NaCI, 5 g/L yeast extract, 10 g/L tryptone) at 37° C. under shaking conditions (200 rpm). The overnight P. aeruginosa culture was diluted to an optical density at 600 nm (OD600) of 0.4, and 1 mL of the culture was taken in a glass Hamilton syringe and printed into the oil-based yield-stress bath at a volumetric flow rate of 50 μL/min via a syringe pump (Harvard PHD ULTRA). The motorized platform was programmed to move at a constant linear speed of 500 mm/min in a serpentine pattern. The arrayed microdroplets of bacteria culture were then allowed to grow for 24 h to form blobs of biofilm. Treatment, staining, and imaging. Citric acid (Sigma-Aldrich) at a concentration of 10mg/mL prepared using LB medium was added to each droplet in varying volumes via a hydrophilically modified glass capillary connected to a syringe pump so that the final concentration in each droplet ranging from 0 to 9 mg/mL propidium iodide (PI) (Sigma-Aldrich) was added to citric acid to a final concentration of 60 μM to visualize the dead bacteria in biofilms. The citric acid—PI mixture was added to the microarrays and incubated for 2 h to allow for the action of citric acid on the bacteria and staining of the dead cells by PI. The droplets were then imaged using a
FluoView 1000 confocal microscope (Olympus) with a 4x objective. Three image channels, GFP 488, Alexa 594, and bright field, were acquired from each droplet. The 3D image stacks of the representative droplets were also acquired to visualize the biofilm structure within the droplets before and after treatment with the citric acid.
It will be appreciated that many further modifications and permutations of various aspects of the described embodiments are possible. Accordingly, the described aspects are intended to embrace all such alterations, modifications, and variations that fall within the spirit and scope of the appended claims.
Throughout this specification and the claims which follow, unless the context requires otherwise, the word “comprise”, and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.
The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10201909264V | Oct 2019 | SG | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/SG2020/050556 | 10/2/2020 | WO |
