Patent Application
20240050950
This disclosure relates to gradient-based microfluidic circuits, devices, and methods for performing an assay.
To decipher the functions and side-effects between an organic small-molecule (approximately <900 Daltons, but even larger molecules may be used) and a biological species (e.g., live eukaryotic and prokaryotic cells), conventional biological assays often use 96 well-plates, in which each well is used to test a different small-molecule concentration (Alsenaid et al., “Biologics and Small Molecules in Patients with Scalp Psoriasis: A Systematic Review,” J. Dermat. Treatment: 1-10 (2020), Mosquera et al., “Cellular Uptake of Nanoparticles Versus Small Molecules: A Matter of Size,” Accounts of Chemical Research 51:2305-2313 (2018); Sarzi-Puttini et al., “Systemic Rheumatic Diseases: From Biological Agents to Small Molecules,” Autoimmunity reviews 18(6):583-592 (2019)). However, sample preparation is time-consuming, costly, and labor-intensive, often requiring large reagent volumes (Lamb et al., “The Connectivity Map: Using Gene-Expression Signatures to Connect Small Molecules, Genes, and Disease,” Science 313:1929-1935 (2006); Stockwell, “Exploring Biology with Small Organic Molecules,” Nature 432:846-854 (2004)). To address these issues, robotics can shorten assay times (Reddy et al., “Point-of-Care Sensors for the Management of Sepsis,” Nature Biomed Eng. 2:640-648 (2018); Wootton et al., “Analog-to-Digital Drug Screening,” Nature 483:43-44 (2012)). However, such systems are generally bulky and expensive, which can impede their use in biological applications, particularly in resource-limited settings (Huang et al., “Smartphone-Based Analytical Biosensors,” Analyst 143(22):5339-5351 (2018); Liu et al., “Point-of-Care Testing Based on Smartphone: The Current State-of-the-Art (2017-2018),” Biosensors and Bioelectronics 132:17-37 (2019); Xu et al., “Discovery and Functional Characterization of a Yeast Sugar Alcohol Phosphatase,” ACS Chem. Biology 13:3011-3020 (2018)).
As an alternative, miniaturization of biological assays using microfluidics may be an ideal solution for improving throughput and lowering costs (Avesar et al., “Rapid Phenotypic Antimicrobial Susceptibility Testing Using Nanoliter Arrays,” Proc. Natl. Acad. Sci. 114(29):E5787-E5795 (2017); Azizi et al., “Nanoliter-Sized Microchamber/Microarray Microfluidic Platform for Antibiotic Susceptibility Testing,” Analytical Chemistry 90(24):14137-14144 (2018); Baltekin et al., “Antibiotic Susceptibility Testing in Less Than 30 Min Using Direct Single-Cell Imaging,” Proc. Natl. Acad. Sci. 114(34):9170-9175 (2017); Campbell et al., “Microfluidic Advances In Phenotypic Antibiotic Susceptibility Testing,” Biomedical Microdevices 18(6):103 (2016); Hong et al., “Antibiotic Susceptibility Determination Within One Cell Cycle at Single-Bacterium Level by Stimulated Raman Metabolic Imaging,” Analytical Chemistry 90(6):3737-3743 (2018); Kao et al., “Gravity-Driven Microfluidic Assay for Digital Enumeration of Bacteria and for Antibiotic Susceptibility Testing,” Lab on a Chip 20(1):54-63 (2020); Kim et al., “Recent Developments of Chip-Based Phenotypic Antibiotic Susceptibility Testing,” BioChip Journal 13(1):43-52 (2019); Li et al., “Adaptable Microfluidic System for Single-Cell Pathogen Classification and Antimicrobial Susceptibility Testing,” Proc. Natl. Acad. Sci. 116(21):10270-10279 (2019); Mohan et al., “A Multiplexed Microfluidic Platform for Rapid Antibiotic Susceptibility Testing,” Biosensors and Bioelectronics 49:118-125 (2013); Yang et al., “All-Electrical Monitoring of Bacterial Antibiotic Susceptibility in a Microfluidic Device,” Proc. Natl. Acad. Sci. 117(20):10639-10644 (2020)). Small-molecule concentration-based biological assays can be performed in microfluidic systems with significantly improved precision (Leonard et al., “Unraveling Antimicrobial Susceptibility of Bacterial Networks on Micropillar Architectures Using Intrinsic Phase-Shift Spectroscopy,” ACS Nano 11:6167-6177 (2017); Li et al., “Adaptable Microfluidic System for Single-Cell Pathogen Classification and Antimicrobial Susceptibility Testing,” Proc. Natl. Acad. Sci. 116:10270-10279 (2019); Syal et al., “Antimicrobial Susceptibility Test with Plasmonic Imaging and Tracking of Single Bacterial Motions on Nanometer Scale,” ACS Nano 10:845-852 (2016)). For example, one of the most studied platforms for this application uses a well-known “Christmas tree” technique (Jang et al., “An Integrated Microfluidic Device for Two-Dimensional Combinatorial Dilution,” Lab on a Chip 11(19):3277-3286 (2011); Kim et al., “A Programmable Microfluidic Cell Array for Combinatorial Drug Screening,” Lab on a Chip 12(10):1813-1822 (2012); Lim et al., “A Microfluidic Spheroid Culture Device with a Concentration Gradient Generator for High-Throughput Screening of Drug Efficacy,” Molecules 23(12):3355 (2018)). This technique produces precise concentrations of a drug in a microfluidic chip, but it needs precision instruments (such as syringe pumps) to robustly control the flowrates of two loading fluids. Moreover, such designs require time-consuming sample-loading protocols that cannot be easily automated, and more importantly, lack enough throughput to enable simultaneous testing of negative controls and a wide concentration range of positive samples in a single test.
The present disclosure is directed to overcoming these and other deficiencies in the art.
One aspect of the present disclosure relates to a microfluidic circuit comprising an inlet port; an outlet port; a main channel fluidically connecting the inlet port and the outlet port; and a series of dead-end microchambers of differing volumes, where each microchamber is individually fluidically connected to the main channel via a side channel.
Another aspect of the present disclosure relates to a microfluidic device comprising a support layer; a substrate layer disposed on the support layer; and one or more microfluidic circuits of the present disclosure, where the one or more circuits are disposed within the substrate layer.
A further aspect of the present disclosure relates to a method for performing an assay. This method involves loading a first reagent solution into the inlet port of a microfluidic device of the present disclosure. A second reagent solution is loaded into the inlet port, and then an isolating solution is loaded into the inlet port. The method further involves detecting an interaction between the first reagent solution and the second reagent solution in one or more of the microchambers.
The present disclosure relates to a novel multi-volume microchamber-based microfluidic (“MVM2”) platform that is designed to produce a spontaneous and broad gradient of small-molecule concentrations within a single test. Antibiotic susceptibility testing and sugar phosphate toxicity (for bacteria and yeast cells, respectively) (Gibney et al., “Common and Divergent Features of Galactose-1-Phosphate and Fructose-1-Phosphate Toxicity In Yeast,” Molecular Biology Cell 29(8):897-910 (2018); Johnston et al., “Nitrate and Phosphate Transporters Rescue Fluoride Toxicity in Yeast,” Chemical Research Toxicology 32(11):2305-2319 (2019); Machado et al., “The Galactose-Induced Decrease in Phosphate Levels Leads to Toxicity in Yeast Models of Galactosemia,” Biochimica et Biophysica Acta (BBA)-Molecular Basis of Disease 1863(6):1403-1409 (2017); Xu et al., “Discovery and Functional Characterization of a Yeast Sugar Alcohol Phosphatase,” ACS chemical biology 13:3011-3020 (2018); which are hereby incorporated by reference in their entirety) were studied as two clinical models to demonstrate the versatility of the MVM2 platform. The loading time, which is the only parameter needed to be controlled by an operator for running the MVM2 platform, was obtained for a wide-range of commercial biological small-molecules in the market including anticancer drugs, antibiotics, and antifungals. Overall, with the MVM2 design, it is possible to rapidly determine precise effects of small-molecules in a broad concentration range with high throughput and low cost, and in a manner that is readily adaptable for automation.
Compared with previous milestones in developing microfluidic platforms for biological assays, the microfluidic circuit and microfluidic device of the present disclosure is, to the best of Applicant's knowledge, the first microfluidics device that is able to: (i) test small-molecules on both eukaryotic and prokaryotic cells; (ii) work in a high-throughput mode with an extended range of small-molecule concentrations (e.g., three orders of magnitude), while also including negative controls; (iii) exploit a low-cost microfluidics chip (˜$1 each) using a facile operation protocol; and (iv) prepare the desired sample concentrations precisely using fluid dynamics with minimal human intervention. Moreover, future integration of this platform with other technologies, such as complementary metal oxide semiconductor imaging, or electrochemical responses, could employ this platform for numerous biological assays, such as cancer cell biology, cell signaling, protein/small-molecule interactions, pesticide analysis, etc. Building from this MVM2 concept, further advanced platforms made possible by the easily modified MVM2 features are envisioned, which could be applied to even broader future biological and non-biological analytical applications.
fully saturates the side-channel and microchamber R1, while others with lower diffusion coefficients are unable to do so (over an unlimited loading time, i.e., ∞, the small-molecule concentration in all microchambers will equal one regardless of the diffusion coefficient). In
can almost saturate the microchamber R1-R5.
The present disclosure relates to gradient-based microfluidic circuits, devices, and methods for performing an assay.
Unless otherwise indicated, the definitions and embodiments described in this and other sections are intended to be applicable to all embodiments and aspects of the present application herein described for which they are suitable as would be understood by a person skilled in the art.
Singular forms “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise. Thus, for example, a reference to “a method” includes one or more methods, and/or steps of the type described herein and/or which will become apparent to those persons skilled in the art upon reading this disclosure. In another example, reference to “a compound” includes both a single compound and a plurality of different compounds.
The term “about” includes being within a statistically meaningful range of a value. Such a range can be within an order of magnitude, preferably within 50%, more preferably within 20%, still more preferably within 10%, and even more preferably within 5% of a given value or range. The allowable variation encompassed by the term “about” depends on the particular system under study, and can be readily appreciated by one of ordinary skill in the art.
The term “and/or” as used herein means that the listed items are present, or used, individually or in combination. In effect, this term means that “at least one of” or “one or more” of the listed items is used or present.
In understanding the scope of the present disclosure, the term “comprising” and its derivatives, as used herein, are intended to be open ended terms that specify the presence of the stated features, elements, components, groups, integers, and/or steps, but do not exclude the presence of other unstated features, elements, components, groups, integers and/or steps. The foregoing also applies to words having similar meanings such as the terms, “including”, “involving”, “having”, and their derivatives. The term “consisting” and its derivatives, as used herein, are intended to be closed terms that specify the presence of the stated features, elements, components, groups, integers, and/or steps, but exclude the presence of other unstated features, elements, components, groups, integers and/or steps. The term “consisting essentially of”, as used herein, is intended to specify the presence of the stated features, elements, components, groups, integers, and/or steps as well as those that do not materially affect the basic and novel characteristic(s) of features, elements, components, groups, integers, and/or steps. In embodiments or claims where the term comprising (or the like) is used as the transition phrase, such embodiments can also be envisioned with replacement of the term “comprising” with the terms “consisting of” or “consisting essentially of.” The methods, kits, systems, and/or compositions of the present disclosure can comprise, consist essentially of, or consist of, the components disclosed.
Certain terms employed in the specification, examples, and claims are collected herein. Unless defined otherwise, all technical and scientific terms used in this disclosure have the same meanings as commonly understood by one of ordinary skill in the art to which this disclosure belongs.
Preferences and options for a given aspect, feature, embodiment, or parameter of the disclosure should, unless the context indicates otherwise, be regarded as having been disclosed in combination with any and all preferences and options for all other aspects, features, embodiments, and parameters of the disclosure.
One aspect of the present disclosure relates to a microfluidic circuit comprising an inlet port; an outlet port; a main channel fluidically connecting the inlet port and the outlet port; and a series of dead-end microchambers of differing volumes, where each microchamber is individually fluidically connected to the main channel via a side channel.
One embodiment of a microfluidic circuit of the present disclosure is illustrated in
Microfluidic circuit 10 also has a series of dead-end microchambers 18A and 18B, each of which is individually fluidically connected to main channel 16 via side channels 20A and 20B. In the embodiment illustrated in
In the embodiment illustrated in
In the microfluidic circuits and devices of the present disclosure, the side channels may have any shape, length, channel width, etc., desirable for a particular application, including, in some embodiments, having essentially no length or shape at all, such as when the side channel essentially constitutes nothing more than an opening from the main channel to the microchamber. Thus, in some embodiments, the microchamber may reside directly adjacent the main channel with only an opening between the main channel and microchamber. Such a structure may be suitable in assays involving test agents larger than small molecules (e.g., proteins or enzymes). In some embodiments, it may be useful to have a side channel of greater or lesser length, with some or no bends or curves (e.g., straight), and with greater or lesser channel width. In some embodiments, all of the side channels of a particular microfluidic circuit are identical in their dimensions, and some embodiments, at least one or some of the side channels of a particular microfluidic circuit vary in one or more ways from other side channels of a microfluidic circuit. In some embodiments, microchambers are staggered along the main channel such that the side channels are not directly opposite each other. Being able to vary the dimensions and/or design of the side channel enables flexibility in achieving desired concentration gradients in the microchambers, and allows adaptation of the microfluidic circuit based on the size of test agents (and other variables) in performing assays.
The embodiment illustrated in
In the embodiment illustrated in
The size of each of the component parts of the microfluidic circuit may vary according to particular application or use. For example, and without limitation, in some embodiments, the side channels may comprise an opening with a width of about 40-100 μm, or 60-80 μm, or about 70 μm, or any particular dimension or range of dimensions therein. In some embodiments, the side channel opening width is 70 μm. The side channels may also comprise a serpentine configuration that forms a switchback configuration (see
The microchambers may comprise a diameter of between about 200-1500 μm, or 250-1130 μm, or 250, 282, 342, 401, 498, 565, 693, 800, 893, 979, 1057, and/or 1130 μm, or any particular dimension or range of dimensions therein. The microchambers may comprise a diameter larger than 1500 μm or smaller than 200 μm. The inlet port may comprise a diameter of about 500-1500 μm. The outlet port may comprises a diameter of about 500-1500 μm. The inlet and outlet ports may be different sizes, and may have a diameter larger than 1500 μm, or smaller than 500 μm.
As illustrated in
In the embodiment illustrated in
The microfluidic circuit of the present disclosure pertains to a fluidic system of ports, channels, and microchambers, all fluidically connected. Since, as discussed in the Examples below, the particular structure of this fluidically connected system enables assays to be performed based on the unique structure of the circuit, the circuit may be used in a variety of contexts or on a variety of platforms. One particular platform where the microfluidic circuit of the present disclosure is useful is a chip-like platform. For example, the microfluidic circuit may be formed into a planar material to create the system of ports, channels, and microchambers, accessible for input or output of fluid only through the ports.
Thus, another aspect of the present disclosure relates to a microfluidic device comprising a support layer; a substrate layer disposed on the support layer; and one or more microfluidic circuits of the present disclosure, where the one or more circuits are disposed within the substrate layer.
In some embodiments, the microfluidic device of the present disclosure includes an aggregation of separate parts, for example, but not limited to, ports, fluid channels, capillaries, joints, chambers, and layers which, when appropriately mated or joined together, form the microfluidic device of the present disclosure. In some embodiments, the microfluidic device may include a top portion, a bottom portion, and an interior portion, one or more of which substantially defines ports, channels, and chambers of the microfluidic device.
In some embodiments, the bottom portion may be a solid support or a substrate that is substantially planar in structure, and which has a substantially flat upper surface. A variety of materials may be used to form the solid support and/or a substrate, which itself is formed on or connected to the solid support. The support and/or substrate materials should be selected based upon their compatibility with known microfabrication techniques, for example, photolithography, 3-D printing, wet chemical etching, laser ablation, air abrasion techniques, injection molding, embossing, and other techniques, or based on the application being used. The support and/or substrate materials are also generally selected for their compatibility with the full range of conditions to which the microfluidic devices may be exposed, including extremes of pH, temperature, salt concentration, and/or application of electric fields, should these be relevant in performing assays using the microfluidic device of the present disclosure.
In some embodiments, suitable support and/or substrate materials include, without limitation, glass, pyrex, glass ceramic, polymer materials, semiconductor materials, and combinations thereof. In some embodiments, the support and/or substrate material may include materials normally associated with the semiconductor industry in which microfabrication techniques are regularly employed, including, e.g., silica based substrates such as glass, quartz, silicon, or polysilicon, as well as other substrate materials, such as gallium arsenide and the like. In the case of semiconductive materials, it will often be desirable to provide an insulating coating or layer, e.g., silicon oxide or silicon nitride, over the support or substrate material, particularly where electric fields are to be applied. In some embodiments, the support layer comprises glass.
Exemplary polymeric materials include, without limitation, plastics such as polymethylmethacrylate (PMMA), polycarbonate, polytetrafluoroethylene (TEFLON™), polyvinylchloride (PVC), polydimethylsiloxane (PDMS), and polysulfone. Other plastics can also be used. Such materials are readily manufactured from microfabricated masters, using well known molding techniques, such as injection molding, embossing or stamping, or by polymerizing the polymeric precursor material within a mold. Such polymeric substrate materials are known for their ease of manufacture, low cost and disposability, as well as their general inertness to most extreme reaction conditions. These polymeric materials may include treated surfaces, for example, derivatized or coated surfaces, to enhance their utility in the microfluidic device or, for example, to provide enhanced fluid direction should that be a needed factor in carrying out assays using microfluidic devices described herein. In some embodiments, the substrate layer comprises PDMS. PDMS is also gas permeable, which allows air to escape into the PDMS substrate from the microchambers during loading of solutions so that the microchambers can be completely filled.
In some embodiments, the material used to build the interior portion, which may at least partially define the microfluidic circuit, is biocompatible and resistant to biofouling. Because the active surface area of the microfluidic device may be only a few μm2, the material used to form the interior portion (i.e., the microfluidic circuit) should have a resolution that enables the structuring of both small cross-sectional area channels (on the order of about 2-3 μm width and about 1-2 μm height) and larger cross-sectional area channels (on the order of about 25 to about 500 μm width and/or height, or other dimensions described herein). Several existing materials, widely used for the fabrication of microfluidic circuits, can address these basic needs.
Two categories can be distinguished among them: those based on glasses, such as glass, Pyrex, quartz, etc. (Ymeti et al., Biosens. Bioelectron 20:1417-1421 (2005), which is hereby incorporated by reference in its entirety); and those based on polymers such as polyimide, photoresist, SU-8 negative photoresist, polydimethylsiloxane (PDMS), 3-D printing, silicone elastomer PDMS (McDonald et al., Electrophoresis 21:27-40 (2000), which is hereby incorporated by reference in its entirety), liquid crystal polymer, Teflon, etc.
While glass materials have good chemical and mechanical resiliency, their high cost and delicate processing make them less frequently used for this kind of application. In contrast, polymers have gained wide acceptance as the materials of choice for fluidics (including microfluidics) applications. Moreover, structuring technologies involved in their use, such as bonding, molding, embossing, melt processing, and imprinting technologies, are now well developed (Mijatovic et al., Lab on a Chip 5:492-500 (2005), which is hereby incorporated by reference in its entirety). An additional advantage of polymer-based microfluidic systems is that valves and pumps made with the same material may be readily integrated (Unger et al., Science 288:113-116 (2000), which is hereby incorporated by reference in its entirety).
PDMS and SU-8 resist are particularly well studied as raw materials for the construction of microfluidic systems. While both of them are optically transparent, their mechanical and chemical comportment are strongly disparate. SU-8 is stiffer (Blanco et al., J Micromechanics Microengineering 16:1006-1016 (2006), which is hereby incorporated by reference in its entirety) than PDMS, and so the structuring techniques of these two materials are different. Their chemical properties are an important aspect for the desired application. They both have a hydrophobic surface after polymerization, which can lead to an attachment of the proteins onto the PDMS walls, and can fill the channel in case of small cross-section. Both the surface of PDMS and of SU-8 can be treated with a surfactant or by plasma to become hydrophilic (Nordstrom et al., J Micromechanics Microengineering 14:1614-1617 (2004), which is hereby incorporated by reference in its entirety). The composition of SU-8 can also be modified before its structuring to become hydrophilic after polymerization (Chen and Lee, J Micromechanics Microengineering 17:1978-1984 (2007), which is hereby incorporated by reference in its entirety). Fouling of the channel surface via nonspecific binding is an obvious concern for any microfluidic application. Anecdotal evidence suggests that SU-8 is less prone to this, but it is important to note that chemical treatment methods are also available for improving the performance of PDMS (Lee and Vörös, Langmuir 21:11957-11962 (2004), which is hereby incorporated by reference in its entirety).
Support and/or substrate materials can also be a combination of a glass or Pyrex base and a polymer lid, which together define the microfluidic circuit. In some embodiments, the microfluidic circuit(s) of the microfluidic device of the present disclosure is fabricated as a mold on a silicon wafer to which a layer of PDMS substrate or other material, without limitation, is applied to form the microfluidic device. The PDMS substrate is then peeled off the mold, and applied to a support material, such as glass, and treated to seal the microfluidic circuit(s), as further described herein, and in the Examples. In some embodiments, the “ceiling” of the microfluidic circuit is formed of the contiguous substrate layer, such as when the PDMS material is poured on a silicon wafer mold. In some embodiments, the microfluidic device is fabricated as microscale grooves or indentations formed into the upper surface of the substrate or bottom surface of the polymer lid using microfabrication techniques. In some embodiments, the lower surface of the top portion of the microfluidic device, which top portion (also referred to as top layer) can comprise a second planar substrate, can be overlaid upon and bonded to the surface of the bottom support or substrate, sealing the channels and/or chambers (the microfluidic circuit) of the device at the interface of these two components. Bonding of portions to form a microfluidic device comprising a microfluidic circuit may be carried out using a variety of known methods, depending upon the nature of the support and/or substrate material. For example, in the case of glass supports/substrates, thermal bonding techniques may be used which employ elevated temperatures and pressure to bond one portion of the device to another portion (e.g., a top portion to a bottom portion). Polymeric substrates may be bonded using similar techniques, except that the temperatures used are generally lower to prevent excessive melting of the substrate material. Alternative methods may also be used to bond polymeric parts of the device together, including acoustic welding techniques, or the use of adhesives, for example, UV curable adhesives.
Like the microfluidic circuit, the microfluidic device of the present disclosure is not limited in its physical dimensions and may have any dimensions that are convenient for a particular application. For the sake of compatibility with current laboratory apparatus, microfluidic devices with external sizes of a standard microscope slide or smaller can be easily made. Other microfluidic devices can be sized such that the device fits a standard size used on an instrument, for example, the sample chamber of a mass spectrometer or the sample chamber of an incubator. The microfluidic circuit within the microfluidic device may have any shape, without limitation, such as rectangular, square, oval, circular, or polygonal. The microfluidic circuit, and/or the microchambers and channels that make the microfluidic circuit in the microfluidic device may have square or round bottoms, V-shaped bottoms, flat bottoms, or U-shaped bottoms, without limitation. The shape of the chamber and/or channel bottoms need not be uniform on a particular chip, but may vary. The chambers in the microfluidic device of the present disclosure may have any width-to-depth ratio, which may vary from chamber to chamber. The microchambers wells, channels, and other associated features in the microfluidic device of the present invention may have any volume or diameter which is compatible with the requirements of the sample volume being used. The microchambers or channels can function as a reservoir, a mixer, or a place where chemical or biological reactions take place. In some embodiments, the biological reactions take place in the microchambers.
One embodiment of a microfluidic device of the present disclosure is illustrated in
Microfluidic device 100 of
Microfluidic circuit 110 of
Microfluidic circuit 110 also has a series of dead-end microchambers 118A and 118B, each of which is individually fluidically connected to main channel 116 via side channels 120A and 120B. In the embodiment illustrated in
In the embodiment illustrated in
In the embodiment illustrated in
Expanded portion 2 of microfluidic device 100 is illustrated in
A perspective view of microfluidic device 100 shown in
In the embodiment of microfluidic device 100 illustrated in
Movement of fluids into and throughout the microfluidic device of the present disclosure can be controlled manually by introducing solutions into the inlet ports and by diffusion, or by pump connected to one or more inlet ports. Alternatively, the introduction of fluids into and throughout the microfluidic device can be controlled automatically using an operating system programmed to regulate the timing of one or more pipette-like dispersion system, and/or one or more valves responsible for regulating the introduction of fluid (e.g., first reagent, second reagent, isolating solution, etc.). An opening can be introduced through the top layer and substrate material at the inlet and outlet ports for this purpose with a tissue culture puncher, as one non-limiting example (see
Because, as discussed below, one embodiment of methods of using the microfluidic device of the present disclosure is sequential in nature, various systems associated with the microfluidic device may be automated and associated with software that runs on a computer and is easily programmable and modifiable, although one appeal of the microfluidic device of the present disclosure is that it is simple to operate manually by simply introducing a fluid into a microfluidic circuit via an inlet port, and permitting solutions to diffuse throughout the microfluidic circuit, including into microchambers to create gradients by simple diffusion. However, computers in microfluidic systems could also be used to control system processes and receive signals for interpretation. For example, the computer can control a robotic sub-system that retrieves samples or reagents from storage as needed. The computer can control specimen stations to designate the order of drawing samples and reagents for receipt into the microfluidic device. Pressure differentials and electric potentials can be applied to microfluidic devices by the computer through computer interfaces known in the art, thereby controlling pump devices and valves to regulate the flow of reagents into and out of the system, although these are not necessary in the carrying out assays using the microfluidic device of the present disclosure. The computer can be a separate sub-system, it can be housed as an integrated part of a multi-assay instrument, or dispersed as separate computers in modular subsystems.
A computer system for controlling processes and interpreting detector signals can be any known in the art. The computer can also include a software program, which, for example, is useful for correlating, analysis, and evaluation of detector signals, evaluation of the detector signals to quantify activity, etc. The computer can be in functional communication with the one or more valves controlling the inflow and outflow of fluids, flow rate controllers to control the rate and direction of flow inside the microfluidic device. The computer can also control power circuits, control mechanical actuators, receive the information through communication lines, store information, interpret detector signals, make correlations, etc.
Systems including the microfluidic device of the present disclosure can include, e.g., a digital computer with data sets and instruction sets entered into a software system to practice the assay methods described herein. The computer can be a personal computer with appropriate operating systems and software control, or a simple logic device, such as an integrated circuit or processor with memory, integrated into the system. Software for interpretation of detector signals is available, or can easily be constructed by one of skill using a standard programming language such as Visualbasic, Fortran, Basic, Java, or the like.
The microfluidic device of the present disclosure can be in fluidic contact with variety of specimen manipulation stations. These specimen stations can be, for example, autosamplers, such as sample carousels holding multiple small molecule libraries in a circular tray that can be rotated sequentially or randomly to align the library containers with one or more pipettors. The pipettors can be on actuated arms that can dip the pipettor tube into the specimen for sampling or delivery.
In some embodiments, the samples or reagents are of very small volume, for example, as is typical of many molecular libraries. Sampling from such libraries, e.g., on microwell plates or microarray slides, is typically accomplished with robotic systems that precisely position the pipettor tip in the micro specimen. In embodiments where the library members are retained in dehydrated form, it can be convenient to sample by ejecting a small amount of solvent from the pipettor to dissolve the specimen for receipt into the microfluidic device of the present disclosure.
Reagents can be any composition useful in assays suitable for being carried out with the microfluidic device of the present disclosure, for example, chemicals or biomolecules capable of interacting with target molecules, controlling the reaction conditions, or generating a detectable signal. Reagents are typically one or more molecules in a solution that can flow into contact with the target in a chamber. Reagents can include a chromophore that reacts with the target to provide a changed optical signal.
Within the microfluidic device are microchambers where the first reagent and second reagent come into contact in the particular concentration defined by the size of the microchamber. These microchambers can also be configured to provide conditions amenable to provide a detectable signal resulting from the contact between targets, if necessary.
Microfluidic devices can also have detection regions that can be monitored by detectors which detect the signals, for example, resulting from cellular growth or density, contact of targets, a signal from a reagent that has reacted with a sample analyte, the absence of a detectable signal (interpretable, e.g., as the absence of sample analyte at a level adequate to generate a signal above the sensitivity of the detector), a signal amplitude related to a quantity of a sample analyte, and/or the like. The detection regions are, in some embodiments, the microchambers of the microfluidic circuit. For example, detector regions can incorporate sensors such as pH electrodes and/or conductivity meter electrodes. Detection regions can comprise one or more microchambers transparent to certain light wavelengths so that light signals, such as, absorbance, fluorescent emissions, chemoluminescence, and the like, can be detected. Detectors can be located in the microfluidic device, or proximate to the device, in an orientation to receive signals resulting from the sample contact with the reagent. Detectors can include, e.g., a nucleic acid sequencer, a fluorometer, a charge coupled device, a laser, a photo multiplier tube, a spectrophotometer, scanning detector, microscope, or a galvo-scanner. Signals detected from interactions of reagents and samples can be, e.g., absorbance of light wavelengths, light emissions, radioactivity, conductivity, refraction of light, etc. The character of signals, such as, e.g., the amplitude, frequency, duration, counts, and the like, can be detected.
Detectors can detect signals from detector regions described by physical dimensions, such as a point, a line, a surface, or a volume from which a signal can emanate. In some embodiments, the detector can scan an image of a surface or volume for signals resulting from interactions of reagents and samples. For example, a detector can contemporaneously image multiple parallel microchambers carrying reaction mixtures from multiple analyses to detect results of several different assays at once.
The detectors can transmit detector signals that express characteristics of resultant signals received. For example, the detector can be in communication with an output device, such as an analog or digital gage, that displays a value proportional to a resultant signal intensity. The detector can be in communication with a computer through a data transmission line to transmit analog or digital detector signals for display, storage, evaluation, correlation, and the like.
In some embodiments, detecting an interaction between the first reagent solution and the second reagent solution comprises detecting a visual signal. In some embodiments, detecting an interaction between the first reagent solution and the second reagent solution comprises detecting a fluorescent signal. In some embodiments, detecting an interaction between the first reagent solution and the second reagent solution comprises detecting a colorimetric signal. In some embodiments, detecting an interaction between the first reagent solution and the second reagent solution comprises detecting a spectrophotometric signal.
A further aspect of the present invention relates to kits that include a microfluidic device of the present disclosure and, optionally, one or more pools of reagents for carrying out assays suitable to the microfluidic device described herein.
Another aspect of the present disclosure relates to a method for performing an assay. This method involves loading a first reagent solution into the inlet port of a microfluidic device of the present disclosure. A second reagent solutions is loaded into the inlet port, and then an isolating solution is loaded into the inlet port. The method further involves detecting an interaction between the first reagent solution and the second reagent solution in one or more of the microchambers.
Turning now to
Thus, in carrying out the method of the present disclosure, the pattern illustrated in
Other variations may also be used based on loading of a biological species, loading of a small molecule test agent, loading an isolating solution, and achieving distribution of the small molecule test agent to detect effectiveness of the small molecule against the biological species at the various concentrations created in the microfluidic device.
In some embodiments, when loading the first reagent into the microfluidic circuit, the first reagent fills the microchambers. Creating an even distribution of first reagent throughout the microfluidic circuit ensures that true concentration gradients of the second reagent are achieved. In some embodiments, the structure of the microchambers or side-channels may be changed to allow non-uniform reagents into the microchambers for further biological or non-biological assays.
In some embodiments, the second reagent solution is loaded into the inlet port(s) of the microfluidic device and allowed to diffuse throughout the circuit and into the microchambers. In some embodiments, a portion of the second reagent solution diffuses into the microchambers, thereby forming a concentration gradient of the second reagent solution within the microchambers from the inlet port to the outlet port. In some embodiments, a third reagent may be loaded prior to the isolating solution. In some embodiments, a fourth reagent may be loaded prior to the isolating solution. In some embodiments, a fifth, a sixth, a seventh, an eighth, or more than eighth reagent may be loaded prior to the isolating solution.
In some embodiments in carrying out the methods described herein, one microfluidic circuit is loaded with the second reagent at a time. In some embodiments in carrying out the methods described herein, one microfluidic circuit is loaded with the second reagent while the inlet and outlet ports of other microfluidic circuits of a microfluidic device are blocked, sealed, or closed. A blocking element may be used for blocking, sealing, or closing inlet and outlet ports of the microfluidic device of the present application. Blocking, sealing, or closing inlet and outlet ports may be carried out by any suitable means including, without limitation, by sealing the ports with tape, inserting a block agent into the ports, or fitting tubing into the ports and sealing the tubing. Blocking may be carried out reversibly to allow access to the inlet and outlet ports at various times during the methods described herein. Inlet and outlet ports may be open or blocked as needed for the loading of the first reagent, the second reagent, and the isolating solution(s). In some embodiments, the microfluidic inlet port and outlet port comprise a blocking element. Similar methods with different loading arrangement may be used depending on the application.
In carrying out the methods of the present application, the isolating solution is used to prevent transport or diffusion of the first reagent solution and second reagent solution from the microchambers. Suitable isolating solutions include, without limitation, a biocompatible oil such as glycerol, vegetable oil, and silicon oil. Other substances may be used, including, for example, those that are more viscous than water and/or are hydrophobic.
In some embodiments in carrying out the methods described herein, the microfluidic device is loaded with a first reagent solution into the inlet port of one or more of the microfluidic circuits, while the outlet ports are blocked (
In further embodiments in carrying out the methods described herein, the microfluidic device is then loaded with a second reagent solution for a specified time, followed by an isolating solution, as shown in
The microfluidic circuits and devices of the present disclosure can be used to perform a variety of assays. Accordingly, the first reagent may be any number of possible substances, including, for example and without limitation, a biological sample. In some embodiments, the biological sample comprises a prokaryotic cell or prokaryotic cell component. In some embodiments, the biological sample comprises a eukaryotic cell or eukaryotic cell component.
Suitable second reagent solutions may comprise an antimicrobial compound. Thus, the method of the present disclosure may be carried out to perform an assay testing the ability of a compound (e.g., small molecule) to act as an antimicrobial compound. Moreover, the method may be carried out to determine an effective or ideal concentration at which an antimicrobial compound may be used to achieve its desired effect.
Antimicrobial compounds are well known in the art. Non-limiting examples of antimicrobial compounds that may be used in the assay method of the present disclosure include, without limitation, Actinomycin D, Actinonin, Aculeacin A, Acycloguanosine (Aciclovir), Adenine 9-β-D-arabinofuranoside (Vidarabine), Alamethicin, L-Alanyl-L-1-aminoethylphosphonic acid (Alafosfalin), Albendazole (Methyl 5-(propylthio)-2-benzimidazolecarbamat), 17-(Allylamino)-17-demethoxygeldanamycin (Tanespimycin), Amastatin, Amikacin, 7-Aminoactinomycin D (7-ADD), 7-Aminocephalosporanic acid (7-ACA), 7-Aminodesacetoxycephalosporanic acid (7-ADCA), (+)-6-Aminopenicillanic acid (6-APA), Amoxicillin, Amphotericin B (Fungizone), Ampicillin (D-(—)-α-Aminobenzylpenicillin), Anhydroerythromycin A, (Erythromycin, or 2BR5PL6H3), Anisomycin (or, Flagecidin), Aphidicolin, Apicidin, Apoptolidin (FU 40A), Apramycin (Nebramycin II), Artesunate, Ascochlorin (Ilicicolin D, NSC 287492), Ascomycin (KD4185000), Azacitidine (Ladakamycin), Azithromycin, Azlocillin (D-α-([Imidazolidin-2-on-1-yl]carbonylamino)benzylpenicillin), Bacitracin, Bafilomycin A1 (4730700), Bafilomycin B1, Bestatin (Ubenimex), Bithionol, Blasticidine (Blasticidin S), Borrelidin, Brefeldin A (Ascotoxin, BFA, Cyanein, Decumbin), Caerulomycin A (Carulomycin A, Cerulomycin), Calcium ionophore III (ANTIBIOTIC A 23187, Calimycin), (S)-(+)-Camptothecin, (Camptothecin), Carbenicillin (α-Carboxybenzylpenicillin), Cefaclor, Cefalexin, Cefazolin, Cefixime, Cefmetazole, Cefoperazone, Cefotaxime ((Z)-Cefotaxime), Cefmetazole, Cefoperazone, Cefsulodin (Ulfaret), Ceftazidime, Ceftriaxone, Cephalexin, Cephalomannine (NSC 318735), Cephalothin (Cefalotin), Cephradine (Cefradine), Cercosporin, Cerulenin, Chloramphenicol, Chlorhexidine, Chloroquine, Chlortetracycline, Chromomycin A3, Chrysomycin A (MFCD07370133), Chrysomycin B (MFCD07370132), Cinoxacin, Clarithromycin, Clindamycin (Cleocin), Clofazimine, Clotrimazole, cloxacillin, Colistin, Concanamycin A (Folimycin), Cordycepin (3′-Deoxyadenosine), Coumermycin A1, Cryptotanshinone (Tanshinone C), Cycloheximide (Actidione, Naramycin A), D-Cycloserine, Cyclosporin A (Antibiotic S 7481F1, Cyclosporine), Cytochalasin D (Zygosporin A, 1632828), Cytochalasin B (Phomin), Dacarbazine ((E)-Dacarbazine), Daptomycin, Daunorubicin (Daunomycin), 10-Deacetylbaccatin III, Demeclocycline, 1-Deoxymannojirimycin, Dichlorophene, Dicloxacillin, Difloxacin, Dihydrostreptomycin, Dimetridazole, Dirithromycin, Doxorubicin, Doxycycline, Duramycin, Econazole, Embelin (Embelic acid, Emberine), Emetine, Enrofloxacin (Baytril), Erythromycin (E-Mycin, Erythrocin), Ethambutol ((+)-S,S-Ethambutol), Etoposide, Florfenicol (Aquafen, Nuflor), Flubendazol (Flumoxanal), Fluconazole, Flumequine, 5-Fluorocytosine (Flucytosine), Flurbiprofen, Formycin A (Adenosine, Formycin, NSC 102811), Fumagillin, Furazolidone, Fusaric acid (5-Butylpicolinic acid), Fusidic acid, G 418, Ganciclovir, Gatifloxacin, Gentamicin, Gliotoxin, gramicidin s, Griseofulvin, Herbimycin A, Honokiol, 8-Hydroxyquinoline, 4-Hydroxytamoxifen ((Z)-Afimoxifene), Hygromycin B (WK2130000), Ikarugamycin, Imipenem, Ionomycin, Irgasan, Itraconazole, Ivermectin Bla, Josamycin, K-252a, Kanamycin, Ketoconazole, Kirromycin (mocimycin), Lactoferricin B (metallibure), Leptomycin A, Leptomycin B, Levamisol (Levamisole), Levofloxacin, Lincomycin, Lomefloxacin, Lysobactin, Magainin I, Mebendazole, Meclocycline, N-Methyl-1-deoxynojirimycin (1524564), Metronidazole, Mevastatin, Miconazole, Minocycline, Mithramycin A (Plicamycin), Mitomycin C (Mitomycin), Monensin, Moxalactam (Latamoxef), Mupirocin, Myxothiazol, Nafcillin, Naftifine, Nalidixic acid, Narasin, Neocarzinostatin (Holoneocarzinostatin, NCS, NSC-69856, Zinostatin), Neomycin, Netilmicin, Netropsin (Congocidin, Sinanomycin), Niclosamide, Nigericin (Antibiotic K178, Antibiotic X464, Azalomycin M, Helexin C, Polyetherin A), Nikkomycin, Nitrofurantoin (Furadoxyl, Nitrofurantoine), Nonactin (Ammonium ionophore), Norfloxacin, Novobiocin, Nystatin (Fungicidin, Mycostatin), Ochratoxin A, Ofloxacin, Oligomycin A, Oxacillin, Oxolinic acid, Oxytetracycline, Paclitaxel, Paromomycin, Patulin, PD 404,182, Pefloxacin, Penicillin G (Benzylpenicillin), Pentamidine, Phenazine, Phleomycin (UNII:BN3E7WJN9X), Phosphomycin (Fosfomycin), Pimaricin (Natamycin), Pipemidic acid, Piperacillin, Pirarubicin (THP), Polymyxin B, Praziquantel, PUROMYCIN, Pyrazinecarboxamide (Pyrazinamide, Pyrazinoic acid amide), Pyronaridine, Pyrrolnitrin, Quinine, 8-Quinolinol (8-Hydroxyquinolin, Oxine), Radicicol, Rapamycin (Sirolimus), Reveromycin A (MFCD00912537), Ribavirin, Ribostamycin, Rifabutin (Ansamycin, Ansatipine (Farmitalia), LM-427, Mycobutin (Farmitalia)), Rifampicin (Rifampin, Rifamycin AMP), Rifapentine (DL 473), Rifaximin (Rifacol), Roxithromycin, Salinomycin, Sisomicin, Sorbic acid, Sordarin, Sparfloxacin, Spectinomycin, Spergualin, Spiramycin, Staurosporine (Staurosporine), Streptomycin, Streptonigrin (Bruneomycin, Nigrin), Streptozocin (Streptozotocin), Prothionamide, and Monensin.
Suitable second reagent solutions may also comprise an anticancer compound or drug. According to some embodiments, when the second reagent solution comprises an anticancer compound or drug, the first biological sample of the first reagent solution is a cancer cell or a component of a cancer cell. Thus, the method of the present disclosure may be carried out to perform an assay testing the ability of a compound (e.g., small molecule) to act as an anticancer drug. Moreover, the method may be carried out to determine an effective or ideal concentration at which an anticancer compound may be used to achieve its desired effect. Anticancer compounds are well known in the art. Non-limiting examples of anticancer compounds that may be used in the assay method of the present disclosure include, without limitation, Abemaciclib (Verzenio), Abiraterone acetate (Zytiga), Acalabrutinib, Afinitor (Everolimus), Aldara (Imiquimod), Alectinib, Alemtuzumab, Alimta (Pemetrexed Disodium), Aliqopa (Copanlisib Hydrochloride), Aloxi (Palonosetron Hydrochloride), Alunbrig (Brigatinib), Ameluz (Aminolevulinic Acid), Amifostine, Anastrozole, Apalutamide, Aprepitant, Arimidex (Anastrozole), Aromasin (Exemestane), Arranon (Nelarabine), Axitinib, Azacitidine (Vidaza), Azedra, Beleodaq (Belinostat), Bendamustine Hydrochloride, Bexarotene (Targretin), Bicalutamide (cosodex), BiCNU (Carmustine), Binimetinib, Bortezomib (Velcade), Bosutinib, Braftovi (Encorafenib), Brigatinib, Busulfan, Cabazitaxel, Calquence (Acalabrutinib), Campath (Alemtuzumab), Camptosar (Irinotecan Hydrochloride), Capecitabine (Xeloda), Carac (Fluorouracil—Topical, Tolak), Carfilzomib (Kyprolis), Carmustine, Ceritinib (Zykadia), Cerubidine (Daunorubicin Hydrochloride), Chlorambucil, Cladribine, Clofarabine, Cobimetinib, Copanlisib Hydrochloride, Copiktra (Duvelisib), Cosmegen (Dactinomycin), Crizotinib (Xalkori), Cyclophosphamide, Cytarabine (Tarabine PFS), Dabrafenib (Tafinlar), Dacarbazine, Dacogen (Decitabine), Dacomitinib (Vizimpro), Dasatinib, Daunorubicin Hydrochloride, Decitabine, Degarelix, Dexamethasone, Dexrazoxane Hydrochloride (Totect, Zinecard), Docetaxel (Taxotere), Doxorubicin, Duvelisib, Leuprolide Acetate, Ellence (Epirubicin Hydrochloride), Eltrombopag Olamine, Emend (Aprepitant), Enasidenib Mesylate, Enzalutamide (Xtandi), Epoetin Alfa, Eribulin Mesylate, Erivedge (Vismodegib), Erleada (Apalutamide), Erlotinib Hydrochloride (Tarceva), Ethyol (Amifostine), Etopophos (Etoposide Phosphate), Everolimus, Evista (Raloxifene Hydrochloride), Evomela (Melphalan Hydrochloride), 5-FU (Fluorouracil Injection), Fareston (Toremifene), Farydak (Panobinostat), Faslodex (Fulvestrant), FEC, Femara (Letrozole), Firmagon (Degarelix), Fludarabine Phosphate, Flutamide, Folotyn (Pralatrexate), Fusilev (Leucovorin Calcium), Gefitinib, Gemcitabine Hydrochloride, Gilotrif (Afatinib Dimaleate), Gleevec (Imatinib Mesylate), Gliadel Wafer (Carmustine Implant), Goserelin, Acetate (Zoladex), Granisetron, Halaven (Eribulin Mesylate), Hemangeol (Propranolol Hydrochloride), Hycamtin (Topotecan Hydrochloride), Hydrea (Hydroxyurea), Ibrance (Palbociclib), Ibritumomab Tiuxetan (Zevalin), Ibrutinib, Iclusig (Ponatinib Hydrochloride), Idarubicin Hydrochloride, Idelalisib (Zydelig), Ifex (Ifosfamide), Imiquimod, Ipilimumab (Yervoy), Istodax (Romidepsin), Ivosidenib (Tibsovo), Ixabepilone, Ixazomib Citrate, Ruxolitinib phosphate, Kisqali (Ribociclib), Lanreotide Acetate, Larotrectinib Sulfate (Vitrakvi), Lenalidomide, Lenvatinib Mesylate, Lomustine, Lorbrena (Lorlatinib), Lupron (Leuprolide Acetate), Lynparza (Olaparib), Marqibo (Vincristine Sulfate Liposome), Matulane (Procarbazine Hydrochloride), Mechlorethamine Hydrochloride, Megestrol Acetate, Mekinist (Trametinib), Melphalan, Mercaptopurine, Methotrexate, Midostaurin, Mitomycin C, Mitoxantrone Hydrochloride, Mozobil (Plerixafor), Mustargen (Mechlorethamine Hydrochloride), Myleran (Busulfan), Navelbine (Vinorelbine Tartrate), Nelarabine, Neratinib Maleate, Neulasta (Pegfilgrastim), Nexavar (Sorafenib Tosylate), Nilandron (Nilutamide), Nilotinib (Tasigna), Ninlaro (Ixazomib Citrate), Odomzo (Sonidegib), Omacetaxine Mepesuccinate, Osimertinib (Tagrisso), Paclitaxel (Taxol), PAD, Palbociclib, Palonosetron Hydrochloride, Panobinostat, Pazopanib Hydrochloride (Votrient), Pegfilgrastim (Zarxio), Pomalidomide, Prednisone, Procarbazine Hydrochloride, Promacta (Eltrombopag Olamine), Purinethol, Raloxifene Hydrochloride, Regorafenib, Ribociclib, Rheumatrex (Methotrexate, Trexall), Rolapitant Hydrochloride, Romidepsin, Rubidomycin (Daunorubicin Hydrochloride), Rydapt (Midostaurin), Sancuso (Granisetron), Somatuline Depot (Lanreotide Acetate), Sonidegib, Stivarga (Regorafenib), Tabloid (Thioguanine), Temodar (Temozolomide), Temsirolimus (Torisel), Thalidomide, Thiotepa, Toremifene, Trabectedin (Yondelis), Treanda (Bendamustine Hydrochloride), Trifluridine and Tipiracil Hydrochloride, Uridine Triacetate (Vistogard), Valrubicin, Vandetanib, Varubi (Rolapitant Hydrochloride), Vemurafenib (Zelboraf), Venclexta (Venetoclax), Vinblastine Sulfate, Vismodegib, Vorinostat (Zolinza), Xospata (Gilteritinib Fumarate), Zofran (Ondansetron Hydrochloride), and Zoledronic Acid (Zometa).
Suitable second reagent solutions may also comprise an antifungal drugs and other small molecules (e.g., for testing on yeast or other fungal species). Thus, the method of the present disclosure may be carried out to perform an assay testing the ability of a compound (e.g., small molecule) to act as an antifungal drug. Moreover, the method may be carried out to determine an effective or ideal concentration at which an antifungal compound may be used to achieve its desired effect. Antifungal compounds are well known in the art. Non-limiting examples of antifungal compounds that may be used in the assay method of the present disclosure include, without limitation, D-fructose, Glucose, Galactose, Antimycin, Bleomycin, 5-Bromo-5-nitro-1,3-dioxane, Cinnamycin, Fengycin (Plipastatin), Filastatin, Filipin, Gentian Violet, Sinefungin, Kasugamycin, Magnolol (2,2′-Bichavicol, 5,5′-Diallyl-2,2′-biphenyldiol), Oligomycin (Oligomycin A), Surfactin, Terconazole, Thiabendazole (2-(4-Thiazolyl)benzimidazole), Thiolutin, Thymol (5-Methyl-2-isopropylphenol), Tioconazole, Tolnaftate, Tubercidin, Terbinafine, Ketoconazole, Fluconazole, Itraconazole, Voriconazole, Caspofungin, and Flucytosine.
In some embodiments in carrying out the methods described herein, the microfluidic device further comprises an in-water bath cavity surrounding the microfluidic circuits. The in-water bath cavity is not fluidically connected to the microfluidic circuits. The in-water bath cavity provides access for a solution such as water, without restriction, to be added through inlet or outlet ports that provide access to the in-water bath cavity, but not the microfluidic circuits (see, e.g.,
The following examples are intended to exemplify the practice of embodiments of the disclosure but are by no means intended to limit the scope thereof.
MVM2 Device Fabrication. The MVM2 device was made following the Microchem Corp. (Newton, MA) instruction for microfluidic device fabrication using soft lithography techniques (Xia et al, “Soft Lithography,” Annual Review of Materials Science 28(1):153-184 (1998); Pajoumshariati et al, “Microfluidic-Based Cell-Embedded Microgels Using Nonfluorinated Oil as a Model for the Gastrointestinal Niche,” ACS Applied Materials & Interfaces 10(11):9235-9246 (2018), Pajoumshariati et al, “A Microfluidic-Based Model for Spatially Constrained Culture of Intestinal Microbiota,” Advanced Functional Materials 28(48):1805568 (2018); and Yaghoobi et al, “Progressive Sperm Separation Using Parallelized, High-Throughput, Microchamber-based Microfluidics,” bioRxiv (2020) doi.org/10.1101/2020.07.31.231373; each of which are hereby incorporated by reference in their entirety). Briefly, SU-8 2050 negative photoresist (Microchem Corp. Newton, MA) was poured on a silicon wafer (ID: 452, UniversityWafer, Boston, MA) and spun-coated at 2100 rpm. Then, the wafer was pre-baked at 65° C. and 95° C. for 3 and 9 min, respectively. The pre-baked SU-8 photoresist was then patterned using a photomask made by CAD/Art Services, Inc. (Bandon, OR) via UV light wavelength with an exposure energy of 120 mJ/cm2 at 365 nm. Then, a post-baking step was followed at 65° C. and 95° C. for 2 and 7 min, respectively, to further permanently stabilize the SU-8 photoresist pattern on the silicon wafer. The uncured SU-8 (non-patterned parts of SU-8) was developed and washed by gently soaking the SU-8 patterned silicon wafer in the SU-8 developer (Microchem Corp. Newton, MA) for 10 min. The mixture of PDMS and its curing agent (10:1) was then poured on the SU-8 deposited wafer and baked for 2 h at 65° C. After peeling the PDMS off the patterned wafer, injection holes (1 mm in diameter) were punched and cleaned by a cellophane tape (3M Scotch Magic, MN, USA), followed by bonding of PDMS to a glass slide by applying oxygen-plasma treatment for 1 min. To further stabilize the bonding strength between PDMS and the glass slide, the device was kept at 65° C. in an oven for 12 h for further stabilization.
Bacteria culture medium, strains, growth, and broth microdilution test. Non-selective Mueller-Hinton (“MET”) culture medium was used for all bacterial culture. In detail, a colony of bacteria pre-cultured on an LB agar plate (Corning, NY) was taken from a freshly streaked plate and suspended into 3 mL of MEI broth, and cultured at 37° C. overnight (˜12 h). The concentration of bacterial suspension was adjusted using a UV-Vis spectrophotometer (NanoDrop, Thermo-Fisher; Wilmington, DE). To do this, the overnight culture medium was diluted by 10-fold serial dilutions in non-selective MEI culture medium to find the appropriate final concentration (1×106 CFU/mL) for the antimicrobial susceptibility testing assay.
Yeast culture medium, strains, and growth. Yeast cell growth and standard laboratory manipulations were performed as described (Guthrie and Fink, “Guide to Yeast Genetics and Molecular and Cellular Biology,” Methods in Enzymol. Part C 351 Gulf Professional Publishing (2002), which is hereby incorporated by reference in its entirety). All media used was either minimal medium (YNB; 0.67% yeast nitrogen base without amino acids plus 2% indicated carbon sources) or rich medium (YP; 2% bacto peptone, 1% yeast extract, 2% indicated carbon sources). Additionally, for fluorescence microscopy, low-fluorescence medium was used (standard minimal medium with 2% glucose, except YNB is prepared without riboflavin or folic acid to reduce background fluorescence) (Sheff and Thorn, “Optimized Cassettes for Fluorescent Protein Tagging in Saccharomyces cerevisiae,” Yeast 21:661-670 (2004), which is hereby incorporated by reference in its entirety). The yeast strains used in this study were DBY12000 and DBY12549. DBY12000 is a wild-type yeast strain (i.e., WT) with a genotype of MATa prototrophic HAP1+ derivative of FY4. DBY12549 or yrKHK is the mutant of the wild-type yeast strain, which is sensitive to fructose and has a genotype of MATa HAP1+ can1Δ::TDH3pr-yrKHK.
The gold standard broth microdilution test was performed by preparing the antibiotic solutions at their final concentrations from a stock solution of each examined antibiotic. A fresh 200 μL volume of each antibiotic solution (prepared in MEI culture medium) was pipetted into each microwell of a 96 MicroWell plate. Note, the concentrations of each antibiotic solution was set at 0.1-1 μg/mL (by 0.1 μg/mL-unit increment between every two consecutive antibiotic concentrations), 1-10 μg/mL (by 1 μg/mL-unit increment between every two consecutive antibiotic concentrations) and 10-100 μg/mL (by 10 μg/mL-unit increment between every two consecutive antibiotic concentrations). Then, 10 μL of each bacterial stock suspension was added to each microwell containing antibiotic solution to reach the appropriate bacterial final concentration (1×106 CFU/mL). The bacteria was incubated in the presence of antibiotics for 20 h, and then the MIC and antimicrobial resistant assay time upon 80% reduction in the OD600-growth curves was measured and compared to the negative control (i.e., without adding any antibiotic to the bacterial suspension). Bacteria were grown at 37° C. shaking for 24 h. Standardized growth curve analysis was performed using a Bioscreen C automated plate reader (Growth Curves USA, NJ) by measuring OD600. All experiments were completed in triplicate and performed twice.
Computational fluid dynamics (CFD) simulations. COMSOL Multiphysics (version 5.3; COMSOL Inc., USA) was used to carry out to obtain the flow velocities and concentration profiles. 2D creeping flow module was used as the flow and boundary condition were considered as: (i) boundary condition: pressure 0 Pa; (ii) wall condition: no slip; (iii) considering the suppression of backflow; and (iv) normal physics-controlled for mesh size. The Navier-Stokes (Eq. 1) and conservation of mass (Eq. 2) equations, in which V denotes the velocity field, ρ is the density of the culture medium, P is pressure, and μ is the dynamic viscosity were solved.
ρ(V·∇V)=−∇P+∇·μ(∇V+(∇V)T (1)
∇·V=0 (2)
Small-molecule diffusion coefficient determination. Based on the spectrophotometry method, 4 mL culture medium was loaded into a cuvette, followed by 80 μL of antibiotics (e.g., nalidixic acid, ampicillin, and cefuroxime plus resazurin as a fluorescent chemical; initial concentration=10 mg/mL) that was gently loaded at the bottom of the cuvette using a chromatography syringe. The diffusion kinetics of the antibiotics were obtained from the UV absorbance of their maximum wavelengths (correlated with concentration) as they diffused up through the cuvette over time.
‘D1×t1=D2×t2’ derivation. To empirically determine the diffusion coefficients of the antibiotics in culture media, the UV absorbance spectrum of the antibiotics diffusing up in a cuvette was measured. These values (i.e., UV absorbance data-points) can be correlated with a mathematical equation. To obtain the theoretical model of mass transport in the cuvette, the mass transfer was considered in a Cartesian geometry due to the rectangular cuboid shape of the cuvette
By substituting t×D with as a variable (and tD=t′, consequently) into the Eq. 3, it can be simplified to Eq. 4:
Two insulated boundary conditions were considered in the ‘y’ direction (the top and bottom layers of solution in the cuvette). The boundary conditions and the initial conditions were considered as follows (Eq. 5 & 6):
Two first terms of the Taylor series were used as initial conditions, as follows:
Using the “Separation of Variables” method, the answer for Eq. 8 can be defined as follows:
C(y,t′)=Y(y)·T(t) (8)
Plugging Eq. 8 into Eq. 4 leads to the following equation:
Eq. 9 can be solved and simplified by using the boundary conditions.
Substituting tD=t′ into Eq. 10 leads to:
Using the initial condition to find a n results in Eq. 12:
For n>>2, the terms of series were negligible. Then, the two first sentences of Eq. 12 were used, which led to Eq. 11. The empirical data obtained with UV-Vis spectrophotometry was correlated with Eq. 11 and the diffusion coefficient for each antibiotic was then calculated:
Imaging and Data Analysis. A ZOE™ fluorescent cell imager (Bio-Rad, CA) was used as an imaging platform to take the images of the microchambers, and ImageJ software was utilized to convert the fluorescent intensity to gray value. The fluorescence of the medium within the wells was calculated by averaging the pixel intensities in a given semi-spherical region. A custom MATLAB script was used to analyze the images.
The MVM2 platform (also referred to herein as a device) features four main-channels in parallel, with openings at the ends of each main-channel (i.e., A1-A4 and B1-B4;
To perform a small-molecule-based biological assay in the MVM2 platform, four main steps to load the reagents are followed. Step-i involves loading a suspension of the biological species (often with a fluorescent chemical indicator) into the MVM2 platform so that it is uniformly distributed throughout (see
Biological suspension loading protocol and steps are shown in
A resazurin red fluorescent solution (representing a biological sample) was used to experimentally illustrate the sample loading in microchamber R1, which features the smallest volume. As shown in
The protocol for small-molecule solution loading is shown in
To produce the low-range gradient-based concentration profile (GCP) of the small-molecule solution, openings A1, A3, A4, B1, B3, and B4 are temporarily blocked and the small-molecule solution is flowed into the main low-range positive channel (
Criteria for microchamber and side-channel geometry design are shown in
Importantly, there are two potential scenarios to make a gradient-based concentration profile (GCP) of the small-molecules in a platform: (a) exposing different amounts of a small-molecule with the same number of a biological species, or (b) exposing the same amount of a small-molecule with different numbers of a biological species. The second method was chosen in the MVM2 platform as it requires exposing the same amounts of a small-molecule (i.e., identical green-color patterns in microchambers of the low, medium, or high range as shown in
Such a GCP is achieved in
Studying two highly potential outcomes in biological small-molecule assay using MVM2 platform is shown in
To validate the hypothesis of small-molecule loading into the multi-volume microchambers via diffusion in the MVM2 platform, a 20% wt/v solution of resazurin (a fluorescent small-molecule) was used. The resazurin diffusion was monitored through a preloaded aqueous phase (Mueller-Hinton culture medium) and it was found that resazurin successfully moved through the side-channel and entered the microchamber (
Small-molecules' self-diffusion is the mechanism for small-molecule loading into microchambers in MVM2 platform as shown in
Control over the small-molecule diffusion into the microchambers is pivotal for obtaining a GCP, which is governed by the loading time (defined as the time period between loading the small molecule into the main channels and flushing the system with oil). For an unlimited loading time (co), the microchambers become fully saturated, leading to the failure of GCP formation (
Small-molecule gradient-based contrition failure within a long (unlimited) loading time in MVM2 platform is shown in
The CFD-based time-dependent normalized concentration profiles—divided by the maximum concentration, C0—along the dashed arrow for microchambers R1 (smallest) and R12 (largest) were obtained (
To investigate the criterion for achieving resazurin GCP in the MVM2 platform (i.e., loading the same amount of resazurin into the multi-volume microchambers at a specific time-point), the concentration profile at t=75 s (pink curve,
As can be seen in
The time-point found by CFD simulations (t=75 s) was used to experimentally verify that resazurin can achieve a GCP in the MVM2 device. After loading the resazurin into microchambers R1-R12 for 75 s (as the loading time), the fluorescence of the microchambers was found to decrease with increasing microchamber size, indicating a successful GCP (
diffusion of small-molecules into the multi-volume microchambers is the key for producing a GCP. However, it is well-known that small-molecules have different diffusion coefficients, which is most impacted by their molecular size. As a result, loading small-molecules with different diffusion coefficients at the same loading time could result in different GCPs in the MVM2 platform, as shown in
The concentration profiles into twelve microchambers was obtained using CFD simulations for the diffusion coefficients 5×10−9 m2/s (
Using an analytical solution for small-molecule mass transport in the side-channels and microchambers and CFD simulations for microchambers R1 and R12 (
as well as the diffusion coefficient of the target small-molecule (e.g., D2).
CFD simulations to confirm the relationship between small-molecule diffusion coefficients and loading times are shown in
However, there is no extensive database available for the diffusion coefficients of biological small-molecules. Therefore, instead the small molecule's molar volume was chosen to investigate its relationship with the loading time, as the molar volume is more readily accessible compared to the diffusion coefficient. Toward this aim, the loading kinetics of different fluorescent dyes, including calcein (
The kinetic for fluorescein dye diffusion into microchamber R1 is studied in
Interrogating the loading kinetics of the fluorescent dyes into the side-channel helped to establish a linear relationship between the small-molecules' molar volumes and loading times (R2=0.9885) in the MVM2 platform, as follows (
Loading time=0.4954×molar volume−3.7623 Eq. 15
To further confirm that Eq. 15 can also apply to non-fluorescent biological small-molecules, a simple method using spectrophotometry was employed. By gently loading a biological small-molecule solution at the bottom of a cuvette preloaded with non-selective Mueller-Hinton culture medium, the maximum absorbance wavelength of the small-molecule was able to be recorded as it diffused up in solution along the cuvette height (
Finding the small-molecule diffusion coefficients and diffusion time is shown in
This experiment allowed an easy correlation between the small molecule's diffusion and its absorbance as it moved upward along the cuvette (mimicking the small molecule diffusion along the side-channels). To test the validity of this methodology for determining the mass transport of small-molecules, three antibiotics were chosen as biological small-molecules (nalidixic acid, cefuroxime, and ampicillin) in addition to resazurin. The results indicated that the small-molecules with similar molar volumes (e.g., resazurin/nalidixic acid and cefuroxime/ampicillin) show very close absorbance curves over time (i.e., similar diffusion patterns in the cuvette solution;
In terms of practical application of the MVM2 platform for different biological assays, water evaporation can become an issue for nanoliter-sized culture media in the microchambers due to the permeability of the PDMS walls (
Techniques to minimize water evaporation from the MVM2 platform for long-term biological suspension cultures are shown in
To further examine the functionality of the MVM2 microfluidic device and due to the importance of worldwide emerging resistance to antibacterial drugs, the susceptibility of a green fluorescent protein (GFP)-labeled E. coli 541-15 to gentamicin (a typical antibiotic) was studied in order to determine the minimum inhibitory concentration (MIC) of an antibiotic. The E. coli 541-15 bacterial suspension (concentration: 1×106 CFU/mL) was first loaded into the microchambers (step i). Resazurin (5 wt %) was also added to the bacterial suspension to allow monitoring of the bacterial cell metabolism through an irreversible resazurin-resorufin enzymatic reduction reaction (i.e., with increasing bacterial growth, the higher resazurin reduction results in greater fluorescent intensity). A GCP of the gentamicin (loading time=170 s; Table 1) was generated in the positive microchambers (step-ii) in the concentration range of 0.1-100 μg/mL, specifically 0.1-1 (low), 1-10 (medium), and 10-100 (high) μg/mL for the three channels. At t=0 h, the negative and positive microchambers feature the same low red fluorescent intensities of resazurin, as expected (
To further confirm these findings based on the bacterial cell metabolism obtained from resazurin reduction, the red fluorescent intensities were correlated with the green fluorescence directly associated with the GFP-labeled bacterial growth in the corresponding microchambers (
As a representative small molecule model (assay), showing a bacterial resistance to antibiotics, the ampicillin-E. coli 541-15 pair assay was also examined. Similarly, bacterial cell metabolism and growth using the resazurin reduction assay was probed and changes in the number of GFP-labeled bacteria during the assay was monitored, as shown in
To further evaluate the platform functionality, isolated bacteria from two clinical scenarios were tested: ((i) ileal mucosa of human patients associated with Crohn's disease in
Clinical models for testing the MVM2 microfluidic device are shown in
To probe the functionality of our MVM2 platform for eukaryotic cells, its long-term cell-culture and growth capability was shown for cancer and yeast cells (the MCF-7 human breast cancer cell line and a Saccharomyces cerevisiae strain, respectively) in
Loading cancer cell line into MVM2 microfluidic device is shown in
Capabilities of yeast growth in MVM2 device for a long-term run are shown in
Then, as an example, the platform was used to study recessive human metabolic diseases, specifically measuring sugar-phosphate toxicity. For these experiments, a strain of S. cerevisiae was chosen in which fructose, but not glucose, is toxic due to constitutive expression of a rat liver ketohexokinase gene (the yrKHK strain, DBY12549). The data obtained using the MVM2 platform precisely pinpointed the binary ‘Yes/No’ sensitivity response of the yrKHK strain to fructose (Yes) and glucose (No), as shown in
In this case, the functionality and validity of the MVM2 platform was used to test for these known “eukaryote cells/sugars” pairs. Consequently, sugars (fructose and glucose) were chosen as the small molecules to show the feasibility of testing small molecules on eukaryotic cells in the MVM2 platform. A wild-type S. cerevisiae, DBY12000, yeast strain (namely, WT) and its genetically modified DBY12549 mutant (called yrKHK) were chosen.. The sensitivity/resistance of WT and yrKHK yeast strains has been reported using conventional biological approaches elsewhere. Briefly, the WT strain is both glucose- and fructose-resistant. But the genetically modified yrKHK strain is, however, fructose sensitive and glucose-resistant.
Sugars were tested in a wide-range of concentrations (0.007%-7%, three orders of magnitude). In this respect, low, medium, and high ranges of sugars featured 0.007-0.07, 0.07-0.7, and 0.7-7% sugar concentration ranges while the negative control-labeled microchambers were not exposed with the tested sugar. The yeast cell density was set at OD600=0.1 (equals to 1×106 cell/mL) for loading into microchambers and then expose with sugars and tested within the sugar concentration ranges of 0.007-7 wt %.
Moreover, a fructose concentration of 1.13 wt % was determined as the critical sensitivity concentration for the yrKHK strain using the MVM2 platform (
Wild-type vs. Glucose (outcome: not sensitive): In
Although preferred embodiments have been depicted and described in detail herein, it will be apparent to those skilled in the relevant art that various modifications, additions, substitutions, and the like can be made without departing from the spirit of the invention and these are therefore considered to be within the scope of the invention as defined in the claims which follow.
This application claims the benefit of U.S. Provisional Patent Application Ser. No. 63/130,172, filed Dec. 23, 2020, which is hereby incorporated by reference in its entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2021/064928 | 12/22/2021 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63130172 | Dec 2020 | US |
