Abstract
There is provided a magnetic digital microfluidic system for performing an assay, the system comprising, abase member comprising at least one magnet disposed thereon; the magnet being configured to immobilise a droplet of reaction mixture doped with magnetic particles; and a droplet manipulator configured to be moveably mountable on the base member, said droplet manipulator comprising at least one test unit, each test unit comprising at least one mixing element for mixing the droplet of reaction mixture; wherein the mixing element is arranged to induce mixing as the droplet manipulator is moved in relation to the base member along an alignment path. There is also provided a method of performing an assay with the magnetic digital microfluidic system as disclosed herein.
Claims
1. A magnetic digital microfluidic system for performing an assay, the system comprising, a base member comprising at least one magnet disposed thereon; the magnet being configured to immobilise a droplet of reaction mixture doped with magnetic particles; and a droplet manipulator configured to be moveably mountable on the base member, said droplet manipulator comprising at least one test unit, each test unit comprising at least one mixing element for mixing the droplet of reaction mixture; wherein the mixing element is arranged to induce mixing as the droplet manipulator is moved in relation to the base member along an alignment path.
2. The system according to claim 1, further comprising a surface for allowing the droplet of reaction mixture to be disposed thereon.
3. The system according to claim 2, wherein the surface is a detachable surface and the droplet manipulator is configured to detachably couple with the detachable surface.
4. The system according to claim 2, wherein the test unit comprises, a first access port for allowing delivery of a sample, magnetic particles, and/or one or more reaction reagents to said surface.
5. The system according to claim 1, wherein the mixing element comprises an array of pillars being arranged to interact with the droplet of reaction mixture to induce mixing.
6. The system according to claim 2, wherein the test unit further comprises a second access port for allowing delivery of a detection reagent to said surface.
7. The system according to claim 1, wherein the test unit further comprises a droplet holder configured to engage and hold the droplet of reaction mixture to facilitate observation.
8. The system according to claim 7, wherein the droplet holder comprises a hydrophilic contact surface for engaging and holding the droplet of reaction mixture.
9. The system according to claim 7, wherein the droplet holder is further configured to facilitate removal of the magnetic particles from the droplet of reaction mixture via movement of the droplet manipulator along the alignment path.
10. The system according to claim 1, wherein the test unit further comprises an observation window configured to facilitate observation of the droplet of reaction mixture.
11. The system according to claim 1, wherein the droplet manipulator comprises a plurality of test units for performing the assay on a plurality of samples simultaneously, each test unit being configured to cooperate with a respective magnet disposed on the base member.
12. The system according to claim 4, further comprising a guide member configured to be detachably couplable to the droplet manipulator, said guide member comprising at least one guide hole configured to be alignable with the first access port, wherein the guide hole is dimensioned such that a droplet dispenser inserted thereto does not contact the surface where the droplet is to be disposed.
13. (canceled)
14. A method of performing an assay with the magnetic digital microfluidic system of claim 1, the method comprising, moving the droplet manipulator in relation to the base member along an alignment path to induce mixing of a droplet of reaction mixture immobilised by the magnet disposed on the base member, wherein the reaction mixture is doped with magnetic particles; and observing changes to the reaction mixture.
15. The method according to claim 14, further comprising, prior to the moving step, disposing a droplet of reaction mixture on a surface that is coupled to the droplet manipulator via a first access port of the test unit.
16. The method according to claim 15, further comprising, subsequent to said moving step, disposing a droplet of detection reagent on said surface via a second access port of the test unit; further moving the droplet manipulator in relation to the base member along an alignment path to merge the droplet of reaction mixture with the droplet of detection reagent; and optionally further moving the droplet manipulator in relation to the base member along an alignment path to further mix the merged droplet.
17. The method according to claim 15, further comprising, prior to the each of said disposing step(s), detachably coupling a guide member to the droplet manipulator; and aligning a guide hole of the guide member to the access port through which the respective disposing step is to be carried out, wherein the guide hole is dimensioned such that a droplet dispenser does not contact the surface of the droplet manipulator when disposing the reaction mixture or detection agent thereon.
18. The method according to claim 17, further comprising, detaching the guide member from the system prior to each of said moving step(s).
19. The method according to claim 14, further comprising, moving the droplet manipulator in relation to the base member along an alignment path to engage and hold the droplet of reaction mixture with a droplet holder; moving the droplet manipulator in relation to the base member along an alignment path to remove the magnetic particles from the droplet of reaction mixture; and observing via an observation window in the test unit, the droplet of reaction mixture that is engaged and held by the droplet manipulator.
20. The method according to claim 14, further comprising performing the assay on a plurality of samples simultaneously on a plurality of test units in the droplet manipulator, wherein each test unit is being configured to cooperate with a respective magnet disposed on the base member.
Description
BRIEF DESCRIPTION OF FIGURES
[0106] FIG. 1A is a schematic perspective view drawing of a system/device for performing an assay in an example embodiment.
[0107] FIG. 1B is a photograph of a base member in the example embodiment.
[0108] FIG. 1C is a schematic bottom view drawing of a droplet manipulator in the example embodiment.
[0109] FIG. 1D is a photograph of the droplet manipulator in the example embodiment.
[0110] FIG. 2A is a schematic bottom view drawing of a droplet manipulator in an example embodiment.
[0111] FIG. 2B is a magnified view of a test unit in the example embodiment.
[0112] FIG. 2C is a schematic perspective view drawing of a guiding piece in an example embodiment.
[0113] FIG. 3A is a schematic perspective view drawing showing sample loading into a system for performing an assay in an example embodiment.
[0114] FIG. 3B is a schematic perspective view drawing showing movement direction of a droplet manipulator relative to a base member in the example embodiment.
[0115] FIG. 3C is a schematic perspective view drawing showing movement direction of droplets relative to the droplet manipulator when the droplet manipulator moves in the direction as shown in FIG. 3B.
[0116] FIG. 3D is a first schematic side view drawing of the system showing movement of the droplet manipulator and relative movement of the droplets in the example embodiment.
[0117] FIG. 3E is a second schematic side view drawing of the system showing movement of the droplet manipulator and relative movement of the droplets in the example embodiment.
[0118] FIG. 3F is a third schematic side view drawing of the system showing movement of the droplet manipulator and relative movement of the droplets in the example embodiment.
[0119] FIG. 4A is a schematic drawing of a system for performing an assay in an example embodiment.
[0120] FIG. 4B is a schematic drawing of the system showing sample loading in the example embodiment.
[0121] FIG. 4C is a schematic drawing of the system showing movement of a droplet manipulator in relation to a base member in the example embodiment.
[0122] FIG. 4D is a schematic drawing of the system showing mixing of a droplet of reaction mixture by the droplet manipulator in the example embodiment.
[0123] FIG. 4E is a schematic drawing of the system showing merging of the droplet of reaction mixture with a droplet of detection reagent in the example embodiment.
[0124] FIG. 4F is a schematic drawing of the system showing movement of the droplet manipulator in relation to the base member in the example embodiment.
[0125] FIG. 4G is a schematic drawing of the system showing mixing of a merged droplet by the droplet manipulator in the example embodiment.
[0126] FIG. 4H is a schematic drawing of the system showing coupling of the merged droplets by the droplet holders in the example embodiment.
[0127] FIG. 4I is a schematic drawing of the system showing removal of magnetic particles from the merged droplets in the example embodiment.
[0128] FIG. 5A is a photograph of a platform for performing a Carba NP assay.
[0129] FIG. 5B is a photograph of the platform showing mixing of samples by moving droplets back and forth under a mixer provided in each detection unit.
[0130] FIG. 5C is a photograph of the platform showing sample droplets merged with reagent droplets.
[0131] FIG. 5D is a photograph of the platform showing merged droplets being mixed under the mixer.
[0132] FIG. 5E is a photograph of the platform showing the merged droplets moved to respective observation windows.
[0133] FIG. 5F is a photograph of the platform showing magnetic particles extracted/moved out from the merged droplets.
[0134] FIG. 6A is a photograph of a digital microfluidic point-of-care platform for performing a Carba NP assay with prepared reagents and magnetic particles placed on the platform.
[0135] FIG. 6B is a photograph of the platform with a 3D printed top plate cover placed on top of the platform.
[0136] FIG. 6C is a photograph of the platform with reaction droplet and detection droplet merged.
[0137] FIG. 6D is a photograph of the platform showing colour changes in the droplets after 30 minutes.
[0138] FIG. 6E is a photograph of the platform showing colour changes in the droplets after 1 hour.
[0139] FIG. 6F is a photograph of the platform showing color changes in the droplets after 2 hours.
[0140] FIG. 6G is a photograph of the platform showing the final results of a Carba NP assay with a test group and a control group labelled.
[0141] FIG. 7 is a photograph of a droplet manipulator showing 12 test units with reaction droplets for different samples.
DETAILED DESCRIPTION OF FIGURES
[0142] Example embodiments of the disclosure will be better understood and readily apparent to one of ordinary skill in the art from the following discussions and if applicable, in conjunction with the figures. It should be appreciated that other modifications related to structural, and optical changes may be made without deviating from the scope of the invention. Example embodiments are not necessarily mutually exclusive as some may be combined with one or more embodiments to form new exemplary embodiments.
[0143] FIG. 1A is a schematic perspective view drawing of a system/device 100 for performing an assay in an example embodiment. The system 100 for performing an assay is a multiplexed CPE detection system comprising a magnetic digital microfluidic system that is capable of conducting multiple Carba NP assays in parallel in droplets. The system 100 comprises four main components, a guide member/guiding piece 102, a droplet manipulator 104, a base member/base plate 106 and a sheet member 108 for disposing one or more droplets of reaction mixture e.g. glass coverslip. The guiding piece 102 and the droplet manipulator 104 can be coupled/combined into a single component. The guiding piece 102 is used to guide loading of a sample during an assay preparation phase, and the other three components 104, 106 and 108 are used to manipulate droplets during the assay.
[0144] FIG. 1B is a photograph of the base member 106 in the example embodiment. The base plate 106 contains an array of magnets e.g. 110 to manipulate droplets using magnetic digital microfluidics. The base plate 106 comprises a first side wall 112, a second side wall 114, and a third side wall 116 surrounding a flat bed 118. The first side wall 112 and the second side wall 114 are disposed at opposite sides of the flat bed 118 and are connected or joined by the third side wall 116. The three side walls 112, 114, 116 restrict movement of the droplet manipulator 104 so that the droplet manipulator does not overshoot, i.e. does not move outside of the base member 106. Magnets e.g. 110 are embedded into holes provided on the flat bed 118. The magnets e.g. 110 interact with magnetic particles added to droplets e.g. droplets of reaction mixture that sit on the coverslip e.g. glass coverslip 108. Together with the droplet manipulator 104, the magnets e.g. 110 control the movement and operation of droplets. The glass coverslip 108 is coated with substances (e.g. Teflon AF) that renders down the surface energy of the coverslip 108. As a result, the droplets could move easily on the surface of the coverslip 108.
[0145] FIG. 1C is a schematic bottom view drawing of the droplet manipulator 104 in the example embodiment. FIG. 1D is a photograph of the droplet manipulator 104 in the example embodiment. The droplet manipulator 104 comprises micro physical structures and chemically modified features to facilitate droplet manipulation. The bottom aspect of the droplet manipulator 104 contains small structures and surface modifications to facilitate droplet manipulation. The droplet manipulator 104 is made of translucent materials so that a user can visualize movement of the magnetic particles and the droplets through the droplet manipulator 104.
[0146] The droplet manipulator 104 comprises a first side wall 120, a second side wall 122, and a third side wall 124 surrounding a base 126. The first side wall 120 and the second side wall 122 are disposed at opposite sides of the base 126 and are connected or joined by the third side wall 124. The three sidewalls 120, 122, 124 comprise slots 128 e.g narrow slots which function as holders for the coverslip 108. The coverslip 108 is arranged to be slided into the slots 126 from an opening side of the droplet manipulator 104.
[0147] The droplet manipulator 104 further comprises a plurality of test units/testing units e.g. 130. A magnified view of one testing unit 130 is also shown in FIG. 1C. The base 126 of the droplet manipulator 104 is divided into two columns of repeating testing units e.g. 130. Each testing unit 130 comprises a first access port/hole 132 and a second access port/hole 134 for delivering fluid e.g. liquid. Reagents such as lysis buffer and magnetic particles are dispensed on top of the coverslip 108 through one access hole e.g. first access hole 132, and detection reagent is dispensed through the other access hole e.g. second access hole 134. Each testing unit 130 further comprises a mixing element e.g. an array of pillars 136 for droplet mixing disposed between the two access holes 132, 134. As the droplet moves through the array of pillars 136, the droplet is stretched and retracted repetitively, which induces mixing inside the droplet. At the end of each testing unit 130, a droplet holder e.g. semi-circular droplet holder 138 is disposed under an observation window e.g. rectangular observation window 140. The entire bottom surface of the droplet manipulator 104 is coated with Teflon. In addition, the semi-circular droplet holder 138 is coated with polydopamine which renders its surface hydrophilic so that it is capable of anchoring the droplet for observation.
[0148] The device 100 is designed for parallel multiplexed analysis of CPE using a droplet based Carba NP assay, capable of analyzing 6 clinical samples (or 12 reactions) concurrently with a simple fluidic operation. All the 12 reactions can be performed at the same time by simply sliding the droplet manipulator 104 from right to left on top of the base plate 106.
[0149] FIG. 2A is a schematic bottom view drawing of a droplet manipulator 200 in an example embodiment. The droplet manipulator 200 comprises 12 units of testing units e.g. 202. FIG. 2B is a magnified view of the test unit 202 in the example embodiment. The magnified view of the test unit 202 provides a closeup of different structures for droplet holding, droplet mixing, and droplet adding. In each testing unit 202, a first access port 204 and a second access port 206 are provided to allow reagents and samples to be added to an underlying surface e.g. glass coverslip. Between the two access ports 204, 206, a mixing element 208 e.g. an array of pillars 208 is provided as mixers to passively mix the fluids by stretching and slinging the droplet. An observation window 210 is provided adjacent to the second access port 206 at the end of the testing unit 202 through which an operator/user could observe the color of droplets. A droplet holder 212 e.g. a semi-circular droplet holder 212 is coated with polydopamine to enhance surface tension (or to provide a surface with relatively high surface tension) to keep the droplet engaged to the droplet holder while the magnetic particles are extracted from the droplet. The glass coverslip was coated with 1% Teflon AF solution by a spin coating method and is arranged to be placed between the droplet manipulator 200 and a base member/platform (compare 106 of FIG. 1A). The base member is embedded with an array of 12 magnets, and the locations of the magnets correspond to the location of the droplets to be disposed on the glass coverslip. FIG. 2C is a schematic perspective view drawing of a guiding piece 214 in an example embodiment. The guiding piece 214 is arranged to be put on top of the droplet manipulator during sample loading, i.e. bacteria transferring process. The guiding piece 214 contains 12 holes e.g. 216 with a pre-determined size/diameter. The locations of the 12 holes e.g. 216 in the guiding piece 214 correspond to the locations of the first access ports e.g. 204 on the droplet manipulator 200, i.e. the initial location where the lysis buffer droplet is disposed on the coverslip. The size of the holes e.g. 216 in the guiding piece 214 restricts how deep a tip of a pipette could be inserted into, ensuring the bacterial colony at the tip of the pipette is fully submerged in the droplet but does not contact the coating on the glass coverslip to prevent scratching of the coating.
[0150] An assay e.g. Carba NP may be performed on the magnetic digital microfluidic platform as disclosed herein in three main steps. FIG. 3A TO FIG. 3C outline the three main steps of parallel multiplexed analysis of CPE (Carbapenemase Producing Enterobacteriaceae) using a droplet-based Carba NP assay.
[0151] FIG. 3A is a schematic perspective view drawing showing sample loading into a system for performing an assay in an example embodiment. FIG. 3B is a schematic perspective view drawing showing movement direction of a droplet manipulator relative to a base member in the example embodiment. FIG. 3C is a schematic perspective view drawing showing movement direction of droplets relative to the droplet manipulator when the droplet manipulator moves in the direction as shown in FIG. 3B.
[0152] FIG. 3D is a first schematic side view drawing of the system showing movement of the droplet manipulator and relative movement of the droplets in the example embodiment. FIG. 3E is a second schematic side view drawing of the system showing movement of the droplet manipulator and relative movement of the droplets in the example embodiment. FIG. 3F is a third schematic side view drawing of the system showing movement of the droplet manipulator and relative movement of the droplets in the example embodiment.
[0153] The Carba NP may be performed on the magnetic digital microfluidic platform in three major steps as follows:
[0154] First, all required reagents are dispensed through the access ports on the droplet manipulator (see FIG. 3A). Two columns of reactions are prepared on the device, with testing reactions in the left column and the control reactions on the right. In each reaction, one lysis buffer droplet and one solution A droplet are added. In the testing reaction, carbapenem (e.g. imipenem) is added to the solution A droplet. Bacterial isolates are added to the lysis buffer droplets using pipette tips. The pipette tip is used to pick the bacterial colony, and the guiding piece ensures the pipette is inserted to a designated depth at the right location.
[0155] Second, the droplet manipulator, which holds the glass coverslip to move together/in tandem with the droplet manipulator, is used to move the lysis buffer droplet to merge with the solution A droplet (see FIGS. 3B, 3D and 3E). During the process, the magnetic particles added to the lysis buffer droplet are pulled by the magnet array which in turn pulls the droplet into motion. Once merged with the solution A droplet, the combined droplet is moved back and forth below the mixer teeth to passively mix the components within. After mixing, the combined droplet is incubated for up to 2 hours.
[0156] Third, the combined droplet is moved to the observation window where there is a polydopamine-coated droplet holder to keep the droplet in position while the magnetic particles are extracted from the droplet (see FIGS. 3C and 3F). All droplets are manipulated at the same time in parallel, all reactions are performed concurrently, and all results are observed in one go from the observation windows in the end.
[0157] FIG. 4A is a schematic drawing of a system 400 for performing an assay in an example embodiment. The system 400 comprises a droplet manipulator 402 slidably mounted on a base member 404. The droplet manipulator 402 is arranged to slide in relation to the base member 404 along an alignment path 406.
[0158] The droplet manipulator 402 comprises a plurality of test units e.g. 408, each test unit 408 comprising a first access port 410 and a second access port 412 for facilitating delivery of fluid onto a surface of a sheet member 414 for disposing droplets. The first access port 410 is arranged to facilitate delivery of a sample, magnetic particles and/or one or more reaction reagents to form a droplet of reaction mixture 416 doped with magnetic particles 418. The second access port 412 is arranged to facilitate delivery of a detection reagent and/or one or more reaction reagents to form a droplet of detection reagent 420. The droplet manipulator 402 further comprises a mixing element 422 positioned between the first access port 410 and the second access port 412. The mixing element 422 comprises a plurality of pillars e.g. overhanging pillars extending from an underside/undersurface of the droplet manipulator 402. The mixing element 422 is arranged to interact with the droplets e.g. 416, 420 disposed on the surface of the sheet member 414 to induce mixing of the droplets e.g. 416, 420. The droplet manipulator 402 further comprises a droplet holder 424 disposed at one end of the test unit 408 adjacent or near to the second access port 412. The droplet holder 424 is configured to engage and maintain/hold a droplet in position under an observation window (not shown) to facilitate observation.
[0159] The base member 404 comprises a first side wall 426 and a second side wall 428 defined at opposite sides of the base member 404. The first side wall 426 and the second side wall 428 define the range of movement of the droplet manipulator 402 along the alignment path 406. The base member 404 further comprises a plurality of magnets e.g. 430 embedded on the base member 404. Each magnet 430 is arranged to immobilise the droplet of reaction mixture 416 doped with magnetic particles 418 within a magnetic field applied by the magnet 430. In the example embodiment, the magnet 430 is arranged to immobilise the droplet of reaction mixture 416 doped with magnetic particles 418 at a position directly above the magnet 430.
[0160] During performance of an assay, the droplet manipulator 402 is placed on top of the base member 404 and is pushed/moved in a first direction 432 to contact/abut against the first side wall 426 (i.e. right wall) of the base member 404. The sheet member e.g. a glass coverslip 414 is slid in between the droplet manipulator 402 and the base member 404 to provide the surface for disposing the droplets. The magnet 430 embedded in the base plate 404 is configured to align with the first access port 410 (i.e. left access hole) of each test unit 408 when the droplet manipulator 402 is abutted against the first side wall 426. The droplet manipulator 402 may comprise six pairs of test units e.g. 408 for parallel testing of multiple samples. The six pairs of test units e.g. 408 may be arranged into two columns on the droplet manipulator 402. The spacing between each test unit 408 is configured to match the spacing between adjacent pipettes in a multiple channel micropipette. All reagents in one column of test units e.g. 408 could be dispensed in together using the multiple channel micropipette. One or more reaction reagents e.g. lysis buffer is dispensed onto the surface of the sheet member 414 e.g. Teflon-coated coverslip through the first access port 410 (i.e. left access hole). The detection reagent is dispensed onto the surface of the sheet member 414 e.g. Teflon-coated coverslip through the second access port 412 (i.e. right access hole). The magnetic particles 418 are dispensed into the droplet of reaction mixture 416 comprising the lysis buffer. All liquids spontaneously form droplets once dispensed onto the surface of the sheet member 414 due to the relatively low surface energy of the surface e.g. Teflon-coated coverslip.
[0161] FIG. 4B is a schematic drawing of the system 400 showing sample loading in the example embodiment. A guide member 434 e.g. guiding piece comprising a plurality of guide holes/guiding holes e.g. 436 is placed/removably coupled on top of the droplet manipulator 402. The guiding holes e.g. 436 are arranged to align with the first access port 410 (i.e. left access hole) when the guide member 434 is coupled to the droplet manipulator 402. A sampler 438 (e.g. a loop or pipette tip) with bacterial isolates from clinical specimens is inserted into the droplet of reaction mixture 416 containing the lysis buffer droplet through the guiding hole 436. The size of the guiding hole 436 is configured such that the tip of the sampler 438 is submerged in the droplet of reaction mixture 416 but does not touch the surface of the sheet member 414 (i.e. surface of the glass coverslip). The size of the guiding hole 436 may be adjusted according to the size of the sampler 438.
[0162] FIG. 4C is a schematic drawing of the system 400 showing movement of the droplet manipulator 402 in relation to the base member 404 in the example embodiment. In the example embodiment, the droplet manipulator 402 is moved in a second direction 440 along the alignment path 406 (i.e. towards the left). The mixing elements e.g. 422 are also moved in tandem with the droplet manipulator 402 in the second direction 440 toward the droplets of reaction mixture e.g. 416. The second direction 440 is opposite to the first direction 432 along the alignment path 406. As the droplet manipulator 402 is moving in the second direction 440, the magnets e.g. 430 immobilise/hold the droplet of reaction mixture 416 (i.e. sample droplet) in position above the magnets e.g. 430. As a result, the droplet of reaction mixture 416 moves along the surface of the sheet member 414 in the first direction 432 to the right towards the droplet manipulator in a relative motion. The direction of motion of the droplet of reaction mixture 416 relative to the surface of the sheet member 414 is depicted as arrows below the base member 404.
[0163] FIG. 4D is a schematic drawing of the system 400 showing mixing of the droplet of reaction mixture 416 by the droplet manipulator 402 in the example embodiment. The droplet manipulator 402 is moved back and forth in the first direction 432 and second direction 440 so that the sample droplets 416 repetitively pass through the pillar array of the mixing element 422 for enhanced mixing. The back and forth motion of the droplet manipulator 402 is depicted in FIG. 4D as arrows 442. After mixing, the sample droplets 416 are incubated to allow the bacterial cells to be fully lysed.
[0164] FIG. 4E is a schematic drawing of the system 400 showing merging of the droplet of reaction mixture 416 with the droplet of detection reagent 420 in the example embodiment. After incubation, the droplet manipulator 402 is moved in the second direction 440 along the alignment path 406 (i.e. to the left). The droplet of detection reagent 420 are also moved in tandem with the droplet manipulator 402 in the second direction 440 toward the droplets of reaction mixture e.g. 416. As the droplet manipulator 402 is moving in the second direction 440, the magnets e.g. 430 immobilise/hold the droplet of reaction mixture 416 (i.e. sample droplet) in position above the magnets e.g. 430. As a result, the sample droplets 416 travel in the first direction 432 (i.e. to the right) in a relative motion to merge with the droplet of detection reagent 420 to form merged droplets e.g. 444.
[0165] FIG. 4F is a schematic drawing of the system 400 showing movement of the droplet manipulator 402 in relation to the base member 404 in the example embodiment. After droplet merging, the droplet manipulator 402 is moved in the first direction 432 along the alignment path 406 (i.e. to the right). The mixing elements e.g. 422 are also moved in tandem with the droplet manipulator 402 in the first direction 432 toward the merged droplets e.g. 444. As the droplet manipulator 402 is moving in the first direction 432, the magnets e.g. 430 immobilise/hold the merged droplets e.g. 444 in position above the magnets e.g. 430. As a result, the merged droplets e.g. 444 travel in the second direction 440 towards the array of pillars of the mixing element 422 (i.e. to the left) in a relative motion.
[0166] FIG. 4G is a schematic drawing of the system 400 showing mixing of the merged droplet 444 by the droplet manipulator 402 in the example embodiment. The droplet manipulator 402 is moved back and forth in the first direction 432 and second direction 440 so that the merged droplets e.g. 444 travel left and right under the array of pillars of the mixing element 422 to facilitate mixing.
[0167] FIG. 4H is a schematic drawing of the system 400 showing coupling of the merged droplets e.g. 444 by the droplet holders e.g. 424 in the example embodiment. After mixing, the droplet manipulator 402 is pushed/moved in the second direction 440 towards, or to contact/abut against the second side wall 428 (i.e. left wall) of the base member 404. As the droplet manipulator 402 is moving in the second direction 440 towards the second side wall 428, the droplet holders e.g. 424 engage and contact the merged droplets e.g. 444.
[0168] FIG. 4I is a schematic drawing of the system 400 showing removal of the magnetic particles e.g. 418 from the merged droplets in the example embodiment. The droplet manipulator 402 is moved in the first direction 432 (i.e. to the right) along the alignment path 406. The merged droplets e.g. 444 which are engaged to the droplet holders e.g. 424 are also moved in tandem with the droplet manipulator 402 in the first direction 432. As a result, the magnetic particles e.g. 418 travel in the second direction 440 (i.e. to the left) in relative motion. As the droplet holders 424 are holding on to the merged droplets e.g. 444, the magnetic particles e.g. 418 are split from the merged droplets e.g. 444 as the droplet manipulator 402 is moved in the first direction 432. The removal of magnetic particles e.g. 418 from the merged droplets e.g. 444 facilitates visualization of the merged droplets e.g. 444. The merged droplets e.g. 444 that are engaged and held by the droplet holders e.g. 424 are positioned directly under the observation window. Prior to observation, the merged droplets e.g. 444 are incubated to allow detection reactions to take place in humidity-controlled environment for a period of time from about 30 min to about 3 hours. The results of the assay may be observed via the observation window e.g. change in color of the detection reagent.
[0169] The system 400 is a magnetic digital microfluidic platform which is suitable for performing various assays which produce results in the form of visual signals. In a Carba NP assay performed on the system 400, the system 400 is first primed with reagents. 10 μL of lysis buffer and 3.5 μL magnetic particles e.g. 418 are dispensed onto the glass coverslip 414 through the left access holes e.g. 410 of both test units in each row of test units e.g. 408, and the droplets form droplets on their own. 10 μL of the detection reagent droplet containing phenol red and 0.1 mM ZnSO.sub.4 are dispensed onto the glass coverslip 414 through the right access holes e.g. 412 of both test units in each row of test units e.g. 408, and the droplets form droplets on their own. The detection reagent droplet in the left test unit contains 6 mg/mL Imipenem antibiotics, whereas the one in the right test unit does not, thereby serving as a control. All of the solutions were prepared before starting the experiment. The results of the assay are shown via the colour of the merged droplets e.g. 444 after incubation. For a sample with bacterial strain that is resistant to the antibiotic tested, a color change is observed in the merged droplet e.g. red to yellow for a phenol red detection reagent. In FIG. 4I, the merged droplet e.g. 444 in the left test unit changed colour as depicted by a change in shade of grey relative to FIG. 4H, while the merged droplet e.g. 444 in the right test unit remained the same colour as depicted by the same shade of grey relative to FIG. 4H. For the assay to be valid, the color of the merged droplet e.g. 444 in the right test unit serving as the control should remain the same colour.
EXAMPLE
[0170] The droplet manipulations required to perform a Carba NP assay on the system/platform have been demonstrated in the various example embodiments.
[0171] FIG. 5A is a photograph of a platform for performing a Carba NP assay. As shown in FIG. 5A, samples, reagents and magnetic particles were dispensed onto respective test units/detection units of the platform to form droplets. Lysis buffer and bacteria sample were added into each detection unit at a first position (see left hand side position of each detection unit in FIG. 5A). Detection reagent (i.e. Solution A with Imipenem for the test group or Solution A without Imipenem for the control group) was added to a second position (see right hand side position of each detection unit in FIG. 5A). After magnetic particles were added into the first position, Carba NP droplet manipulation was performed as shown in the subsequent FIGS. 5B to 5F. FIG. 5B is a photograph of the platform showing mixing of the samples by moving the droplets back and forth under a mixer provided in each detection unit. FIG. 5C is a photograph of the platform showing the sample droplets merged with the reagent droplets. FIG. 5D is a photograph of the platform showing the merged droplets being mixed under the mixer. FIG. 5E is a photograph of the platform showing the merged droplets moved to respective observation windows. FIG. 5F is a photograph of the platform showing magnetic particles extracted/moved out from the merged droplets.
[0172] The device/platform was first primed with reagents. 10 μL of lysis buffer and 3.5 μL of magnetic particles were dispensed onto the glass coverslip through the left access holes of both detection units in each row, and the droplets formed droplets on their own. 10 μL of the detection reagent droplet containing phenol red and 0.1 mM ZnSO.sub.4 were dispensed onto the glass coverslip through the right access holes of both detection units in each row, and the droplets formed droplets on their own. The detection reagent droplet in the left detection unit contained 6 mg/mL Imipenem antibiotics, whereas the one in the right detection unit did not contain Imipenem antibiotics. All of the solutions were prepared before starting the experiment.
[0173] The multiplexed Carba NP assays were carried out. Twenty-four droplets containing reagents and magnetic particles were filled in the holes by using multichannel pipette. Five CPE samples (1.sup.st-5.sup.th) and one negative sample (6.sup.th), counting from top to bottom respectively, were picked and immersed in lysis buffer droplets by using the 3D printed top plate cover to hold the sample tips. After a simple slide of the platform, the yellow color change was observed (Table. 1 and FIG. 6A-FIG. 6G). Table 1 shows a list of samples of bacteria isolates from the top to bottom row and the color changes shown after the 2 hours incubation on the Digital Microfluidic Point-of-Care Platform.
TABLE-US-00001 TABLE 1 List of samples of bateria isolates and results of the Carba NP assay Sol A + No. Samples Imi Sol A 1 Escherichia coli NCTC (IMP-type) Yellow Red 2 Escherichia coli 6013499989 (blaKPC+) Yellow Red 3 Klebsiella pneumonia 2073318014 (blaNDM+) Orange Red 4 Klebsiella pneumonia ATT BAA-1705 (KPC+) Orange Red 5 Escherichia coli MBRL 235 (NDM+) Yellow Red 6[—] Escherichia coli- C3 (6123-57039) Red Red
[0174] FIG. 6A to FIG. 6G are a series of photographs taken at various time points of performing a Carba NP assay using the platform as disclosed herein.
[0175] FIG. 6A is a photograph of a digital microfluidic point-of-care platform for performing a Carba NP assay with the prepared reagents and magnetic particles placed on the platform. As shown in FIG. 6A, the samples in the test group were loaded into the detection units on the left side of the platform (as demarcated by the rectangle in solid line) while the samples in the control group were loaded into the detection units on the right side of the platform (as demarcated by the rectangle in dotted line). 10 μL of lysis buffer and 3.5 μL of magnetic particles were dispensed onto the glass coverslip through the left access holes of each detection units to form droplets (see 1.sup.st and 3.sup.rd columns of the platform in FIG. 6A). 10 μL of the detection reagent droplet containing phenol red and 0.1 mM ZnSO.sub.4 were dispensed onto the glass coverslip through the right access holes of each detection unit to form red colour droplets (see 2.sup.nd and 4.sup.th columns of the platform in FIG. 6A).
[0176] FIG. 6B is a photograph of the platform with a 3D printed top plate cover placed on top of the platform. The top plate cover or guide member comprising 12 guide holes was detachably coupled to the platform such that each of the 12 guide holes were aligned to the left access hole of each detection unit. The guide holes are dimensioned such that a tip of a dispenser such as a droplet dispenser, e.g. pipette tip of a pipetting device is allowed to deliver fluid e.g. fluid containing a sample, but is not allowed to contact the surface where the droplet e.g. droplet of reaction mixture is to be disposed. This may prevent the tip of the droplet dispenser from scratching and/or damaging the surface/substrate, which may impede movement of droplets over the surface. Five CPE samples (1.sup.st-5.sup.th rows of the platform) and one negative sample (6.sup.th row of the platform), counting from top to bottom respectively, were picked and immersed in the lysis buffer droplets by using the 3D printed top plate cover to hold the sample tips.
[0177] FIG. 6C is a photograph of the platform with reaction droplet and detection droplet merged. The magnetic particles are extracted out of the merged droplet after moving the platform left and right. As shown in FIG. 6C, the extracted magnetic particles were positioned on the 1.sup.st and 3.sup.rd columns of the platform while the merged droplets were positioned on the 2.sup.nd and 4.sup.th columns of the platform. As the photograph of FIG. 6C was taken immediately after the reaction droplets and detection droplets were merged, the merged droplets still display the red colour of the phenol red indicator.
[0178] FIG. 6D is a photograph of the platform showing color changes in the droplets after 30 minutes. The test group sample in the 2.sup.nd row of the platform changed colour from red to yellow and the test group sample in the 5.sup.th row of the platform changed colour from red to orange after 30 minutes of merging the reaction droplets and detection droplets. The rest of the droplets remained red in colour after 30 minutes of merging the reaction droplet and detection droplet.
[0179] FIG. 6E is a photograph of the platform showing color changes in the droplets after 1 hour. The test group sample in the 2.sup.nd row of the platform remained yellow and the test group sample in the 5.sup.th row of the platform changed colour from orange to yellow after 1 hour of merging the reaction droplets and detection droplets. In addition, the test group sample in the 1.sup.st row of the platform changed colour from red to yellow after 1 hour of merging the reaction droplets and detection droplets. The rest of the droplet remained red in colour after 1 hour of merging the reaction droplets and detection droplets.
[0180] FIG. 6F is a photograph of the platform showing color changes in the droplets after 2 hours. The test group samples in the 1.sup.st, 2.sup.nd and 5.sup.th rows of the platform remained yellow after 2 hours of merging the reaction droplets and detection droplets. In addition, the test group samples in the 3.sup.rd and 4.sup.th rows of the platform changed colour from red to orange after 2 hours of merging the reaction droplets and detection droplets. The test group sample in the 6.sup.th row remained red in colour after 2 hours of merging the reaction droplet and detection droplet.
[0181] FIG. 6G is a photograph of the platform showing the final results of the Carba NP assay with the test group and control group labelled. The test units on the left column belong to the test group which has an antibiotic agent (Imipenem antibiotics) added. The test units on the right column belong to the control group without addition of the antibiotic agent. The bacterial strains of the samples and the final colours of the respective merged droplets after 2 hours of merging the reaction droplets and detection droplets are summarised in the above Table 1. For the assay to be valid, the color of the droplets in the control group should remain red (see 4.sup.th column of the platform).
[0182] FIG. 7 is a photograph of a droplet manipulator showing 12 test units with reaction droplets for different samples. The results of the assay were based on color changes in accordance to different bacterial strains. The genotypes of the bacterial strains were characterized using molecular techniques. The first five strains, which comprised of two species with 3 CRE subgroups (IMP, KPC and NDM) are CRE.sub.+ (CRE positive) (CRE=carbapenem-resistant enterobacteriaceae, IMP=Imipenem-resistant Pseudomonas, KPC=Klebsiella Pneumoniae Carbapenemase, NDM=New Delhi Metallo-beta-lactamase). The last strain was a CRE-(CRE negative) E. Coli (Escherichia coli) strain. Each strain was tested in a pair of testing units in one row. The left unit contained antibiotics for testing. The right unit was antibiotic-free and served as a control. In the left testing units, the color of the top 5 droplets changed from red to yellow, indicating resistance to carbapenems. The color of the last droplet remained red, suggesting that the strain was susceptible to carbapenems. For the assay to be valid, the color of the control droplets in the right column should remain red.
Applications
[0183] Embodiments of the disclosure provided herein provide a system and method of performing an assay. In various embodiments, an assay may be performed by adding samples and reagents required for the assay into test units provided in a droplet manipulator.
[0184] Advantageously, embodiments of the disclosed system and method provide flexible fluidic control as the reaction mixture is performed on a surface disposed on the droplet manipulator. Accordingly, in various embodiments, the samples and reagents do not have to follow pre-defined fluid flow paths such as those found in conventional channel-based microfluidic platforms. Embodiments of the disclosed system and method also provide flexibility in the types of assays which can be performed. Reagents for various assays can be easily introduced onto the surface of the droplet manipulator via the access ports provided in each testing unit of the droplet manipulator.
[0185] Even more advantageously, embodiments of the disclosed system and method may be capable of simplifying assay workflow and reduces the assay time by testing multiple samples (i.e. multiplexed detection) on a magnetic digital microfluidic platform with just a single action. As the reaction of the assay may be performed in the microdroplet form, consumption of reagents is significantly reduced. For example, the magnetic digital microfluidic system may be capable of conducting multiple assays e.g. Carba NP assays in parallel in droplets. This may overcome the problems of conventional detection assays such as the Carba NP assay, which are tedious to perform and typically only analyze one clinical isolate at a time, which is time-consuming in diagnostic settings where a large number of clinical isolates need to be tested. The magnetic digital microfluidic platform may be applied in multiplexed diagnostics of infections caused by carbapenemase resistance in gram-negative bacilli.
[0186] Even more advantageously, embodiments of the disclosed system and method can be operated manually or by other means that do not require an external power source, which is an important consideration for point-of-care applications in resource-limited environments where electricity is not readily available. Embodiments of the disclosed system and method may also be compatible with automated control systems to provide a high throughput system for performing assays.
[0187] It will be appreciated by a person skilled in the art that other variations and/or modifications may be made to the embodiments disclosed herein without departing from the spirit or scope of the disclosure as broadly described. For example, in the description herein, features of different exemplary embodiments may be mixed, combined, interchanged, incorporated, adopted, modified, included etc. or the like across different exemplary embodiments. The present embodiments are, therefore, to be considered in all respects to be illustrative and not restrictive.
