Patent Application
20250033058
The general technical field is point-of-care sample-to answer devices that perform assays in microfluidic cartridges and components thereof that are used to control the sequential dispensing of reagents and other assay steps performed by point-of-care sample-to-answer devices.
Point of Care (POC) devices can bring rapid molecular diagnostic testing at the site of patient care. Such sample-to-answer devices and associated procedures commonly utilize molecular amplification techniques like PCR or isothermal amplification, which require control over the sequence of reagent dispensing and other assay parameters.
Typically, liquid reagents are stored in a reagent blister on a microfluidic cartridge with an opening to allow the liquid to flow into the cartridge. The opening is sealed with a rupturable membrane or layer. Ordinarily, the rupturable membrane must be torn or ruptured and the blister crushed to dispense liquid reagent into the cartridge.
Crushing reagent blisters to dispense reagent requires force delivered by structures configured to deform the reagent blister under pressure. This operation is commonly powered by individual geared motors controlling each actuating structure. However, certain complex cartridge designs e.g., with multiple reagents to dispense from multiple reagent blisters sometimes according to a predetermined dispensing sequence, require increased instrument complexity including, for example, increasing the number of motors and drive assemblies required. This increases the size, cost, and portability of the instrument.
Assays performed on sample-to-answer devices often perform molecular amplification on a microfluidic cartridge depending on the assay. Most of these technologies use molecular amplification techniques like PCR or isothermal amplification, which rely on heating (or in the case of PCR, thermal cycling) of the reaction for the amplification.
Most sample-to-answer devices utilize resistive based heaters. In a resistive heat method, an electric current is passed through a resistor element. This current is converted into heat at the resistor that is used to heat the amplification reaction. One issue with this method is the need for precise contact between the reaction chamber on the microfluidic cartridge and resistive heater element. Any loss of contact can lead to poor heat conduction into the amplification chamber and hence a failed amplification. To overcome this issue, few technologies on the market implant the resistive heater elements as part of their disposable cartridge. Alternate approaches involve adding pressure to the amplification well causing it to bulge outwards and improve contact with a heater element.
Another challenge with the contact-based approach is temperature control. In such a setup the temperature sensor is placed on the heater element (instead of the fluid). This approach needs a predictive algorithm to determine the temperature of the fluid in the amplification chamber that is based on the heater element temperature and the ambient temperature. This could lead to inaccurate temperature control.
PCR thermal cycling technologies use “heat zones” whereby PCR reagents are moved between the heat zones during the thermal cycling process. While this is a rapid technique, it requires pumps and accurate fluid movement controls to ensure the temperatures are held for the appropriate cycle times.
In accordance with the present invention, various embodiments of the apparatus for controlling assay processes performed in a microfluidic cartridge are disclosed.
In one embodiment, an apparatus for controlling assay processes in a microfluidic cartridge is provided. In some embodiments, the apparatus can be powered by a single motor. In one embodiment, the single motor can be a stepper motor, a servo motor, or a gear motor. In another embodiment, the single motor contains one gear and is configured to operate at one speed.
In another embodiment, the microfluidic cartridge can contain one or more reagent filled blisters, a fluidic channel, and one or more wells or chambers. In another embodiment, the microfluidic cartridge can contain a lateral flow strip. In yet another embodiment, the microfluidic cartridge can contain one or more flow through blisters. The one or more flow through blisters can contain an inlet valve and an outlet valve. In yet another embodiment, the one or more reagent filled blisters comprise crush blisters.
The apparatus can further include a drive belt assembly. In one embodiment, the drive belt assembly can contain a single drive belt. In another embodiment, the drive belt assembly can contain one axle extending from the single motor.
The apparatus can further include a clutch assembly comprising one or more clutches configured to rotate about a central axis of rotation. In some embodiments, each clutch can contain a drive shaft centrally positioned within each of the one or more clutches on the central axis of rotation, and wherein rotation of said one or more clutches is driven by said drive belt assembly. In one embodiment, the clutch assembly contains a plurality of clutches. In another embodiment, the clutch assembly can contain three clutches. In other embodiments, the one or more clutches are electromagnetic clutches. In some embodiments, a single axle extending from the single motor is affixed to only one of the plurality of clutches to power rotation of the drive belt assembly.
The apparatus can further include an actuation mechanism comprising one or more actuation elements mounted to said drive shaft configured to engage the microfluidic cartridge and actuate one or more assay processes. In one embodiment, the one or more actuation elements can comprise one or more press plates. In another embodiment, the one or more press plates can contain one or more protrusions configured for physical engagement with said microfluidic cartridge. In some embodiments, the one or more protrusions comprise a first shape configured to engage and open said inlet valve and outlet valve on the one or more flow through blisters. In other embodiments, the one or more protrusions comprise a second shape configured to deform said one or more crush blisters to force said reagent out of said crush blister. In another embodiment, the drive shaft can contain a threaded portion for mounting said one or more actuation elements.
The apparatus can further include a series of spatially arranged permanent magnets positioned proximate the microfluidic cartridge. In some embodiments, the microfluidic cartridge can contain metal particles (e.g., metal beads) for binding to target analytes and moving the target analytes through the microfluidic cartridge. In other embodiments, the permanent magnets can be configured to move the metal particles through the microfluidic cartridge by magnetic force. In one embodiment, the spatially arranged permanent magnets can be affixed to a rotating wheel positioned adjacent to and substantially coplanar with the microfluidic cartridge.
In another embodiment, a method for controlling assay processes in a microfluidic cartridge is provided. The method can include the step of providing an apparatus configured to be programmed with one or more sequence files corresponding to one or more assays. The apparatus can further include the following components: a single motor; a drive belt assembly comprising a single drive belt; a clutch assembly comprising a plurality of clutches configured to rotate about a central axis of rotation, each clutch comprising a drive shaft centrally positioned within each of the one or more clutches on the central axis of rotation, and wherein rotation of said plurality of clutches is driven by said drive belt assembly; and an actuation mechanism comprising one or more actuation elements mounted to said drive shaft configured to engage the microfluidic cartridge and actuate one or more assay processes. In some embodiments, the method can include the step of programming the apparatus with one or more sequence files corresponding to the one or more assays. In another embodiment, the method can include the additional steps of inserting the microfluidic cartridge into said apparatus and initiating performance of the assay using the apparatus. In some embodiments, the one or more assay processes comprises polymerase chain reaction, magnetic bead based movement of a target analyte through said microfluidic cartridge, and/or lateral flow strip detection. In some embodiments, the detection procedure can be any of lateral flow strip detection, real time optical florescence detection, optical microarray detection, and electrochemical detection.
In one embodiment, the apparatus monitors and controls reaction temperatures, for example, in polymerase chain reaction. Thus, the apparatus used can include a microfluidic cartridge, an actuator plate, and a temperature control unit comprising a heating element, a heat dissipating element, and a temperature sensor. In some embodiments, the temperature sensor comprises an IR temperature sensor.
In another embodiment, the microfluidic cartridge comprises an amplification chamber wherein an assay reaction, such as PCR, is performed. In another embodiment, the heating element comprises an inductive coil element. In yet another embodiment, the inductive coil element comprises a bifilar coil. In yet another embodiment, the heating element can be mounted to a rotating wheel that can move (e.g., rotate) with respect to the cartridge. This movement will present the heating element in proximity to the amplification well when heating is required and remove the heating element during cooling to facilitate rapid heating and cooling. In a further embodiment, the heating element and said amplification chamber are not in physical contact leaving a vacant gap therebetween.
In some embodiments, the heat dissipating element can also be mounted to the rotating wheel. In another embodiment, the heat dissipating element comprises a heat sink. In yet another embodiment, the heat sink is comprised of aluminum, ferrous metals, copper (combinations and alloys of the same), carbon-derived materials in combination with aluminum, and/or natural graphite composite materials.
In yet another embodiment, the heat dissipating elements performance can further be enhanced using fans and/or a water pump to introduce convective cooling either with air or a coolant fluid. In a further embodiment, a thermoelectric cooler (TEC) with or without the combination of a fan/fans and heat sinks can be utilized to enhance the performance of the heat dissipating element.
In some embodiments, the temperature control unit further comprises a heat storage target. In another embodiment, the heat storage target is positioned inside said amplification chamber or in contact with the wall of the amplification chamber to facilitate heat transfer into the fluid contained within the amplification chamber. In another embodiment, the heat storage target is positioned inside the amplification chamber so that the reagents being amplified is in direct contact with the front face and/or the back face(s) of the heat storage target. In yet another embodiment, the heat storage target forms a pocket to envelop the reagents being amplified. This is accomplished by having a recess within the target and allowing the reagents to flow within said recess. This in turn increases the surface area in contact with the fluid, allowing for faster heat transfer. In yet another embodiment, the heat storage target may comprise of fins that will increase the surface area of contact between the target and fluid being heated. In yet another embodiment, the heat storage target is comprised of metal. In some embodiments, the metal is aluminum, aluminum alloys, ferrous metals and their alloys, copper, and or combinations thereof. In yet another embodiment, the metal target may be coated with a polymer to modify its thermal inertial properties. In a further embodiment, the heat storage target is comprised of a material comprising predetermined thermal inertia properties. In another embodiment, the heat storage target material comprises high thermal inertia properties. The heat storage target may be bonded in the amplification chamber with an adhesive or welded to the plastic substrate of the microfluidic cartridge. In yet another embodiment, the heat storage target is suspended inside the amplification chamber. In some embodiments, the temperature control unit is capable heating and cooling rates of said amplification reagent of up to about 15° C. per second. In yet another embodiment, the device is capable of reaction speeds of up to about 40 cycles of PCR in under 15 mins.
In one embodiment, the apparatus is incorporated into a sample-to-answer and/or point-of-care device for carrying out sample-to-answer diagnostic assays. Thus, in one embodiment, a sample-to-answer and/or point-of-care device for the performance of diagnostic assays comprising the apparatus for controlling assay processes in a microfluidic cartridge is contemplated.
Having thus described the presently disclosed subject matter in general terms, reference will now be made to the accompanying Figures which disclose representative embodiments of the invention.
heat storage target contained therein.
The presently disclosed subject matter now will be described more fully hereinafter with reference to the accompanying Figures, in which some, but not all embodiments of the presently disclosed subject matter are shown. The presently disclosed subject matter may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will satisfy applicable legal requirements. Indeed, many modifications and other embodiments of the presently disclosed subject matter set forth herein will come to mind to one skilled in the art to which the presently disclosed subject matter pertains having the benefit of the teachings presented in the foregoing descriptions and the associated Figures. Therefore, it is to be understood that the presently disclosed subject matter is not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims.
Referring to
Referring to
In one embodiment, the clutch assembly 101 can contain one or more clutches 104 driven by a power source. In some embodiments, the clutch assembly 101 can be electromagnetically controlled. A single clutch 104 is shown in
With continued reference to
Referring to the embodiment illustrated in
It should be noted that single motor potentiated rotation of a plurality of clutches producing controlled actuation of a plurality of actuation elements greatly reduces the size, complexity, cost, and power needs/consumption of the sample-to-answer diagnostic instrument. Such a design results in a more compact and portable instrument without sacrificing diagnostic assay speed, accuracy, and efficiency. Thus, precision control over a predetermined reagent dispensing sequence (adjustable depending on the diagnostic assay) from a single microfluidic cartridge 116 using a single power source provides a technical advantage over known sample-to-answer devices.
Referring to the embodiment illustrated in
For example, some protrusions 113 are configured (size, shape, and overall design) to engage and open certain inlet and outlet port valves on a flow through blister 138 (shown in
In this manner, the sample-to-answer instrument can be programmed in a predetermined manner such that each of the one or more actuation elements 112 is actuated forwards or backwards to engage and/or disengage the one or more blisters in the correct sequence needed depending on the diagnostic assay being performed. In this system, the amount of pressure applied to the blister can also be controlled by, for example, controlling the distance traveled by the press plate or the length of the protrusion used. Programmable software driven microfluidic systems have been described in, for example, Mezic et al., US/2009/0038938A1, Microfluidic Central Processing Unit and Microfluidic Systems Architecture.
It should be noted that actuation elements 113 can further contain one or more permanent magnets configured to engage and translocate analyte bound magnetic beads through the fluidic channels of a microfluidic cartridge 116. Magnetic beads with ionizable groups that have a net positive charge at a given pH are used for binding DNA. An example of such as type of magnetic bead is the ChargeSwitch Magnetic bead (Life Technologies, Inc. Carlsbad, Calif.). DNA binds to such a type of magnetic bead in the presence of a binding buffer capable of creating a net positive charge on the bead and DNA is eluted in the presence of an elution buffer capable of creating a net neutral or negative charge on the bead. In this embodiment, ChargeSwitch magnetic beads that cause DNA to bind to them in the presence of a buffer having a pH<5 and elute from them in the presence of a buffer having a pH >8 are used. The magnetic beads are sequentially moved through all the wells on the microfluidic cartridge by methods described in this disclosure. DNA is first allowed to bind to the magnetic beads in the presence of a binding buffer. The beads are then moved in and out of two wells containing wash buffer to wash the impurities from the beads. The impurities are left behind in the wash buffer solution. The washed beads are then moved to a well containing elution buffer where the DNA is eluted from the beads into the elution buffer. The eluted DNA may then be used to hydrate lyophilized reagents for a Nucleic Acid Amplification Test (NAAT). In some embodiments, the magnetic beads may be eluted directly into the amplification well. One or more heaters is turned on as part of the automation sequence to supply the temperatures for amplification.
In some embodiments, the actuation elements 112 or the microfluidic cartridge 116 is configured to rotate while remaining substantially coplanar with the actuation element or microfluidic cartridge (depending on which one is able to rotate). Alternatively, as shown in
With continued reference to
The sample-to-answer device also controls and regulates the temperature of a reagent. Even more specifically, the sample-to-answer device can regulate, heat, cool, and/or measure the temperature of reagents used for molecular amplification processes, such as PCR. In one embodiment, the sample to answer device applies a non-contact approach to heating and measuring temperature.
Referring now to
Alternatively, the heating element can be a resistive heating element comprising a resistor embedding in a block of thermally conducting material such as a metal, metal oxide or metal alloy. The heating element may also be a resistive thin-film heating element or a peltier element. In other embodiments the heating element is a positive temperature coefficient self-regulating element. The heating element may be integrated as part of the microfluidic device and intended as a single use, disposable element. In an additional embodiment, a phase change material may be used to generate the heat energy for lysis and sample transfer. Phase change materials are widely used for a variety of applications requiring thermal energy storage and have been developed for use across a broad range of temperatures (−40° C. to more than 150° C.). Phase change materials are advantageous because they offer high-density energy storage and store heat within a narrow temperature range. Additionally they are inexpensive, non-toxic and do not require electrical energy for generating heat. As such, they are an appealing choice for point-of-care settings and for single-use devices that require heat energy. In an additional embodiment a phase change material contained in a sealed pouch is used to form a jacketed sheath around the sample extraction container. The jacket of phase change material may either be present as part of the microfluidic device or as part of the sample extraction container. The act of connecting the sample extraction container to the microfluidic device works to create a nucleation site that in turn activates the phase change material, causing it to rise in temperature and heat the sample. This may be accomplished by packaging a metal piece in the pouch containing the phase change material, which snaps when the sample extraction container is connected to the microfluidic device. The phase change material may also be activated by an external actuator present on the actuating element in the microfluidic device. In some embodiments, a suitable phase change material may be activated to cool the sample so as to prevent its degradation. In some embodiments, the heating element may serve to heat lyse and transfer the sample to the fluidic well on the microfluidic devices, as well as to run a NAAT on the microfluidic device.
Referring to
As shown in
Referring to the embodiment shown in
Accurate control of the reagent temperature during thermal cycling is critical for successful polymerase chain reaction and requires accurate temperature monitoring and measurement. Various temperature monitoring techniques are known by those of skill in the art-some more effective than others. In one embodiment, monitoring and measurement can be carried out by one or more sensors or other means that do not contact the measurement target.
As discussed above, in one embodiment, an infrared (IR) temperature sensor 124 can be positioned proximate the microfluidic cartridge's amplification chamber 121—within its field of view. Amplification chambers 121 can be made of materials that permit IR radiation wave penetration such as, in one example, a thin polycarbonate which is relatively transparent to IR radiation. In this manner, the IR sensor can measure the temperature of the fluid inside the amplification chamber 121 for a “true closed loop” temperature control. In other words, a heating element 123 (e.g., bifilar coil) and temperature sensor 124 positioned on opposite sides of the amplification chamber 121 containing a heat storage target 125 and reaction reagent creates a closed loop temperature control system.
In some embodiments, other temperature sensors may be suitable depending on the system requirements. For example, thermocouple sensors, thermistor sensors, resistance temperature detectors, or a semiconductor based sensor may be used in some embodiments.
In one embodiment, the heating element and temperature sensor can be on either side of the amplification chamber as long as the heating element is proximate to the heat storage target, wherever it is positioned within the amplification chamber, and the temperature sensor is proximate the reagent. In some embodiments, the heat storage target is positioned on the surface of the amplification chamber closest to the heating element, as illustrated in
Due to the low thermal inertia of the heat storage target 125 material (e.g., metal target), effective temperature control for PCR thermal cycling (either 2 step or 3 step) can be accomplished via single heating element (e.g., inductive coil). The amount of heat produced by the single heating element 123 can itself be controlled to maintain different temperatures in the amplification chamber. In other embodiments, more than one heating element 123 can be used in some embodiments.
Referring to
Referring to
With continued reference to the embodiment shown in
In yet another embodiment shown in
The following is an example of an assay involving PCR amplification and lateral flow analysis of the amplified product carried out using the apparatus for controlling assay processes performed in a sample-to-answer device. The description below provides a practical example of important aspects of the invention discussed above.
CT and NG cells were spiked in pooled negative vaginal swab samples. CT serovar E spiked at 1.2 IFU/mL, NG WHO-L (ciprofloxacin resistant) at 5 CFU/mL, NG ATCC 430669 (ciprofloxacin sensitive) at 106 CFU/mL. Samples were lysed and purified with charge switch magnetic beads using Applicant's proprietary system. The master mix contains multiplex 5 primers mix to amplify CT, NG, gyrA (ciprofloxacin resistant marker), human GAPDH for sample adequacy control, 1× platinum II PCR buffer (thermos), 5.5 mM MgCl2, 10 U platinum II taq HS DNA polymerase, 120 mM Tris buffer pH 8.8, 0.75× platinum GC enhancer, and 2 μg/μL BSA. Following sample preparation step, amplification was initiated by heating first to 95° C. for 2 minutes to activate the hot start DNA polymerase, then 40 thermal cycling between 95° C. for 15 seconds, and 62.5° C. for 30 seconds. Then amplified products were analyzed by gel electrophoresis. First 4 μl of amplified product was mixed with 1 μL of 5× loading dye, then 3 μL was added to the gel well and run for 13 minutes at 175 V. Following gel analysis, amplified samples were further analyzed by lateral flow strips. Samples were digested by lambda exonuclease enzyme to generate single stranded DNA before being applied to the lateral flow assay. Both gel and lateral flow analysis showed amplification of the corresponding targets. See
The microfluidic cartridge 116 shown in
Various methods of detection can be used for detecting the amplified products, including visual detection using pH sensitive dyes or metal-sensitive indicators, electrochemical detection, optical detection using intercalating dyes or fluorescent probes, turbidimetry, lateral flow strip detection. In this example, a lateral flow strip is used to detect the amplified nucleic acids. Biotin and FAM/FITC modified FIP and BIP primers respectively may be used in the LAMP reaction. A sandwich format lateral flow test may be used. The amplified product may be mixed with a dilution or running buffer before lateral flow strip detection. A valve may be present and actuated as part of the assay automation sequence to allow the amplified products to flow on the lateral flow strip. Alternatively, a septum may be pierced to allow the amplified product to flow on the lateral flow strip.
Applicant's instrument embodying the apparatus set forth herein was used to interact with the microfluidic cartridge to automate a sample to answer test. The instrument included the cartridge feed module, blister/reagent dispense module, sample preparation and amplification module, and the detection module. Sample was added into the lysis chamber and the cartridge was partially inserted into the instrument. An RFID tag reader in the instrument read the RFID tag on the cartridge to identify the cartridge type. This is important for the instrument to select the appropriate assay specific sequence file to run.
Assay steps performed by the Applicant's instrument in this example are outlined by the example sequence file below in Table 2 with additional explanation below Table 2.
The cartridge feed module accepts the microfluidic cartridge 116 and pulls it into the instrument. This module positions the cartridge inside the instrument so the remaining modules on the instrument may interact with the cartridge. In the sequence file step 1: the feed motor turns on and the cartridge 116 is pulled inside the instrument (Dir FM value is +1). See Table 2.
Next, the blister/reagent dispense module (e.g., actuator mechanism 103) activates plate 1 (e.g., 112a) which ruptures the blister seals (e.g., seals for the magnetic bead blister, wash blister 1, wash blister 2 and the amplification buffer blister) and opens the fluidic pathway from the reagent blisters to the respective chambers on the cartridge.
The air pump 137 is now turned ON for 30 s. See
Next, the blister/reagent dispense module (e.g., actuator mechanism 103) actives the plate 2 (e.g., press plate 112b). This plate impinges on the reagent blisters causing them to deform/crush and thereby dispensing the reagents contained within them into the cartridge. This fills the cartridge, i.e., the wash buffer blisters dispense into wash chamber 1 and 2, the amplification blister dispenses into the amplification chamber 121 and the oil blister fills the primary channel of the cartridge. Specifically, Step 2: Actuator motor 110 (Act Mtr) value is set to 1—motor is turned ON, Direction (Dir AM) value is set to 1—travel is towards the cartridge, Plate 1 value is set to 1—clutch 1 is activated that moves plate 1. Step 3: Air Pump 137 value is set to 1 that turns ON the air pump, HoldTime value set to 30 so the air pump is turned ON for 30 seconds. Step 4: Actuator motor (Act Mtr) value is set to 1—motor is turned ON, Direction (Dir AM) value is set to 1—travel is towards the cartridge, Plate 2112b value is set to 1—clutch 2104b is activated that moves plate 2112b. See Table 2, steps 2-4.
At this point, as illustrated in
The following steps (for example, see Table 2, steps 7-12) describe how DNA from 650 μL of the sample is concentrated on the magnetic bead particles, purified by washing steps and finally eluted into 50 μL of solution.
In this example, amplification on the cartridge 116 is performed by thermal cycling of the fluid in the amplification chamber. The heating is performed by induction heating and cooling is done by a heat sink that is cooled with a Thermoelectric cooler. Table 2, steps 64-73 illustrates the PCR thermal cycling sequence file. Generally, the steps are as follows:
Following amplification, the PCR product is moved from the amplification chamber to the lateral flow strip 133 on the cartridge. See
While the example above describes the use of polymerase chain reaction (PCR) specifically, other NAATs are also contemplated. Recent advances in isothermal amplification assay technologies have simplified the instrumentation requirements for performing NAATs.
In this example, we spiked CT and NG cells in pooled negative vaginal swab samples. CT serovar E spiked at 1.2 IFU/mL, NG WHO-L (ciprofloxacin resistant) at 5 CFU/mL, NG ATCC 430669 (ciprofloxacin sensitive) at 106 CFU/mL. Samples were lysed and purified with charge switch magnetic beads using NovelDx proprietary system. The master mix contains multiplex 5 primers mix to amplify CT, NG, gyrA (ciprofloxacin resistant marker), human GAPDH for sample adequacy control, 1× platinum II PCR buffer (thermos), 5.5 mM MgCl2, 10 U platinum II taq HS DNA polymerase (Thermo), 120 mM Tris buffer pH 8.8, 0.75× platinum GC enhancer, and 2 μg/μL BSA. Following sample preparation step, amplification was started by heating first to 95° C. for 2 minutes to activate the hot start DNA polymerase, then 40 thermal cycling between 95° C. for 15 seconds, and 62.5° C. for 30 seconds. Then amplified products were analyzed by gel electrophoresis using FlashGel™ System (Lonza). First 4 μL of amplified product was mixed with 1 μL of 5× FlashGel™ loading dye (Lonza), then 3 μL was added to the gel well and run for 13 minutes at 175 V. Following gel analysis, amplified samples were further analyzed by lateral flow strips. Samples were digested by lambda exonuclease enzyme to generate single stranded DNA before being applied to the lateral flow assay. Both gel and lateral flow analysis showed amplification of the corresponding targets.
To demonstrate that NovelDx platform is capable of rapid amplification of a specific target sequence, we used two primers specific for SARS-CoV-2 to amplify 92 and 112 bp targets. We used 2× ready mix One Step PrimeScript™ III RT-PCR Kit (Cat. #RR600B, Takara Bio), enhanced with fast SpeedStar DNA polymerase (TakataBio). The total volume 50 μL of amplification mix consisted of; 14.6 μL water, 25 μL One Step PrimeScript™ III RT-PCR mix, 5 μL primer1 and primer2, (1 μM each), 0.4 μL SpeedStar DNA Polymerase (5 U/μL), 5 μL of gRNA (1000, 100 or 10 copies/reaction). 50 μL was added to the amplification chamber of the cartridge and heated at 55° C. for 2 min for the reverse transcription step to synthesize cDNA heated to 95° C. for 10 seconds to deactivate the RT enzyme and activate the hot start DNA polymerase. Then PCR amplification for 40 cycles between 95° C. for 1 second, and 65° C. for 3 seconds, with a total RT PCR amplification time of around 10.5 minutes. Then amplified products were analyzed by gel electrophoresis using FlashGel™ System (Lonza). First 4 μL of amplified product was mixed with 1 μL of 5× FlashGel™ loading dye (Lonza), then 3 μL was added to the gel well and run for 13 minutes at 175 V. Gel analysis shows two bands below and above the 100 bp DNA marker for 1000 copies and 100 copies of gRNA marker correspond to 92 and 108 bp amplification products. Very faint bands can be seen for the 10 copies, while no bands can be seen for the NTC samples.
Based on the above disclosure, it should be apparent to one of ordinary skill in the art that the apparatus for controlling assay processes in a microfluidic cartridge as disclosed above is configured for use in point-of-care sample-to-answer devices or instruments. Thus, the invention described herein also covers point-of-care devices or instruments (or sample-to answer devices or instruments) that include the apparatus for controlling assay processes in a microfluidic cartridge.
Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this presently described subject matter belongs.
“Nucleic acid” as used herein means a polymeric compound comprising covalently linked subunits called nucleotides. A “nucleotide” is a molecule, or individual unit in a larger nucleic acid molecule, comprising a nucleoside (i.e., a compound comprising a purine or pyrimidine base linked to a sugar, usually ribose or deoxyribose) linked to a phosphate group.
“Polynucleotide” or “oligonucleotide” or “nucleic acid molecule” are used interchangeably herein to mean the phosphate ester polymeric form of ribonucleosides (adenosine, guanosine, uridine or cytidine; “RNA molecules” or simply “RNA”) or deoxyribonucleosides (deoxyadenosine, deoxyguanosine, deoxythymidine, or deoxycytidine; “DNA molecules” or simply “DNA”), or any phosphoester analogs thereof, such as phosphorothioates and thioesters, in either single-stranded or double-stranded form. Polynucleotides comprising RNA, DNA, or RNA/DNA hybrid sequences of any length are possible. Polynucleotides for use in the present invention may be naturally-occurring, synthetic, recombinant, generated ex vivo, or a combination thereof, and may also be purified utilizing any purification methods known in the art. Accordingly, the term “DNA” includes but is not limited to genomic DNA, plasmid DNA, synthetic DNA, semisynthetic DNA, complementary DNA (“cDNA”; DNA synthesized from a messenger RNA template), and recombinant DNA (DNA that has been artificially designed and therefore has undergone a molecular biological manipulation from its natural nucleotide sequence).
“Amplify,” “amplification,” “nucleic acid amplification,” or the like, refers to the production of multiple copies of a nucleic acid template (e.g., a template DNA molecule), or the production of multiple nucleic acid sequence copies that are complementary to the nucleic acid template (e.g., a template DNA molecule).
The terms “top,” “bottom,” “over,” “under,” and “on” are used throughout the description with reference to the relative positions of components of the described devices, such as relative positions of top and bottom substrates within a device. It will be appreciated that the devices are functional regardless of their orientation in space.
“Bead,” with respect to beads on a droplet actuator, means any bead or particle that is capable of interacting with a droplet on or in proximity with a droplet actuator. Beads may be any of a wide variety of shapes, such as spherical, generally spherical, egg shaped, disc shaped, cubical, amorphous and other three dimensional shapes. The bead may, for example, be capable of being subjected to a droplet operation in a droplet on a droplet actuator or otherwise configured with respect to a droplet actuator in a manner which permits a droplet on the droplet actuator to be brought into contact with the bead on the droplet actuator and/or off the droplet actuator. Beads may be provided in a droplet, in a droplet operations gap, or on a droplet operations surface. Beads may be provided in a reservoir that is external to a droplet operations gap or situated apart from a droplet operations surface, and the reservoir may be associated with a flow path that permits a droplet including the beads to be brought into a droplet operations gap or into contact with a droplet operations surface. Beads may be manufactured using a wide variety of materials, including for example, resins, and polymers. The beads may be any suitable size, including for example, microbeads, microparticles, nanobeads and nanoparticles. In some cases, beads are magnetically responsive; in other cases beads are not significantly magnetically responsive. For magnetically responsive beads, the magnetically responsive material may constitute substantially all of a bead, a portion of a bead, or only one component of a bead. The remainder of the bead may include, among other things, polymeric material, coatings, and moieties which permit attachment of an assay reagent. Examples of suitable beads include flow cytometry microbeads, polystyrene microparticles and nanoparticles, functionalized polystyrene microparticles and nanoparticles, coated polystyrene microparticles and nanoparticles, silica microbeads, fluorescent microspheres and nanospheres, functionalized fluorescent microspheres and nanospheres, coated fluorescent microspheres and nanospheres, color dyed microparticles and nanoparticles, magnetic microparticles and nanoparticles, superparamagnetic microparticles and nanoparticles (e.g., DYNABEADS® particles, available from Invitrogen Group, Carlsbad, Calif.), fluorescent microparticles and nanoparticles, coated magnetic microparticles and nanoparticles, ferromagnetic microparticles and nanoparticles, coated ferromagnetic microparticles and nanoparticles. Beads may be pre-coupled with a biomolecule or other substance that is able to bind to and form a complex with a biomolecule. Beads may be pre-coupled with an antibody, protein or antigen, DNA/RNA probe or any other molecule with an affinity for a desired target.
“Immobilize” with respect to magnetically responsive beads, means that the beads are substantially restrained in position in a droplet or in filler fluid on a droplet actuator. For example, in one embodiment, immobilized beads are sufficiently restrained in position in a droplet to permit execution of a droplet splitting operation, yielding one droplet with substantially all of the beads and one droplet substantially lacking in the beads.
“Magnetically responsive” means responsive to a magnetic field. “Magnetically responsive beads” include or are composed of magnetically responsive materials. Examples of magnetically responsive materials include paramagnetic materials, ferromagnetic materials, ferrimagnetic materials, and metamagnetic materials. Examples of suitable paramagnetic materials include iron, nickel, and cobalt, as well as metal oxides, such as Fe304, BaFe12019, CoO, NiO, Mn203, Cr203, and CoMnP.
When a liquid in any form (e.g., a droplet or a continuous body, whether moving or stationary) is described as being “on”, “at”, or “over” an electrode, array, matrix or surface, such liquid could be either in direct contact with the electrode/array/matrix/surface, or could be in contact with one or more layers or films that are interposed between the liquid and the electrode/array/matrix/surface. In one example, filler fluid can be considered as a film between such liquid and the electrode/array/matrix/surface.
Following long-standing patent law convention, the terms “a,” “an,” and “the” refer to “one or more” when used in this application, including the claims. Thus, for example, reference to “a subject” includes a plurality of subjects, unless the context clearly is to the contrary (e.g., a plurality of subjects), and so forth.
Throughout this specification and the claims, the terms “comprise,” “comprises,” and “comprising” are used in a non-exclusive sense, except where the context requires otherwise. Likewise, the term “include” and its grammatical variants are intended to be non-limiting, such that recitation of items in a list is not to the exclusion of other like items that can be substituted or added to the listed items.
For the purposes of this specification and appended claims, unless otherwise indicated, all numbers expressing amounts, sizes, dimensions, proportions, shapes, formulations, parameters, percentages, parameters, quantities, characteristics, and other numerical values used in the specification and claims, are to be understood as being modified in all instances by the term “about” even though the term “about” may not expressly appear with the value, amount or range. Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are not and need not be exact, but may be approximate and/or larger or smaller as desired, reflecting tolerances, conversion factors, rounding off, measurement error and the like, and other factors known to those of skill in the art depending on the desired properties sought to be obtained by the presently disclosed subject matter. For example, the term “about,” when referring to a value can be meant to encompass variations of, in some embodiments, ±100% in some embodiments ±50%, in some embodiments ±20%, in some embodiments ±10%, in some embodiments ±5%, in some embodiments ±1%, in some embodiments ±0.5%, and in some embodiments ±0.1% from the specified amount, as such variations are appropriate to perform the disclosed methods or employ the disclosed compositions.
All publications, patent applications, patents, and other references (including references to specific commercially available products or product lines) mentioned in the specification are indicative of the level of those skilled in the art to which the presently disclosed subject matter pertains. All publications, patent applications, patents, and other references are herein incorporated by reference to the same extent as if each individual publication, patent application, patent, and other reference was specifically and individually indicated to be incorporated by reference. It will be understood that, although a number of patent applications, patents, and other references are referred to herein, such reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art.
Although the foregoing subject matter has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be understood by those skilled in the art that certain changes and modifications can be practiced within the scope of the appended claims.
This application is a 35 U.S.C. § 371 national phase non-provisional application of PCT Application Serial No. PCT/US2022/048636 filed on Nov. 1, 2022, which claims priority to U.S. Provisional Application Ser. Nos. 63/274,507 and 63/274,510, each of which were filed Nov. 1, 2021, the disclosures of which are incorporated by reference as if fully set forth herein.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2022/048636 | 11/1/2022 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63274507 | Nov 2021 | US | |
| 63274510 | Nov 2021 | US |
