WO2024178146A1 - Dispositifs de pcr rapide et cartouches d'échantillon destinées à être utilisées avec ceux-ci - Google Patents

Dispositifs de pcr rapide et cartouches d'échantillon destinées à être utilisées avec ceux-ci Download PDF

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Publication number
WO2024178146A1
WO2024178146A1 PCT/US2024/016765 US2024016765W WO2024178146A1 WO 2024178146 A1 WO2024178146 A1 WO 2024178146A1 US 2024016765 W US2024016765 W US 2024016765W WO 2024178146 A1 WO2024178146 A1 WO 2024178146A1
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WIPO (PCT)
Prior art keywords
sample
temperature
sample analysis
cartridge
analysis cartridge
Prior art date
Application number
PCT/US2024/016765
Other languages
English (en)
Inventor
Ernest TEMPLIN
Yakov KAPLAN
Branden Wolner
Wiam TURKI-JUDEH
Said BOGATYREV
Michael Thomas
Michael White
Bruce Sargeant
Ross DEHMOOBED
Kyle NEGUS
Marvin MACHARIA
Ramiro ECHEVERRIA
Mark Kelleher
Original Assignee
Sensible Diagnostics Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Sensible Diagnostics Inc. filed Critical Sensible Diagnostics Inc.
Publication of WO2024178146A1 publication Critical patent/WO2024178146A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/416—Systems
    • G01N27/447—Systems using electrophoresis

Definitions

  • the present disclosure relates generally to a sample analysis cartridge and a sample analysis device, and more specifically to a device for performing sample analysis on a sample in a sample analysis cartridge, such as a RT-qPCR analysis for detecting the presence of one or more nucleic acid sequences in a biological sample.
  • the present disclosure is directed to a system for detecting a nucleic acid, the system comprising the sample analysis cartridge and devices described herein, and components thereof.
  • the present disclosure is directed to methods of loading the sample analysis cartridge and methods of analyzing a sample for the presence of a nucleic acid using the sample analysis cartridge and devices described herein.
  • the cartridge includes a cartridge body.
  • the cartridge body includes a fluidic circuitry that is capable of being sealed and pressurized.
  • the fluidic circuitry includes a sample load chamber, a reaction chamber in fluid communication with the sample load chamber, and a venting port comprising a swellable plug.
  • the venting port is in fluid communication with the reaction chamber.
  • the swellable plug is configured to swell responsive to contact with a liquid to seal the venting port and form a closed system within the fluidic circuitry.
  • the cartridge body also includes two or more surfaces forming boundaries of the reaction chamber, the two surfaces being configured for optical analysis of the reaction chamber.
  • the cartridge body also includes two opposing surfaces forming boundaries of the reaction chamber, the two opposing surface configured to be deformable.
  • the sample analysis cartridge also includes a cap configured to operably engage with the cartridge body to seal the fluidic circuitry at or in proximity to the sample load chamber.
  • the cap includes a plunger. Operably engaging the cap with the cartridge body causes the plunger to enter at least a portion of the cartridge body and provide positive pressure to the fluidic circuitry.
  • a method of loading the sample analysis cartridge with a sample includes obtaining a sample analysis cartridge.
  • the method also includes introducing the sample into the sample load chamber of the sample analysis cartridge.
  • the method also includes operably engaging the cap with the cartridge body to pressurize the fluidic circuitry and force the sample through at least a portion of the fluidic circuitry.
  • the sample analysis cartridge is maintained in a substantially upright position during the introducing, the operable engaging, and for a period of time after the operable engaging to allow for the swellable plug to contact the sample and swell to seal the venting port.
  • a device for analyzing a sample in a sample analysis cartridge includes a sample analysis cartridge receiving port configured to receive and position the sample analysis cartridge such that a reaction chamber of the cartridge is in a predetermined position relative to the receiving port.
  • the device also includes a thermocycling module comprising a first temperature switching unit and a second temperature switching unit.
  • the first temperature switching unit includes a first temperature- regulated block and a second temperature-regulated block each comprising a contact surface.
  • the first temperature switching unit is configured to apply the contact surface of the first temperature-regulated block to a first surface of the reaction chamber and the contact surface of the second temperature-regulated block to a second opposing surface of the reaction chamber.
  • the second temperature switching unit includes a third temperature-regulated block and a fourth temperature-regulated block each comprising a contact surface.
  • the second temperature switching unit is configured to apply the contact surface of the third temperature- regulated block to the second opposing surface of the reaction chamber and the contact surface of the fourth temperature-regulated block to the first opposing surface of the reaction chamber.
  • the device further includes an optical analysis module configured to subject the reaction chamber to an excitation light and detect an emission light from the reaction chamber.
  • the device includes one or more control units configured to provide instructions to control the thermocycling module and the optical analysis module.
  • a system for detecting the presence or absence of one or more nucleic acids within a sample includes a sample analysis cartridge, such as the sample analysis cartridge of any one of claim 1-77, and a device, such as the device of any one of claim 85-109.
  • a method of analyzing a sample for the presence or absence of a nucleic acid includes loading a sample into the sample analysis cartridge of any one of claims 1-77. The method further includes operably engaging the cap to seal the fluidic circuitry. The method further includes inserting the sample analysis cartridge into the device of any one of claims 85-109. Yet further, the method includes performing one or more thermocycles on the sample. Performing the one or more thermocycles includes actuating at least one of the first temperature switching unit and the second temperature switching unit. Further, the method includes emitting an excitation light toward the reaction chamber. Yet further, the method includes detecting an emission light from the reaction chamber. The presence or absence of the nucleic acid is determined based on the emission light.
  • FIG. 1A and FIG. IB illustrate an exemplary sample analysis cartridge 100.
  • FIG. 1A illustrates an isometric front view of an exemplary sample analysis cartridge 100 including a cartridge body 110 and a cap 150.
  • the cartridge body 110 includes an elongated blade feature.
  • the cap 150 includes a handle 152 and a plunger 154.
  • FIG. IB illustrates an isometric back view of the same exemplary sample analysis cartridge 110, showing the fluidic circuitry 111 of the cartridge body 110.
  • FIG. 2 illustrates an isometric front view of an exemplary sample analysis cartridge 200, showing the central layer 112, the first outer layer 114 and the second outer layer 116 of the cartridge body.
  • the fluidic circuitry of the cartridge body includes an entry port 220, a sample load chamber 222, a chamber 224, a reaction chamber 226, a venting port 232 including including a swellable plug 234, and an overflow chamber 228.
  • the sample analysis cartridge 200 also includes a cap 250, which includes a handle 252, a plunger 254.
  • the cap 250 can be attached to the cartridge body by mating an attachment feature on the cap (e.g, knob 256) with an attachment feature on the cartridge body (e.g, the opening 240 in the cartridge body that includes tines 242).
  • FIG. 3A and FIG. 3B illustrate a central layer of an exemplary sample analysis cartridge.
  • FIG. 3A illustrates an isometric front view of the central layer 312.
  • FIG. 3B illustrates an isometric back view of the central layer 312.
  • FIG. 4 illustrates an isometric view of an exemplary cap 450 of a sample analysis cartridge, the cap featuring a handle 452, a plunger 454, and an attachment feature 456.
  • FIG. 5 illustrates an isometric view of an exemplary plunger 554 that can be included in a cap of a sample analysis cartridge.
  • FIG. 6 illustrates an isometric view of the external aspects of an exemplary sample analysis device 600.
  • the device includes a housing 610 and a sample analysis cartridge receiving port 612 for receiving and positioning a sample analysis cartridge 601.
  • FIG. 7A and FIG. 7B illustrate isometric views of an exemplary thermocycling module 700 that can be included in a device for analyzing a sample in a sample analysis cartridge (e.g. , sample analysis cartridge 701 that includes a reaction chamber 702 and a handle 703).
  • the thermocycling module 700 includes a first temperature switching unit 720 that includes a first temperature-regulated block 722 and a second temperature-regulated block 724, and a second temperature switching unit 730 that includes a third temperature-regulated block 726 and a fourth temperature-regulated block 728.
  • the thermocycling module 700 also includes an actuator 740 operably connected to one or more of the first temperature switching unit 720 and the second temperature switching unit 730.
  • FIG. 7A illustrates the exemplary thermocycling module 700 in a first position.
  • FIG. 7B illustrates the exemplary thermocycling module 700 in a second position.
  • FIG. 8A, FIG. 8B, and FIG. 8C illustrate side views of an exemplary thermocycling module 800 that can be included in a device for analyzing a sample in a sample analysis cartridge (e.g, sample analysis cartridge 801 that includes a reaction chamber 802).
  • the thermocycling module 800 includes a first temperature switching unit 820 that includes a first temperature-regulated block 822 and a second temperature-regulated block 824, and a second temperature switching unit 830 that includes a third temperature-regulated block 826 and a fourth temperature-regulated block 828.
  • the thermocycling module 800 also includes an actuator 840 operably connected to one or more of the first temperature switching unit 820 and the second temperature switching unit 830.
  • FIG. 8A, FIG. 8B, and FIG. 8C illustrate side views of an exemplary thermocycling module 800 that can be included in a device for analyzing a sample in a sample analysis cartridge (e.g, sample analysis cartridge 801 that includes a reaction chamber 802).
  • the thermocycling module 800 includes a first temperature switching
  • FIG. 8A illustrates the exemplary thermocycling module 800 in a first “open” position.
  • FIG. 8B illustrates the exemplary thermocycling module 800 in a first “closed” position.
  • FIG. 8C illustrates the exemplary thermocycling module 800 in a second “closed” position.
  • FIG. 9 illustrates a side view of an exemplary optical analysis module 900 that can be included in a device for analyzing a sample in a sample analysis cartridge (e.g., sample analysis cartridge 901 that includes a reaction chamber 902 and a handle 903).
  • the optical analysis module 900 includes an emitter unit 980 including one or more light sources 982, one or more lenses 984 and one or more filters 984 optically coupled to the one or more light sources 982, and optionally a focusing lens 988 configured to combine and focus excitation light from the various independent light sources 982 onto the reaction chamber.
  • the optical analysis module 900 also includes an emission detector 992 arranged on a chip 990 and optically coupled to a lens 994 and a mechanically switchable optical filter 996 oriented to receive light emissions from the reaction chamber 902.
  • FIG. 10 illustrates a block diagram of an exemplary control unit 1000 that can be included in a device for analyzing a sample in a sample analysis cartridge.
  • the control unit 1000 includes one or more processors 1010 and a memory 1020 storing one or more programs 1022.
  • the one or more programs 1020 are configured to be executed by the one or more processors 1010 and include instructions 1024 for controlling various aspects of the thermocycling module and the optical analysis module and for performing various steps of a sample analysis method.
  • FIG. 11 illustrates a flowchart of an exemplary method of loading a sample analysis cartridge 1100.
  • the method 1100 includes the steps of: obtaining a sample analysis cartridge 1101; introducing the sample into the sample load chamber of the sample analysis cartridge 1102; and operably engaging the cap with the cartridge body to pressurize the fluidic circuitry and force the sample through at least a portion of the fluidic circuitry 1103.
  • FIG. 12 illustrates a flowchart of an exemplary method of analyzing a sample for the presence or absence of a nucleic acid 1200.
  • the method 1200 includes the steps of: loading a sample into a sample analysis cartridge 1201; operably engaging the cap to seal the fluidic circuitry of the cartridge 1202; inserting the sample analysis cartridge into a device 1202; performing one or more thermocycles 1204; emitting an excitation light toward the reaction chamber; and detecting an emission light from the reaction chamber 1205.
  • FIG. 13A and FIG. 13B provide graphs showing data from various SARS-CoV-2 assays conducted on samples deposited in an exemplary sample analysis cartridge and analyzed using an exemplary device.
  • FIG. 13A provides graphs showing data from a SARS-CoV-2 assay conducted on a positive sample, i.e., a sample that has tested positive for the presence of one or more nucleic acids associated SARS-CoV-2
  • FIG. 13B provides graphs showing data from a SARS-CoV-2 assay conducted on a control sample that is negative for SARS-CoV- 2.
  • FIG. 14 provides graphs showing results from optical readings of a sample containing decreasing amounts of reagents.
  • FIG. 15 provides graphs showing the selective excitation of decreasing titrations of various fluorescent dyes, demonstrating the multiplex capabilities of a sample analysis device. More specifically, the graphs show optical readings of decreasing titrations of fluorescent dyes Cai-Orange 560, Cai-Red 635 and Quaser-705.
  • the fluorescent dyes may be incorporated into fluorescent probes included in reagents and used to detect one or more particular nucleic acids in a sample.
  • Various platforms and devices are provided for amplifying the amount of a nucleic acid in a sample and detecting and/or quantifying the desired nucleic acid in the sample. For instance, devices may perform a polymerase chain reaction to amplify a specific targeted nucleic acid in a sample, and then perform an optical detection method to determine the quantity of the nucleic in the sample.
  • PCR Polymerase chain reaction
  • PCR is performed in a small reaction mixture comprising a nucleic acid template, or a biological sample containing the nucleic acid template, as well as various reagents, such as: 1) primers, or two small strands of synthetic DNA that are designed to be complementary to the beginning and end of the target sequence; 2) a mixture of deoxynucleoside trisphosphates (dNTPs); 3) a PCR reaction buffer containing Mg ++ ; and 4) a DNA polymerase enzyme.
  • the typical process of PCR comprises performing a number of thermocycles comprising steps at high and low temperatures, for instance:
  • thermocycling uses a Peltier element to heat and cool a temperature block that houses the samples.
  • An important parameter of a heating element for PCR thermocycling is its maximum ramp rate, or the maximum rate at which the heating block can change temperature.
  • the maximum rate of a conventional thermocycler e.g, a thermocycler with Peltier heating elements
  • the ramp rate is a key limiting variable to the efficiency of a PCR reactions during thermocycling.
  • a conventional device requires about two to three hours to perform 30 thermocycles of a typical PCR reaction.
  • RT-qPCR is a highly sensitive molecular biology method that includes the amplification of cDNA reverse-transcribed from RNA.
  • U.S. Pub. No. 2021/0164062A1 which is hereby incorporated by reference in its entirety.
  • RT-qPCR combines the elements of two PCR-based techniques that are performed in sequential stages: reverse transcription PCR, followed by qPCR.
  • Reverse transcription PCR allows for the use of RNA as a nucleic acid template and uses an enzyme called reverse transcriptase to create complementary DNA (cDNA).
  • Real-time PCR, or quantitative PCR allows for the real-time quantification of the amplified nucleic acid products from the cDNA template, typically through the optical detection of a fluorescent reporter bound to the products.
  • RT-qPCR relies on the fundamental principles of PCR, with two additions: 1) a non-cyclic reverse transcription (RT) stage prior to thermocy cling, and 2) the incorporation of a fluorescent reporter to allow for real-time detection and quantification of the amplified nucleic acid product during the thermocy cling steps.
  • the non-cyclic steps of the RT stage typically includee denaturing the RNA at about 65°C, incubating the sample on ice, allowing random hexamers to anneal to the template at a temperature of about 25°C, synthesizing cDNA with reverse transcriptase at a temperature of about 50°C, and removing the initial RNA template.
  • the qPCR stage proceeds similarly to the PCR thermocy cling steps described previously, with the addition of a fluorescent reporter that binds to the amplified nucleic acid product.
  • the fluorescence signal of the reporter can be detected at every cycle, and the abundance of the nucleic acid product can be quantified through analysis of the fluorescence amplification curves generated by repeated readings of the sample.
  • a sample e.g, a biological fluid sample
  • the microfluidics cartridge is inserted into a device for performing RT-qPCR.
  • the cartridge may direct an amount of the sample into a chamber where thermocycling and optical analysis of the sample takes place.
  • a sample In some conventional devices, a sample must be mixed with various RT-qPCR reagents prior to depositing the mixed sample and reagent into the microfluidics cartridge, which creates additional pre-processing steps and introduces added time and complexity to sample analysis. Individualized mixing steps may introduce further variability and nonuniformity into sample analysis and may fail to completely mix the sample and the one or more reagents prior to performance of RT-qPCR. Further, pre-processing of a sample may leave a user of a microfluidics cartridge or RT-qPCR device susceptible to contact with hazardous materials, such as infectious diseases present in a biological sample or various reagents intended to be mixed with the sample.
  • thermocycling typically, conventional PCR devices place Peltier heating elements in continuous contact with a sample-containing vessel (e.g., a microfluidic cartridge) to modulate the temperature of a sample during thermocycling for PCR.
  • a sample-containing vessel e.g., a microfluidic cartridge
  • Continuous contact Peltier heating elements may take longer to heat or cool a sample to a desired thermocycling temperature, increasing the amount of time required to complete a single thermocycle and to perform multiple thermocycles.
  • the temperature of conventional heating elements must be precisely controlled to avoid overshooting a desired temperature and overheating a sample during thermocycling.
  • PCR methods occur at approximately atmospheric pressure.
  • the relatively low or atmospheric pressure in conventional microfluidic cartridges may make it easier for bubbles to form in a fluid sample inside the cartridge, introducing additional sources of nonuniformity into sample analysis.
  • Standard microfluidics cartridges may be unable to withstand increased pressures, making them unsuitable for conducting PCR or RT- qPCR at pressures above atmospheric pressure.
  • the present disclosure provides, in some aspects, an improved rapid PCR device and a cartridge for use therewith, along with associated methods for loading the cartridge and analyzing a sample using the device.
  • a sample analysis cartridge In some aspects, provided herein is a device for analyzing a sample in a sample analysis cartridge.
  • a system for detecting a nucleic acid In yet further aspects, provided herein is a method of loading a sample into a sample analysis cartridge. In still further examples, provided herein is a method of analyzing a sample to determine the presence or absence of a nucleic acid.
  • the present disclosure is based at least in part on the inventors’ unique insights and findings regarding the design and configuration of a sample analysis cartridge and a device for analyzing a sample.
  • the exemplary sample analysis cartridges described herein have been designed with several advantages compared to standard microfluidics cartridges.
  • the fluidic circuitry of the cartridge is configured to be sealed to form a closed system in the fluidic circuitry of the cartridge that can be pressurized.
  • Providing positive pressure to the sample analysis cartridge prior to thermocycling and optical readings may advantageously reduce the occurrence of gas bubbles in fluid (e.g, a fluid biological sample and one or more reagents) inside the sample cartridge.
  • Pressurization may also cause the sample to advance through at least a portion of the fluidic circuitry to more easily position the sample in the chamber of the circuitry where thermocycling and optical readings occur (e.g, a reaction chamber). Pressurizing the fluidic circuitry may also result in enhanced mixing of the sample with one or more reagents as the sample and reagent mixture moves through the circuitry to the reaction chamber.
  • the fluidic circuitry may also result in enhanced mixing of the sample with one or more reagents as the sample and reagent mixture moves through the circuitry to the reaction chamber.
  • one or more surfaces of the reaction chamber of the cartridge could be flexible or deformable, such that the reaction chamber bulges or expands responsive to positive pressure inside the fluidic circuitry.
  • one or more surfaces of the reaction chamber configured to contact heating elements of a thermocycling module may be deformable and configured to bulge outward responsive to positive pressure inside the fluidic circuitry. The outward bulging and deformability of the surfaces allows for more uniform contact between the surfaces of the reaction chamber and the heating elements of the thermocycling module (e.g, one or more temperature-regulated blocks), while reducing the occurrence of small air gaps between the surface of the reaction chamber and the heating elements that can interfere with heat transfer to the sample.
  • compression of the deformable surfaces of the reaction chamber during thermocycling may improve the mixing of the sample with one or more reagents, leading to improved signal in an RT-qPCR analysis.
  • the heating elements when one or more heating elements are placed in contact with the bulging deformable surface of the pressurized reaction chamber during thermocycling, the heating elements may at least partially compress the surfaces of the reaction chamber.
  • the compression of the pressurized reaction chamber causes the sample and reagent mixture to flow and mix within the fluidic circuitry, resulting in increased occurrences of binding (e.g., annealing of primers with a nucleic acid sequence in the sample) and other desired reactions during RT-qPCR.
  • the exemplary device for sample analysis described herein is also designed with several advantages over conventional PCR devices.
  • the device uses mechanical switching of heating elements to thermocycle a sample, instead of heating a sample using more conventional continuous-contact Peltier heating elements.
  • a thermocycling method that involves mechanical switching, either the heating element(s) or the cartridge (or both) is movable, and the temperature of a sample in a reaction chamber of the cartridge is modulated by moving a heating element at a particular desired temperature to contact the reaction chamber.
  • mechanical switching relies on the physical movement of the heating elements and/or cartridge, the device can achieve more rapid temperature switching compared to continuous -contact heating elements, whose thermocycling speed is limited by the ramp rate of the heating element (e.g, a Peltier heating element).
  • mechanical switching allows for sub-second switching of the heating elements contacting the reaction chamber.
  • Faster temperature switching may improve the speed of a thermocycle, allowing the device to complete more thermocycles and amplify nucleic acids in a sample more quickly than traditional PCR devices.
  • thermocycling may further improve the efficiency of thermocycling by allowing for increased rates of heat transfer between the heating elements and a sample within the reaction chamber.
  • the elements are typically maintained at the same temperature as the desired temperature of the sample during a particular phase of a thermocycle.
  • the temperature of the continuous -contact heating elements is set to the desired sample temperature and maintained in contact with the reaction chamber for a period of time sufficient for the sample to reach the temperature of the heating elements.
  • Continuous-contact heating elements that are maintained at higher or lower temperature than the desired sample temperature may risk burning the sample or otherwise interfering with thermocycling.
  • heating elements that are mechanically switched may be maintained at temperatures even higher or lower than a desired sample temperature during a given phase of a thermocycle. For instance, during a relatively high temperature phase of a thermocycle, the heating elements may be maintained at an even higher temperature; and during relatively lower phases of a thermocycle, the heating elements may be maintained at even lower temperature.
  • increasing the differential between the temperature of the heating elements and the sample temperature will increase the rate of heat transfer, allowing for even faster temperature changes during a thermocycle. Accordingly, mechanical switching can allow for even more rapid thermocycling of a sample with less risk of overheating.
  • thermocycling can allow for more precise control of a sample temperature and a more favorable temperature curve during thermocycling compared to conventional PRC devices.
  • thermal inertia can cause temperature overshoots and make it difficult to precisely control the temperature and ramp rate (z.e., the rate of temperature change) of the heating elements used to modulate the sample temperature.
  • each of the heating elements can be maintained at a single predetermined temperature, and the temperature curve of the sample can be precisely controlled by moving a particular heating element(s) to contact the reaction chamber, which may result in a more desirable temperature curve compared to the temperature curve of a continuous -contact Peltier heating element.
  • thermocycling by mechanical switching may produce a more sinusoidal temperature curve compared with thermocycling using continuous-contact Peltier heating elements.
  • maintaining the heating elements at a single predetermined temperature may improve the efficiency of the device by reducing the power required to change the temperature of each heating element during a series of thermocycles.
  • a sample analysis cartridge such as a microfluidics cartridge for handling a biologic fluid sample.
  • the exemplary cartridge is configured for flowing a sample through a series of fluidic channels and chambers, where pre-processing and mixing of the sample takes place.
  • the sample analysis cartridge may be inserted into a device for sample analysis (e.g. , the exemplary device 600 shown in FIG. 6, including the exemplary components of a device shown in FIGS. 7A-7B, FIGS. 8A-8C, FIG. 9, and FIG. 10) for detecting the presence, absence, or amount of a nucleic acid within a sample in the cartridge, e.g, through an RT-qPCR method.
  • the sample includes biological material, such as biological material from a human, a bacterium, a plant, an animal, or an environment.
  • the sample is a biologic fluid sample from a human patient, e.g., a saliva sample, a mucus sample, or a blood sample.
  • FIGS. 1A-1B illustrate an isometric view of an exemplary sample analysis cartridge 100 for use with a device for analyzing a sample.
  • the sample analysis cartridge 100 includes a cartridge body 110 that contains a fluidic circuitry 111.
  • the fluidic circuitry 111 includes a number of channels and chambers arranged in fluid communication.
  • the fluidic circuitry 111 provides a pathway for flowing a fluid that had been deposited in the cartridge body 110 (e.g, a fluid biological sample and one or more reagents) into a reaction chamber, where thermocycling and/or optical analysis of a sample is conducted.
  • the fluidic circuitry 111 can include any number of chambers (/.£., volumes of space where fluid can collect within the cartridge), channels (e.g, microfluidic channels that connect the various chambers) and other features for improving sample handling and analysis.
  • the exemplary sample analysis cartridge 100 has been assembled by operably engaging a cap 150 and attaching a handle 152 to a cartridge body 110.
  • FIG. 1A provides a front isometric view of the exemplary sample analysis cartridge 100.
  • FIG. IB provides a back isometric view of the same exemplary sample analysis cartridge 100.
  • the sample analysis cartridge 100 includes a cap 150, which includes a handle 152, a plunger 154, and an attachment mechanism 156 for securing the cap to the sample analysis cartridge.
  • the cap 150 can be operably engaged with the cartridge body 110 to seal an entry port of the fluidic circuitry 111, provide a positive pressure to the circuitry, and to form a closed system within the circuitry prior to sample analysis.
  • operably engaging the cap 150 with the cartridge body includes attaching a handle 152 of the cap to the cartridge body, e.g, by mating an attachment feature 156 of the cap 150 and/or handle 152 with the cartridge body 110.
  • the cartridge body of the sample analysis cartridge 100 includes a distal end and a proximal end, and the body may be shaped in an elongated blade-like shape with an elongated distal end that is configured for insertion into a sample analysis device (e.g, configured for insertion into a receiving port of the cartridge, such as the receiving port 612 described with respect to FIG. 6).
  • the distal end of the cartridge body 110 is the end that is generally farthest from a user of the sample analysis cartridge 100 when the cartridge is held by a user, z.e., the end that is opposite the handle 152.
  • the distal end may have a relatively narrow width, e.g, with a width between 0.5mm to 15mm, between 0.5mm to 1.5mm, or between 0.5mm and 1.2mm.
  • the distal end of the cartridge body 110 may include at least a portion of the fluidic circuitry 111 of the sample analysis cartridge.
  • a proximal end of the cartridge body 110 is the end that is generally closest to a user of the sample analysis cartridge 100 when the cartridge is held by a user and may remain at least partially outside of a sample analyzer device during a qPCR method.
  • the proximal end of the cartridge body 110 is configured to be held be a user of the cartridge during sample loading and the insertion of the cartridge into a sample analysis device.
  • the proximal end of the cartridge body 110 is attachable to a handle 152 via one or more attachment features 156 on the cartridge body or handle.
  • the exemplary cartridge body 110 includes an elongated blade feature.
  • the elongated blade feature is a portion of the distal end of the cartridge body 110 that is relatively narrower and configured for insertion into a sample analysis device.
  • the elongated blade feature is sized to permit insertion of the cartridge body into a device used for sample analysis.
  • the thickness of the elongated blade feature could be approximately equal to or just less than the width of a receiving port of an analysis device (e.g, slightly less than the width of the receiving port of the device to allow a small spatial tolerance for inserting the cartridge).
  • the thickness of the elongated blade feature could be between approximately 0.5mm and 15mm, between 0.5mm to 1.5mm, or between 0.5mm and 1.2mm.
  • the fluidic circuitry 111 of the cartridge 100 may be oriented along an invisible plane defined by the elongated blade feature of the cartridge body 110, with at least a portion of the fluidic circuitry positioned within the elongated blade feature.
  • the fluidic circuitry 111 of the cartridge 100 is configured substantially in-line with an imaginary plane defined by the elongated blade feature.
  • the elongated blade feature reduces the thickness of the portion of the cartridge body 110 that contains portions of the fluidic circuitry (e.g, the reaction chamber) and is inserted into the sample analysis device, thereby reducing the distance between a sample in the cartridge and elements of the device used for qPCR, such as thermal heating blocks and an optical analyzer.
  • Other advantages of the elongated blade feature of the cartridge body 110 are also anticipated.
  • the cartridge body 110 may be formed of any desired material, for instance, a rigid or semi-rigid polymeric or plastic material. More specifically, the cartridge body 110 could be formed at least in part from a polypropylene, a polycarbonate, a CoC, a CoP, a polyurethane, a nylon material, or a copolymer or composite thereof. In some aspects, the material of the cartridge body is configured to withstand temperatures typically used for thermocy cling during a standard qPCR method (z.e., the material is capable of withstanding those temperatures without deforming or degrading the cartridge).
  • the material of the cartridge body 110 is configured to withstand elevated pressures (e.g, pressures up to about 25 psi) inside the fluidic circuitry for at least the duration of a typical RT-qPCR method.
  • the material is at least partially deformable responsive to positive pressures within the fluidic circuitry and/or external forces applied to the cartridge body.
  • the material could be configured to compress and/or expand by a small amount and substantially return to its previous shape after being compressed or expanded.
  • the material of the cartridge body 110 is configured to expand or bulge responsive to increased pressure inside the fluidic circuitry.
  • An exemplary cartridge body may be formed by depositing one or more materials in a plurality of layers to form a fluidic circuitry in the cartridge body.
  • FIG. 2 illustrates an exploded isometric view of an exemplary sample analysis cartridge 200, such as the sample analysis cartridge 100 depicted in FIGS. 1A-1B, showing the one or more layers that form the cartridge body.
  • the cartridge body includes at least three layers, e.g, a central layer 212 and two outer layers (e.g, a first outer layer 214 and a second outer layer 216) adjacent to opposite sides of the central layer.
  • the cartridge body is formed from two or fewer layers (e.g, a central layer and a single outer layer adjacent to the central layer) or from more than three layers (e.g. , a central layer and more than two outer layers adjacent to sides of the central layer), or some other configuration.
  • the various layers of the cartridge body may be bonded to each other using heat, pressure, sonication, laser welding, adhesive, chemical bonding, or organic bonding.
  • FIGS. 3A-3B provide isometric views of a central layer 312 of an exemplary sample analysis cartridge.
  • FIG. 3A shows a front isometric view of a central layer 312, while FIG. 3B shows a back isometric view of the same central layer.
  • the central layer 312 of the cartridge body includes at least a portion of the fluidic circuitry.
  • the central layer may be manufactured using an injection molding, CNC, or a casting technique that preserves space for the various channels and chambers of the fluidic circuitry.
  • the fluidic circuitry may be arranged along an imaginary plane defined by the central layer 310 (e.g, the same imaginary plane that is defined by the elongated blade feature, described supra).
  • outer layers 214, 216 of the cartridge body may form surfaces (e.g, exterior walls) of the various chambers or channels included in the fluidic circuitry, such as the sample load chamber 222, the reaction chamber 226, a mixing or reagent chamber 224, an overflow chamber 228, and/or the channels therebetween.
  • the various layers of the cartridge body are formed from the same material, for instance, the same polymer material or plastic material.
  • the one or more layers of the cartridge body are formed from any number of different materials.
  • the central layer 212 of the cartridge body is formed from a first plastic material
  • the outer layers 214, 216 are formed from a second plastic material different from the first plastic material.
  • the second plastic material may be deformable to permit surfaces of the reaction chamber 226 to expand, bulge, deform, and compress responsive to positive pressure inside the fluidic circuitry and external forces applied to the cartridge (e.g, forces applied by thermal heating elements of a sample analysis device).
  • the first plastic material may be relatively more rigid than the second material to prevent deformation of the central layer 210 and the overall cartridge shape during pressurization and thermocycling.
  • the outer layer(s) 214, 216 of the cartridge body include one or more thin polymer films that are affixed to the central layer 212.
  • surfaces of the fluidic circuitry e.g, the deformable surfaces of the reaction chamber 226, surfaces of the channels, or surfaces of other chambers of the fluidic circuitry
  • the thickness of the outer layer(s) 214, 216 thin polymer films permits the outer layers to deform responsive to positive pressure within the fluidic circuitry and/or responsive to external forces applied to the outer surface of the cartridge body (e.g, forces applied by a temperature-regulated blocks in contact with the reaction chamber during thermocycling).
  • the outer layers could have a thickness between 0.05mm and 3mm.
  • the outer layer(s) 214, 216 may be configured to bulge responsive to positive pressure provided to the fluidic circuitry by a plunger 254 when the cap 250 is operably engaged with the cartridge body.
  • the central layer 212 is relatively thicker than the outer layers 214, 216 of the cartridge body.
  • the central layer 212 may have a thickness between 0.5mm and 1.2mm.
  • the cartridge body includes a variety of channels, chambers, and other features arranged in fluid communication to form a fluidic circuitry in the cartridge body.
  • the fluidic circuitry provides a path along which a fluid (e.g, a fluid biological sample and one or more reagents) can travel through the cartridge body before or during sample analysis.
  • a fluid e.g, a fluid biological sample and one or more reagents
  • the fluidic circuitry includes an entry port 220, a sample load chamber 222, a reaction chamber 226, a venting port 232, an overflow chamber 228, and an exit port 230.
  • the fluidic circuitry is capable of being sealed and pressurized.
  • the fluidic circuitry may be sealed by operably engaging a cap 250 with the cartridge body to seal an entry port 220 of the circuitry and/or via the swelling of a swellable plug 234 within a venting port 232 upstream of the exit port 230.
  • the fluidic circuitry is capable of being pressurized to greater than atmospheric pressure.
  • the fluidic circuitry may be configured to be pressurized up to at least 8 psi, up to at least 14 psi, up to at least 25 psi, or some other positive pressure.
  • the fluidic circuitry of the cartridge body includes at least one entry port 220 and, in some aspects, at least one exit port 230 downstream of the entry port.
  • upstream and downstream relate to the general direction of fluid flow through the fluidic circuitry of the cartridge. For instance, after sample loading and prior to conducting a sample analysis method, a fluid sample in the cartridge body and one or more reagents will generally flow downstream through the fluidic circuitry.
  • the entry port 220 defines the most upstream portion of the circuitry, z.e., where a sample is deposited into the fluidic circuitry, while an exit port 230 or overflow chamber 228 represents the most downstream portion of the circuitry.
  • the entry port 220 and the exit port 230 provide for fluid communication between the fluidic circuitry and an environment outside the circuitry to allow for sample loading and the flow of fluid into and out of the circuitry (z.e., the entry and exit port may be vented to atmosphere).
  • an entry port 220 to the fluidic circuitry is provided as part of or in proximity to a sample load chamber 222, such that a sample or one or more reagents deposited into the entry port 220 are loaded into the sample load chamber 222.
  • the exit port 230 includes the venting port 232 (z.e., the venting port 232 vents to atmosphere and acts as an exit port 230 of the fluidic circuitry).
  • the venting port 232 z.e., the venting port 232 vents to atmosphere and acts as an exit port 230 of the fluidic circuitry.
  • the exit port 230 is disposed inside or proximate to an overflow chamber 228.
  • the fluidic circuitry may be sealed at its distal end, such that the circuitry includes only an entry port 220 and no exit port at its downstream terminus. In such examples, the fluidic circuitry may terminate at a downstream overflow chamber 228 or at some other sealed channel or chamber.
  • the fluidic circuitry includes a sample load chamber configured for receiving a sample deposited into the sample analysis cartridge 200.
  • the sample load chamber 222 is positioned upstream of the remaining chambers of the fluidic circuitry and generally above the remaining fluidic circuitry with respect to the standard orientation of the cartridge and to gravity.
  • the sample load chamber 222 include an entry port 220 to the fluidic circuitry.
  • the entry port 222 of the sample load chamber may be oriented upward with respect to the standard orientation of the cartridge 200 and to gravity, such that a sample can be dropped into the sample load chamber (e.g, by gravity, a pipette, or a dropper) to load the cartridge.
  • the sample load chamber 222 may be approximately cylindrical or barrel shaped.
  • the sample load chamber 222 could be a cylinder having a diameter between 1mm and 5mm, between 5mm and 6mm, approximately 6mm, or greater than 6mm.
  • the length of the sample load chamber 222 i.e., the distance between an upper entry or entrance port 220 of the chamber and a lower exit port or capillary stop of the chamber) could be approximately 12mm, between 10mm and 15mm, or any other desired length to fit a sample and one or more reagents.
  • the volume of the sample load chamber 228 is such that a sample and one or more reagents can be deposited inside the chamber.
  • the volume of the sample load chamber 222 could be configured to fit a sample having a volume between approximately 40uL and approximately 60uL.
  • a portion of the sample load chamber 222 includes a conical shape configured to direct fluid (e.g, a fluid sample and/or one or more reagents) into downstream aspects of the fluidic circuitry.
  • fluid e.g, a fluid sample and/or one or more reagents
  • the sample load chamber 222 is configured for receiving a sample, for instance, a fluid sample including biological material.
  • the sample is of, or derived from, an individual, such as a human. Further, in some examples the sample undergoes preprocessing or purification before loading of the sample into the sample load chamber 222. For instance, cells of the sample could be lysed prior to depositing the sample into the sample load chamber 222, or, as described infra, the sample may be mixed with one or more reagents 260 prior to loading of the sample into the cartridge.
  • the sample load chamber 222 is configured to retain liquid within the chamber 222 via a capillary stop when the fluidic circuitry is at atmospheric pressure.
  • the capillary stop is created by narrowed portion of the chamber located at the downstream end of the sample load chamber 222.
  • a capillary stop could include a narrow port, orifice, or channel leading from the downstream end of the sample load chamber 222.
  • the cross-sectional area of the capillary stop creates surface tension in a fluid deposited in the sample load chamber 222, the surface tension being sufficient to prevent the liquid from evacuating the sample load chamber 222 and advancing into downstream aspects of the fluidic circuitry at atmospheric pressure.
  • the capillary stop could be approximately 1mm in diameter, or less than 1mm in diameter.
  • the capillary stop can be interrupted by providing a sufficient positive pressure to the sample loading chamber 222.
  • operably engaging the cap 250 with the cartridge body provides positive pressure to the fluidic circuitry sufficient to interrupt the capillary stop and allow liquid to evacuate the sample load chamber 222.
  • the sample analysis cartridge 200 includes one or more reagents 260.
  • the sample analysis cartridge 200 could be provided to a consumer (e.g, to a physician, a patient, or some other user of the cartridge) pre-loaded with one or more reagents.
  • the one or more reagents 260 are disposed in a portion of the fluidic circuitry.
  • the one or more reagents could be disposed in the sample load chamber 222 and/or proximate to the entry port 220 such that a sample loaded into the sample load chamber cartridge begins to mix with the one or more reagents pre-loaded in the chamber.
  • the sample load chamber 222 may further include a reagent retention feature configured to prevent the one or more reagents 260 disposed in the chamber from exiting through an entrance of the chamber (e.g, through the entry port 220).
  • the reagent retention feature includes a plastic, metal, or fabric film that covers the entrance of the sample load chamber 222 (e.g, entry port 220) and can be peeled away by a user of the cartridge to provide access to the sample load chamber 222 (e.g, to permit loading of a sample into the sample load chamber).
  • the reagent retention features includes a filter positioned between the entry port 220 of the sample load chamber 222 and the one or more reagents 260 disposed within the chamber.
  • the one or more reagents 260 may be disposed in another location(s) within the fluidic circuitry, such as a reagent chamber (e.g, chamber 224), a mixing chamber, a channel or port of the circuitry, or in the reaction chamber 226.
  • a reagent chamber e.g, chamber 224
  • a mixing chamber e.g., a mixing chamber
  • a channel or port of the circuitry e.g., a mixing chamber, a channel or port of the circuitry, or in the reaction chamber 226.
  • the one or more reagents 260 may be disposed in the sample analysis cartridge 200 in any form suitable for mixing with a sample.
  • the reagents may be disposed in a solid form (e.g, solid pellet form, powder form, or as a surface coating) or as a liquid (e.g, as a liquid droplet, a liquid surface coating or some other liquid formulation).
  • the one or more reagents are lyophilized (e.g, freeze-dried or otherwise dehydrated).
  • the one or more reagents are provided in a powder form or in a solid reagent bead or pellet form.
  • the dry volume or wet volume of the one or more reagents 260 may be between about luL and about 20uL. Additionally or alternatively, the one or more reagents 260 could be included in a coating on one or more surfaces of the fluidic circuitry, e.g., included as a surface coating in one or more chambers or channels of the fluidic circuitry.
  • the cartridge body and fluidic circuitry do not include the one or more reagents 260.
  • the sample analysis cartridge 200 could be provided to a consumer without one or more reagents 260 disposed inside the cartridge body or fluidic circuitry, and the one or more reagents 260 may be provided to a user physically separated from the cartridge body in a kit or assembly.
  • a user of the device mixes the one or more reagents 260 and the sample before loading the mixed sample and reagents into the sample analysis cartridge 200.
  • the one or more reagents 260 could include a PCR master mix.
  • the PCR master mix may be configured to quantitatively detect the presence of one or more specific target nucleic acid sequences in a sample.
  • the PCR master mix is configured to quantitatively detect the presence of between 1 and 10 specific nucleic acid sequences, between 1 and 20 specific nucleic acid sequences, between 1 and 35 specific nucleic acid sequences, between 1 and 50 specific nucleic acid sequences, and between 1 and 100 specific nucleic acid sequences.
  • the one or more reagents are configured to quantitatively detect the presence of even greater or fewer numbers of specific nucleic acid sequences.
  • the PCR master mix may be configured to quantitatively detect the presence of 5 or fewer nucleic acids, for instance, 4 nucleic acids, 3 nucleic acids, 2 nucleic acids or a single target nucleic acid.
  • the one or more reagents 260 could include one or more of the following: (1) a target nucleic acid sequence (e.g., a DNA or DNA template), or biological sample containing the target nucleic acid sequence; (2) primers, or two small strands of synthetic nucleic acids that are designed to be complementary to the beginning and end of the target nucleic acid template; (3) a mixture of deoxynucleoside trisphosphates (dNTPs); (4) a PCR reaction buffer containing Mg ++ ; and (5) a nucleic acid polymerase enzyme (e.g., a DNA polymerase or RNA polymerase enzyme).
  • a target nucleic acid sequence e.g., a DNA or DNA template
  • dNTPs deoxynucleoside trisphosphates
  • the one or more reagents 260 could further include one or more of the following: (1) a RNA template from a biological sample, (2) a reverse transcriptase enzyme, (3) random hexamer primers for synthesis of cDNA, (4) an appropriate qPCR reaction buffer containing dNTPs, (5) a fluorescent reporter, (6) at least one forward primer and at least one reverse primer specific for the target nucleic acid sequence, and (7) DNA polymerase.
  • the reporter is a fluorescent dye that binds double-stranded nucleic acids.
  • the reporter is a hybridization probe that uses fluorescence resonance energy transfer (FRET) chemistry. Additional and alternative reagents are also anticipated.
  • FRET fluorescence resonance energy transfer
  • the fluidic circuitry could include additional chambers having specified functions that aid in sample handling, processing, and analysis.
  • a chamber 224 of the fluidic circuitry could serve as, e.g. , a reagent chamber for depositing one or more reagents pre-loaded into the fluidic circuitry, or a mixing chamber for improving the mixing of fluid that flows through the circuitry.
  • the fluidic circuitry includes a reagent chamber and one or more regents (e.g., the one or more reagents 260) are disposed in the reagent chamber and intended to mix with a sample before the sample and reagent mixture undergoes sample analysis in the reaction chamber 226.
  • the one or more reagents 260 are in powder form.
  • the one or more reagents 260 could be provided in any suitable form, such as a solid pellet form, two or more solid pellet forms, a surface coating, a liquid, or some other reagent formulation.
  • the chamber may be positioned downstream of the sample load chamber 222 and below the sample load chamber with respect to the standard orientation of the sample analysis cartridge 200 and to gravity (/.£., such that gravity and the action of the plunger 254 forces fluid in a direction from the sample load chamber 222 into the reagent chamber, causing mixing of a sample with the one or more reagents 260).
  • a reagent chamber is in fluid communication with the sample load chamber 222 via an orifice or via a microfluidic channel.
  • the reagent chamber includes one or more protrusions (e.g, ridges or bumps) extending from at least one outer surface of the chamber (e.g, an outer wall of the chamber) and configured to maintain one or more reagents disposed in the chamber away from the outer surface of the chamber (e.g. , in order to prevent overheating of the reagents when heat is applied to the cartridge).
  • a reagent chamber may additionally include one or more mixing features configured to increase the turbulence of fluid that flows through the chamber and facilitate the mixing of a sample with one or more reagents in the chamber.
  • the reagent chamber includes one or more pillars extending between surfaces of the chamber, or one or more solid free-floating particles configured to promote turbulence in the chamber.
  • the cartridge body includes one or more mixing features configured to promote turbulence in fluid that flow through the fluidic circuitry and promote mixing of a sample and one or more reagents 260.
  • the mixing features could include variably-sized and/or shaped protrusions extending from one or more surfaces of the fluidic circuitry, e.g, pins, barriers, wings, pillars or other protrusions. Additionally, or alternatively, the mixing features could include free-floating solid beads or other shapes disposed in chambers or channels of the fluidic circuitry.
  • mixing features facilitates increased mixing of a sample (e.g, a biological sample deposited in liquid form) with one or more reagents 260 disposed in the fluidic circuitry. Improved mixing of the sample and reagents may advantageously result in improved signal-to-noise for optical detection of the one or more target nucleic acid sequences.
  • one or more mixing features could be included in a dedicated mixing chamber of the fluidic circuitry (e.g, chamber 224).
  • the mixing chamber could, for instance, include one or more pillars extending between surfaces of the chamber, or one or more solid free-floating particles configured to promote turbulence in the chamber.
  • the mixing chamber could be positioned downstream and in fluid communication with the sample load chamber 222 (and, optionally, a reagent chamber) and below one or both chambers with respect to the standard orientation of the cartridge and to gravity.
  • a mixing chamber may additionally be positioned upstream and in fluid communication with a reaction chamber 226, such that adequate mixing of the sample and one or more reagents occurs prior to the sample entering the reaction chamber 226.
  • the mixing chamber is approximately spherical. However, in other examples the mixing chamber could be a rectangular prism, conic, or some other shape.
  • the fluidic circuitry includes a channel providing for fluid communication between the mixing chamber and the reaction chamber 226.
  • mixing features could be included on additional surfaces of the fluidic circuitry.
  • the sample load chamber 222 or a reagent chamber could include mixing features disposed inside the chambers or protruding from outer surfaces (z.e., outer walls) of the chambers.
  • mixing features could be included in channels, ports, or orifices positioned between the various chambers of the fluidic circuitry.
  • one or more mixing features could be included in a channel positioned between and providing for fluid communication between the sample load chamber 222 and the reaction chamber 226.
  • the fluidic circuitry of the cartridge body also includes a reaction chamber.
  • the reaction chamber is configured to provide a volume into which a sample and one or more reagents 260 can flow to undergo thermocy cling and/or optical detection processes, e.g, as part of a qPCR method.
  • the reaction chamber may include at least two opposing deformable surfaces configured for heat transfer from one or more temperature-regulated blocks, and at least two different surfaces configured for optical analysis.
  • the reaction chamber 226 is positioned downstream and in fluid communication with the sample load chamber 222 and, if included in the fluidic circuitry, a reagent chamber and/or mixing chamber (e.g, chamber 224).
  • a reagent chamber and/or mixing chamber e.g, chamber 224.
  • the reaction chamber 226 is positioned downstream of the sample load chamber 222, a reagent chamber, and/or a mixing chamber with respect to the flow of a sample through the fluidic circuitry.
  • the reaction chamber 226 is separated from other portions of the fluidic circuitry by way of one or more orifices, channels, or fluidic chambers.
  • the reaction chamber 226 may be positioned at or proximate to a distal end of the sample analysis cartridge 200 (z.e., near a distal end of the elongated blade feature of the cartridge body discussed supra).
  • the reaction chamber 226 includes a volume that can be filled with a sample and one or more reagents 260, and at least one surface forming a boundary of the reaction chamber (z.e., at least one outer wall of the chamber).
  • the reaction chamber 226 may have any desired shape, for instance, an approximately spherical shape, a rectangular prismatic shape, a cubic shape, a conic shape, a segment of a cylinder, or some other shape.
  • the volume within the reaction chamber 226 is defined by a cut-out in the central layer 210 of the cartridge body.
  • the volume of the reaction chamber 226 is about the same volume as an average volume of a sample inserted into the fluidic circuitry, for instance, between about 40uL and about 60uL. However, in other examples the volume of the reaction chamber 226 could be larger or smaller than the average volume of a sample inserted into the circuitry, for example, the volume of the reaction chamber could be approximately 20uL, or even smaller than 20uL. In some aspects, the proportions of the reaction chamber 226 are optimized to promote a desired rate of heat transfer to a sample in the chamber, while allowing sufficient thickness for optical analysis through distal side edges of the chamber (z.e., the optical analysis surfaces).
  • the surface area of the reaction chamber 226 could be related to the thickness of the chamber (i.e., a thickness of one of the optical analysis surfaces or the distance between the opposing deformable surfaces) in a ratio of approximately 20: 1, approximately 10: 1, or some other ratio.
  • each of the opposing deformable surfaces of the reaction chamber 226 is approximately square shaped with a surface area of about 20mm 2 , or between about 10mm 2 and about 50mm 2 .
  • the thickness of the reaction chamber i.e., a width of one of the optical analysis surfaces, represented by the distance between the two opposing deformable surfaces
  • Surfaces of the reaction chamber 226 may be defined by at least one of the central layer 212, the first outer layer 214 and the second outer layer 216 of the cartridge body.
  • two opposing lateral surfaces of the reaction chamber 226 could be defined by the first and second outer layers 214, 216 of the cartridge body.
  • the reaction chamber 226 includes two opposing lateral surfaces forming boundaries of the chamber and configured to be deformable (the “deformable surfaces”). The deformable surfaces may be oriented substantially in parallel to one another and aligned with a plane defined by the elongated blade feature of the cartridge body.
  • the deformable surfaces are formed from the outer layers 214, 216 of the cartridge body.
  • the deformable surfaces include thin polymeric films affixed to a central layer 212 of the cartridge body.
  • the thickness of each of the deformable surfaces may be between approximately 0.05mm and approximately 3mm.
  • the deformable surfaces are configured for contacting a contact surface of a temperature-regulated block.
  • each of the two opposing and parallel deformable surfaces is configured to engage with a respective contact surface of a temperature-regulated block of a temperature switching unit, such as either of the temperature switching units shown in FIGS. 7A-7B and FIGS. 8A-8C and described herein, when the sample analysis cartridge 200 is inserted into a sample analysis device.
  • the deformable nature of the surfaces may advantageously improve contact with the contact surfaces of the temperature regulated blocks.
  • the deformable surfaces are configured to bulge responsive to positive pressure within the fluidic circuitry and to compress responsive to contact with the temperature- regulated blocks of a clamping temperature unit.
  • the bulging and compression of the reaction chamber 226 surfaces may facilitate more uniform mixing of a sample and one or more reagents 260 within the fluidic circuitry.
  • the reaction chamber 226 additionally includes two or more surfaces forming boundaries of the chamber and configured for optical analysis of the chamber (the “optical analysis surfaces”).
  • the reaction chamber may include a first optical analysis surface configured for receiving excitation light (e.g, excitation light emitted from a light source of an optical analysis module) and a second optical analysis surface configured to transmit light emissions from the reaction chamber 226 (e.g, light emissions produced by a fluorescent reporter-tagged nucleic acid in a sample in the reaction chamber).
  • the optical analysis surfaces include distal-end surfaces formed by the central layer 212 of the cartridge body.
  • the optical analysis surfaces may be oriented such that, when the sample analysis cartridge 200 is inserted into a sample analysis device, the optical analysis surfaces are aligned with elements of an optical analysis module of the device. More particularly, when the sample analysis cartridge 200 is inserted into the device, an emitter module may be oriented substantially in the direction of the first optical analysis surface and configured to emit excitation light toward the first surface, and a detector module may be oriented substantially in the direction of a second optical analysis surface and configured to receive light emissions transmitted through the second surface. In some examples, the first optical analysis surface is substantially perpendicular to the second optical analysis surface.
  • the first and second optical analysis surfaces may be perpendicular and forming a distal end edge of the cartridge body, e.g, positioned at a distal tip of the elongated blade feature.
  • the reaction chamber 226 is a rectangular prism-shaped chamber, and the two or more surfaces configured for optical analysis are substantially perpendicular to the two opposing surfaced configured to be deformable.
  • the reaction chamber 226 is a rectangular prism-shaped chamber, and the two or more optical analysis surfaces form a distal tip of the cartridge body (e.g, a distal tip of the elongated blade feature), while the deformable surfaces form lateral sides of the cartridge body (e.g, lateral sides of the elongated blade feature).
  • the material of the optical analysis surfaces is configured to permit the transmission of light into and out of the reaction chamber 226.
  • the optical analysis surfaces could include a transparent or translucent polymer.
  • the optical analysis surfaces are defined by a central layer 212 of the cartridge body (e.g. , the distal tip of the central layer). Accordingly, the thickness of the optical analysis surfaces may be relatively small, for instance, between about 0.5mm and about 1.2mm.
  • the optical analysis surfaces are formed from different material than the cartridge body.
  • the optical analysis surfaces could be formed from a polymer that permits a relatively greater amount of light to pass through to the sample compared to the material of the cartridge body, or a polymer that provides relatively increased rigidity compared to the material of the deformable surfaces.
  • the cartridge body also includes a thermal restrictor (e.g., 236) configured to reduce heat transfer between the reaction chamber and the remainder of the cartridge body and fluidic circuitry.
  • the thermal restrictor is configured to reduce the rate of heat transfer from the reaction chamber to other areas of the cartridge body by providing an area with a relatively lower heat transfer coefficient compared to the material of the cartridge body.
  • a thermal restrictor 236 could be a chamber in the cartridge body including a vacuum, a chamber including air or some other gas, or an area of the cartridge body formed from a different material having a relatively lower heat transfer coefficient compared to the material of the cartridge body.
  • the thermal restrictor could be a cut-out (z.e., an open aperture) through the cartridge body that surrounds or abuts at least a portion of the reaction chamber 226.
  • the thermal restrictor 236 may also improve the flexibility of the cartridge body, and more particularly the flexibility of the distal tip of the elongated blade feature containing the reaction chamber 226.
  • the fluidic circuitry further includes a venting port downstream and in fluid communication with the reaction chamber.
  • the venting port may selectively permit gasses to evacuate the sample analysis cartridge, while substantially preventing liquids in the cartridge from flowing through the venting port.
  • a swellable plug in the venting port that swells responsive to liquid contact may function as a shut-off value, selectively permitting air and other gasses to evacuate the fluidic circuitry through the venting port prior to swelling, while substantially preventing the flow of fluid through the venting port after a liquid (e.glust a liquid sample flowed through the fluidic circuitry) contacts the swellable plug.
  • the venting port and swellable plug may allow all or substantially all of the gasses inside the fluidic circuitry to evacuate the fluidic circuitry before thermocycling and sample analysis begins inside the reaction chamber.
  • the venting port 232 may be positioned above the reaction chamber 226 relative to a standard orientation of the sample analysis cartridge 200 and to gravity.
  • the venting port 232 provides a fluid connection between the reaction chamber 226 and the environment exterior to the circuitry (i. e. , the venting port 232 could be vented to atmosphere).
  • the venting port 232 provides a fluid connection between the reaction chamber 226 and additional portions of fluidic circuitry downstream of the reaction chamber 226 (e.g. , additional channels, chambers, or other features, such as an overflow chamber 228).
  • the venting port 232 includes a swellable plug 234 that is configured to swell responsive to contact with a liquid to seal the venting port 232.
  • the plug absorbs an amount of the liquid and expands within the venting port 232 to seal the port and prevent liquid flow through the port.
  • the swellable plug 234 absorbs a volume of liquid.
  • the volume of liquid absorbed by the swellable plug 234 in various aspects, could be between about 1 uL and 10 uL or between about 1 uL and 5 uL. In some examples, the volume of liquid absorbed by the swellable plug is less than 1 uL.
  • the swellable plug 234 is configured to seal the venting port 232 without allowing any of the liquid to flow through the venting port 232.
  • the swellable plug 234 may absorb an amount of liquid to swell and seal the venting port 232 before any amount of the liquid flows downstream through the venting port 232.
  • a trace amount of liquid may flow through the venting port 232 before the swellable plug 234 swells to seal the port 232, e.g, an amount of liquid that is less than 5 uL, or less than luL.
  • the material of the swellable plug 234 is porous to permit the flow of gas through the port before the plug has swelled responsive to liquid contact.
  • the material of the swellable plug 234 is hydrophilic to produce the desired absorbance and swelling responsive to liquid contact.
  • the material of the swellable plug 234 could include a frit material, i.e., a granulated and absorbent ceramic composition.
  • the material of the swellable plug 234 could include cellulose or a derivative of cellulose, e.g., methylcellulose. Yet further, the material of the swellable plug 234 could include a polymeric composition, e.g., a polyethylene or a copolymer thereof. In a particular example, the material of the swellable plug 234 includes a mixture of a polymer and cellulose, for instance, a sintered mix of carboxymethylcellulose and polyethylene. In more particular example, the swellable plug 234 includes a sintered mix of approximately 50% carboxymethylcellulose and approximately 50% polyethylene.
  • the ratio of carboxymethylcellulose and polyethylene in the swellable plug 234 could be about 4:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, or some other ratio or combination of a cellulose and a polymer.
  • the swellable plug 234 swells and seal the venting port 232 responsive to contact with a liquid sample deposited into the sample analysis cartridge 200 and/or one or more reagents 260 mixed with the liquid sample. Accordingly, in some aspects, upon loading of a sample into the sample load chamber 222, the swellable plug 234 is in an unswelled state in which gas in the fluidic circuitry may flow freely through the venting port 232. Operably engaging the cap 250 with the cartridge body causes the plunger 254 to force at least a portion of gasses within the fluidic circuitry to evacuate the circuitry through the venting port.
  • the plunger 254 also forces the sample and one or more reagents 260 to advance at least partially through the fluidic circuitry. As the sample advances through the fluidic circuitry, the swellable plug 234 may continue to permit gas to evacuate the fluidic circuitry through the venting port 232 until the plug swells responsive to liquid contact. In some examples, the plunger 254 forces substantially all of the gasses within the fluidic circuitry to evacuate the circuitry through the venting port 232 before the swellable plug 234 swells to seal the venting port 232.
  • the sample when the sample flows through the reaction chamber 226 and enters the venting port 232, the sample contacts at least a portion of the swellable plug 234 and the swellable plug absorbs a small amount of the liquid in the sample to swell and seal the venting port 232.
  • fluids are preventing from flowing through the venting port.
  • the closed system inside the fluidic circuitry may be pressurized, e.g. , by providing a pressure by way of a plunger 254 inserted into the circuitry via engagement of a cap 250.
  • the swellable plug 234 when swelled to seal the venting port 232, inhibits fluid flow through the venting port 232 at pressures up to about 25 psi.
  • the swellable material of the plug 234, when swelled may inhibit fluid flow through the venting port 232 at even higher pressures, for instances, pressures up to 50 psi or 100 psi.
  • the swellable plug 234 of the venting port 232 may fail and permit liquid to advance through the venting port. For instance, a pressure in the fluidic circuitry may rise to a level above which the swellable plug 234 can no longer prevent liquid flow through the venting port 232.
  • thermocycling in the reaction chamber 226 and the heating of a liquid sample may disrupt or impede the functionality of the swellable plug 234.
  • a manufacturing or user error could result in a failure of the swellable plug 234. In these circumstances, it is desirable to avoid liquids inside the sample analysis cartridge 200 escaping the fluidic circuitry and, e.g, creating a risk of contamination or damaging a device in which the sample analysis cartridge 200 has been inserted.
  • the fluidic circuitry includes an overflow chamber configured to receive liquid that flows through the venting port 232, such as a liquid sample mixed with one or more reagents 260.
  • the overflow chamber 228 may prevent excess liquid from escaping the circuitry in the case that the swellable plug 234 fails.
  • the overflow chamber 228 is positioned downstream and in fluid connection with the reaction chamber 226 and other elements of the fluidic circuitry such that it receives fluids flowed through the fluidic circuitry (e.g. , a liquid sample, one or more reagents, and any gasses in the fluidic circuitry).
  • the overflow chamber 228 is the downstream terminus of the fluidic circuitry (z. e. , the fluid pathway of the fluidic circuitry ends at the overflow chamber 228).
  • the fluidic circuitry further includes an overflow channel positioned between the reaction chamber 226 and the overflow chamber 228.
  • the overflow channel may be relatively longer relative to other channels of the fluidic circuitry.
  • the overflow channel may include other features, e.g, one or more zig zags or setbacks, that increase the length of the channel to provide more volume for liquid flowing from the reaction chamber 226.
  • an overflow chamber 228 of the fluidic circuitry could be sealed.
  • the overflow chamber 228 could be a closed chamber at the downstream terminus of the fluidic circuitry configured to receive fluids flowed through the circuitry.
  • a sealed overflow chamber may advantageously allow the fluidic circuitry to withstand greater levels of positive pressure because the differential between the pressure upstream of the venting port 232 and the pressure downstream of the port will be reduced compared to an example including an exit port 230 vented to atmosphere. Such a configuration may allow the swellable plug 234 to withstand relatively greater positive pressures inside the fluidic circuitry without breaking the seal inside the venting port 232.
  • the entry of a liquid sample into the overflow chamber 228 may cause an increase in pressure in the overflow chamber 228 as fluid (z.e., both the gas that was already present in the fluidic circuitry prior to loading of a sample, as well as a liquid sample flowing into the chamber) begins to accumulate in the chamber.
  • the gasses trapped in the overflow chamber 228 may create an air spring at the downstream terminus of the fluidic circuitry.
  • the air spring may advantageously facilitate the mixing of the sample and the one or more reagents 260.
  • the air spring may facilitate the back and forth mixing of a sample within the fluidic circuitry.
  • the overflow chamber could be vented to the environment outside of the sample analysis cartridge 200 (z.e., vented to atmosphere) by way of one or more exit ports (e.g, exit port 230) positioned in or proximate to the overflow chamber 228.
  • the exit port 230 of the overflow chamber may be positioned above the chamber with respect to the standard orientation of the sample analysis cartridge 200 and to gravity 200, such that liquid flowed into the chamber (e.g, a liquid sample and one or more reagents) will generally accumulate inside the chamber instead of flowing through the exit port 230.
  • liquid may flow through the exit port 230 when the volume of liquid flowed through the circuitry is greater than volume of the overflow chamber 228.
  • the volume of the overflow chamber 228 provides sufficient space to receive liquids flowed through the fluidic circuitry.
  • the volume of the overflow chamber 228 may be greater than or equal to an average volume of a sample received by the sample analysis cartridge 200.
  • the volume of the overflow chamber 228 is between lOuL and 20uL, between 20uL and 40uL, between 40uL and 60uL, greater than 40uL, or greater than 60uL.
  • the volume of the overflow chamber 228 is approximately 45uL.
  • the overflow chamber 228 provides excess space compared to the volume of a sample.
  • the volume of the overflow chamber 228 could be at least 2x or at least 3x greater than the average volume of a sample received by the cartridge 200.
  • a sample analysis cartridge 200 could include any number of chambers without departing from the present disclosure. Further, it is anticipated that certain chambers could be combined and the functions merged, for instance, a reagent chamber that includes elements for mixing, or a loading chamber that includes one or more reagents. Still further, the various chambers may be connected via a network of channels, orifices, ports, and other structures that may be arranged in any desired configuration. Such fluidic circuitry elements may be reconfigured, rearranged, added to, or subtracted from without departing the scope of the present disclosure.
  • the sample analysis cartridge 200 additionally includes a cap configured to seal an entry port of the cartridge.
  • a cap configured to seal an entry port of the cartridge.
  • fluid is prevented from flowing out through an entry port of the fluidic circuitry and a closed system can be formed within the fluidic circuitry.
  • FIG. 4 illustrates a front isometric view of an exemplary cap 450, which includes a handle 452, a plunger 454, and a securement mechanism (e.g. , knob 456) for attaching the cap to the cartridge body.
  • the cap 250 is configured to operably engage with the cartridge body to seal the fluidic circuitry.
  • FIGS. 1A-1B illustrate an example sample analysis cartridge 200 operably engaged with the cap, while FIG. 2 provides an exploded view of the cap and cartridge body.
  • the cap 250 may operably engage with the cartridge body by mating with a complementary portion of the cartridge body.
  • the cap 250 may be configured to mate with an opening of the fluidic circuitry (e.g., the entry port 220) or the sample load chamber 222.
  • Mating could include, in various aspects, placing the cap 250 over an opening in the fluidic circuitry, at least partially inserting the cap 250 into an opening the fluidic circuitry, sliding the cap 250 to cover an opening in the circuitry, or some other means of creating a seal over an opening or entry port 220.
  • operably engaging the cap 250 causes at least a portion of the cap (e.g. , a plunger 254) to enter the fluidic circuitry.
  • the cap 250 is configured to seal the fluidic circuitry at or in proximity to the sample load chamber 222.
  • the cap 250 may be configured to seal the fluidic circuitry at or in proximity to any desired portion of the fluidic circuitry, for instance, at or proximate to an upstream entry port 220 of the fluidic circuitry, at or proximate to a downstream exit port 230, or proximate to some other portion of the fluidic circuitry.
  • operably engaging the cap 250 with the cartridge body includes applying a force to the cap.
  • the force applied to operably engage the cap 250 may be such that an average human can operably engage the cap without excess force, e.g. , the force may be less than an amount specified by the ASTM standards.
  • the cap 250 may operably engaged with the cartridge body by twisting or rotating the cap (e.g., a twist-on cap), pushing the cap (e.g, via a snap fit with an entry port of the cartridge body), sliding the cap, squeezing the cap, or otherwise applying a force to the cap.
  • each of the cartridge body and the cap 250 include respective complementary attachment mechanisms, and operably engaging the cap with the cartridge body includes mating the attachment mechanism of the cap with the complementary attachment mechanism of the cartridge body.
  • the cartridge body may include an aperture (e.g, the aperture 240 including tines 242 shown in FIG. 2), and the cap 250 includes a protrusion or knob 256 that can be mated with the aperture to operably engage the cap.
  • operably engaging the cap 250 with the cartridge body causes a tactile feedback feature(s) to indicate to a user that the cap is property positioned relative to the cartridge body.
  • operably engaging the cap 250 with the cartridge body may produce a “click” or “snap” or some other sound to indicate to a user that the cap is operably engaged.
  • the sample analysis cartridge 200 includes a visual indicator that indicates to a user when the cap 250 has been operably engaged with the cartridge body.
  • the cap 250 is physically attached to the cartridge body.
  • the cap 250 may be formed from a portion of the cartridge body and, optionally, from the same material as the cartridge body.
  • the cap 250 may be operably engaged with the cartridge body by, e.g, bending the cap to cover an entry port of the fluidic circuitry or applying a force to mate the cap with an entry port.
  • the cap 250 is provided separate from the cartridge body (e.g, included in an assembly or kit with the cartridge body) and may be operably engaged by attaching the cap to the cartridge body to seal the fluidic circuitry and form a sample analysis cartridge.
  • operably engaging the cap 250 with the cartridge body could be reversible such that the cap can be repeatedly attached and detached from the cartridge body without damaging the cap or sample analysis cartridge 200.
  • the cap 250 and/or cartridge body includes a securement mechanism that prevents removal of the cap when the cap is operably engaged with the cartridge body. Preventing removal of the cap 250 once the fluidic circuitry has been sealed may provide several advantages, including decreasing the risk of contaminating a sample that is being analyzed, decreasing the risk of contact with a sample by a user of the device (e.g. , decreasing the risk that a sample containing an infectious disease leaves the cartridge), preventing positive pressure within the fluidic circuitry from ejecting the cap, and other advantages.
  • operably engaging the cap 250 may include securing the cap to the cartridge body via the securement mechanism.
  • the twisting could be one-way directional such that twisting in the reverse direction does not remove the cap.
  • the cap 250 may be secured to the cartridge body using clips or an adhesive.
  • the cap 250 is secured by mating a portion of the cap with a portion of the cartridge body.
  • the cap 250 includes a knob 256
  • the cartridge body includes a tined opening 240
  • operably engaging the cap with the cartridge body includes inserting the knob into the tined opening 240 and securing the cap in place using the tines 242.
  • the tines 242 or other protrusions in the opening may be oriented in a direction to prevent removal of the cap 250 when it is in an operably engaged position.
  • Other locking mechanisms are also anticipated.
  • the sample analysis cartridge 200 further includes a handle. Providing a handle may aid a user in holding and maneuvering the sample analysis cartridge 200 while depositing the sample into the cartridge and/or inserting the cartridge into a sample analysis device.
  • the cartridge body includes the handle 252, i.e., a handle that is integral to the cartridge body and optionally formed from the same material as the cartridge body.
  • the cap 250 includes the handle 252, and operably engaging the cap 250 with the cartridge body includes attaching the handle to the cartridge body.
  • the handle 252 is attachable to the cartridge body at or in proximity to the proximal end of the cartridge body.
  • the handle 254 may be attached in any manner described above with respect to the cap 250 (e.g, pushing, bending, turning, or by inserting a knob into an opening).
  • the handle 254 or cap 250 can be attached in two stages. For instance, in one aspect the handle 254 can be pivoted at a first (/.e., pre-engagement) location, but once the handle is in a second engaged location (e.g, held by the in tines 242 in the opening 240 in the cartridge body) it can no longer pivot, thereby preventing the handle and cap 250 from being removed.
  • the handle 254 is opaque to prevent light from entering the distal end of the cartridge and interfering with optical readings when the cartridge is inserted into the device.
  • the handle could be formed form a black or an opaque polymer.
  • a distal portion of the cartridge body may be formed from a black or opaque polymer.
  • a cover may be provided with the sample analysis cartridge 200 and the cover is configured to cover the proximal end of the cartridge when the cartridge is inserted in a sample analysis device to prevent light from escaping the device.
  • the cap 250 includes a plunger 254 configured to enter at least a portion of the cartridge body and provide positive pressure to the fluidic circuitry when the cap 250 is engaged with the cartridge body.
  • FIG. 5 illustrates a front perspective view of an exemplary plunger 554 that could be included in a cap of a sample analysis cartridge (e.g., the cap 250 and sample analysis cartridge 200 of FIG. 2).
  • the plunger 554 is formed from a deformable polymeric material, such as a TPE or silicone rubber.
  • the material of the plunger 554 may be more compliant or deformable relative to the material of the cartridge body, such that the plunger deforms when it is at least partially inserted into the cartridge body and does not cause the cartridge body to deform.
  • the plunger 254 includes one or more ridges arranged circumferentially around the plunger and configured to create a hydraulic seal against walls of the fluidic circuitry (e.g, a sample load chamber 222 of the circuitry) and to reduce friction to facilitate the entry of the plunger into the cartridge body.
  • operably engaging the cap 250 with the cartridge body causes the plunger 254 to enter at least a portion of the cartridge body.
  • the plunger may enter at least a portion of the sample load chamber 222 or another portion of the fluidic circuitry proximate to the entry port 220 of the circuitry.
  • the plunger may displace a portion of fluid (e.g, a liquid sample mixed with one or more reagents 260) in the fluidic circuitry and force the fluid further downstream in the circuitry.
  • the plunger 254 forms a hydraulic seal against a wall of the cartridge body to seal the fluidic circuitry.
  • the plunger 254 may be configured to create a seal against walls of the sample load chamber 222, against walls of an entry port 220 of the cartridge body, or against another portion of the fluidic circuitry.
  • operably engaging the cap 250 with the cartridge body causes the plunger 254 to provide positive pressure to the fluidic circuitry.
  • the plunger 254 when the plunger 254 enters at least a portion of the cartridge body and fluidic circuitry, it creates a hydraulic seal and forces fluids through the circuitry until a closed system is formed when the swellable plug 234 seals the venting port 232. Once sealed, a positive pressure builds up inside the fluidic circuitry as the plunger 254 continues to advance through the circuitry during engagement of the cap 250. Pressurization of the fluidic circuitry provides several advantages compared to non-pressurized sample analysis cartridges. For example, the positive pressure provided by the plunger may reduce the incidence of bubbles in a liquid sample, improve contact between thermal elements and surfaces of the reaction chamber 226, and facilitate sample mixing with the one or more reagents 260.
  • the plunger 254 is configured to provide a positive pressure of about 8 psi to about 10 psi to the fluidic circuitry. However, the plunger 254 may provide more or less positive pressure to the fluidic circuitry, for example, between about 5 psi and 15 psi, or between 1 psi and 20 psi. Greater or lesser pressures are also anticipated.
  • the plunger 254 may be configured to provide a positive pressure sufficient to interrupt the capillary stop and allow liquid to evacuate the sample load chamber 222. The positive pressure provided by the plunger 254 may also force a sample deposited in the sample load chamber 222 at least partially through the fluidic circuity.
  • the positive pressure from the plunger may force at least a portion of the sample into the reaction chamber 226 and in contact with the swellable plug 234 of the vent port 232 (z'.e., to cause the swellable plug to swell and seal the venting port, thereby creating a closed system within the fluidic circuitry).
  • operably engaging the cap 250 with the cartridge body causes the plunger 254 to force at least a portion of gasses within the fluidic circuitry to evacuate the circuitry through the venting port 232 before the swellable plug 234 swells to seal the venting port.
  • the plunger 254 forces substantially all of the gasses initially within the fluidic circuitry to evacuate through the venting port 232, leaving substantially only the sample within the fluidic circuitry (and, as described below, a portion of air trapped by the plunger which forms an air spring).
  • operationally engaging the cap 250 with the cartridge body causes a portion of air to become trapped in the fluidic circuitry by the plunger 234.
  • the plunger 254 may trap at least a portion of air or another gas within the fluidic circuitry near an entry port 220 or the sample load chamber 222.
  • the gas trapped in the fluidic circuitry may act as an air spring on the upstream sides of a sample within the circuitry to facilitate mixing of the sample.
  • the volume of air trapped in this portion of the fluidic circuitry may be approximately equal to the volume of the sample load chamber 222 minus the volume of a sample deposited in the chamber.
  • the plunger 254 traps about 5mm of air inside the upstream portion of the fluidic circuitry. Air trapped by the plunger 254 in upstream elements of the fluidic circuitry may create an air spring that forces the sample downstream in the fluidic circuitry and promotes sample mixing. When paired with an air spring created in downstream elements of the fluidic circuitry (e.g, an air spring created within an overflow chamber 228), the dual air springs promote mixing by causing the sample to flow back and forth through the fluidic circuitry when external forces are applied to the deformable surfaces of the reaction chamber 226.
  • the air springs may be particularly useful when the reaction chamber is compressed by heating elements of a sample analysis device, such as one or more temperature-regulated blocks. Compression provided to the reaction chamber 226 surfaces forces the sample toward one or more of the air springs, and, responsive to being forced against an air spring, the air spring will provide a reciprocal pressure to the sample that causes it to flow back and forth in the fluidic circuitry. Such a configuration may result in increased mixing of a sample with one or more reagents 260, thereby increasing the accuracy and uniformity of sample analysis.
  • FIG. 6, FIGS. 7A-7B, FIGS. 8A-8C, FIG. 9, and FIG. 10 illustrate various aspects of an exemplary device for analyzing a sample in a sample analysis cartridge received by the device, such as the exemplary sample analysis cartridge 100 depicted in FIGS. 1A-1B or the exemplary sample analysis cartridge 200 depicted in FIG. 2.
  • the device may be configured for performing a method of nucleic acid amplification and/or analysis on a sample in the cartridge to determine the presence, absence, or amount of one or more of the nucleic acids contained in a sample (e.g, one or more nucleic acid sequences, such as a specific DNA or RNA sequences or a plurality of DNA or RNA sequences).
  • method includes a form of PCR.
  • the form of PCR could include real-time PCR (qPCR), reverse-transcription real-time PCR (RT-qPCR), multiplex PCR, nested PCR, hot start PCR, long-range PCR, digital droplet PCR, assembly PCR, asymmetric PCR, in situ PCR, colony PCR, or touch down PCR.
  • method includes an optical detection method, such as a method for detecting or quantifying a number of fluorescent reporters in the sample correlated with a number of the one or more target nucleic acids in the sample.
  • the device may be configured for quantifying an amount of RNA transcripts in the sample to detect the presence, absence, and/or amount of a viral RNA in the sample.
  • the quantification of RNA using RT-qPCR may be used for the detection of infectious diseases, diagnosis of human and animal infections, detection of food pathogens, gene expression analysis, microarray validation, and RNAi validation.
  • the method could be applied for the detection of antibiotic resistant microbes, evaluation of emerging novel infections, early detection of biothreat agents, genotyping, diagnosis of human and animal disease, genetically modified organism testing, plant genotyping for breeding, food pathogen detection, forensic investigations, mutagenesis, cloning, methylation analysis, lineage tracing, identification of agricultural cultivars, agricultural seed quality control, and identification of fishery products.
  • the device performs real-time quantitative PCR (z.e., RT qPCR) on a sample to detect the presence or absence of one or more nucleic acids associated with, e.g, a disease or health state, a genotype or phenotype of the sample, the presence or absence of a bacteria in the sample, the environmental source of a sample, or to determine some other aspect related to the sample.
  • RT qPCR real-time quantitative PCR
  • FIG. 6 depicts the external aspects of a device 600, including a housing 610 and a sample cartridge receiving port 612 configured to receive and position a sample analysis cartridge 601 inserted into the device.
  • FIGS. 7A-7B, FIGS. 8A-8C, FIG. 9, and FIG. 10 depict various components that may be included in internal aspects of a device.
  • FIGS. 7A-7B depict isometric views of an exemplary thermocycling module 700 of a device.
  • FIGS. 8A-8C depict side-view images of an exemplary thermocycling module 800 that can be included in a device, showing the module in various states of a thermocycle.
  • FIG. 9 depicts a side-view image of an exemplary optical analysis module 900 that can be included in a device.
  • FIG. 10 depicts a block diagram of an exemplary control unit 1000 of a device.
  • an exemplary device 600 for analyzing a sample in a sample cartridge 601 includes a housing 610 and a sample analysis cartridge receiving port 612.
  • the housing 610 is configured to surround and enclose aspects of the device, such as aspects included in a thermocycling module and an optical analysis module of the device.
  • the housing 610 includes a sample cartridge receiving port 612.
  • the housing 610 of the device could include one or more vents for dissipating heat from the internal aspects of the device.
  • the housing 610 could be opaque or otherwise formed from a material that prevents light transmission through the housing (e.g, to prevent light emitted from an optical analysis module of the device from escaping the internal aspects of the device 600).
  • a sample analysis cartridge receiving port 612 is configured to receive and position a sample analysis cartridge 601 inserted into the device, such that a reaction chamber of the cartridge is in a predetermined position relative to the receiving port.
  • the sample cartridge receiving port 612 is positioned such that a sample analysis cartridge 601 inserted in the receiving port is maintained in an upright orientation with respect to the standard orientation of the cartridge and to gravity (e.g, with an entry port or a sample loading chamber of the cartridge oriented upward).
  • at least a portion of the sample analysis cartridge 601 e.g. , a handle 602 or a cap of the cartridge
  • sample cartridge receiving port 612 is positioned on a front face of the housing 610 of the device 600. However, in other examples the receiving port 612 could be positioned on a top face of the device, on a side or back face of the device, or in some other location.
  • an exemplary sample analysis device further includes various components that may be included internal to the device.
  • the device 600 may include at least a thermocy cling module and an optical analysis module, for instance the modules shown in respective FIGS. 7A-7B, FIGS. 8A-8C and FIG. 9
  • the receiving port 612 is configured to position the sample analysis cartridge 601 such that a reaction chamber of the cartridge is in a predetermined position relative to a thermocycling module or an optical analysis module of the device.
  • the receiving port 612 could be configured to position a sample analysis cartridge 601 such that the reaction chamber is positioned to contact one or more temperature-regulated blocks of the thermocycling module.
  • the receiving port 612 is configured to position a sample analysis cartridge 601 such that a first and second temperature modulation unit of the of the thermocycling module are in point symmetry around a center point of a reaction chamber of the cartridge.
  • the receiving port 612 is configured to position a sample analysis cartridge 601 such that such that excitation light produced by the optical analysis module is incident on a reaction chamber of the cartridge and/or optical emissions from the reaction chamber can be detected at an emission detector of the optical analysis module.
  • An exemplary device for analyzing a sample in a sample analysis cartridge also includes a thermocycling module for modulating the temperature of a sample.
  • FIGS. 7A-7B provide isometric views of an exemplary thermocycling module 700 that can be included in a device, such as the exemplary device 600 shown in FIG. 6.
  • FIG. 7A depicts the thermocycling module 700 in an “open” position, i.e., with none of the temperature-regulated blocks 722, 724, 726, 728 of the temperature switching units contacting the reaction chamber 702 of a sample analysis cartridge 701 inserted into the device.
  • FIG. 7B depicts the thermocycling module 700 in a first “closed” position, z.e., with temperature-regulated blocks (e.g, blocks 724, 728) contacting opposing surfaces of the reaction chamber 702.
  • the thermocycling module 700 is configured to modulate the temperature of a sample in a reaction chamber 702 of a sample analysis cartridge 701 received by the device to perform one or more thermocycles.
  • performing a thermocycle on a sample includes modulating the temperature of the sample between at least a first temperature and a second temperature.
  • the first temperature could be sufficient to denature DNA contained within the sample
  • the second temperature could be sufficient to permit primers (e.g, primers present in one or more reagents mixed with the sample) to bind to DNA in the sample.
  • Repeated heating and cooling may facilitate interactions between a sample mixed with one or more reagents, for instance, the denaturing of DNA in the sample and the binding of primers to the DNA in the sample, resulting in an increased abundance of nucleic acids in the sample.
  • Thermocycling of a sample may take place as part of a sample analysis method performed by the device, e.g, as part of RT-qPCR method for detecting one or more nucleic acids.
  • the thermocycling module 700 is configured to modulate the temperature of a sample between at least a first temperature and a second temperature.
  • the second temperature is different from the first temperature.
  • the first temperature could be sufficient to denature DNA contained within a sample in the reaction chamber 702, and in some examples is between about 94°C to about 96°C.
  • the second temperature could be sufficient to permit primers (e.g. , primers present in one or more reagents mixed with the sample) to anneal with the denatured DNA, and may be between about 55°C and about 70°C.
  • thermocycling module 700 is configured to modulate the temperature of a sample between three or more temperatures, e.g, a first temperature sufficient to denature DNA, a second temperature sufficient to permit primers to bind to the DNA, and a third temperature that facilitates the elongation of a new DNA stand from the annealed primer at a temperature optimal for DNA polymerase activity.
  • the third temperature is between about 70°C and about 75°C.
  • the thermocycling module 700 may repeatedly modulate the temperature of the sample between at least a first temperature and a second temperature (z.e., perform one or more thermocycles on the sample).
  • the device may be configured to thermocycle the sample any desired number of times during a sample analysis method. For instance, the device could be configured to thermocycle the sample at least one time, between 1 and 10 times, between 10 and 20 times, between 20 and 50 times, approximately 45 times, or greater than 50 times. However, the device may be configured to perform even greater or lesser thermocycles on the sample without departing from the present disclosure.
  • the device continues to thermocycle the sample until the presence or absence of a specific nucleic acid sequence can be determined by an optical analysis module of the device, z.e., until amplification of the nucleic acid by thermocycling has produced enough of the nucleic acids to be detectable by the optical analysis module.
  • the thermocycling module 700 includes a plurality of heating elements (e.g, temperature-regulated blocks 722, 724, 726, 728) that can be contacted with a reaction chamber 702 of a sample analysis cartridge 701 to modulate the temperature of a sample in the reaction chamber and improve mixing of the sample with one or more reagents.
  • the one or more temperature-regulated blocks include a first temperature- regulated block 722, a second temperature-regulated block 724, a third temperature-regulated block 726, and a fourth temperature-regulated block 728 arranged into a respective first temperature switching unit 720 and second temperature switching unit 730.
  • the temperature switching units 720, 730 are operably connected to one or more actuators 740 configured to actuate the temperature switching units to contact one or more of the temperature-regulated blocks 722, 724, 726, 728 with opposing surfaces of the reaction chamber 702 (e.g, the opposing surfaces configured to be deformable of the reaction chamber described supra with respect to FIG. 2).
  • an exemplary thermocycling module 700 includes a include a first temperature-regulated block 722, a second temperature-regulated block 724, a third temperature-regulated block 726, and a fourth temperature-regulated block 728.
  • the thermocycling module 700 is configured to apply one or more temperature-regulated blocks 722, 724, 726, 728 to the reaction chamber 702 by, e.g, contacting a contact surface of at least one of the temperature- regulated block with an outer surface of the reaction chamber 702 (e.g, one of the opposing and deformable surfaces described above with respect to FIG. 2).
  • the temperature of a sample can be efficiently cycled between two or more temperatures by controlling the temperature of one or more temperature-regulated blocks 722, 724, 726, 728 and contacting the outer surface of the reaction chamber 702 with the temperature-regulated blocks.
  • one or more of the temperature-regulated blocks 722, 724, 726, 728 is maintained at the first temperature, and one or more of the temperature-regulated blocks 722, 724, 726, 728 is maintained at the second temperature.
  • the thermocycling module 700 could include two temperature-regulated blocks at a first temperature, and two temperature-regulated blocks at a second temperature.
  • the first temperature and the second temperature may remain substantially constant during a thermocycling phase of a sample analysis method, e.g., during a thermocycling step for RT-qPCR.
  • one or more of the first or second temperatures may be changed during the operation of the device.
  • the first and the second temperature may be approximately room temperature during an initial phase of the sample analysis method (e.g, during a mixing phase).
  • the first temperature could be sufficient to denature DNA contained within the sample
  • the second temperature could be sufficient to permit primers (e.g, primers present in one or more reagents mixed with the sample) to bind to DNA in the sample.
  • the temperature-regulated blocks 722, 724, 726, 728 include a resistive heating element.
  • the thermocycling module may include one or more electrically conductive wires connected to and configured to supply electricity to the temperature-regulated blocks.
  • the temperature-regulated blocks 722, 724, 726, 728 include Peltier heating elements.
  • each of the temperature-regulated blocks 722, 724, 726, 728 includes a respective contact surface configured to contact an outer surface of the reaction chamber 702 of a sample analysis cartridge 701 inserted into the sample analysis device.
  • the contact surface of each temperature-regulated block 722, 724, 726, 728 may be shaped to engage with an outer surface of the reaction chamber 702.
  • the contact surfaces could be substantially flat, substantially convex, substantially concave, slanted, or having some other shape.
  • each temperature-regulated block 722, 724, 726, 728 is shaped to provide maximum thermal contact with the reaction chamber 702 of the cartridge 701 and to minimize thermal energy transfer to additional portions of the cartridge 701, e.g., additional chambers, channels, ports, and other aspects of the fluidic circuitry and other portions of the cartridge body.
  • the thermocycling unit 700 may be used for both thermocycling as well as facilitating mixing of the sample and one or more reagents.
  • the outer surfaces of a reaction chamber 702 of a sample analysis cartridge 701 are deformable surfaces, and compressing the deformable surfaces can facilitate mixing of a sample in the cartridge.
  • contacting one or more temperature-regulated blocks 722, 724, 726, 728 with the outer surfaces of the reaction chamber 702 e.g., one or more of the first and second opposing surfaces could include compressing the deformable surface(s) of the reaction chamber.
  • contacting one or more of the temperature-regulated blocks 722, 724, 726, 728 with an outer surface of the reaction chamber 702 could include moving the temperature-related block(s) to a temperature application contact position or a pulse contact position relative to the reaction chamber 702.
  • a contact surface of the temperature-regulated block is positioned in contact with a surface of the reaction chamber 702 to allow for heat transfer between the block and a sample in the reaction chamber.
  • a contact surface of the temperature-regulated block is positioned in contact with an outer surface of the reaction chamber 702 and the temperature-regulated block at least partially compresses the outer surface of the reaction chamber.
  • the temperature-regulated blocks are arranged in temperature switching units, for instance, a first temperature switching unit 720 and second temperature switching unit 730.
  • the first temperature switching unit 720 and the second temperature switching unit 730 can be actuated to contact one or more temperature-regulated blocks with one or more outer surfaces of the reaction chamber 702.
  • performing one or more thermocycles includes actuating at least one of the first temperature switching unit 720 and the second temperature switching unit 730 to apply their respective temperature-regulated blocks to the deformable opposing surfaces of the reaction chamber.
  • the first temperature switching unit 720 includes a first temperature-regulated block 722 and a second temperature-regulated block 724 and is configured to apply the contact surface of the first temperature-regulated block 722 and the contact surface of the second temperature-regulated block 724 to opposing surfaces of the reaction chamber 702. More specifically, the first temperature switching unit 720 is configured to apply the contact surface of the first temperature-regulated block 822 to contact a first surface of the reaction chamber 802, and a contact surface of the second temperature-regulated block 724 to contact a second opposing surface of the reaction chamber 702.
  • the second temperature switching unit 730 includes a third temperature-regulated block 726 and a fourth temperature-regulated block 728 and is configured to apply the contact surface of the third temperature-regulated block 726 and the contact surface of the fourth temperature-regulated block 728 to opposing surfaces of the reaction chamber 702. More specifically, the second temperature switching unit 730 is configured to apply a contact surface of the fourth temperature-regulated block 728 to contact a first surface of the reaction chamber 702, and a contact surface of the third temperature-regulated block 724 to contact a second opposing surface of the reaction chamber 702.
  • thermocycling module 700 may be configured to apply two or more temperature-regulated blocks 722, 724, 726, 728 to the reaction chamber simultaneously by actuating the first temperature switching unit 720 and/or the second temperature switching unit 730 such that the contact surfaces of two respective temperature- regulated blocks are contacting ⁇ opposing surfaces of the of the reaction chamber 702 (e.g., with a contact surface of first block 722 contacting a first side of the reaction chamber and a contact surface of the second block 724 or the third block 726 contacting a second opposing side of the reaction chamber). More or fewer temperature switching units are also anticipated. For instance, in an exemplary device that modulates a sample between three temperatures, three temperature switching units could be provided, each of the temperature switching units including two or more temperature-regulated blocks. Additional configurations are also anticipated.
  • the temperature switching units 720, 730 are positioned such that actuating each of the respective units causes at least one of the temperature-regulated blocks 722, 724, 726, 728 to be placed in contact with the reaction chamber 702.
  • the first temperature switching unit 720 and the second temperature switching unit 730 are positioned in point symmetry around a center point of the reaction chamber 702 of the sample analysis cartridge 701.
  • the first temperature switching unit is positioned above the reaction chamber 702, and the second temperature switching unit is positioned an equal distance below the reaction chamber 702.
  • the device includes at least one actuator operably connected to and configured to actuate various portions of the device, including at least one of the temperature switching units 720, 730.
  • the actuator 740 includes a stepper motor.
  • actuating the first and/or second temperature switching units 720, 730 includes contacting at least one of the temperature- regulated blocks of the unit with a surface of the reaction chamber 702, z.e., applying a contact surface of the temperature regulated block to the first surface or second opposing surface of the reaction chamber.
  • actuating the first and/or second temperature switching units 720, 730 may include clamping or pivoting one or more of the respective units.
  • an actuator 740 is operably connected to and configured to actuate both the first temperature switching unit 720 and the second temperature switching unit 730.
  • the actuator 740 could be configured to actuate (e.g., pivot or clamp) the first temperature switching unit and the second temperature switching unit in a synchronized motion to conduct one or more thermocycles.
  • each of the temperature switching units 720, 730 is actuated by an independent actuator, for instance, a first actuator operably connected to and configured to actuate the first temperature switching unit 720 and a second actuator operably connected to and configured to actuate the second temperature switching unit 730.
  • the independent actuators may be synchronized by a control unit of the device to actuate (e.g, pivot or clamp) the first temperature switching unit and the second temperature switching unit in a synchronized motion to conduct one or more thermocycles.
  • FIGS. 8A-8C illustrate a thermocy cling module 800 of a sample analysis device in various states of a thermocycle (i. e. , at various times during the performance of a thermocycle on a sample in a reaction chamber 802 of a sample analysis cartridge 801 inserted into a device).
  • FIG. 8A depicts an exemplary thermocy cling module 800 in an “open” position, i.e., with none of the temperature-regulated blocks 822, 824, 826, 828, contacting a sample analysis cartridge.
  • FIG. 8B depicts an exemplary thermocy cling module 800 in a first “closed” position, in which the first temperature regulated block 822 is contacting a first surface of the reaction chamber 802 and the third temperature-regulated block 826 is contacting a second opposing surface of the reaction chamber 802.
  • FIG. 8C depicts an exemplary thermocycling module 800 in a second “closed” position, in which the contact surface of the second temperature-regulated block 824 is contacting the second opposing surface of the reaction chamber and the contact surface of the fourth temperature-regulated block 828 is contacting the first surface of the reaction chamber 802.
  • actuating a temperature switching unit causes the unit to pivot on an axis to position the contact surface of a temperature-regulated block in contact with an outer surface of the reaction chamber 802.
  • the first temperature switching unit 820 could be pivotable around a first axis
  • the second temperature switching unit 830 could be pivotable around a second axis.
  • the first axis is different from the second axis.
  • the actuator 840 is operably connected to and configured to pivot the first temperature switching unit 820 in either a first direction (e.g., clockwise) or a second direction (e.g, counterclockwise).
  • the actuator 840 is operably connected to and configured to pivot the second temperature switching unit 830 in either the first direction (e.g, clockwise) or the second direction (e.g. , counterclockwise).
  • a first actuator could be operably connected to and configured to pivot the first temperature switching unit 820, and a second actuator operably connected to and configured to pivot the second temperature switching unit 830.
  • actuating the first temperature switching unit 820 to pivot in a first direction causes the first contact surface of the first temperature-regulated block 822 to contact a first surface of the reaction chamber 802.
  • Actuating the first temperature-switching unit 820 in a second direction causes the contact surface of the second temperature-regulated block 824 to contact a second opposing surface of the reaction chamber 802.
  • actuating the second temperature switching unit 830 in a first direction causes the contact surface of the third temperature-regulated block 826 to contact the second opposing surface of the reaction chamber 802.
  • Actuating the second temperature switching unit 830 in a second direction causes the contact surface of the fourth temperature-regulated block 828 to contact the first surface of the reaction chamber 802.
  • the first temperature switching unit 820 and the second temperature switching unit 830 are rotationally coupled such that the actuator 840 is configured to pivot both the first temperature switching unit 820 and the second temperature switching unit 830 synchronously in either the first direction or the second direction.
  • the actuator 840 is configured to pivot both the first temperature switching unit 820 and the second temperature switching unit 830 synchronously in either the first direction or the second direction.
  • Conducting a thermocycle may therefore comprise using the actuator 840 to pivot the first temperature switching unit 820 and the second temperature switching unit 830 in a synchronized motion such that: at a first time, the first temperature-regulated block 822 of the first temperature switching unit 820 contacts a first surface of the reaction chamber 802, and the third temperature-regulated block 826 of the second temperature switching unit 830 contacts the second opposing surface of the reaction chamber 802; and, at a second time, the fourth temperature-regulated block 828 of the second temperature switching unit 830 contacts the first surface of the reaction chamber 802, and the second temperature-regulated block 824 of the first temperature switching unit 820 contacts the second opposing surface of the reaction chamber 802.
  • each of the temperature switching units 820, 830 includes one temperature-regulated block maintained at a first temperature and one temperature-regulated block maintained at a second temperature.
  • the first temperature-regulated block 822 of the first temperature switching unit 820 and the third temperature-regulated block 826 of the second temperature switching unit 830 may be maintained at a first temperature
  • the second temperature-regulated block 824 of the first temperature switching unit 820 and the third temperature-regulated block 826 of the second temperature switching unit 830 may be maintained at a second temperature different from the first temperature.
  • actuating a temperature switching unit causes the unit to clamp around the sample analysis cartridge to position the contact surface of each of its temperature- regulated blocks in contact with opposing surfaces of the reaction chamber 801.
  • the actuator 840 is operably connected to and configured to clamp the first temperature switching unit 820 and/or the second temperature switching unit 830.
  • a first actuator could be operably connected to and configured to clamp the first temperature switching unit 820, and a second actuator operably connected to and configured to clamp the second temperature switching unit 830.
  • actuating the first temperature switching unit 820 causes the unit to clamp such that the first temperature-regulated block 822 and the second temperature- regulated block 824 contact opposing surface of the reaction chamber 802 (e.g, with the first temperature-regulated block 822 contacting the first surface of the reaction chamber 802 and the second temperature-regulated block 824 contacting the second opposing surface of the reaction chamber 802).
  • actuating the second temperature switching unit 830 causes the unit to clamp such that the third temperature-regulated block 826 and the fourth temperature-regulated block 828 contact opposing surfaces of the reaction chamber 802 (e.g, with the fourth temperature-regulated block 828 contacting the first surface of the reaction chamber 802 and the third temperature-regulated block 826 contacting the second opposing surface of the reaction chamber 802).
  • Conducting a thermocycle may therefore comprise using the actuator 840 to clamp the first temperature switching unit 820 and the second temperature switching unit 830 in a synchronized motion such that: at a first time, the first temperature-regulated block 822 of the first temperature switching unit 820 contacts a first surface of the reaction chamber 802, and the second temperature-regulated block 822 of the first temperature switching unit 820 contacts the second opposing surface of the reaction chamber 802; and, at a second time, the fourth temperature-regulated block 828 of the second temperature switching unit 830 contacts the first surface of the reaction chamber 802, and the third temperature-regulated block 826 of the second temperature switching unit 830 contacts the second opposing surface of the reaction chamber 802.
  • each of the respective temperature switching units 820, 830 may correspond to either a first temperature or a second temperature.
  • the first temperature-regulated block 822 and the second temperature-regulated block 824 of the first temperature switching unit 820 may be maintained at a first temperature.
  • the third temperature-regulated block 826 and the fourth temperature-regulated block 828 of the second temperature switching unit 830 may be maintained at a second temperature different from the first temperature.
  • An exemplary device also includes an optical analysis module configured to perform an optical analysis on a sample in a cartridge that has been received by the sample analysis device (e.g. in order to perform RT-qPCR on the sample).
  • the optical analysis module may be configured to emit excitation light toward the reaction chamber of the sample analysis cartridge, and detect light emitted from the cartridge to determine the presence, absence, or amount of a nucleic acid in the sample.
  • the optical analysis module may be configured to emit light in a wavelength band that corresponds to the excitation range of a fluorescent reporter probe and is configured to excite the fluorescent reporter (e.g., emit that particular wavelength band of light, and/or optionally filter the light to the particular wavelength band), and detect light emissions emitted by the fluorescent reporter.
  • the device is configured for quantifying light emissions from a number of different fluorescent reporter probes introduced to the sample via one or more reagents (z.e., by detecting fluorescence emissions in various fluorescent bands emitted by the respective reporter probes and readable by the optical detector).
  • each of the fluorescent reporter probes may be configured to target and bind to a different specific nucleic acid sequence in the sample to permit simultaneous detection and/or quantification of multiple nucleic acids in a sample.
  • the device may be configured to quantify light emissions from at least one fluorescent reporter probe, at least 2 fluorescent reporter probes, at least 3 fluorescent reporter probes, at least 4 fluorescent reporter probes, or exactly 4 fluorescent reporter probes, where light emissions from each fluorescent reporter probe are related to the presence, absence, or amount of a specific nucleic acid in a sample.
  • reagents having additional or fewer fluorescent reporters are also envisioned for detecting and/or quantifying greater or fewer nucleic acids in a sample.
  • FIG. 9 illustrates an exemplary optical analysis module 900 that can be included in a sample analysis device, such as device 600 shown in FIG. 6.
  • the optical analysis module 900 is c69onfigured to subject the reaction chamber 902 of the cartridge to an excitation light and to detect an emission light from the reaction chamber.
  • the amount and characteristics of emission light detected at an emission detector 992 can indicate various aspects about a sample in the reaction chamber, such as the presence, absence, or amount of one or more nucleic acids in the sample.
  • the exemplary optical analysis module 900 includes one or more light sources 982 for producing an excitation light in the direction of the reaction chamber 902 and an emission detector 992 for detecting the emission light from the reaction chamber.
  • the optical analysis module 900 includes one or more light sources 982, and in some aspects includes an array of light sources.
  • subjecting the reaction chamber 902 to an excitation light includes emitting excitation light from one or more of the light sources 982.
  • each of the light sources 982 is optically coupled with a lens 984 configured to direct light from each of the one or more light sources 918 onto the reaction chamber 902.
  • each of the light sources 982 is further optically coupled with a filter 986 positioned between the lens 984 and reaction the chamber 902 of a sample analysis cartridge 901.
  • the filter 986 is configured to transmit only a desired portion of excitation light emitted from the one or more light sources 982.
  • each of the filters 986 is configured to transmit a desired range of wavelengths of excitation light toward the reaction chamber 902, e.g, a range of wavelengths corresponding to an excitation spectrum of a fluorescent reporter configured to bind to specific nucleic acids in the sample.
  • the one or more light sources 982 and optically coupled lens(es) 984 and filter(s) 986 are arranged in a respective optical emission module 980 configured to emit excitation light toward an optical analysis surface of the reaction chamber 902.
  • the one or more light sources 982, the lens(es) 984 and the filter(s) 986 are arranged along a pathway that directs excitation light from the one or more light sources 982 onto an optical analysis surface of the reaction chamber 902.
  • each of the one or more light sources 912 is independently configurable from the others by adjusting various aspects of the light source and the lens 914 and filter 916 optically coupled with the light source 912.
  • the optical analysis module 900 further includes a focusing lens 988 configured to combine and focus excitation light from the various independent light sources 912 onto the reaction chamber, z'.e., after the excitation light from the one or more light sources 982 has passed through the lens(es) 984 and filter(s) 986 and before the light reaches the reaction chamber 902.
  • the optical analysis module 900 further includes an emission detector 922 for quantifying light emissions received from the reaction chamber 902.
  • the emission detection 992 is optically coupled with a lens 992 and a mechanically switchable optical filter 926 (e.g. , a filter wheel).
  • the lens 924 is configured to receive light emissions from the reaction chamber 902 and direct the emissions toward the emission detector 992.
  • the mechanically switchable optical filter 996 is positioned between the lens 994 and the optical detector 992, such that light emissions from the reaction chamber pass through the lens and filter before reaching the optical detector. Accordingly, in one aspect, the lens 994 and optical filter 996 are arranged along a pathway that directs light emissions from an optical analysis surface of the reaction chamber 902 onto the optical detector 992.
  • one or more components is included on a chip 990.
  • the emission detector 922 may be disposed on a chip 990 oriented such that the detector receives emissions light transmitted through one of the optical analysis sides of the reaction chamber 902.
  • the various components of the optical analysis module 900 are oriented with particular pathways for emitting light toward a sample in a reaction chamber 902 of a sample analysis cartridge, and for receiving light emissions from the reaction chamber.
  • the emitter module 910 is oriented such that the pathway of excitation light is perpendicular to a pathway of the emission light received by the emission detector 982, e.g., the one or more light sources 982 or the focusing lens 988 may be oriented at a 90 degree angle relative to the emission detector 982 such that a pathway of light emitted toward the reaction chamber from the one or more light sources is approximately perpendicular to the pathway of light emissions detected by the emission detector.
  • the one or more light sources 992 and the emission detector 982 may be oriented toward respective optical analysis surfaces of the reaction chamber.
  • the emitter module 910 could be positioned such that the one or more light sources 912 direct light toward a first optical analysis surface of the reaction chamber 902 (e.g, a first distal edge of the cartridge body described above with respect to the analysis chamber of FIG. 2)
  • the emission detector 982 could be positioned such that the detector received light emissions from a second optical analysis surface of the reaction chamber (e.g, a second distal edge of the cartridge body that is approximately perpendicular with respect to the first distal edge).
  • a pathway of the excitation light and a pathway of the emission light of the optical analysis module 900 does not intersect with a motion path of the first and second clamping temperature units of the thermocycling module.
  • Other orientations of the optical analysis module 900 are also anticipated.
  • the sample analysis device further includes a control unit configured to control various aspects of the thermocycling module and the optical analysis module, e.g, to change various settings of the device and to initiate various steps of a sample analysis method.
  • a control unit 1000 includes one or more processors 1010 and a memory 1020 storing one or more programs 1022.
  • the one or more programs 1022 are configured to be executed by the one or more processors 1010.
  • Each of the one or more programs 1022 includes instructions 1024 readable by the processor 1010.
  • the processor 1010 could be a computer processor within the device, an external remote computer system configured to communicate with the device, a networked or cloud computing system, or some other processing system.
  • the one or more programs 1022 include instructions 1024 for setting the temperature of the first temperature-regulated block, the second temperature-regulated block, the third temperature-regulated block, and the fourth temperature-regulated block (e.g, the temperature-regulated blocks 722, 724. 726, and 728 depicted in FIGS. 7A-7B or the corresponding temperature-regulated blocks 822, 824, 826, 828 depicted in FIGS. 8A-8C) to maintain each of the temperature-regulated blocks at a desired temperature during thermocycling.
  • the respective temperatures of the various blocks are independently controllable, and the control unit 1000 is configured modulating the temperature of each of the block independently to one or more different temperatures.
  • the temperature of various pairs of blocks may be controlled together.
  • the controller is configured to set the temperature of the first temperature-regulated block and the third temperature-regulated block at a first temperature, and the second temperature-regulated block and the fourth temperature-regulated block to a second temperature.
  • the control unit 1000 is configured to set the temperature of the first temperature-regulated block and the second temperature-regulated block to a first temperature, and the second temperature-regulated block and the fourth temperature-regulated block to a second temperature.
  • the temperature of the temperature-regulated blocks can be selected by way of a user interface associated with the device.
  • one or more programs 1022 include instructions 1024 for actuating the respective first temperature switching unit and the second temperature switching unit (e.g, units 720 and 730 of FIGS. 7A-7B and/or units 820 and 830 of FIGS. 8A-8C).
  • the one or more programs 1022 could include instructions 1024 for pivoting the first temperature switching unit and the second temperature unit in either a first direction or a second direction.
  • the one or more programs 1022 could include instructions 1024 for clamping the first temperature switching unit and the second temperature unit.
  • the one or more programs 1022 could include instructions 1024 for actuating the temperature switching units in a synchronized motion, e.g, to pivot the first temperature switching unit and the second temperature switching unit in a first direction and then in a second direction, or to clamp the first temperature switching unit and then to clamp the second temperature switching unit.
  • the one or more programs 1022 include instructions 1024 for performing one or more thermocycles using the first temperature switching unit and the second temperature switching unit.
  • Various aspects of the thermocy cling may be specified by the instructions 1024.
  • one or more of the programs 1022 could include instructions for performing a particular number of thermocycles or duration of time to perform thermocycles, for a particular rate of performing a thermocycle or a series of thermocycles, or for a particular duration of various steps in a single thermocycle.
  • the instructions 1024 include instructions for performing at least one thermocycle, 10 or more thermocycles, between 20 and 50 thermocycles, approximately 45 thermocycles, greater than 40 thermocycles, or another amount of thermocycles selected by a user of the device.
  • the number of thermocycles can be selected by a user of the device, e.g, on a user interface of the device.
  • the instructions 1024 for performing a thermocycle also include instructions 1024 for subjecting the reaction chamber to an excitation light and detecting light emissions from the reaction chamber using the optical analysis module, such that each thermocycle of the method also includes taking a reading from the optical analysis module.
  • one or more programs 1022 include instructions 1024 for performing repeated thermocycles and/or readings by the optical analysis module until the presence or absence of a nucleic acid can be determined, e.g, via RT-qPCR.
  • the instructions 1024 could also include instructions for controlling various aspects of the optical analysis module (e.g, optical analysis module 900 depicted in FIG. 9). More particularly, the instructions 1024 could include instructions for subjecting the reaction chamber to an excitation light, by emitting excitation light by way of the one or more light sources of the optical analysis module. In another aspect, the instructions 1024 could include instructions for detecting a light emissions from the reaction chamber at an emission detector of the optical analysis module. One or more programs 1022 could further include instructions 1024 for mechanically switching one or more of the filters coupled to the one or more light sources or a mechanically switchable optical fiber coupled to the emission detector.
  • the instructions 1024 could include instructions for controlling various aspects of the optical analysis module (e.g, optical analysis module 900 depicted in FIG. 9). More particularly, the instructions 1024 could include instructions for subjecting the reaction chamber to an excitation light, by emitting excitation light by way of the one or more light sources of the optical analysis module. In another aspect, the instructions 1024 could include instructions for detecting a light emissions from the
  • the one or more programs 1022 include instructions 1024 for determining the presence or absence of a nucleic acid in the sample based on the light emissions from the reaction chamber detected at the optical detector.
  • the instructions 1024 could include quantifying an amount of light detected at the optical detector and, based on the amount of light detected at the optical detector, determining the presence or absence of a nucleic acid.
  • the instructions 1024 are for determining the presence, absence, or amount of a nucleic acid based on a series of readings of the optical analysis module (z.e., over a series of successive emissions of excitation light and detection of light emissions that may take place as part of a thermocycle).
  • the one or more programs 1022 include further instructions 1024 for diagnosing a disease or determining a health state based on the presence or absence of the one or more nucleic acid sequences.
  • the one or more programs 1022 include instructions 1024 for transmitting data, for instance, data relating to the presence or absence of one or more nucleic acids, or data relating to a diagnosis or heath state determined based on the optical analysis.
  • the instructions 1024 could be instructions for transmitting data to a display, to another computer system (e.g, a computer system for reporting a diagnosis or health state), to a physician, to a patient, or to some other recipient.
  • a program 1022 may include instructions 1024 configured to be executed by a processor 1010 in any desired order.
  • a particular program 1022 could include instructions 1024 for: at a first time, setting the temperature of the temperature regulated blocks; at a second time, performing one or more thermocycles using the first temperature switching unit and the second temperature switching unit; and, at a third time, subjecting the reaction chamber to an excitation light and detecting light emissions from the reaction chamber.
  • a program 1022 could include one or more instructions 1024 configured to be performed simultaneously or in a repeated sequence.
  • the instructions 1024 could include actuating one or more of the temperature switching units and one or more optical readings simultaneously, or in a repeated sequence, e.g, as part of a plurality of thermocycles.
  • control unit 1000 includes one or more programs 1022 stored in the memory 1020 and including instructions 1024 for performing a predetermined sample analysis method.
  • various pre-set programs 1022 could be included in the memory 1020 with instructions 1024 for processing various sample types according to the content or source of the sample, the expected nucleic acid content of the sample, or the specific nucleic acid(s) quantified or detected via the sample analysis.
  • various pre-set program 1022 are displayed on a user interface associated with the device, and a user selects a particular pre-set program 1022 on the user interface.
  • control unit 1000 is configured for controlling multiple devices to perform sample analyses in parallel.
  • the one or more programs 1022 include instructions 1024 readable by a processor 1010 and configured to be executed by the processors 1010 of multiple devices to control a plurality of devices to conduct a sample analysis.
  • Various aspects of the devices could be controlled independently from other devices (e.g. , various settings of the sample analysis, for instance, settings corresponding to a particular sample type or nucleic acid detected by the particular device), while certain steps (e.g, thermocy cling) are conducted in parallel between the multiple devices.
  • the multiple devices may be controlled by a single control unit 1000 configured to execute instructions 1024 to control the thermocycling module and optical analysis module of the multiple devices. Additional multi-device control units are anticipated.
  • the present disclosure also describes system for determining the presence, absence, or amount of a nucleic acid, the system including a sample analysis cartridge (such as the sample analysis cartridge described above with respect to FIGS. 1A-1B and FIG. 2) and a device (such as the device described above with respect to FIG. 6, FIGS. 7A-7B, FIGS. 8A- 8C, FIG. 9, and FIG. 10).
  • the nucleic acid is from SAR-CoV-2, or a variant thereof.
  • the system may be provided in a kit or assembly that is assembled by a user of the system, such as a patient or a physician.
  • a device may be provided to a user in a kit or assembly including one or more sample analysis cartridges. The user may then be instructed to insert the cartridge into the device (z'.e., such that the device receives and positions the cartridge) to perform one or more sample analyses using the device.
  • the device and the sample analysis cartridge may be provided separately, e.g, with one user of the system obtaining the sample analysis cartridge and another user of the system obtaining or possessing the device.
  • the device is possessed by a first user (e.g, a physician, a health care worker, a scientist, a laboratory, a clinic, or a mobile health clinic), and the sample analysis cartridge is provided to second user, such as a patient.
  • the second user may be instructed to deposit a sample into the sample analysis cartridge and seal the cartridge (e.g, by operably engaging a cap with the cartridge body) before bringing the cartridge to the first user.
  • the first user may obtain the sample analysis cartridge from the second user and insert the sample analysis cartridge into the device to perform one or more methods of sample analysis using the device.
  • the sample analysis cartridge is a single-use cartridge configured to be disposed after using the cartridge to analyze a sample using the device.
  • the sample analysis cartridge is provided in a packaging, such as a sterilized packaging.
  • the sample analysis cartridge may be provided disassembled, z.e., with the cartridge body and the cap of the cartridge provided in a non-engaged state and optionally in separate packaging.
  • one or more reagents are provided with the sample analysis cartridge and a user of the system deposits the one or more reagents in the sample analysis cartridge or mixes the one or more reagents with a sample before depositing the mixed sample and reagents into the sample analysis cartridge.
  • the one or more reagents may be provided in a packaging separate from the packaging of the sample analysis cartridge, or may be included in a sealed container within the packaging of the sample analysis cartridge.
  • the device of the system is transportable.
  • one or more devices may be provided in a mobile health clinic, e.g, a temporary clinic, a vehicle, or some other mobile platform for transporting the device to users.
  • the sample analysis cartridge may be provided at the mobile health clinic, or may be provided to users separately (e.g, prior to the transportation of the device to users of the clinic).
  • the mobile health clinic may assemble the system by inserting sample analysis cartridges from users into the device to conduct a sample analysis method.
  • the system could include multiple devices.
  • a system could include a plurality of devices operably connected and configured from conducting sample analyses in parallel.
  • the plurality of devices is controlled by a single control unit, such as the control unit described above in relation to FIG. 10.
  • each of the plurality of devices includes a respective control unit.
  • each of the plurality of devices is configured to receive and position a respective sample analysis cartridge and to conduct an independent sample analysis method on a sample in the respective cartridge.
  • the plurality of devices could be configured for conducting sample analyses simultaneously or could be configured to operate a plurality of independent sample analyses, z.e., a plurality of analyses proceeding at different times or having different settings on each of the plurality of devices for performing the sample analysis.
  • the present disclosure further describes an exemplary method of loading a sample into a sample analysis cartridge, such as any of the exemplary cartridges 100 and 200 depicted in respective FIGS. 1A-1B and FIG. 2 and described herein.
  • an exemplary method 1100 includes, at a first step 1101, obtaining a sample analysis cartridge.
  • the sample analysis cartridge may be obtained with a device for analyzing a sample, such as the device illustrated in FIG. 6 and described supra.
  • the sample analysis cartridge may be obtained in a system, a kit, or an assembly with a sample analysis device.
  • the sample analysis cartridge may be obtained separate from a sample analysis device, e.g. , by purchasing a single-use sample analysis cartridge which can be inserted into a device and is then disposed or discarded after the sample analysis method is performed by the device.
  • An exemplary method 1100 further includes, at a second step 1102, introducing the sample into the sample load chamber of the sample analysis cartridge.
  • introducing the sample into the sample load chamber includes depositing the sample into an entry port or sample loading chamber of the cartridge.
  • the sample may be introduced by dropping, pipetting, or inserting the sample into the sample load chamber or entry port.
  • the sample is mixed with one or more reagents, such as a PCR master mix, prior to introducing the sample into the sample load chamber.
  • introducing the sample into the sample analysis cartridge could include mixing the sample with one or more reagents (e.g., one or more reagents provided separately from the cartridge or included in a kit or assembly including the cartridge).
  • the sample analysis cartridge includes one or more reagents in a fluidic circuitry of the cartridge and is configured to mix the sample with the one or more reagents as the sample flows through the fluidic circuitry.
  • the sample analysis cartridge could include a retention feature configured to retain the one or more reagents within the sample analysis cartridge (e.g, in a sample load chamber of the cartridge).
  • introducing the sample into the sample load chamber includes removing the retention feature, e.g, by peeling the retention feature from the cartridge body.
  • an exemplary method 1100 includes, at a third step 1103, operably engaging the cap with the cartridge body to pressurize the fluidic circuitry and force the sample through at least a portion of the fluidic circuitry.
  • operably engaging the cap with the cartridge body includes applying a force between the cap and the cartridge body, e.g, pressing the cap, twisting the cap, or otherwise applying a force to create a seal between the cap and the cartridge body.
  • the cap includes a plunger and operably engaging the cap causes the plunger to enter at least a portion of the sample analysis cartridge (e.g, the sample load chamber or another portion of the fluidic circuitry) and provide a positive pressure to (/. e.
  • pressurize the fluidic circuitry.
  • operably engaging the cap creates a hydraulic seal between the plunger and the sample analysis cartridge.
  • advancing the plunger through at least a portion of the sample analysis cartridge e.g, through the sample load chamber or another portion of the fluidic circuitry
  • maintaining the hydraulic seal increases the positive pressure within the fluidic circuitry.
  • operably engaging the cap forces the sample through at least a portion of the fluidic circuitry.
  • operably engaging the cap may force at least a portion of the sample into a reaction chamber of the sample analysis cartridge and/or may force at least a portion of the sample to contact the swellable plug in the venting port of the cartridge.
  • the swellable plug may swell to seal the venting port responsive to contact with the sample.
  • the sealing of the venting port by the swellable plug creates a closed system within the fluidic circuitry (the closed system being sealed at one end by the hydraulic seal between the plunger and the cartridge, and at the other end by the swelling of the swellable plug in the venting port).
  • the pressure in the fluidic circuitry is between about 1 psi to about 20 psi, or between about 8psi to about lOpsi.
  • the sample analysis cartridge is maintained in a substantially upright position during the introducing of the sample, the operable engaging of the cap, and for a period of time after the operable engaging to allow for the swellable plug to contact the sample and swell to seal the venting port.
  • the sample analysis cartridge is configured to inserted into a device for sample analysis, and a receiving port of the device maintains the cartridge in an upright position.
  • the sample analysis cartridge is maintained in a substantially upright position during a sample analysis method, e.g., a sample analysis method performed by a device into which the cartridge is inserted.
  • the present disclosure further describes an exemplary method 1200 of analyzing a sample for the presence or absence of a nucleic acid.
  • the method could be a RT- qPCR method.
  • the nucleic acid is from SAR-CoV-2, or a variant thereof.
  • the method 1200 may be implemented using any of the sample analysis cartridges and devices described herein, such as the exemplary sample analysis cartridges 100 and 200 described in relation to respective FIGS 1A-1B and FIG. 2 and the exemplary device 600 described in relation to FIG. 6 featuring the exemplary components described in relation to FIGS. 7A-7B, FIGS. 8A-8C, FIG. 9, and FIG. 10
  • the exemplary method 1200 includes loading a sample into the sample analysis cartridge, such as either of the sample analysis cartridges 100 or 200 described herein with respect to FIGS. 1A-1B and FIG. 2.
  • Loading the sample could include, for instance, depositing the sample into an entry port or sample loading chamber of the cartridge.
  • loading the sample into the sample analysis cartridge includes mixing the sample with one or more reagents prior to loading the sample into the cartridge, or peeling a retention feature of the cartridge. Further steps for loading the sample analysis cartridge are described supra with respect to the second step 1102 of the method 1100 of FIG. 11, and such steps may additionally be incorporated into step 1201 of the method 1200.
  • the exemplary method 1200 includes operably engaging the cap with the cartridge body to seal the fluidic circuitry.
  • the operable engagement of the cap with the cartridge body is described in more detail supra, for instance, in relation to the cap 250 and plunger 254 of FIG. 2, and with respect to the third step 1103 of the method 1100 of FIG. 11. Such details are additionally incorporated into step 1202 of the method 1200.
  • the method 1200 includes inserting the sample analysis cartridge into a device, such as any of the exemplary devices described herein with respect to FIG. 6, FIGS. 7A-7B, FIGS 8A-8C, FIG. 9, and FIG. 10.
  • inserting the sample analysis cartridge into the device could include inserting the sample analysis cartridge into a receiving port of the device.
  • inserting the sample analysis cartridge could include inserting a portion of the cartridge into the device, e.g., a distal end of the cartridge or at least a portion of an elongated blade feature of the cartridge.
  • the method 1200 includes performing one or more thermocycles.
  • step 1204 includes performing a plurality of thermocycles, for instance, 10 or more thermocycles, between 20 and 50 thermocycles, approximately 45 thermocycles, greater than 40 thermocycles, or another amount of thermocycles selected by a user of the device.
  • performing one or more thermocycles includes actuating one or more of a first temperature switching unit and a second temperature switching unit to contact one or more temperature-regulated blocks with a reaction chamber of the sample analysis cartridge.
  • performing a thermocycle includes actuating the first temperature switching unit and the second temperature switching unit in a synchronized motion, for instance, by clamping or pivoting the temperature switching units to contact the reaction chamber in a reciprocal motion.
  • performing one or more thermocycles could include: at a first time, contacting at least one temperature-regulated block with the reaction chamber to modulate the temperature of a sample in the chamber to a first temperature; and, at a second time, contacting at least one temperature-regulated block with the reaction chamber to modulate the temperature of a sample in the chamber to a second temperature.
  • thermocycles 7A-7B and FIGS. 8A-8C are additionally incorporated into step 1204 of the method 1200.
  • the performance of one or more thermocycles by a control unit of the device is described in relation to FIG. 10 and is additionally incorporated into step 1204 of the method 1200.
  • the method 1200 includes emitting an excitation light toward the reaction chamber of the sample analysis cartridge.
  • step 1205 could include using one or more light sources of an optical analysis module to emit an excitation light toward a sample in the reaction chamber.
  • Step 1205 may further include adjusting various aspects of the optical analysis module using, e.g, a control unit of the device.
  • step 1105 may further include mechanically switching one or more filters or lenses optically coupled to the one or more light sources.
  • the method 1200 includes detecting an emission light from the reaction chamber to analyze the sample for the presence of the nucleic acid.
  • step 1106 could include detecting an emission light at an emission detector of an optical analysis module.
  • Step 1106 may further include adjusting various aspects of the optical analysis module using, e.g, mechanically switching a mechanically switchable optical filter or a lens optically coupled to the emission detector. Additional aspects pertaining to the emission of excitation light and the detection of emission light from the reaction chamber are described above in relation to FIG. 9 and such aspects may be incorporated into steps 1205 and 1206 of the method 1200
  • thermocycle an optical reading is taken during each thermocycle.
  • steps 1204, 1205, and 1206 may be performed simultaneously or in sequence.
  • performing a plurality of thermocycles could include repeating one or more of steps 1204, 1205, and 106 a plurality of times in sequence.
  • the method 1200 further includes determining the presence or absence, or amount of a nucleic acid based on the emission light.
  • the method 1200 could include determining the presence, absence, or amount based on, e.g, the quantity of emission light detected at the emission detector, a frequency of emission light detected at the emission detector, a quantity of emission light detected at the emissions detector at a particular time or over a particular period of time or series of readings by the optical analysis module, or based on some other aspect of the emission light. Other methods of optical detection are also anticipated.
  • FIGS. 13A-13B, FIG. 14, and FIG. 15 provide various graphs showing results from a sample analysis performed using a device and a sample analysis cartridge according to the present disclosure.
  • FIG. 13A provides an example of an optical reading of a positive sample (e.g. , a sample positive for a nucleic acid, such as a nucleic acid associated with S ARS- CoV-2). The experiment was conducted by spiking 1,250 copies per mL contrived SARS- CoV-2 into a nasal matrix derived from negative patient sample.
  • a positive sample e.g. , a sample positive for a nucleic acid, such as a nucleic acid associated with S ARS- CoV-2
  • the experiment was conducted by spiking 1,250 copies per mL contrived SARS- CoV-2 into a nasal matrix derived from negative patient sample.
  • FIG. 13B provides an example of an optical reading of a negative sample. The experiment was conducted by using a nasal swab from a negative patient in which the patient swabs five (5) times in each nostril followed by inserting the swab into the collection buffer and swirling for ten (10) seconds.
  • a portion of the sample is subsequently placed on the cartridge with a lyobead reagent, the cartridge is inserted into the device, and a sample analysis (e.g., RT-qPCR analysis) is run on the sample.
  • a sample analysis e.g., RT-qPCR analysis
  • FIG. 13B provides data of a negative sample as demonstrated by only HEX amplification (green, shown in the lower graphs), which represents amplification of human target gene RNase P, without amplification of FAM (blue, shown in the upper graphs).
  • FIG. 14 provides data showing results from optical readings of a sample containing decreasing amounts of reagents.
  • the first peak represents data from a sample containing the relatively greatest amount of reagent, while the second and third peak provides data from samples containing relatively lesser amounts of reagent.
  • increased reagent availability e.g, increased amount of reagent in the sample, or improved reagent/sample binding and other interactions caused by increased mixing of the reagents with the sample
  • FIG. 15 provides a series of graphs demonstrating the multiplex capabilities of the device.
  • Each of the graphs shows a series of optical readings performed by a device on various titrations of fluorescent dye that may be incorporated into fluorescent reporter probes for detecting nucleic acids in a sample.
  • the titration of the fluorescent dye is decreased over the series of readings shown in the graphs, resulting in the stairstep shape of the optical reading.
  • To selectively excite the various fluorescent dyes light is emitted toward the dyes in a wavelength range corresponding to the excitation range of a particular fluorescent dye.
  • FIG. 1 In the three graphs shown in FIG.
  • the uppermost graph shows the selective excitation of fluorescent dye Cai-Red 635 (orange)
  • the middle graphs shows the selective excitation of fluorescent dye Cai-Orange 560 (green)
  • the bottom graph shows the selective excitation of fluorescent dye Quaser-705 (red).
  • Each of the fluorescent dyes may be incorporated into a fluorescent reporter probe designed to selectively bind with a particular target nucleic acid in a sample to permit the optical detection or quantification of the target nucleic acid.
  • the optical system emits excitation light toward the sample and detects fluorescence emissions from the fluorescent probes bound to the nucleic acids.
  • the magnitude of a particular fluorescence signal in an optical reading correlates with the amount of a fluorescent reporter probe that has bound to a target nucleic acid, indicating the presence, absence, or amount of the nucleic acid in the sample.
  • nucleic acids can be detected or quantified in a sample in parallel by mixing the sample with fluorescent reporter probes configured to bind with a plurality of nucleic acids in the sample. Each of the plurality of nucleic acids can then be detected or quantified by selectively emitting light in a wavelength range corresponding to the excitation range of a particular fluorescent reporter probe in the sample, and then measuring the fluorescence emissions from the various fluorescent reporter probes in the sample.
  • FIG. 11 and FIG. 12 illustrate two exemplary methods, the steps of each method may be rearranged, reordered, removed, or modified without departing from the subject invention.
  • Reference to “about” a value or parameter herein includes (and describes) variations that are directed to that value or parameter per se. For example, description referring to “about X” includes description of “X.”

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Abstract

La présente invention concerne, selon certains aspects, un dispositif de PCR rapide amélioré et une cartouche d'analyse d'échantillon destinée à être utilisée avec celui-ci. Dans d'autres aspects, la présente invention concerne un procédé de chargement d'une cartouche d'analyse d'échantillon, un procédé d'analyse d'un échantillon pour déterminer la présence, l'absence ou la quantité d'un acide nucléique, et un système pour détecter la présence, l'absence ou la quantité d'un ou de plusieurs acides nucléiques à l'intérieur d'un échantillon
PCT/US2024/016765 2023-02-22 2024-02-21 Dispositifs de pcr rapide et cartouches d'échantillon destinées à être utilisées avec ceux-ci WO2024178146A1 (fr)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20120329144A1 (en) * 2010-01-04 2012-12-27 Keumcheol Kwak Sample analysis cartridge and sample analysis cartridge reader
US20180304260A1 (en) * 2017-04-21 2018-10-25 Mesa Biotech, Inc. Fluidic Test Cassette
US20190178787A1 (en) * 2009-12-07 2019-06-13 Meso Scale Technologies, Llc Assay Cartridges and Methods of Using the Same
US20190256890A1 (en) * 2014-05-21 2019-08-22 IntegenX, Inc. Fluidic cartridge with valve mechanism

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20190178787A1 (en) * 2009-12-07 2019-06-13 Meso Scale Technologies, Llc Assay Cartridges and Methods of Using the Same
US20120329144A1 (en) * 2010-01-04 2012-12-27 Keumcheol Kwak Sample analysis cartridge and sample analysis cartridge reader
US20190256890A1 (en) * 2014-05-21 2019-08-22 IntegenX, Inc. Fluidic cartridge with valve mechanism
US20180304260A1 (en) * 2017-04-21 2018-10-25 Mesa Biotech, Inc. Fluidic Test Cassette

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