Devices and Methods for Nucleic Acid Identification in Samples

A device, method, and non-transitory computer readable medium storing instructions for nucleic acid identification of material in a sample includes a microfluidics system, including a movable cartridge, and a heat source. The movable cartridge assembly includes at least one target-specific set of reagent components and a set of RCA-LAMP reaction components deposited on the surface. Moreover, the at least one target-specific set of reagent components includes at least one target specific padlock probe reagent component. The set of RCA-LAMP reaction components includes: at least one polymerase buffer component, at least one polymerase enzyme with strand displacement activity, a betaine additive, a TETRONIC additive, a sequence-specific probe, dNTPs, and a primer mix. The primer mix includes both a forward inside primer and a backward inside primer specific to a backbone of the at least one target specific padlock probe reagent component.

TECHNICAL FIELD

This disclosure relates to devices, methods, and non-transitory computer readable media storing instructions for the amplification and identification of genetic materials that may be present in samples.

BACKGROUND

One of the primary methods used to identify the presence of an organism is the mass amplification of some or all of its genetic material. When originally developed, amplification reactions relied on thermocycling (repeated heating and cooling) certain polymerases to amplify a target organism's genetic material (i.e., polymerase chain reaction, “PCR”). Thermocycling requires dedicated laboratory equipment and a unique primer set for each target to be detected. It is also relatively slow and expensive.

Since the development of thermocycling amplification, efforts have been made to increase the speed and sensitivity of genetic material amplification, reduce its cost, and make it easier to perform. Rolling circle amplification (“RCA”) using padlock probes is one of those reactions. It has been used for nearly thirty years and is advantageous because it is an isothermal method (i.e., does not require thermocycling) that can detect the genetic material of specific organisms. Also, it does not require as much assay development time because it uses a universal set of reagents that recognizes the padlock probe backbone.

Loop-mediated amplification (“LAMP”) is a biochemical reaction developed more recently. It has gained wide usage because it is an isothermal reaction, and it is relatively fast (five to twenty minutes). While it is advantageous from a speed standpoint, it is disadvantageous from a development standpoint. A typical stand-alone LAMP reaction can require a minimum of six primer sets. These sets can be time-consuming to develop, and that time can be overwhelming if many different targets need to be analyzed.

SUMMARY

Disclosed herein are devices, methods, and non-transitory computer readable media storing instructions for the amplification and identification of genetic samples that retain the advantages of both RCA and LAMP, while removing their disadvantages.

According to an exemplary embodiment of the present disclosure, a device for nucleic acid identification of material in a sample includes a microfluidics system and a heat source, where the microfluidics system further includes a movable cartridge assembly. In an embodiment, the movable cartridge assembly includes a surface configured to receive the sample at a sample location, at least one target-specific set of reagent components deposited on the surface at a target location, and at least one set of RCA-LAMP reaction components deposited on the surface at an RCA-LAMP location. Moreover, in an embodiment, the at least one target-specific set of reagent components includes: at least one target-specific padlock probe reagent component, at least one target probe-associated ligase enzyme component, and at least one target probe-associated set of ligase buffer components. In an embodiment, the at least one set of RCA-LAMP reaction components includes: at least one polymerase buffer component, at least one polymerase enzyme with strand displacement activity, a betaine additive, a TETRONIC additive, a sequence-specific probe, dNTPs, and a primer mix. Further still, in an embodiment, the primer mix includes a forward inside primer specific to a backbone of the at least one target-specific padlock probe reagent component, and a backward inside primer specific to the backbone of the at least one target-specific padlock probe reagent component.

According to another exemplary embodiment of the present disclosure, a device for biological identification of a sample includes the device of the previous embodiment, and further includes a second target-specific set of reagent components deposited on the surface at a second target location. Moreover, in an embodiment, the second target-specific set of reagent components includes: a second target-specific padlock probe reagent component, the at least one target probe-associated ligase enzyme component, and the at least one target probe-associated set of ligase buffer components. Further still, in an embodiment, the second target-specific padlock probe reagent component includes the backbone of the at least one target-specific padlock probe reagent component.

According to a further exemplary embodiment, a method for nucleic acid identification of material in a sample includes providing a microfluidics system, where the microfluidics system includes a movable cartridge assembly. In an embodiment, the movable cartridge assembly includes a surface configured to receive the sample at a sample location, at least one target-specific set of reagent components deposited on the surface at a target location, and at least one set of RCA-LAMP reaction components deposited on the surface at an RCA-LAMP location. Moreover, in an embodiment, the at least one target-specific set of reagent components includes: at least one target-specific padlock probe reagent component, at least one target probe-associated ligase enzyme component, and at least one target probe-associated set of ligase buffer components. In an embodiment, the at least one set of RCA-LAMP reaction components includes: at least one polymerase buffer component, at least one polymerase enzyme with strand displacement activity, a betaine additive, a TETRONIC additive, a sequence-specific probe, dNTPs, and a primer mix. Further still, in an embodiment, the primer mix includes a forward inside primer specific to a backbone of the at least one target-specific padlock probe reagent component, and a backward inside primer specific to the backbone of the at least one target-specific padlock probe reagent component. In an embodiment, the method further includes: transporting an aliquot of the sample received on the surface to the target location, applying heat to the target location, transporting an aliquot of RCA-LAMP reaction components on the surface to the target location, and applying heat to the target location.

According to another exemplary embodiment, a method for nucleic acid identification of material in a sample includes the method of the previous embodiment, where the movable cartridge assembly further includes a second target-specific set of reagent components deposited on the surface at a second target location. Moreover, in an embodiment, the second target-specific set of reagent components includes: a second target-specific padlock probe reagent component, the at least one target probe-associated ligase enzyme component, and the at least one target probe-associated set of ligase buffer components. Further still, in an embodiment, the second target-specific padlock probe reagent component includes the backbone of the at least one target-specific padlock probe reagent component. In an embodiment, the method further includes: transporting a second aliquot of the sample received on the surface to the second target location, applying heat to the second target location, transporting a second aliquot of RCA-LAMP reaction components on the surface to the second target location, and applying heat to the second target location.

According to a further exemplary embodiment, a non-transitory computer readable medium storing instructions that when executed by a digital microfluidics system cause the digital microfluidics system to perform a method for nucleic acid identification of material in a sample, the digital microfluidics system including a heat source, the method including transporting an aliquot of the sample received on a surface to a target location, applying heat to the target location, transporting an aliquot of RCA-LAMP reaction components on the surface to the target location, and applying heat to the target location. In an embodiment, the digital microfluidics system includes a movable cartridge assembly. In an embodiment, the movable cartridge assembly further includes a surface configured to receive the sample at a sample location, at least one target-specific set of reagent components deposited on the surface at a target location, and at least one set of RCA-LAMP reaction components deposited on the surface at an RCA-LAMP location. Moreover, in an embodiment, the at least one target-specific set of reagent components includes: at least one target-specific padlock probe reagent component, at least one target probe-associated ligase enzyme component, and at least one target probe-associated set of ligase buffer components. In an embodiment, the at least one set of RCA-LAMP reaction components includes: at least one polymerase buffer component, at least one polymerase enzyme with strand displacement activity, a betaine additive, a TETRONIC additive, a sequence-specific probe, dNTPs, and a primer mix. Further still, in an embodiment, the primer mix includes a forward inside primer specific to a backbone of the at least one target-specific padlock probe reagent component, and a backward inside primer specific to the backbone of the at least one target-specific padlock probe reagent component.

According to another exemplary embodiment, a non-transitory computer readable medium storing instructions that when executed by a digital microfluidics system cause the digital microfluidics system to perform a method for nucleic acid identification of material in a sample includes the non-transitory computer readable medium storing instructions of the previous embodiment, where the movable cartridge assembly further includes a second target-specific set of reagent components deposited on the surface at a second target location. Moreover, in an embodiment, the second target-specific set of reagent components includes: a second target-specific padlock probe reagent component, the at least one target probe-associated ligase enzyme component, and the at least one target probe-associated set of ligase buffer components. Further still, in an embodiment, the second target-specific padlock probe reagent component includes the backbone of the at least one target-specific padlock probe reagent component. In an embodiment, the method further includes: transporting a second aliquot of the sample received on the surface to the second target location, applying heat to the second target location, transporting a second aliquot of RCA-LAMP reaction components on the surface to the second target location, and applying heat to the second target location.

According to further exemplary embodiments, a device, method, or non-transitory computer readable medium storing instructions consistent with the current disclosure can be any of the previous embodiments where: (a) the ligase enzyme component is a Taq DNA ligase enzyme component; and/or (b) the set of ligase buffer components are a set of Taq DNA ligase buffer components. Alternatively, a device, method, or non-transitory computer readable medium storing instructions consistent with the current disclosure can be any of the previous embodiments where: (a) the ligase enzyme component is an RNA ligase enzyme component; and/or (b) the set of ligase buffer components are a set of RNA ligase buffer components.

According to further exemplary embodiments, a device or method consistent with the current disclosure can be any of the previous embodiments where the microfluidics system is a digital microfluidics system.

Further still, a device, method, or non-transitory computer readable medium storing instructions consistent with the current disclosure can be any of the previous embodiments where: (a) the sequence-specific probe is an oligonucleotide strand displacement probe; (b) the at least one polymerase buffer component is a Bst3 polymerase buffer component; and/or (c) the at least one polymerase enzyme with strand displacement activity is a Bst3 polymerase enzyme.

Moreover, in an embodiment, a device, method, or non-transitory computer readable medium storing instructions consistent with the current disclosure can be any of the previous embodiments where the heat source can be configured to heat fluid located at the first and/or second target location to approximately 95 degrees Celsius, and, after cooling, to approximately 65 degrees Celsius.

In a further embodiment, a device, method, or non-transitory computer readable medium storing instructions consistent with the current disclosure can be any of the previous embodiments where movable cartridge assembly is a consumable cartridge such as—but not limited to—a “single use” cartridge.

Moreover, in an embodiment, a device, method, or non-transitory computer readable medium storing instructions consistent with the current disclosure can be any of the previous embodiments further including a source of excitation radiation, and/or a camera system for monitoring possible fluorescence emanating from the surface of the movable cartridge assembly. Further still, the source of excitation radiation can be an LED configured to emit at 495 nm (or any other appropriate excitation wavelength).

In a further embodiment, a device, method, or non-transitory computer readable medium storing instructions consistent with the current disclosure can be any of the previous embodiments where the microfluidics system is configured to transport an aliquot of the sample received on the surface to the target location and is further configured to transport a second aliquot of the sample received on the surface to the second target location. Further still, the microfluidics system can be further configured to transport an aliquot of RCA-LAMP reaction components on the surface to the target location and is further configured to transport a second aliquot of RCA-LAMP reaction components on the surface to the second target location.

In a further embodiment, a device, method, or non-transitory computer readable medium storing instructions consistent with the current disclosure can be any of the previous embodiments where the at least one target-specific set of reagent components deposited on the surface at the target location are printed at the target location, and/or where the second target-specific set of reagent components deposited on the surface at the second target location are printed at the second target location. Further still, a device, method, or non-transitory computer readable medium storing instructions consistent with the current disclosure can be any of the previous embodiments where the at least one set of RCA-LAMP reaction components deposited on the surface at the RCA-LAMP location are deposited in a dried form. Such an embodiment can further include a means or step to hydrate the dried RCA-LAMP reaction components.

DETAILED DESCRIPTION

Exemplary embodiments are described with reference to the accompanying drawings. In the figures, which are not necessarily drawn to scale, the left-most digit(s) of a reference number identifies the figure in which the reference number first appears. Wherever convenient, the same reference numbers are used throughout the drawings to refer to the same or like parts. While examples and features of disclosed principles are described herein, modifications, adaptations, and other implementations are possible without departing from the spirit and scope of the disclosed embodiments. Also, the words “comprising,” “having,” “containing,” and “including,” and other similar forms are intended to be equivalent in meaning and be open ended in that an item or items following any one of these words is not meant to be an exhaustive listing of such item or items, or meant to be limited to only the listed item or items. It should also be noted that as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise.

Embodiments of the present disclosure relate generally to devices, methods, and non-transitory computer readable media storing instructions for the amplification and identification of genetic materials that may be present in samples by combining aspects of RCA and LAMP. In an embodiment, a padlock probe (an aspect of RCA) is a linear piece of single-stranded DNA and/or RNA. Its 5′ and 3′ arms are complementary to the genetic sequence of the target, and universal primer/probe binding sites are constructed between the arms. In the presence of the target, in an embodiment, the padlock probe arms anneal to the target genetic material in such a way that the ends of the arms become adjacent (causing the padlock probe to form a circular shape). Once it forms this circular shape, the padlock probe arms can be covalently linked with a ligase enzyme. This transforms it from a linear strand into a circular strand. Once the padlock probe is in a circular conformation, two LAMP primers (the forward inside primer, “FIP,” and the backward inside primer, “BIP”) can bind to it and begin creating a complementary strand that initially facilitates a hyper-branched RCA reaction. Once the first constituents of the hyper-branched RCA reaction are produced, FIP and BIP transition from an RCA reaction to a rapid LAMP reaction whose products can be detected in under twenty-five minutes. Devices, methods, and non-transitory computer-readable media storing instructions consistent with the present disclosure couples RCA and LAMP with a microfluidic device, such as a digital microfluidics (“DMF”) system. One of ordinary skill in the art will appreciate that devices, methods, and non-transitory computer-readable media storing instructions consistent with the current disclosure can also be implemented using platforms based upon other kinds of microfluidic flow, including but not limited to: pressure-based, electrophoretic-based, gravitational-based, centrifugal-based, capillary-based, thermal-based, magnetophoretic, acoustic-based and electrowetting.

In accordance with embodiments of the present disclosure, there may be provided a device with a microfluidics system, such as a digital microfluidics system, and a heat source.

FIG. 1illustrates an exemplary device100consistent with the present disclosure. Device100can include a DMF processor system110. Connected to DMF processor system110are: DMF transport grid couplers115, a coupler128to a combination display125and user interface127, a coupler178to a lamp170, a coupler188to a camera system180, and a coupler198to a heating/cooling device190. Device100also includes a slot152that is configured to accommodate movable cartridge assembly150. Alternatively, or in addition, slot152can be configured to accommodate movable cartridge assembly160.

Movable cartridge assembly150includes DMF transport grid interface155, which is configured to couple with DMF transport grid couplers115. Likewise, movable cartridge assembly160includes DMF transport grid interface165, which is configured to couple with DMF transport grid couplers115. One of ordinary skill in the art associated with DMF systems would appreciate that DMF transport grid interface155and DMF transport grid interface165can be consistent with PCB board edge connectors. Likewise, one of ordinary skill in the art associated with DMF systems would appreciate that DMF transport grid couplers115can be consistent with PCB board edge connector sockets.

FIG. 2provides an alternate perspective of the device ofFIG. 1without movable cartridge assembly150and without movable cartridge assembly160. Consistent with the present disclosure, lamp170, camera system180and heating/cooling device190can be situated to one side of slot152. Moreover, consistent with the present disclosure, heating/cooling device190can be situated beneath slot152, and lamp170and camera system180can be situated above slot152but within the housing of device100. Consistent with an embodiment of the present disclosure, when either movable cartridge150or movable cartridge160is situated within slot152(not shown inFIG. 2), lamp170and camera system180can be situated above a transparent plate (described further below) that forms a portion of movable cartridge150or movable cartridge160in a preferred embodiment. In an embodiment, camera system180can incorporate additional optical elements including, without limitation, filters as appropriate.

FIG. 4illustrates an exploded cross-section view of movable cartridge assembly150ofFIG. 3. Also shown inFIG. 4are: cross-section location10A-B with perspective indicator, cross-section location11C-D with perspective indicator, and cross-section location12E-F with perspective indicator. (As stated above, cross-sections of movable cartridge assembly150associated with these locations and perspectives are shown, respectively, inFIGS. 10, 11, and 12.)FIG. 4also depicts (in exemplary order): top frame352, transparent plate490, spacer492, DMF Board450, and back frame458. Again, shown as part of DMF Board450is DMF transport grid interface155. As depicted inFIG. 4, openings311,312,313, and314in top frame352for assembly fasteners line up with openings411,412,413, and414(respectively) in back frame458. Consistent with the present disclosure, openings311,312,313, and314for assembly fasteners and openings411,412,413, and414for assembly fasteners can accommodate fasteners (such as, but not limited to, bolts) for structurally maintaining the movable cartridge assembly150in the relative order shown inFIG. 4.

FIGS. 5-9illustrate top views of components of the movable cartridge assembly ofFIG. 4, where each ofFIGS. 5-9include cross-section locations10A-B,11C-D, and12E-F.

FIG. 5depicts a top view of top frame352. Consistent with the present disclosure, top frame352includes opening366to RCA-LAMP location, opening368to sample location, and opening370to transport and reaction area. In a preferred embodiment, top frame352can be composed of rigid material such as, but not limited to, FR-4 that (when secured to the back frame458—which is composed of like material) structurally maintains the relative order of the components of the movable cartridge assembly150and any necessary gaps between the components. In one embodiment, opening370can be approximately 56 mm×25 mm, opening368can be approximately 9 mm×39 mm, opening366can be approximately 45 mm×9 mm, with the entire structure of top frame352lying within a rectangle that can be approximately 100 mm×57 mm. In an embodiment, top frame352can have a thickness of the order of millimeters (i.e., approximately 1 mm if relatively flat, or ((not shown)) approximately 2 mm if shaped to envelope over, such as provide a shaped housing, for transparent plate490, spacer492, and DMF Board450). In a preferred embodiment, where spacer492is a conductor, top frame352can generally be composed of non-conducting material.

FIG. 6depicts a top view of transparent plate490. In an embodiment, transparent plate490can be formed of a glass plate with a thin layer of indium tin oxide on the “bottom” side (from the perspective ofFIG. 6) to render it both transparent and conductive. Generally, in an embodiment, transparent plate490can be composed of any transparent conductive material. As used herein “transparent” means transmissive to incident radiation without appreciable scattering (or blocking) of the incident radiation, where the incident radiation can include any probing, excitation, and/or fluorescent radiation of interest (as described further below). In an embodiment, for example, transparent material can provide for approximately 60% or higher transmission of relevant incident radiation, and, in a preferred embodiment, highly transparent material can provide for approximately 75% or approximately 85% or higher transmission of relevant incident radiation. With regard to conductivity, in an embodiment, the sheet resistance of transparent plate490can be in a range from approximately 1 Ω/sq (“Ohms per square”) to approximately 200 Ω/sq. Transparent plate490can have a thickness in the millimeter range and, in a preferred embodiment, can have a thickness of approximately 1.1 millimeters. One of ordinary skill in the art would appreciate that the bottom surface of transparent plate490, in the transport and reaction area associated with microfluidic flow, and below any thin conductive coating, can be provided with a hydrophobic coating. In an embodiment, hydrophobic coating on transparent plate490can include Teflon, cytonix fluropel 1101V-FS, or Cytop CT L 809 M applied in an even method through spray, spin, dip or blot methods such that the thickness of the hydrophobic coating is between approximately 100-10,000 nanometers. One of ordinary skill in the art would also appreciate that where the bottom surface of transparent plate490is away from the transport and reaction area associated with microfluidic flow, and where transparent plate490is configured to make contact with a conductor that is part of the circuit in the digital microfluidics system responsible for controlling microfluidic flow (such as in a select region where transparent plate490is configured to make direct electrical contact with spacer492, described below), the bottom surface of transparent plate490can be masked off so that the thin conductive layer in transparent plate490can make direct contact with the conductor, and thereby ensure that transparent plate490is part of the circuit in the digital microfluidics system.

FIG. 7depicts a top view of spacer492. In an embodiment spacer492can be manufactured out of conductive material, including, but not limited to, stainless steel and copper. Furthermore, in an embodiment where the conductive electrodes in the transport and reaction area on the DMF Board450associated with microfluidic flow (described below), are approximately 2.6 mm×2.6 mm squares, then spacer492can be approximately 210-270 micrometers thick to set the appropriate gap between the “top” surface of the DMF Board450and the “bottom” surface of transparent plate490. In a preferred embodiment, spacer492can be configured to be approximately 230 micrometers thick. One of ordinary skill in the art would appreciate that the thickness of spacer492(and, therefore, the gap thickness between the “top” surface of the DMF Board450and the “bottom” surface of transparent plate490) scales with the size of the approximately square conductive electrodes in the transport and reaction area on the DMF Board450associated with microfluidic flow.

FIG. 8depicts a top view of DMF Board450. Consistent with the present disclosure, DMF Board450includes DMF transport grid interface155. Also shown inFIG. 8are DMF electrode locations805. As described above, in an embodiment, the size of the electrodes at the electrode locations805can be approximately 2.6 mm×2.6 mm squares. One of ordinary skill in the art would appreciate that the electrodes at certain “reservoir” locations on the surface of DMF Board450, such as sample location880and RCA-LAMP location855, can generally be larger polygons composed of the base square electrode size. Moreover, one of ordinary skill in the art associated with DMF systems would appreciate the locations805represent grid-like locations on the surface of DMF Board450where fluid portions can be manipulated to move within movable cartridge assembly150according to the electronics of DMF Board450and any programming logic supported by a connected DMF processor system110. As with the “bottom” of transparent plate490, one of ordinary skill in the art would appreciate that the “top” surface of DMF Board450in the transport and reaction area, and in the “reservoir” areas described above, can be provided with a hydrophobic coating. In an embodiment, hydrophobic coating on DMF Board450can include Teflon, cytonix fluropel 1101V-FS, or Cytop CT L 809 M applied in an even method through spray, spin, dip or blot methods such that the thickness of the hydrophobic coating is between approximately 100-10,000 nanometers.

Also shown inFIG. 8are first target location810, second target location820, third target location830, and fourth target location840. Located near or on an electrode location805in the first target location810is first target-specific set of reagent components815. Likewise, located near or on an electrode location805in the second target location820is second target-specific set of reagent components825. Located near or on an electrode location805in the third target location830is third target-specific set of reagent components835. Similarly, located near or on an electrode location805in the fourth target location840is fourth target-specific set of reagent components845.

In one embodiment consistent with the present disclosure, the first target-specific set of reagent components815, the second target-specific set of reagent components825, the third target-specific set of reagent components835, and the fourth target-specific set of reagent components845are deposited on the surface of DMF Board450over the hydrophobic coating. Preferably, in an embodiment, the first target-specific set of reagent components815, the second target-specific set of reagent components825, the third target-specific set of reagent components835, and the fourth target-specific set of reagent components845are deposited (and preferably printed) on the surface of DMF Board450in a dried state. Consistent with the present disclosure, one of ordinary skill in the art can select the reagent components to be deposited or printed at first target location810, second target location820, third target location830, and fourth target location840(which are the padlock probes) based upon the targeted nucleic acids. The selection of reagents based upon the targeted nucleic acid is well known in the art as disclosed, for example, and without limitation, in the article “Padlock Probe Assay for Detection and Subtyping of Seasonal Influenza” by F. Neumann, et al.Clinical Chemistry, vol. 64, no. 12, pp. 1704-1712 (Dec. 1, 2018), where target-specific reagent components are selected in a padlock-probe-based method to identify influenza-positive samples. In an embodiment, for example, first target-specific set of reagent components815are selected based upon a first target nucleic acid such that the first target-specific set of reagent components815include the padlock probe target sequence appropriate to the first target nucleic acid. Likewise, in an embodiment, the second target-specific set of reagent components825are selected based upon a second target nucleic acid (which can be different from the first target nucleic acid) such that the second target-specific set of reagent components825include the padlock probe target sequence appropriate to the second target nucleic acid. Similarly, in an embodiment, third target-specific set of reagent components835are selected based upon a third target nucleic acid (which can be different from both the first target nucleic acid and the second target nucleic acid) such that the third target-specific set of reagent components835include the padlock probe target sequence appropriate to the third target nucleic acid. Further still, in an embodiment, fourth target-specific set of reagent components845are selected based upon a fourth target nucleic acid (which can be different from the first, second, and third target nucleic acids) such that the fourth target-specific set of reagent components845include the padlock probe target sequence appropriate to the fourth target nucleic acid.

FIG. 8also depicts sample location880and RCA-LAMP location850. In an embodiment, located on an electrode location805in the RCA-LAMP location850over the hydrophobic coating are RCA-LAMP reaction components855. Moreover, in an embodiment consistent with the present disclosure, RCA-LAMP reaction components855are deposited at the RCA-LAMP location850in a dried state. Consistent with the present disclosure, the RCA-LAMP components855are “universal” reagents in the sense that the reagents in the RCA-LAMP components855are selected to function with each of the first target-specific set of reagent components815, the second target-specific set of reagent components825, the third target-specific set of reagent components835, and the fourth target-specific set of reagent components845regardless of the target nucleic acid each of the target-specific set of reagent components (815,825,835, and845in the present disclosure) are specifically selected to identify using the padlock probe technique. The “universal” reagents in the RCA-LAMP components855include (where the reagents flagged with an asterisk, “*”, are those that directly associate with the padlock probe backbone in the target-specific set of reagent components): ligase buffer; ligase enzyme*; RCA-LAMP buffer; TETRONIC; betaine; oligonucleotide strand displacement (OSD) probe*; deoxynucleotidetriphosphates (dNTPs); forward inside primer* (FIP); backward inside primer* (BIP); and Bst3* (polymerase with strand displacement activity).

Consistent with the present disclosure, the surface of DMF Board450at sample location880is accessible through both an opening in the spacer492and the opening368in top cover352. This allows a sample to be provided on the surface of DMF Board450at sample location880through the top of movable cartridge assembly150. Likewise, consistent with the present disclosure, the dried RCA-LAMP reaction components855on the surface of DMF Board450at RCA-LAMP sample location850is accessible through an opening in the spacer492and the opening366in top cover352. This allows fluid to be provided to the surface of DMF Board450through the top of movable cartridge assembly150at RCA-LAMP location850, in order to hydrate any dried RCA-LAMP reaction components855. Also depicted inFIG. 8is electrode connector801, which, consistent with an embodiment herein, can be connected to electrical “ground” through the DMF transport grid interface155, and can also make contact with the spacer492(which is conductive), and which, in turn, can make contact with a portion of the bottom of transparent plate490, where the hydrophobic coating has not been applied (through masking, for example) so that the conductive layer portion of transparent plate490is thereby connected to electrical “ground.”

FIG. 9depicts a top view of back frame458. Also shown inFIG. 9are openings411,412,413, and414, discussed earlier in connection withFIG. 4. In a preferred embodiment, back frame458can be composed of rigid material such as, but not limited to, FR-4 that (when secured to the top frame352as described above) structurally maintains the relative order of the components of the movable cartridge assembly150and any necessary gaps between the components. In one embodiment, the entire structure of back frame458lies within a rectangle that is approximately 100 mm×60 mm. In an embodiment, back frame458can have a thickness of the order of millimeters (i.e., approximately 1 mm if relatively flat). In a preferred embodiment, where spacer492is a conductor, back frame458can generally be composed of non-conducting material.

FIGS. 10-12illustrate cross-section views of portions of the exemplary movable cartridge assembly ofFIGS. 3 and 4.

FIG. 10shows a cross-section view at location10A-B, which includes a cross-section view of DMF Board450within first target location810. Consequently,FIG. 10includes a view of first target-specific set of reagent components815on hydrophobic coating1070. DMF Board450further includes dielectric1060, substrate1050, electrodes1005and electrode connectors1007(where electrode connectors1007, in turn, connect to the DMF transport grid interface155and are under control of DMF processor system110). Spacer492is shown, as well as transparent plate490, which can include glass layer1095, conductive layer1090, and hydrophobic coating1080. Dielectric1060can include material such as, but not limited to, parylene-C and can be formed in a layer with a thickness from approximately 2-20 microns.

FIG. 11shows a cross-section view at location11C-D, which includes a cross-section view of DMF Board450within RCA-LAMP location850. Consequently,FIG. 11includes a view of RCA-LAMP reaction components855on hydrophobic coating1070. As inFIG. 10, DMF Board450further includes dielectric1060, substrate1050, electrodes1005and electrode connectors1007(where electrode connectors1007, in turn, connect to the DMF transport grid interface155, and are under control of DMF processor system110). Spacer492is shown, as well as transparent plate490, which includes glass layer1095, conductive layer1090, and hydrophobic coating1080.

FIG. 12shows a cross-section view at location12E-F, which includes a cross-section view of DMF Board450within sample location880. As inFIGS. 10 and 11, DMF Board450further includes dielectric1060, substrate1050, electrodes1005and electrode connectors1007(where electrode connectors1007, in turn, connect to the DMF transport grid interface155, and are under control of DMF processor system110). Spacer492is shown, as well as transparent plate490, which includes glass layer1095, conductive layer1090, and hydrophobic coating1080.

FIG. 14illustrates an exploded cross-section view of movable cartridge assembly160ofFIG. 13. Also shown inFIG. 14are: cross-section location20A-B with perspective indicator, cross-section location21C-D with perspective indicator, and cross-section location22E-F with perspective indicator. (As stated above, cross-sections of movable cartridge assembly160associated with these locations and perspectives are shown, respectively, inFIGS. 20, 21, and 22.)FIG. 14also depicts (in exemplary order): top frame1352, transparent plate1490, spacer1492, DMF Board1450, and back frame1458. Again, shown as part of DMF Board1450is DMF transport grid interface165. As depicted inFIG. 14, openings1311,1312,1313, and1314in top frame1352for assembly fasteners line up with openings1411,1412,1413, and1414(respectively) in back frame1458. Consistent with the present disclosure, openings1311,1312,1313, and1314for assembly fasteners and openings1411,1412,1413, and1414for assembly fasteners can accommodate fasteners (such as, but not limited to, bolts) for structurally maintaining the movable cartridge assembly160in the relative order shown inFIG. 14.

FIGS. 15-19illustrate top views of components of the movable cartridge assembly ofFIG. 14, where each ofFIGS. 15-19include cross-section locations20A-B,21C-D, and22E-F.

FIG. 15depicts a top view of top frame1352. Consistent with the present disclosure, top frame1352includes opening1366to RCA-LAMP location, opening1368to sample location, and opening1370to transport and reaction area. In a preferred embodiment, top frame1352can be composed of rigid material such as, but not limited to, FR-4 that (when secured to the back frame1458—which is composed of like material) structurally maintains the relative order of the components of the movable cartridge assembly160and any necessary gaps between the components. In one embodiment, opening1370can be approximately 56 mm×25 mm, opening1368can be approximately 9 mm×9 mm, opening1366can be approximately 9 mm×9 mm, with the entire structure of top frame1352lying within a rectangle that can be approximately 100 mm×57 mm. In an embodiment, top frame1352can have a thickness of the order of millimeters (i.e., approximately 1 mm if relatively flat, or ((not shown)) approximately 2 mm if shaped to envelope over, such as provide a shaped housing, for transparent plate1490, spacer1492, and DMF Board1450). In a preferred embodiment, where spacer1492is a conductor, top frame1352can generally be composed of non-conducting material.

FIG. 16depicts a top view of transparent plate1490. In an embodiment, transparent plate1490can be formed of a glass plate with a thin layer of indium tin oxide on the “bottom” side (from the perspective ofFIG. 16) to render it both transparent and conductive. Generally, in an embodiment, transparent plate1490can be composed of any transparent conductive material. In an embodiment, for example, transparent material can provide for approximately 60% or higher transmission of relevant incident radiation, and, in a preferred embodiment, highly transparent material can provide for approximately 75% or approximately 85% or higher transmission of relevant incident radiation. With regard to conductivity, in an embodiment, the sheet resistance of transparent plate1490can be in a range from approximately 1 Ω/sq to approximately 200 Ω/sq. Transparent plate1490can have a thickness in the millimeter range and, in a preferred embodiment, can have a thickness of approximately 1.1 millimeters. One of ordinary skill in the art would appreciate that the bottom surface of transparent plate1490, in the transport and reaction area associated with microfluidic flow, and below any thin conductive coating, can be provided with a hydrophobic coating. In an embodiment, hydrophobic coating on transparent plate1490can include Teflon, cytonix fluropel 1101V-FS, or Cytop CT L 809 M applied in an even method through spray, spin, dip or blot methods such that the thickness of the hydrophobic coating is between approximately 100-10,000 nanometers. One of ordinary skill in the art would also appreciate that where the bottom surface of transparent plate1490is away from the transport and reaction area associated with microfluidic flow, and where transparent plate1490is configured to make contact with a conductor that is part of the circuit in the digital microfluidics system responsible for controlling microfluidic flow (such as in a select region where transparent plate1490is configured to make direct electrical contact with spacer1492, described below), the bottom surface of transparent plate1490can be masked off so that the thin conductive layer in transparent plate1490can make direct contact with the conductor, and thereby ensure that transparent plate1490is part of the circuit in the digital microfluidics system.

FIG. 17depicts a top view of spacer1492. In an embodiment spacer1492can be manufactured out of conductive material, including, but not limited to, stainless steel and copper. Furthermore, in an embodiment where the conductive electrodes in the transport and reaction area on the DMF Board1450associated with microfluidic flow (described below), are approximately 2.6 mm×2.6 mm squares, then spacer1492can be approximately 210-270 micrometers thick to set the appropriate gap between the “top” surface of the DMF Board1450and the “bottom” surface of transparent plate1490. In a preferred embodiment, spacer1492can be configured to be approximately 230 micrometers thick. One of ordinary skill in the art would appreciate that the thickness of spacer1492(and, therefore, the gap thickness between the “top” surface of the DMF Board1450and the “bottom” surface of transparent plate1490) scales with the size of the approximately square conductive electrodes in the transport and reaction area on the DMF Board1450associated with microfluidic flow.

FIG. 18depicts a top view of DMF Board1450. Consistent with the present disclosure, DMF Board1450includes DMF transport grid interface165. Also shown inFIG. 8are DMF electrode locations1805. As described above, in an embodiment, the size of the electrodes at the electrode locations1805can be approximately 2.6 mm×2.6 mm squares. One of ordinary skill in the art would appreciate that the electrodes at certain “reservoir” locations on the surface of DMF Board1450, such as sample location1880and RCA-LAMP location1855, can generally be larger polygons composed of the base square electrode size. Moreover, one of ordinary skill in the art associated with DMF systems would appreciate the locations1805represent grid-like locations on the surface of DMF Board1450where fluid portions can be manipulated to move within movable cartridge assembly160according to the electronics of DMF Board1450and any programming logic supported by a connected DMF processor system110. As with the “bottom” of transparent plate1490, one of ordinary skill in the art would appreciate that the “top” surface of DMF Board1450in the transport and reaction area, and in the “reservoir” areas described above, can be provided with a hydrophobic coating. In an embodiment, hydrophobic coating on DMF Board1450can include Teflon, cytonix fluropel 1101V-FS, or Cytop CT L 809 M applied in an even method through spray, spin, dip or blot methods such that the thickness of the hydrophobic coating is between approximately 100-10,000 nanometers.

Also shown inFIG. 18are first target location1810, second target location1820, third target location1830, and fourth target location1840. Located near or on an electrode location1805in the first target location1810is first target-specific set of reagent components1815. Likewise, located near or on an electrode location1805in the second target location1820is second target-specific set of reagent components1825. Located near or on an electrode location1805in the third target location1830is third target-specific set of reagent components1835. Similarly, located near or on an electrode location1805in the fourth target location1840is fourth target-specific set of reagent components1845.

In one embodiment consistent with the present disclosure, the first target-specific set of reagent components1815, the second target-specific set of reagent components1825, the third target-specific set of reagent components1835, and the fourth target-specific set of reagent components1845are deposited on the surface of DMF Board1450over the hydrophobic coating. Preferably, in an embodiment, the first target-specific set of reagent components1815, the second target-specific set of reagent components1825, the third target-specific set of reagent components1835, and the fourth target-specific set of reagent components1845are deposited (and preferably printed) on the surface of DMF Board1450in a dried state. Consistent with the present disclosure, one of ordinary skill in the art can select the reagent components to be deposited or printed at first target location1810, second target location1820, third target location1830, and fourth target location1840(which are the padlock probes) based upon the targeted nucleic acids. The selection of reagents based upon the targeted nucleic acid is well known in the art as disclosed, for example, and without limitation, in the article cited earlier, “Padlock Probe Assay for Detection and Subtyping of Seasonal Influenza” by F. Neumann, et al.Clinical Chemistry, vol. 64, no. 12, pp. 1704-1712 (Dec. 1, 2018), where target-specific reagent components are selected in a padlock-probe-based method to identify influenza-positive samples. In an embodiment, for example, first target-specific set of reagent components1815are selected based upon a first target nucleic acid such that the first target-specific set of reagent components1815include the padlock probe target sequence appropriate to the first target nucleic acid. Likewise, in an embodiment, the second target-specific set of reagent components1825are selected based upon a second target nucleic acid (which can be different from the first target nucleic acid) such that the second target-specific set of reagent components1825include the padlock probe target sequence appropriate to the second target nucleic acid. Similarly, in an embodiment, third target-specific set of reagent components1835are selected based upon a third target nucleic acid (which can be different from both the first target nucleic acid and the second target nucleic acid) such that the third target-specific set of reagent components1835include the padlock probe target sequence appropriate to the third target nucleic acid. Further still, in an embodiment, fourth target-specific set of reagent components1845are selected based upon a fourth target nucleic acid (which can be different from the first, second, and third target nucleic acids) such that the fourth target-specific set of reagent components1845include the padlock probe target sequence appropriate to the fourth target nucleic acid.

FIG. 18also depicts sample location1880and RCA-LAMP location1850. In an embodiment, located on an electrode location1805in the RCA-LAMP location1850over the hydrophobic coating are RCA-LAMP reaction components1855. Moreover, in an embodiment consistent with the present disclosure, RCA-LAMP reaction components1855are deposited at the RCA-LAMP location1850in a dried state. Consistent with the present disclosure, the RCA-LAMP components1855are “universal” reagents in the sense that the reagents in the RCA-LAMP components1855are selected to function with each of the first target-specific set of reagent components1815, the second target-specific set of reagent components1825, the third target-specific set of reagent components1835, and the fourth target-specific set of reagent components1845regardless of the target nucleic acid each of the target-specific set of reagent components (1815,1825,1835, and1845in the present disclosure) are specifically selected to identify using the padlock probe technique. The “universal” reagents in the RCA-LAMP components1855include (where the reagents flagged with an asterisk, “*”, are those that directly associate with the padlock probe backbone in the target-specific set of reagent components): ligase buffer; ligase enzyme*; RCA-LAMP buffer; TETRONIC; betaine; oligonucleotide strand displacement (OSD) probe*; deoxynucleotidetriphosphates (dNTPs); forward inside primer* (FIP); backward inside primer* (BIP); and Bst3* (polymerase with strand displacement activity).

Consistent with the present disclosure, the surface of DMF Board1450at sample location1880is accessible through both an opening in the spacer1492and the opening1368in top cover1352. This allows a sample to be provided on the surface of DMF Board1450at sample location1880through the top of movable cartridge assembly160. Likewise, consistent with the present disclosure, the dried RCA-LAMP reaction components1855on the surface of DMF Board1450at RCA-LAMP sample location1850is accessible through an opening in the spacer1492and the opening1366in top cover1352. This allows fluid to be provided to the surface of DMF Board1450through the top of movable cartridge assembly160at RCA-LAMP location1850, to hydrate any dried RCA-LAMP reaction components1855. Also depicted inFIG. 18is electrode connector1801, which, consistent with an embodiment herein, can be connected to electrical “ground” through the DMF transport grid interface165, and can also make contact with the spacer1492(which is conductive), and which, in turn, can make contact with a portion of the bottom of transparent plate1490, where the hydrophobic coating has not been applied (through masking, for example) so that the conductive layer portion of transparent plate1490is thereby connected to electrical “ground.”

FIG. 19depicts a top view of back frame1458. Also shown inFIG. 19are openings1411,1412,1413, and1414, discussed earlier in connection withFIG. 14. In a preferred embodiment, back frame1458can be composed of rigid material such as, but not limited to, FR-4 that (when secured to the top frame1352as described above) structurally maintains the relative order of the components of the movable cartridge assembly160and any necessary gaps between the components. In one embodiment, the entire structure of back frame1458lies within a rectangle that is approximately 100 mm×60 mm. In an embodiment, back frame1458can have a thickness of the order of millimeters (i.e., approximately 1 mm if relatively flat). In a preferred embodiment, where spacer1492is a conductor, back frame1458can generally be composed of non-conducting material.

FIGS. 20-22illustrate cross-section views of portions of the exemplary movable cartridge assembly ofFIGS. 13 and 14.

FIG. 20shows a cross-section view at location20A-B, which includes a cross-section view of DMF Board1450within first target location1810. Consequently,FIG. 20includes a view of first target-specific set of reagent components1815on hydrophobic coating2070. DMF Board1450further includes dielectric2060, substrate2050, electrodes2005and electrode connectors2007(where electrode connectors2007, in turn, connect to the DMF transport grid interface165and are under control of DMF processor system110). Spacer1492is shown, as well as transparent plate1490, which can include glass layer2095, conductive layer2090, and hydrophobic coating2080. Dielectric2060can include material such as, but not limited to, parylene-C and can be formed in a layer with a thickness from approximately 2-20 microns.

FIG. 21shows a cross-section view at location21C-D, which includes a cross-section view of DMF Board1450within RCA-LAMP location1850. Consequently,FIG. 21includes a view of RCA-LAMP reaction components1855on hydrophobic coating2070. As inFIG. 20, DMF Board1450further includes dielectric2060, substrate2050, electrodes2005and electrode connectors2007(where electrode connectors2007, in turn, connect to the DMF transport grid interface165, and are under control of DMF processor system110). Spacer1492is shown, as well as transparent plate1490, which includes glass layer2095, conductive layer2090, and hydrophobic coating2080.

FIG. 22shows a cross-section view at location22E-F, which includes a cross-section view of DMF Board1450within sample location1880. As inFIGS. 20 and 21, DMF Board1450further includes dielectric2060, substrate2050, electrodes2005and electrode connectors2007(where electrode connectors2007, in turn, connect to the DMF transport grid interface165, and are under control of DMF processor system110). Spacer1492is shown, as well as transparent plate1490, which includes glass layer2095, conductive layer2090, and hydrophobic coating2080.

FIG. 23is a schematic diagram of a data processing system2300for managing the mixing and evaluating of samples. The system2300can include a processor2310, a memory module2315, a storage device2320, an input interface127, a display device125, a microfluidics hydrating and transport module2340, a target probe reagent reaction module2360, an RCA-LAMP reaction module2365, and an excitation/fluorescence module2370. System2300can also include a hydrating and transport grid interface2342, a heating/cooling interface2362, and a lamp/camera interface2372. The system2300can include additional, fewer, and/or different components than those listed above. The type and number of listed devices are exemplary only and not intended to be limiting.

The processor2310can be a central processing unit (“CPU”) or a graphic processing unit (“GPU”). The processor2310can execute sequences of computer program instructions to perform various processes that will be explained in greater detail below. The memory module2315can include, among other things, a random access memory (“RAM”) and a read-only memory (“ROM”). Generally, memory module2315can be a non-transitory computer readable medium. The computer program instructions can be accessed and read from the ROM, or any other suitable memory location, and loaded into the RAM for execution by the processor2310. The processor2310can include one or more printed circuit boards, and/or a microprocessor chip.

The storage device2320can include any type of mass storage suitable for storing information. For example, the storage device2320can include one or more hard disk devices, optical disk devices, or any other storage devices that provide data storage space. For example, the storage device2320can store data related to a data processing process, such as the processing of data received from the camera system180, and any intermediate data created during the data processing process. The storage device2320can also include analysis and organization tools for analyzing and organizing data and/or information contained therein.

In an embodiment, the hydrating and transport grid interface2342is configured to provide the electrical impulses (or the instructions for impulses) to the DMF Board450, the DMF Board1450, transparent plate490, and transparent plate1490(as appropriate) to cause microfluidic flow to occur in the gap between the surface of DMF Board450and the bottom of transparent plate490, or between the surface of DMF Board1450and the bottom of transparent plate1490(as appropriate). In another embodiment, the hydrating and transport grid interface2342can be configured for two-way communication between the DMF Board450, the DMF Board1450, the device100, and the system2300. For example, consistent with one embodiment, the hydrating and transport grid interface2342can be configured to receive data from the DMF Board450(or the DMF Board1450) and/or device100and store the data into the storage device2320. As described above, the hydrating and transport grid interface2342can be further configured to send control instructions to the DMF Board450(or to the DMF Board1450) to initiate and terminate movement of fluid aliquots over the DMF Board450(or the DMF Board1450). The hydrating and transport grid interface2342can be further configured to send control instructions to the DMF Board450(or the DMF Board1450) and/or device100initiate and terminate hydration operations. For example, device100may include a fluid reservoir (not shown) coupled to the hydrating and transport grid interface2342, where the fluid reservoir is configured to provide fluid to RCA-LAMP location850(or RCA-LAMP location1850) upon the receipt of control instructions from the hydrating and transport grid interface2342. In an embodiment the digital microfluidics hydrating and transport module2340can be configured to manage data and processing instructions associated with the transport of fluid aliquots over the DMF Board450, the DMF Board1450, and associated with hydrating dried components that may be deposited or printed on the DMF Board450and the DMF Board1450. Software consistent with one embodiment of the current disclosure for controlling microfluidic flow in the gap between the surface of DMF Board450and the bottom of transparent plate490, or between the surface of DMF Board1450and the bottom of transparent plate1490(as appropriate), as well as portions of hardware consistent with one embodiment of the current disclosure, is available under the platform name OPENDROP from GAUDILABS at: https://gaudishop.ch/index.php/product/opendrop-v4-digital-microfluidics-platform/.

In an embodiment, the heating/cooling interface2362can also be configured for two-way communication between the DMF Board450, the DMF Board1450, the heating/cooling device190, and the system2300. Consistent with one embodiment, the heating/cooling interface2362can be configured to receive data from the DMF Board450(or the DMF Board1450) (or sensors adjacent to DMF Board450and/or DMF Board1450) and/or the heating/cooling device190and store the data into the storage device2320. The heating/cooling interface2362can be further configured to send control instructions to the heating/cooling device190to initiate and terminate heating and cooling operations.

In an embodiment the target probe reagent reaction module2360can be configured to manage data and processing instructions associated with the annealing and ligation of target-specific set of reagent components and aliquots of a sample. Further still, in an embodiment, the RCA-LAMP reaction module2365can be configured to manage data and processing instructions associated with the RCA-LAMP process at the target locations.

Consistent with the present disclosure, the lamp/camera interface2372can be configured for two-way communication between the camera system180and the system2300. Consistent with one embodiment, the lamp/camera interface2372can be configured to receive data from the camera system180and store the data into the storage device2320. The lamp/camera interface2372can be further configured to send control instructions to lamp170and camera system180to initiate and terminate excitation radiation and to initiate and stop camera operations for monitoring fluorescence.

In an embodiment the excitation/fluorescence module2370can be configured to manage data and processing instructions associated with the provision of excitation radiation and the monitoring of fluorescence from the target locations.

The system2300can be accessed and controlled by a user using the input interface127. The input interface127can be available for the user to input information into data processing system2300, and can include, for example, a keyboard, a mouse, a touch screen and/or optical or wireless computer input devices. The user can input control instructions via the input interface127to control the operation of the DMF processing system110, the heating/cooling device190, the lamp170, and/or the camera system180.

The system2300can also provide visualized information to the user via the display125. For example, the display125can include a computer screen and make available a graphical user interface (“GUI”) to the user. The display125can display an image of the target locations on DMF Board450(or DMF Board1450) during an RCA-LAMP process. Consistent with another embodiment, the display125can also display an abbreviated inspection report, or a simple indicator, to the user indicating certain characteristics of biological items identified in a provided sample.

FIG. 24illustrates a top view of DMF Board450consistent with exemplary operation of device100with movable cartridge assembly150. (Although the description of operation of device100with movable cartridge assembly150is provided below withFIGS. 24-33, one of ordinary skill in the art should immediately understand and appreciate the operation of device100with movable cartridge assembly160.)

FIG. 24illustrates the view of DMF Board450previously provided inFIG. 8. However, inFIG. 24, sample2410has been introduced to the surface2450of DMF Board450. Consistent with the present disclosure, as described above, sample2410may be introduced to the surface2450of DMF Board450from the top of movable cartridge assembly150through opening368.

Consistent with the present disclosure, sample2410is a liquid. In one embodiment, sample2410can include an addition of between 5 and 20%, by volume, of a hydrophobic oil, immiscible with water, such as polydimethyl siloxane (PDMS), or a similar fluid. One of ordinary skill in the art would appreciate that the addition of between 5 and 20%, by volume, of such a hydrophobic oil can minimize evaporation of sample droplet during heating.

FIG. 25illustrates a view of surface2450, and depicts how a single aliquot (or series of aliquots) of sample2410can be transported across surface2450using digital microfluidics to arrive at target locations. For example, arrow2515depicts a possible route of aliquot2510to first target location810; arrow2525depicts a possible route of aliquot2510to second target location820; arrow2535depicts a possible route of aliquot2510to third target location830; and arrow2545depicts a possible route of aliquot2510to fourth target location840.

FIG. 26provides a cross section view of digital microfluidics system2600, and depicts the movement of aliquot2510according to arrow2515. As shown inFIG. 26, electrode1005is not provided with an electrical charge (relative to electrical “ground”). Electrode2605, however, is provided with a charge through its associated electrode connector1007. The presence of the electrical charge on electrode2605causes aliquot2510to move in the direction of electrode2605. Consistent with DMF processing systems, after aliquot2510is over electrode2605, then that electrode can be returned to “ground,” and the next subsequent electrode in the direction of arrow2515is provided a charge relative to “ground.” In this way, aliquot2510is transported across surface2450ofFIG. 25.

FIG. 27shows a cross-section view at location10A-B, which includes a cross-section view of DMF Board450within first target location810, and also shows aliquot2510being transported over first target-specific set of reagent components815on hydrophobic coating1070. As has been shown previously, DMF Board450further includes dielectric1060, substrate1050, electrode1005and electrode connectors1007(where electrode connectors1007, in turn, connect to the DMF transport grid interface155, and are under control of DMF processor system110). Spacer492is shown, as well as transparent plate490, which includes glass layer1095, conductive layer1090, and hydrophobic coating1080. Furthermore, heating/cooling device190is shown as being located beneath first target location810.

As shown inFIG. 27, consistent with the present disclosure, electrode1005is not provided with an electrical charge (relative to electrical “ground”). Electrode2605, however, is provided with a charge through its associated electrode connector1007. The presence of the electrical charge on electrode2605causes aliquot2510to move in the direction of electrode2605over first target-specific set of reagent components815on hydrophobic coating1070. In this way, when aliquot2510is over electrode2605, then that electrode can be returned to “ground” (which is depicted inFIG. 28), and aliquot2510fromFIG. 27mixes with first target-specific set of reagent components815fromFIG. 27to form first mixture2815, depicted inFIG. 28.

Consistent with the present disclosure, and as depicted inFIGS. 27 and 28, the aliquot2510of sample2410has been transported onto a pre-designated electrode located in the first target location810, where the first target-specific set of reagent components815have been deposited or printed (which include a target-specific padlock probe, a ligation enzyme, and buffer components necessary for a ligation reaction to proceed).

Once aliquot2510reaches the first target location810, aliquot2510will absorb the deposited (or printed) reagents815so that they become part of the liquid sample2815.

Consistent with the present disclosure, heating/cooling device190provides heat2892to first mixture2815until first mixture2815reaches a temperature of approximately 95 degrees Celsius. This allows the padlock probe to access the nucleic acid sequence of the sample2410. The first mixture2815is then cooled (or allowed to cool), and if the nucleic acid sequence of the sample matches the target-specific complementary sequences of the padlock probe, the padlock probe will form a circular confirmation. The ligase then covalently connects the two padlock probe target-specific arms so that the circular confirmation becomes permanent. Consistent with the present disclosure, if the sample2410does not contain the target genetic material, ligation of the padlock probe does not occur.

FIG. 29illustrates a view of surface2450, and depicts how a single aliquot2951(or series of aliquots2952,2953, and/or2954) of hydrated RCA-LAMP Reaction Components2955can be transported across surface2450using digital microfluidics to arrive at target locations. For example, arrow2916depicts a possible route of first aliquot2951to first target location810; arrow2926depicts a possible route of aliquot2952to second target location820; arrow2936depicts a possible route of aliquot2953to third target location830; and arrow2946depicts a possible route of aliquot2954to fourth target location840. Consistent with the present disclosure, first aliquot2951, second aliquot2952, third aliquot2953, and fourth aliquot2954can consist of less than 10 microliters of hydrated RCA-LAMP Reaction Components2955.

Further still, consistent with the present disclosure, if RCA-LAMP Reaction Components855are deposited in a dried form, then RCA-LAMP Reaction Components855can be hydrated prior to the operation depicted inFIG. 29through the introduction of fluid introduced to the surface2450of DMF Board450at RCA-LAMP location850. Consistent with the present disclosure, as described above, fluid may be introduced to the surface2450of DMF Board450at RCA-LAMP location850from the top of movable cartridge assembly150through opening366. Such fluid for purposes of hydrating the RCA-LAMP components855can be introduced when sample2410is introduced, or later. Moreover, device100may include a fluid reservoir coupled to and under control of microfluidics hydrating and transport module2340for just this purpose.

After first aliquot2951, second aliquot2952, third aliquot2953, and fourth aliquot2954have been transported to first target location810, second target location820, third target location830, and fourth target location840(respectively) using digital microfluidics system2600, each of the aliquots mixes with the fluid already present in each of the target locations. As shown inFIG. 29, these previously present mixtures include first mixture,2815, second mixture2925, third mixture2935, and fourth mixture2945.

Consistent with the present disclosure, the fluid mixtures in each of the target locations is heated again, but only to approximately 65 degrees Celsius. These mixtures include: a mixture of first aliquot2951and first mixture2815at first target location810—which becomes first mixture3015depicted inFIG. 30; a mixture of second aliquot2952and second mixture2925at second target location820—which becomes second mixture3025depicted inFIG. 30; a mixture of third aliquot2953and third mixture2935at third target location830—which becomes third mixture3035depicted inFIG. 30; and a mixture of fourth aliquot2954and fourth mixture2945at fourth target location840—which becomes fourth mixture3045depicted inFIG. 30. At this temperature, the LAMP primers specific to the padlock probe backbone (which are common for all target-specific padlock probes) and polymerase bind to the ligated padlock probe and begin the LAMP reaction. As the reaction progresses, an oligonucleotide strand displacement (OSD) probe binds to a loop region of the LAMP products. This causes the OSD quencher to be removed from the probe, which allows for unquenched fluorescence from the fluorophore attached to the OSD probe. As more LAMP amplification occurs, more LAMP loop products are made, and more OSD probes bind and create more fluorescence.

FIG. 30illustrates a view of surface2450, and depicts how final mixtures3015,3025,3035, and3045at the target locations810,820,830, and840(respectively) may be irradiated by excitation radiation3010from lamp170to determine if fluorescence3065is produced. The fluorescence of the mixtures inFIG. 30can be monitored in real-time with camera system180, where camera system180can include an appropriate filter. Again, if the sample2410does not contain the target genetic material, ligation of the padlock probe does not occur. Thus, the LAMP reaction cannot commence in the absence of ligated padlock probe, and samples2410without target genetic material cannot fluoresce.

FIG. 31shows a cross-section view at location10A-B, which includes a cross-section view of DMF Board450within first target location810, and also shows first aliquot2951being transported to mix with first mixture2815on hydrophobic coating1070. As has been shown previously, DMF Board450further includes dielectric1060, substrate1050, electrode1005and electrode connectors1007(where electrode connectors1007, in turn, connect to the DMF transport grid interface155, and are under control of DMF processor system110). Spacer492is shown, as well as transparent plate490, which includes glass layer1095, conductive layer1090, and hydrophobic coating1080. Furthermore, heating/cooling device190is shown as being located beneath first target location810.

As shown inFIG. 31, consistent with the present disclosure, electrode1005is not provided with an electrical charge (relative to electrical “ground”). Electrode2605, however, is provided with a charge through its associated electrode connector1007. The presence of the electrical charge on electrode2605causes aliquot2951to move in the direction of electrode2605to mix with first mixture2815. In this way, when aliquot2951is over electrode2605, then that electrode can be returned to “ground” (which is depicted inFIG. 28), and aliquot2951fromFIG. 30mixes with first mixture2815fromFIG. 30to form first mixture3015, depicted inFIG. 32.

Consistent with the present disclosure, and as depicted inFIGS. 31 and 32, heating/cooling device190provides heat3292to first mixture3015until first mixture3015reaches a temperature of approximately 65 degrees Celsius.FIG. 32also depicts a perspective associated with view33A, shown inFIG. 33.

FIG. 33illustrates a cross section view of a portion of DMF Board450, and depicts how first mixture3015at the first target location810may be irradiated by excitation radiation3010from lamp170to determine if fluorescence3065is produced.

Consistent with the current disclosure, a single sample2410can be analyzed for different targets through spatial multiplexing where a different padlock probe is located at each of the first target location810, the second target location820, the third target location830, and the fourth target location840. Similarly, different samples can be independently analyzed (for the same or different targets) at different locations on the same platform. One of ordinary skill in the art would appreciate that movable cartridge assembly160, for example, provides reservoir locations in addition to sample location1880and RCA-LAMP location1850, that can accommodate additional samples. This spatial multiplexing is enabled through sample and reactant transport via digital microfluidics. The sequence-specific probe described above, which results in fluorescence, is one method of LAMP detection consistent with the current disclosure. In another embodiment consistent with the current disclosure, bivalent metal ions, such as magnesium, calcium or manganese, can be added so that they are present when the final mixtures (3015,3025,3035, and3045) are formed. In this alternative embodiment, these ions can form complexes with a LAMP byproduct (pyrophosphate), which will precipitate as LAMP proceeds. This turbidity of the precipitate can be measured, or observed, to determine whether a LAMP reaction has occurred, thereby confirming identification of a target nucleic acid. Further still, in another embodiment, methods of LAMP detection can include the use of DNA-intercalating dyes such as SYBR Green, EvaGreen, SYBR Gold, SYBR Safe, berberine, etc.

Consistent with the current disclosure, in an embodiment, the padlock probe backbone can include: 1) a sequence-specific probe binding site; 2) a B1 primer binding site, a B2 primer binding site; 3) a complement sequence to an F1 primer binding site, a complement sequence to an F2 primer binding site, and a complement sequence to a loop primer binding site.

FIGS. 34 and 35depict flowcharts consistent with methods for nucleic acid identification of material in a sample disclosed herein.

FIGS. 34 and 35include a step3405of providing a microfluidics system with a movable cartridge assembly. Consistent with the current disclosure, such a microfluidics system can include digital microfluidics system2600with movable cartridge assembly150and/or movable cartridge assembly160.FIGS. 34 and 35next depict dispensing a sample onto a movable cartridge assembly (step3410), followed by transporting an aliquot of the sample received on the surface to the target location (step3415and/or step3515). Consistent with the present disclosure,FIGS. 34 and 35next depict the step of mixing an aliquot of the sample (for example, sample2410) and a first target-specific set of reagent components (for example, components815) at a first target location (step3420and/or step3520). After an aliquot of the sample is mixed with the first target-specific set of reagent components at the first target location (for example, first target location810),FIGS. 34 and 35depict the step of annealing and ligation of the first target-specific set of reagent components mixed with the aliquot of the sample by heating the mixture to approximately 95 degrees Celsius followed by cooling (step3445and/or step3545).

Consistent with the present disclosure, the methods ofFIGS. 34 and 35can also include the step of hydrating RCA-LAMP reaction components on the movable cartridge assembly (step3450). One of ordinary skill in the art would appreciate the step3450does not have to follow step3445(or step3545), but can occur earlier, such as before step3410.

Consistent with the present disclosure, the methods ofFIGS. 34 and 35include the step of transporting an aliquot of hydrated RCA-LAMP reaction components on the movable cartridge assembly to the first target location (step3455and/or step3555). Step3460(and/or step3560) includes mixing the aliquot of hydrated RCA-LAMP reaction components with the mixture present at the first target location—previously provided by step3445(and/or step3545).

Step3485(and/or step3585), consistent with the present disclosure provides for heating the fluid at the first target location to approximately 65 degrees Celsius.

An embodiment may also include providing excitation radiation to the first target location (step3490and/or step3590), and monitoring fluorescence from the first target location (step3495and/or step3595).

Consistent with a further embodiment,FIG. 35includes a further step of transporting a second aliquot of the sample received on the surface to a second target location (step3525).FIG. 35further depicts the step of mixing the second aliquot of the sample (for example, sample2410) and a second target-specific set of reagent components (for example, components825) at a second target location (step3530). After the aliquots of the sample are mixed with their respective target-specific set of reagent components at their respective locations,FIG. 35depicts the step of annealing and ligation of the first target-specific set of reagent components mixed with the first aliquot of the sample and annealing and ligation of the second target-specific set of reagent components mixed with the first second of the sample by heating each mixture to approximately 95 degrees Celsius followed by cooling (step3545).

Consistent with the present disclosure, the method ofFIG. 35can also include the step of hydrating RCA-LAMP reaction components on the movable cartridge assembly (step3450). One of ordinary skill in the art would appreciate the step3450does not have to follow step3545, but can occur earlier, such as before step3410.

Consistent with the present disclosure, the method ofFIG. 35includes the further step of transporting a second aliquot of hydrated RCA-LAMP reaction components on the movable cartridge assembly to the second target location (step3565). Step3570includes mixing the second aliquot of hydrated RCA-LAMP reaction components with the mixture present at the second target location—previously provided by step3545.

Step3585, consistent with the present disclosure provides for heating the fluid at the second target location to approximately 65 degrees Celsius.

An embodiment may also include providing excitation radiation to the first target location (step3590), and monitoring fluorescence from the second target location (step3596).

One of ordinary skill in the art would appreciate that the embodiments disclosed herein can be used in many settings to identify biological targets using any sample type (once purified) including, but not limited to: POC diagnostics, population screening, emergency response situations, bio/chemical defense, food testing, environmental testing, and biological testing in zero-gravity settings.

The foregoing description has been presented for purposes of illustration. It is not exhaustive and is not limited to precise forms or embodiments disclosed. Modifications and adaptations of the embodiments will be apparent from consideration of the specification and practice of the disclosed embodiments. While certain components have been described as being coupled to one another, such components may be integrated with one another or distributed in any suitable fashion.