Patent Application
20230078644
The present invention is directed to devices for and methods of isolating, concentrating, amplifying, and detecting nucleic acids in biological samples such as saliva, blood, or urine samples and, more particularly, to such devices or methods including solid-state membranes and microfluidic reaction chambers and methods of collecting and processing such biological samples.
A prior art isothermal nucleic acid amplification reactor with an integrated solid-state membrane is shown in U.S. Pat. No. 9,796,176 (“the '176 patent”). The '176 patent discloses a microfluidic cassette that integrates nucleic acid capture, concentration, purification, isothermal amplification, and real-time fluorescence detection into a single reaction chamber. As shown in
One drawback associated with the above-described prior art is that the solid-state membrane is fixedly mounted within a fixed fluid conduit to the reaction chamber, and thus the biological sample, lysate mixture and wash buffers are first introduced into the reaction chamber, and then wicked through the membrane and absorbed by the absorbent sink pad. As a result, the volumes of the biological sample, lysate mixture and wash buffers are limited by the capacity of the reaction chamber and absorbent sink pad. As indicated above, the total volume of the reaction chamber is about 20 μl. This limits the ability to add higher sample volumes in order to increase the ability to detect targets that may be in small or low concentrations in the original sample. The small volume limits the amount of sample which can be tested and decreases the ability of the test to detect dilute or low concentration nucleic acid targets. Yet another potential drawback is that the reaction chamber may contain dry reagents encapsulated in a low-melting point paraffin for release during the heating and nucleic acid amplification step. Because the lysed sample and the wash buffers must all flow through the reaction chamber, the lyophilized reagents must be sealed in paraffin to prevent premature hydrolyzation and the loss of reagent before the reaction. The paraffin may upset the purity of the reagents and reduce the sensitivity of the assay. In addition, the encapsulated reagents contained within the reaction chamber may further limit the available volume of the reaction chamber for the above-described fluids required for the preceding steps. As a result, smaller volumes of biological samples may be passed across the membrane, and lesser amounts of target nucleic acids may be captured, purified and amplified, than desired. Yet another drawback is that the capture, purification and amplification of lesser amounts of targeted nucleic acids than desired, may lead to less sensitive detection of, and testing for, such targeted nucleic acids. In other words, it would be desirable for a device or method to allow for greater volumes of biological samples to be passed across such a membrane, to in turn allow for the capture of greater amounts of targeted nucleic acids to thereby improve the ability to detect such nucleic acids. It would also be desirable to have a system that does not require paraffin or like sealant to prevent hydrolyzation.
Some in vitro diagnostic molecular biology devices contain blisters filled with liquid reagents. These blisters are usually formed with a semi-stiff cold formed top dome and a more flexible lidding film that is added and heat sealed after filling. The blister must be compressed with an actuator that deforms the top film and in turn expands the flexible lidding film so that is comes into contact with a rupture pin. The force required to perform this action can be greater than desired, especially in the case of a human or manually-actuated blister. The top blister is then continued to be compressed and further deformed until all or most of the liquid is expelled into the microfluidic device.
As indicated above, a solid-state, glass fiber membrane is commonly used to capture RNA from a sample solution. In that process ethanol is used to promote RNA retention to the membrane and water is used to release the RNA from the membrane and deliver the captured RNA to a LAMP reaction chamber where amplification occurs in a process known as elution. For amplification to occur, it is critical that substantially no ethanol is delivered to the amplification chamber. It is challenging to do that because some ethanol is retained in the solid-state membrane and released into the water during elution. This is referred to as ethanol poisoning. One way to eliminate ethanol poisoning is to first dry the ethanol from the membrane before elution. However, in a lab on chip system, it can be difficult or challenging to dry the membrane before elution.
There are two primary means of making visual detection of RNA amplification. One is color change which means that the sample's spectral absorption changes, and accordingly, the color of light reflected by the sample also changes. In a simplified example, if a sample which reflects green light changes to absorb more green light, the sample will appear less green to an observer. When the change in absorption is within the visual spectrum, a human observer may be able to detect the change. Sometimes an illumination light is used such that the incoming light is filtered by the medium and then directed to the observer. Another means of visual detection is by fluorescence. In this case, the sample is exposed to light that stimulates fluorescence and the observer selectively observes the fluorescence. When the stimulating light is ultraviolet, human eyes can perform the observation, as the ultraviolet light will not interfere. However, if the stimulating light and the fluorescent light have sufficiently similar wavelengths, human eyes are not able to detect the presence of the fluorescent light with accuracy, and a tool is required to aid the human filter out the stimulating light. One means of suppressing the observation of the stimulating light is by the use of sharp cutoff or passband filters, which selectively allow only certain wavelengths of light to pass through. A passband filter could thus allow the fluorescent light to pass through while blocking the stimulating light, even if the wavelengths are too close for the human eye to discern the difference. However, passband filters can be expensive, particularly when the two wavelengths are closely matched.
There are several known methods for the collection of human saliva. One method is referred to as a drool method where the patient spits directly into a cup or collection vessel. After the saliva is collected, a transfer device, such as a pipette, is required to meter out saliva from the collection vessel. Then, another mechanism is required to push the transferred sample into a lysis reservoir, and yet another mechanism may be required to seal the lysis reservoir after the sample is transferred therein. Another method uses an absorbent pad to collect the saliva. The saliva sample is transferred from the pad to a preservation buffer by allowing the absorbent pad to soak in the preservation buffer for several minutes. After the saliva is transferred to the preservation buffer, the same sequence of steps as mentioned above may be required to transfer the sample into a lysis reservoir and to seal the reservoir.
The foregoing steps relating to the preparation of saliva samples are a potential source of testing error, and require a trained operator or relatively complex processes to mitigate risks related to cross-contamination among samples and to ensure containment of harmful pathogens that may be contained within the samples. In addition, such processes of preparing biological samples involve interaction with hazardous chemical mixtures, including lysing agents which break down cell membranes. Due to these limitations, prior art devices are generally not suitable for use by the general public, limiting their utility and the market for such devices.
It is an object of the present invention, and/or of the currently preferred embodiments thereof, to overcome one or more of the above-described drawbacks and/or disadvantages of the prior art.
In accordance with a first aspect, the present invention is directed to a device for detecting nucleic acids in a biological sample. The device comprises a sample port for receiving therein a biological sample; a solid-state membrane configured to capture nucleic acids in the biological sample passed across the membrane; a sample conduit in fluid communication between the sample port and the solid-state membrane; a lysis station in fluid communication with the sample conduit and including a lysis agent therein; a wash station in fluid communication with at least one of the sample conduit or the solid-state membrane and including a wash solution therein; an elution station in fluid communication with at least one of the sample conduit or the solid-state membrane and including an eluent therein; a waste chamber located downstream of the solid-state membrane; and one or more reaction chambers located downstream of the solid-state membrane. The sample port, lysis station and sample conduit are configured to mix the sample and lysis agent to form a sample-lysis mixture, pass the sample-lysis mixture across the solid-state membrane to capture nucleic acids in the biological sample therein, and receive the remainder of the sample-lysis mixture in the waste chamber. The wash station is configured to introduce the wash solution into at least one of the sample conduit or solid-state membrane following the sample-lysis mixture to purify nucleic acids captured on the solid-state membrane. The wash solution from the solid-state membrane is received in the waste chamber. The elution station is configured to pass the eluent across the solid-state membrane, elute captured nucleic acids from the solid-state membrane, and pass the captured nucleic acids into one or more reaction chambers configured for amplifying and detecting the captured nucleic acids therein.
Some embodiments of the present invention further comprise (i) a lysis leg extending in fluid communication between the lysis station and the sample conduit and configured to direct a flow of the lysis agent from the lysis station into the sample conduit, and (ii) a wash leg extending in fluid communication between the wash station and the sample conduit at a point upstream relative to the lysis leg and configured to direct a flow of the wash solution from the wash station into the sample conduit behind the sample-lysis mixture. In some such embodiments, (i) the wash leg is in fluid communication with the sample conduit at a sample-wash junction located adjacent to the sample port and configured to allow a substantial portion of the sample to flow into the sample conduit downstream of the sample-wash junction prior to introducing the wash solution through the wash leg and into the sample conduit; and (ii) the lysis leg is in fluid communication with the sample conduit at a sample-lysis junction located downstream of the sample-wash junction and configured to allow the lysis agent to mix with the sample and form the sample-lysis mixture and the wash solution to flow into the sample conduit behind or upstream of the sample-lysis mixture.
Some embodiments of the present invention further comprise a static mixer in fluid communication between the sample-lysis junction and the solid-state membrane to mix the sample and lysis agent and form a sample-lysis mixture prior to passage across the solid-state membrane. In some such embodiments, the static mixer is defined by a plurality of axially-spaced recesses or grooves formed in the sample conduit. In some embodiments, the axially-spaced recesses or grooves are formed in a bottom wall of the sample conduit and are shaped such that a first part of each groove imparts a first rotational component to a fluid flowing therethrough in a first direction, and a second part of each groove imparts a second rotational component to the fluid flowing therethrough in a second direction that is different than the first direction, to thereby facilitate mixing the sample and lysis agent into the sample-lysis mixture. In some such embodiments, the first part of each recess or groove is oriented approximately at an acute angle relative to the second part of each recess or groove. In some embodiments, the first part of each recess or groove is shorter than then second part of each recess or groove. In some such embodiments, the plurality of axially-spaced recesses or grooves define an approximate herringbone shape.
In some embodiments of the present invention the lysis station includes a sealed lysis agent chamber containing the lysis agent, and a lysis actuator movable between a non-actuated position and an actuated position. In the actuated position the lysis agent is released from the lysis chamber through the lysis leg and into the sample conduit where the lysis agent is mixed with the sample into the sample-lysis mixture. In some such embodiments, the wash station includes a sealed wash chamber containing the wash solution, and a wash actuator movable between a non-actuated position and an actuated position. In the actuated position the wash solution is released from the wash chamber through the wash leg and into the sample conduit behind or upstream of the sample-lysis mixture. In some such embodiments, the lysis actuator is manually actuated and movable from the non-actuated position to the actuated position, and the lysis chamber includes a frangible or breakable wall that is breakable by movement of the lysis actuator in the actuated position to release the lysis agent from the lysis chamber into the lysis leg. In some such embodiments, the wash actuator is manually actuated and movable from the non-actuated position to the actuated position, and the wash chamber includes a frangible or breakable wall that is breakable by movement of the wash actuator in the actuated position to release the wash solution from the wash chamber into the wash leg.
In some embodiments of the preset invention the lysis actuator is a lysis plunger and the lysis chamber includes a lysis blister containing the lysis agent therein. Movement of the lysis plunger from the non-actuated to the actuated position causes the lysis plunger to break the lysis blister and release the lysis agent into the lysis leg. In some such embodiments, the wash actuator is a wash plunger and the wash chamber includes a wash blister containing the wash solution therein. Movement of the wash plunger from the non-actuated to the actuated position causes the wash plunger to break the wash blister and release the wash solution into the wash leg.
Some embodiments of the present invention further comprise a second wash station in fluid communication with the sample conduit that includes a second wash solution therein. The second wash station is configured to introduce the second wash solution into the sample conduit following the other wash solution and to pass the second wash solution across the solid-state membrane to purify nucleic acids captured therein. The second wash solution passes across the solid-state membrane and is received in the waste chamber.
In some embodiments of the present invention, the second wash station includes a sealed second wash chamber containing the second wash solution. A second wash leg is in fluid communication between the second wash station and the sample conduit downstream of the other wash leg and is configured to direct a flow of the second wash solution from the second wash station into the sample conduit. A second wash actuator is movable between a non-actuated position and an actuated position. In the actuated position the second wash solution is released from the second wash chamber through the second wash leg and into the sample conduit, is passed across the solid-state membrane to purify nucleic acids captured therein, and is received in the waste chamber. In some embodiments of the present invention, the second wash actuator is manually actuated and movable from the non-actuated position to the actuated position, and the second wash chamber includes a frangible or breakable wall that is breakable by movement of the second wash actuator in the actuated position to release the second wash solution from the second wash chamber into the second wash leg. In some such embodiments, the second wash actuator is a plunger and the second wash chamber includes a second wash blister containing the second wash solution therein. Movement of the plunger from the non-actuated position to the actuated position causes the plunger to break the second wash blister and release the second wash solution into the second wash leg.
In some embodiments of the present invention, the elution station includes a sealed eluent chamber containing the eluent, an elution leg extending in fluid communication between the elution station and the solid-state membrane, and an elution actuator movable between a non-actuated position and an actuated position. In the actuated position the eluent is released from the eluent chamber through the elution leg and across the solid-state membrane to elute captured nucleic acids from the solid-state membrane and pass the captured nucleic acids into the reaction chamber. In some such embodiments, the elution actuator is manually actuated and movable from the non-actuated position to the actuated position, and the eluent chamber includes a frangible or breakable wall that is breakable by movement of the elution actuator in the actuated position to release the eluent from the eluent chamber into the elution leg. In some embodiments, the elution actuator is a plunger and the eluent chamber includes an eluent blister containing the eluent therein, and movement of the plunger from the non-actuated to the actuated position causes the plunger to break the frangible or breakable wall and release the eluent into the elution leg.
Some embodiments of the present invention further comprise a lysis-wash actuator fixedly connected to the lysis plunger and to the wash plunger and manually depressible to move the lysis plunger and the wash plunger from the non-actuated position to the actuated position. Some such embodiments further comprise a plunger mount connected to the lysis-wash actuator, a plunger spring defining a plunger spring force and mounted between the lysis-wash actuator and the plunger mount, and a latch. Upon manually moving the lysis-wash actuator from a non-actuated position to a first actuated position, the latch secures the lysis-wash actuator in the first actuated position where the lysis plunger engages the lysis blister and the wash plunger engages the wash blister, and the plunger spring drives the plunger mount from the first actuated position to a second actuated position. The lysis plunger and wash plunger break the frangible or breakable walls of the lysis agent chamber and the wash solution chamber, respectively, and the plunger spring drives the lysis plunger and the wash plunger into the respective chambers to release the lysis agent and the wash solution into the lysis leg and wash leg, respectively. In some such embodiments, the latch includes a locking tab and a latch spring coupled to the locking tab. During movement of the lysis-wash actuator between the non-actuated positon and the first actuated position, the latch spring urges the locking tab into contact with the lysis-wash actuator but allows relative movement thereof. In the first actuated position, the latch spring urges the locking tab into a locked position securing the lysis-wash actuator in the first actuated position.
Some embodiments of the present invention further comprise a base station for receiving the device. The base station includes a lock-release bar moveable between (i) a locked position where it prevents movement of the lysis-wash actuator and (ii) a release position where it allows movement of the lysis-wash actuator into the first actuated position. The device is movable relative to the base station between a non-operational position and an operational position. During movement of the device between the non-operational position and the operational position, the lock-release bar is moved from the locked position to the release position to thereby allow the lysis-wash actuator to move into the first actuated position when the device is located in the operational position. Some embodiments further comprise a lock-release spring coupled to the lock release bar and urging the lock release bar in a direction from the release position to the locked position. Upon removal of the device from the base station, the lock-release spring drives the lock-release bar from the release positon to the locked position, and the lock-release bar drives the lysis-wash actuator upwardly from the first actuated positon to the non-actuated position.
Some embodiments of the present invention further comprise a base station for receiving the device. The base station includes a ramp, a sled mounted on the ramp, and a heater mounted on the sled. Upon receiving the device into the base station, the sled is connectable to the device and movable therewith between a non-operational position and an operational position. During movement between the non-operational position and the operational position, the heater is moved from a non-operational positon out of contact with the device to an operational position in contact with the device and adjacent to the reaction chamber for incubating the captured nucleic acids within the reaction chamber. In some embodiments, the reaction chamber(s) are preheated by activating the heater prior to filling the reaction chamber(s), such as upon movement of the heater into the operational position. Some embodiments of the present invention further comprise a heater spring mounted between the sled and the heater and urging the heater into contact with the device in the operational position. In some embodiments, the sled includes a tang on a distal end thereof, and the device includes a connecting recess on a distal end thereof. Upon inserting the device into the base station, the tang is partially received in the connecting recess. During movement between the non-operational position and the operational position, the tang is more fully received in the connecting recess. In some embodiments, the base station includes a spring-biased latch engageable with the connecting recess in the operational position to releasably retain the device in the operational positon.
Some embodiments of the present invention further comprise a waste chamber vent in fluid communication between the waste chamber and ambient atmosphere. The waste chamber vent defines an open condition and a closed condition. In the open condition fluid passing across the solid-state membrane is received within the waste chamber. In the closed condition fluid passing across the solid-state member is prevented from passing into the waste chamber. In some such embodiments, during passage of the sample-lysis mixture and wash solution across the solid-state membrane, the waste chamber vent is in the open condition and the sample-lysis mixture and the wash solution passing across the solid-state membrane flow into the waste chamber and are prevented from flowing into the reaction chamber.
In some embodiments of the present invention, the elution station includes a first sealed eluent chamber containing a first eluent; a first elution leg in fluid communication between the first elution station and the solid-state membrane; a second sealed eluent chamber containing a second eluent; and a second elution leg in fluid communication between the second elution station and the solid-state membrane. An elution actuator is movable between a non-actuated position and an actuated position. In the actuated position the first and second eluents are released from the first and second eluent chambers and into the first and second elution legs. In some such embodiments, the elution actuator substantially simultaneously releases the first and second eluents from the first and second eluent chambers, and the second elution leg is longer than the first elution leg to thereby allow the first eluent to pass across the solid-state membrane prior to passage of the second eluent across the solid-state membrane.
In some embodiments of the present invention, the wash solution and/or lysis agent leaves an evaporative contaminant on the solid-state membrane after passage therethrough. The first elution leg contains a volume of air or other gas therein such that upon releasing the first eluent into the first elution leg, the volume of air or other gas in the first elution leg is passed across the solid-state membrane and substantially evaporates the evaporative contaminant and thereby prevents contamination of the first eluent and captured nucleic acids received within the reaction chamber. In some such embodiments, the evaporative contaminant is ethanol, and the volume of air or other gas in the first elution leg is sufficient to substantially evaporate the ethanol in and about the solid-state membrane prior to passage of the first eluent across the membrane to thereby prevent ethanol poisoning of the reaction chamber.
In some embodiments of the present invention, the elution actuator is manually actuated and movable from the non-actuated position to the actuated position. Each of the first and second eluent chambers includes a frangible or breakable wall that is breakable by movement of the elution actuator in the actuated position to release the first and second eluents into the first and second elution legs, respectively. In some such embodiments, movement of the elution actuator into the actuated position partially dispenses the first and second eluents into the first and second elution legs, respectively. During such partial dispensing, the waste chamber vent is in the open condition to thereby allow any wash solution and/or air with evaporative contaminants in or about the solid-state membrane to flow into the waste chamber and not into the reaction chamber. After partially dispensing the first and second eluents and flowing any remaining wash solution in or about the solid-state membrane into the waste chamber, the waste vent is in the closed condition to thereby direct the first eluent and captured nucleic acids from the solid-state membrane into the reaction chamber.
Some embodiments of the present invention comprise a waste vent seal movable between (i) an open position allowing fluid to flow out of the waste chamber vent and thereby allow fluid to flow into the waste chamber, and (ii) a closed position sealing the vent and thereby preventing fluid from flowing into the waste chamber. In some such embodiments, the waste vent seal is mounted on the elution actuator. In some embodiments, the elution actuator includes a manually-engageable portion, a plunger mount, a first elution plunger engageable with the first eluent chamber, a second elution plunger engageable with the second eluent chamber, a plunger spring defining a plunger spring force and mounted between the manually-engageable portion and the plunger mount, and a latch. Upon manually moving the manually-engageable portion from a non-actuated position to a first actuated position, the latch secures the manually-engageable portion in the first actuated position where the first elution plunger partially dispenses the first eluent chamber and the second elution plunger partially dispenses the second eluent chamber. Then, the plunger spring drives the plunger mount from the first actuated position to a second actuated position under the plunger spring force to further dispense the first and second eluents from the first and second eluent chambers, respectively. Some embodiments further comprise a waste vent seal spring urging the waste vent seal in a direction from the open position to the closed position. In some such embodiments, the waste vent seal is mounted on the plunger mount, and the waste vent seal spring is mounted between the waste vent seal and the plunger mount. Upon movement of the manually-engageable portion into the first actuated position, the plunger spring and the waste vent seal spring urge the waste vent seal into the closed position to thereby seal the waste chamber vent.
Some embodiments of the present invention further comprise a reaction chamber valve in fluid communication between the solid-state membrane and the reaction chamber. The reaction chamber valve (i) is closed to prevent fluid flow into the reaction chamber when a fluid pressure between the solid-state membrane and the reaction chamber valve is below a valve-opening pressure, or (ii) is open to allow fluid flow into the reaction chamber when the fluid pressure between the solid-state membrane and the reaction chamber valve is above the valve-opening pressure. In some embodiments, movement of the waste chamber vent seal into the closed position causes the fluid pressure between the solid-state membrane and reaction chamber valve to exceed the valve-opening pressure and thereby allow fluid flow from the solid-state membrane into the reaction chamber.
Some embodiments of the present invention further comprise a first reaction chamber and a first reaction chamber valve in fluid communication between the solid-state membrane and the first reaction chamber. The first reaction chamber valve defines a first valve-opening pressure. The device further comprises a second reaction chamber and a second reaction chamber valve in fluid communication between the solid-state membrane and the second reaction chamber. The second reaction chamber valve defines a second valve-opening pressure that is greater than the first valve opening pressure, to thereby cause the eluted nucleic acids to first flow into the first reaction chamber and then flow into the second reaction chamber. In some such embodiments, each reaction chamber valve is a Laplace, Laplace-type or burst valve.
Some embodiments of the present invention further comprise a first reaction chamber vent in fluid communication between the first reaction chamber and ambient atmosphere. The first reaction chamber vent allows gas but substantially prevents liquid flow therethrough, to thereby flow eluted nucleic acids into the second reaction chamber after filling the first reaction chamber. Some embodiments further comprise a second reaction chamber vent in fluid communication between the second reaction chamber and ambient atmosphere. The second reaction chamber vent allows gas but substantially prevents liquid flow therethrough to thereby prevent liquid from flowing into the second reaction chamber upon filling the second reaction chamber with liquid. In some such embodiments, each of the first and second reaction chamber vents includes a hydrophobic vent membrane that allows gas but substantially prevents liquid flow therethrough. Some embodiments further comprise (i) a first reconstitution chamber in fluid communication between the first reaction chamber valve and the first reaction chamber, and (ii) a second reconstitution chamber in fluid communication between the second reaction chamber valve and the second reaction chamber.
Some embodiments of the present invention comprise a plurality of actuators. Each actuator is manually movable from a non-actuated position to an actuated position. Each of the lysis station, wash station and elution station includes a sealed chamber including a frangible or breakable wall and containing therein the lysis agent, wash solution or eluent, respectively. Upon movement of each actuator from the non-actuated to the actuated position, one or more of the frangible or breakable walls is broken to release at least one of the lysis agent, wash solution and/or eluent from its respective sealed chamber. In some embodiments, one or more of the actuators includes a plunger engageable with a respective sealed chamber. The plunger defines an axial direction of movement and includes first and second chamber-engaging surfaces. The first chamber-engaging surface defines a first width or diameter. The second chamber-engaging surface extends outwardly relative to the first chamber-engaging surface in the axial direction of movement and defines a second width or diameter that is less than the first width or diameter. Movement of the actuator from the non-actuated to the actuated positon moves the second chamber-engaging surface into engagement with the respective sealed chamber and breaks the frangible or breakable wall. In some embodiments the first chamber-engaging surface defines a first diameter, and the second chamber-engaging surface defines a second diameter, wherein the second diameter is less than the first diameter. In some such embodiments, the second diameter is at least about two times and preferably about three times less than the first diameter. In some embodiments, the first chamber-engaging surface is defined by at least one first radius of curvature, the second chamber-engaging surface is defined by at least one second radius of curvature, and the least one first radius of curvature is greater than the at least one second radius of curvature. In some embodiments, each of the lysis station, wash station and elution station includes a piercing member engageable with the respective frangible or breakable wall upon moving a respective actuator from the non-actuated position to the actuated position. The piercing member facilitates breaking the wall, and each second chamber-engaging surface includes a recess therein for receiving the piercing member at least partially therein. In some embodiments, each of the lysis station, wash station and elution station includes a recessed surface located below the respective sealed chamber for receiving the lysis agent, wash solution or eluent upon breaking the respective wall. An outlet is located at approximately the lowest point of the recessed surface and is in fluid communication with the lysis leg, wash leg or elution leg, respectively. In some embodiments, each recessed surface defines a plurality of elongated grooves therein in fluid communication with the outlet and angularly spaced relative to each other to facilitate the flow of fluid into the outlet.
In some embodiments of the present invention, the solid-state membrane includes an inlet side and an outlet side. The device further comprises a membrane inlet located on the inlet side of the solid-state membrane and in fluid communication between the sample conduit and elution station, on the one hand, and the solid-state membrane, on the other hand. A membrane outlet is located on the outlet side of the solid-state membrane in fluid communication between the solid-state membrane and the waste chamber or reaction chamber. The membrane inlet defines a plurality of inlet fluid channels configured to facilitate a flow of fluid across the inlet side of the solid-state membrane, and the membrane outlet includes a plurality of fluid outlet channels therein configured to facilitate a flow of fluid across the outlet side of the solid-state membrane. In some such embodiments, the inlet fluid channels include a plurality of radially-extending inlet fluid channels angularly spaced relative to each other, and the outlet fluid channels include a plurality of radially-extending outlet fluid channels angularly spaced relative to each other. In some embodiments, the inlet fluid channels include at least one annularly extending inlet fluid channel intersecting at least a plurality of the radially-extending inlet fluid channels, and the outlet fluid channels include at least one annularly extending outlet fluid channel intersecting at least a plurality of the radially-extending outlet fluid channels.
Some embodiments of the present invention further comprise a body including the at least one reaction chamber therein. At least a portion of the body including the reaction chamber(s) is substantially transparent, and includes a substantially transparent top surface extending over the reaction chamber(s) and two substantially transparent side surfaces extending downwardly from the top surface along opposite sides of the reaction chamber(s) relative to each other. The reaction chamber(s) is visually observable in a viewing direction through the substantially transparent top surface. A stimulating light source is located adjacent to a substantially transparent side surface configured to transmit stimulating light through the side surface and reaction chamber(s) in a direction substantially lateral to the viewing direction. In some embodiments, the index of refraction of the substantially transparent body and the index of refraction of the fluid in the reaction chamber(s) are configured to facilitate the passage of the stimulating light from the body into the reaction chamber(s) to generate fluorescing light in the reaction chamber. The fluorescing light is emitted in substantially all directions and is observable in the viewing direction through the top surface of the body. Preferably, there is an observable difference to the human eye between the stimulating light and the fluorescing light to facilitate the ability of an observer to view the fluorescing light and distinguish it from any observed stimulating light. In some embodiments, the stimulating light defines a first wavelength, and the fluorescing light defines a second wavelength, wherein the first wavelength is less than the second wavelength. In some such embodiments, the first wavelength is within the range of about 425 nm to about 550 nm. In some embodiments, the first wavelength is about 470 nm and the second wavelength is about 510 nm. In some embodiments of the invention, the top surface of the body is substantially smooth or polished and the side surfaces of the body are substantially smooth or polished to facilitate maintaining the stimulating light within the substantially transparent body.
In some embodiments of the invention, the body is substantially transparent throughout. The body defines integrally molded therein the sample conduit, a lysis leg extending in fluid communication between the lysis station and the sample conduit and configured to direct a flow of the lysis agent from the lysis station into the sample conduit, a wash leg extending in fluid communication between the wash station and the sample conduit at a point upstream of the lysis leg and configured to direct a flow of the wash solution from the wash station into the sample conduit behind the sample-lysis mixture, an elution leg extending in fluid communication between the elution station and the solid-state membrane, the reaction chamber and the waste chamber.
Some embodiments of the present invention further comprise a saliva collection swab for collecting saliva thereon and receivable within the sample port for introducing the saliva directly into the sample port and sample conduit for mixture therein with the lysis agent. In some such embodiments, the saliva collection swab includes a plunger depressible against the saliva collection swab within the sample port to release saliva from the collection swab into the sample port and sample conduit. In some such embodiments, at least one of the saliva collection swab or the sample port includes a locking tab, and the other of the saliva collection swab or sample port includes a corresponding locking recess or aperture configured to receive the locking tab and retain the swab within the sample port with the plunger depressed against the swab to facilitate release of saliva therefrom and into the sample port.
Some embodiments of the present invention further comprise a reaction chamber vent in fluid communication between the reaction chamber and ambient atmosphere. The reaction chamber vent defines a venting length extending between the reaction chamber and ambient atmosphere and a venting cross-sectional area. The venting cross-sectional area is sufficiently small compared to the venting length to create a saturation gradient between the reaction chamber and ambient atmosphere to slow the evaporation of liquid from, and prevent the entry of atmospheric air into, the reaction chamber.
Some embodiments of the present invention further comprise a body defining therein the sample conduit, a lysis leg extending in fluid communication between the lysis station and the sample conduit and configured to direct a flow of the lysis agent from the lysis station into the sample conduit, a wash leg extending in fluid communication between the wash station and the sample conduit upstream of the lysis leg and configured to direct a flow of the wash solution from the wash station into the sample conduit behind the sample-lysis mixture, an elution leg extending in fluid communication between the elution station and the solid-state membrane, and a viewing window overlying the reaction chamber and allowing visual observation of the reaction chamber therethrough in a viewing direction. A heater is mounted to the body adjacent to the reaction chamber and configured to heat the reaction chamber. A stimulating light source is configured to transmit stimulating light into the reaction chamber in a direction lateral to the viewing direction. A power source, such as a battery, a plug, or a receptacle, is connected to the heater and light source and configured to provide power thereto.
In accordance with another aspect, the present invention is directed to a method for detecting nucleic acids in a biological sample. The method comprises:
receiving a biological sample through a sample port and into a sample conduit in fluid communication between the sample port and a solid-state membrane for capturing nucleic acids in the biological sample and amplifying and detecting the captured nucleic acids therein in at least one reaction chamber;
introducing a lysing agent into the sample conduit, mixing the lysing agent with the sample to form a sample-lysis mixture, passing the sample-lysis mixture across the solid-state membrane and capturing nucleic acids in the biological sample therein, preventing the flow of the sample-lysis mixture that passes across the solid-state membrane into the at least one reaction chamber, and receiving the remainder of the sample-lysis mixture that passes across the solid-state membrane in a waste chamber;
introducing a wash solution into at least one of the sample conduit or solid-state membrane following the sample-lysis mixture, passing the wash solution across the solid-state membrane and purifying nucleic acids captured from the sample-lysis mixture therein, preventing the flow of the wash solution into the reaction chamber, and receiving the wash solution that passes through the solid-state membrane in the waste chamber; and
introducing an eluent across the solid-state membrane and eluting captured nucleic acids from the solid-state membrane, substantially preventing the captured nucleic acids from flowing into the waste chamber, directing the captured nucleic acids into the at least one reaction chamber, and amplifying and detecting the captured nucleic acids in the at least one reaction chamber.
Some embodiments of the present invention further comprise introducing a second wash solution into the sample conduit following the other wash solution and passing the second wash solution across the solid-state membrane and purifying nucleic acids captured therein, preventing the flow of the second wash solution into the reaction chamber, and receiving the second wash solution that passes through the solid-state membrane in the waste chamber.
Some embodiments of the present invention further comprise closing a vent to the waste chamber after receiving the lysis agent and wash solution therein, and opening an inlet valve to the reaction chamber(s) for directing the captured nucleic acids from the solid-state membrane therein.
In some embodiments, the wash solution and/or lysis agent leaves an evaporative contaminant on the solid-state membrane after passage therethrough, and the method further comprises flowing air or other gas across the solid-state membrane prior to passage of the eluent therethrough and substantially evaporating the evaporative contaminant and preventing contamination of the eluent and captured nucleic acids.
In some embodiments each reaction chamber defines a viewing direction for visually observing an interior thereof, and the method further comprises directing stimulating light into the reaction chamber lateral to the viewing direction, generating fluorescing light in substantially all directions and allowing for visual observation in the viewing direction a greater amount of the fluorescing light as compared to the stimulating light.
One advantage of the invention, and/or of embodiments thereof, is that the sample-lysis mixture and wash solution or buffers are not introduced into the reaction chamber. Rather, they bypass the reaction chamber and are introduced into the separate waste chamber. As a result, the volumes of the biological sample, lysate mixture and wash buffers are not limited by the capacity of the reaction chamber, as in the above-described prior art. Yet another advantage is that the need to encapsulate the dry reagents in a low-melting point paraffin and the associated drawbacks thereof can be avoided. As a result, the device and method of the invention can allow for greater volumes of biological samples to be passed across the membrane, and greater amounts of targeted nucleic acids to be captured, purified and amplified. This can, in turn, lead to a more sensitive detection of, and testing for, targeted nucleic acids.
Yet another advantage of the invention, and/or of embodiments thereof, is that less force may be required to depress the blisters and rupture the flexible lidding film or other breakable or frangible walls than in the above-described prior art. For example, the manually-engageable actuators or plungers may include the first and second chamber-engaging surfaces, where the second chamber-engaging surface defines a smaller width or diameter than the first chamber-engaging surface. These features may take the form of a “bump on a bump” configuration. A significant advantage is that these features can reduce the manual force required to depress the blisters and rupture the films or other frangible or breakable walls in order to release the liquids therefrom, as compared to the above-described prior art.
A still further advantage of the invention, and/or of embodiments thereof, is that the elution leg of the elution chamber can contain a sufficient volume or air or other gas that is passed over the solid-state membrane prior to passage of the eluent therethrough. As a result, substantially all residual ethanol can be evaporated by the air or other gas and the ethanol poisoning encountered in the above-described prior art can be avoided. Yet another advantage is that this configuration can be implemented in a lab on chip system or device.
A still further advantage of the invention, and/or of embodiments thereof, is that the use of sharp cutoff or passband filters to suppress the observation of the stimulating light, and their associated costs, can be avoided. Rather, the stimulating light may be transmitted through the substantially transparent body in a direction substantially lateral or perpendicular to the direction of viewing the reaction chamber(s), thus geometrically separating the different lights and facilitating the ability to visually observe only, or primarily, the fluorescing light.
Yet another advantage of the invention, and/or of embodiments thereof, is that the saliva or other biological sample is inserted into the sample port to seal the sample port and deposit the saliva or other biological sample directly into the sample conduit where it is mixed with a lysing agent prior to passage across the solid-state membrane. As a result, the relatively complex processing of samples, and the associated risks and disadvantages thereof, as encountered in the above-described prior art can be avoided.
Still another advantage of the invention, and/or of embodiments thereof, is that preheating the reaction chamber or chambers can cause the reaction to occur more quickly and/or and reduce the time required for the reaction to complete. As a result, in a clinical setting, the number of tests that may be performed in a given time period can be increased and, in any setting, the wait time for a patient or user to receive results can be reduced.
Other advantages of the present invention, and/or of the embodiments thereof, will become more readily apparent in view of the following detailed description and accompanying drawings.
In
The device 10 is used by placing a sample, such as saliva, into the sample collection device 14, inserting the sample collection device 14 into the body 12, and inserting the body 12 into the base station 16 before pressing a first button 18, a second button 20 and a third button 22. After waiting a period of time for a reaction to complete, the results will be visible in the reaction chamber 24 of the body 12, which can be seen through a reaction chamber window 26 in the base station 16. In at least some embodiments of the device, a first indicator 28 will indicate when to press the first button 18, a second indicator 30 will indicate when to press the second button 20, and a third indicator 32 will indicate when to press the third button 22. These indicators may be LEDs which illuminate to indicate a button should be pressed, other light sources, or other devices capable of changing color or brightness. A reaction complete indicator 34 may indicate when the reaction has completed, which in some embodiments will be approximately twenty minutes after the third button 22 is depressed (view “7” of
The cartridge or body 12 comprises a sample port 36, multiple microfluidic conduits, multiple stations with associated blisters 49 and manually-engageable actuators 156, a solid-state membrane 78, a plurality of reconstitution chambers 48 and associated reaction chambers 24 in fluid communication therewith, and a waste chamber or sump 80. The illustrated embodiment includes five stations (
As described further below, the sample port 36, lysis station “2” and sample conduit 35 are configured to mix the sample 60 and lysis agent contained within a lysis buffer 70 to form a sample-lysis mixture 76, pass the sample-lysis mixture 76 across the solid-state membrane 78 to capture nucleic acids in the biological sample 60 therein, and receive the remainder of the sample-lysis mixture 76 in the waste chamber 80. The first wash station “1” is configured to introduce the first wash solution 66 into the sample conduit 35 following the sample-lysis mixture 76, pass the first wash solution 66 across the solid-state membrane 78 to purify nucleic acids captured therein, and receive the first wash solution 66 from the solid-state membrane 78 in the waste chamber 80. The second wash station “3” is configured to introduce the second wash solution 82 into the sample conduit 35 following the first wash solution 66, pass the second wash solution 82 across the solid-state membrane 78 to purify nucleic acids captured therein, and receive the second wash solution 82 from the solid-state membrane 78 in the waste chamber 80. The elution station “5” is configured to pass the eluent 86 across the solid-state membrane 78, elute captured nucleic acids from the solid-state membrane 78, and pass the captured nucleic acids initially into the first reconstitution chamber 48c and first reaction chamber 24c for amplifying and detecting the captured nucleic acids, and then into the second reconstitution chamber 48b and second reaction chamber 24b for amplifying and detecting the captured nucleic acids.
Each of the first wash blister 40, second wash blister 42, elution blister 44, and negative control blister 46 contain respective liquids as indicated above. When sufficient pressure is applied to the top 144 of each blister 49, the frangible or breakable wall of the blister bottom 146 ruptures, and the liquid therein enters the respective microfluidic conduits of the body 12, pushing the sample 60 along the sample conduit 35 and eventually pushing the eluted nucleic acids into one or more reaction chambers 24. In some embodiments of the device, there are three or more reaction chambers comprising a negative control reaction chamber 24a, a positive control reaction chamber 24b, and one or more test reaction chambers 24c. When the test is complete, the color of the test reaction chamber 24c will be compared to the color of the control chambers 24a, 24b to determine whether a positive or negative result is indicated (view “8” of
The mixer fluid channel 74 shown on the body 12 is a herringbone passive mixer; a magnified detail of a herringbone mixer is shown in
The herringbone mixer used herein is defined by a plurality of axially-spaced recesses or grooves 75 formed in the sample conduit 35, where the axially-spaced recesses or grooves 75 are formed in a bottom wall of the sample conduit 35. As can be seen, the recesses or grooves 75 are shaped such that a first part of each groove 75 imparts a first rotational component to a fluid flowing therethrough in a first direction, and a second part of each groove imparts a second rotational component to the fluid flowing therethrough in a second direction that is different than the first direction, to thereby facilitate mixing the sample 60 and lysis buffer 70 into the sample-lysis mixture. As also shown, the first part of each recess or groove is oriented approximately at an acute angle relative to the second part of each recess or groove, and the first part of each recess or groove is shorter than then second part of each recess or groove. The plurality of axially-spaced recesses or grooves 75 define an approximate herringbone shape. In the illustrated embodiment, the first part is about ⅓ the overall length of the respective groove and the second part is about ⅔ the overall length of the respective groove. Also in the illustrated embodiment, approximately equal amounts of the saliva sample and lysis buffer are introduced into the sample conduit, and the passive mixer creates a substantially homogenous mixture of these components in an approximately 1:1 ratio. However, as may be recognized by those of ordinary skill in the pertinent art based on the teachings herein, other relative amounts of components may be mixed as desired or otherwise required.
To ensure the sample 60 is adequately lysed, i.e. that cell walls within the sample 60 are sufficiently disrupted to ensure that a sufficient amount of nucleic acids therein are released into solution, it is important to ensure that a sufficient quantity of the lysing agent within the lysis buffer 70 mixes with the sample 60. Accordingly, in a microfluidic environment, the flow rate of the sample 60 relative to the flow rate of the lysis buffer 70 is preferably controlled. In the illustrated embodiment, the flow rate of the lysis buffer 70 is determined primarily by the rate at which the lysis blister 38 empties and the flow rate of the sample 60 is determined primarily by the rate at which the first wash blister 40 empties. Accordingly, the rate at which the lysis blister 38 empties relative to the rate at which the first wash blister 40 empties are preferably within a desired or predetermined range. The present inventors have found that with uniform source volumes for the two blisters 38, 40, and approximately equal pressures applied to the two blisters 38, 40, altering the length of the microfluidic conduits provides sufficiently precise control of the liquids entering the passive mixer to ensure the intended fluids are adequately mixed. In the illustrated embodiment, the first wash blister 40 and lysis blister 38 are emptied at approximately equal rates by applying approximately equal pressures to the blisters. As described below, such equal pressures can be applied by using dual plungers driven substantially simultaneously by a common drive spring or springs into the blisters.
Next, as shown in
Finally, the third button 22 is depressed, in turn depressing the elution buffer blister 44 and the negative control blister 46. As shown in
The waste chamber vent 102 is connected to the waste chamber 80 through a tortuous waste chamber vent conduit 104 from the far end of the waste chamber 80 to a location approximately between the elution buffer blister 44 and the negative control blister 46. This elongated path places the waste chamber vent 102 in a location where it can be closed by pressing the third button 22. In the microfluidic environment of the device, closing the waste chamber vent 102 substantially prevents the entry of further fluids into the waste chamber 80. Opening the reaction chamber vent allows fluids to enter the reaction chamber conduit 100 while at about the same time sealing the waste chamber 80, directing fluids into the reaction chambers 24.
A hydrophobic valve sits at the beginning or middle of the waste chamber vent conduit preventing liquids in the waste chamber from exiting the device. Because the liquids in the chamber can be hazardous chemicals or biological waste, preventing escape of the fluids is important. Liquids may not pass the hydrophobic valve, but some aerosolized particles may. Because the gas inside the chassis is vented and is likely saturated with water and other volatiles at a rate related to the vapor pressure of the water or other volatiles and the temperature of the surface from which the evaporation is to occur, and because the air at the exit of the vent tube is at the local ambient composition, there is a changing vapor concentration gradient within the device leading from the vent to the outside air. All gas from that ambient location back to the evaporation surface has a variable and increasing level of vapor comprising water and other volatiles. The time to equilibrium is equal to the tube length squared divided by the diffusivity of the molecule that is evaporating. If the length of the tube is great enough and diffusivity low enough, the evaporating molecules will not exit the device until the measurement is over. Based on the diffusivity of water vapor in air of 0.24 cm2/second, if the duration of a test is 20 minutes, the leading edge of the diffusing vapor will have progressed a distance of about 17 cm. If the tube length is only 10 cm, the time for the diffusing molecules to reach the exit is about 7 minutes. In any test with diffusing molecules which should not escape, or in which interference by outside air is problematic, the length of the vent tube may be calibrated appropriately to ensure that volatiles do not escape before the test completes and that the outside air does not interfere with the test.
For this reason, long vent tubes with small cross-sectional areas are added to the distal side of the vent, leading to an eventual opening to the environment or test room conditions. Any water molecules that permeate the vent membrane transportation will be slowed by diffusion that is limited by the small cross section of the tube. A saturation gradient will be developed between the source of water molecules, the vent membrane distal end, and the exit to the atmosphere. This diffusion gradient will slow evaporation and limit the entry of air into the reaction chamber. Because the mass flow is very low, the cross-sectional area of the tube can be extremely small without creating a viscous pressure drop or other adverse flow consequences; the practical limitation on tube cross-sectional area is set by fabrication capability. Preferably, it is made as narrow as practicable given the technologies used to make the chassis. For instance, a passage that is preferably within the range of about 75 um to about 125 um wide by about 15 to about 35 um tall, and more preferably about 100 um wide by about 25 um tall, would suffice and is within the ability of known manufacturing techniques.
Initially, before the chassis is filled with liquid, the gas in the chassis is simply air. The vent tube gas remains as “pure” air up until the moment the fluid within the chassis is added. For a period of time from adding the fluid until there is a steady vapor concentration gradient within the device, the concentration of evaporating molecules is changing. This period of time can be estimated based on the following formula:
Time to equilibrium is equal to the tube length squared divided by the diffusivity of the molecule that is evaporating.
If the length of the tube is great enough and diffusivity low enough, the evaporating molecules will not exit the device until the measurement is over.
In at least some embodiments, the reaction chamber vents are closed by covering the chambers with a plastic film or foil. Because the plastic film traps the air, the vent is closed. In such environments the reaction chamber vents may be opened by piercing the film with, for example, a snake tooth shaped piercing agent. In the embodiment shown, the waste chamber vent located between the elution buffer blister and the control blister terminates a waste chamber vent path. In some such embodiments, the reaction chamber vents may be connected to a reaction chamber vent path terminating close to the waste chamber vent. In this way, a single button press could rupture the blisters, close the waste chamber, and open the reaction chamber vent. In other embodiments, the reaction chamber vent is never sealed; instead a Laplace valve is used to direct flow. The Laplace valve will direct fluids first into the waste chamber and then into the reaction chambers. The Laplace valve connected to reaction chamber conduit 100 will be opened by closing the waste chamber vent; by blocking flow to the reaction chamber the pressure in the valve will increase such that fluids will flow down the reaction chamber conduit.
The elution buffer 86 will remove adsorbed DNA or RNA from the membrane 78; such DNA or RNA will enter the elution buffer 86 and proceed into the reaction chamber conduit 100 for amplification and detection. The reaction chamber conduit 100 contains reconstitution chambers 48b, 48c and reaction chambers 24b, 24c. The reconstitution chambers 48 contain dried reagents needed to complete the amplification reaction, and are arranged in a manner such that the reagents will be dissolved into the elution buffer 86 with any eluted DNA or RNA therein. In at least some embodiments, the dried reagents take the form of lyophilized beads. The reagents also may be “warm start” reagents, which are optimized for use in a warm environment. When using “warm start” reagents, the reaction chambers are preferably preheated prior to introducing the reaction fluids therein, as described further below. In
As shown in
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A flexible circuit board 126 shown in
In
The device shown in
The body 12 may be substantially transparent throughout, and at least a portion of the body 12 including the reaction chamber 24 is substantially transparent, and includes a substantially transparent top surface 138 extending over the reaction chambers 24 and two substantially transparent side surfaces 136 extending downwardly from the top surface 138 along opposite sides of the reaction chambers 24 relative to each other. Each reaction chamber 24 is visually observable in a viewing direction through the substantially transparent top surface 138, and the device further comprises a stimulating light source 128 located adjacent to a substantially transparent side surface 136 configured to transmit stimulating light through the side surface 136 and reaction chambers 24 in a direction 134 substantially lateral or substantially perpendicular to the viewing direction. In some embodiments, the index of refraction of the substantially transparent body 12 and the index of refraction of the fluid in the reaction chambers 24 are configured to facilitate the passage of the stimulating light from the body 12 into the reaction chambers 24 to generate fluorescing light in the reaction chambers 24. The fluorescing light is emitted in substantially all directions 142 and is observable in the viewing direction through the top surface 138 of the body. As indicated above, preferably there is an observable difference to the human eye between the stimulating light and the fluorescing light to facilitate the ability of an observer to view the fluorescing light and distinguish it from any observed stimulating light.
The top surfaces of the body and the side surfaces of the body are smooth or polished to facilitate maintaining the stimulating light within the substantially transparent body. Ideally, the surfaces are optically smooth with respect to the wavelength and angle(s) of the stimulating light. No surface is absolutely smooth, if for no other reason than matter is composed of molecules in motion. An approximate criterion for smoothness is the Rayleigh criterion. A surface is reckoned to be optically smooth if d<λ/(8 cos θ), where d is the surface roughness (e.g., root-mean-square roughness height measured from a reference plane), λ is the wavelength of the incident illumination, and θ is the angle of incidence of this illumination. Thus, a surface that is smooth at some wavelengths is rough at others, or that is rough at some angles of incidence is smooth at others (e.g., near-grazing angles).
In at least some embodiments, such as those shown in
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As shown typically in
In
In at least some embodiments of the invention, a stem seal 164 is used to close the waste sump vent 102 after the third button 22 is pressed. The stem seal 164 closes the waste sump vent 102 after a sufficient quantity of air has passed through the membrane 78 and into the waste sump 80 such that the membrane 78 has been adequately dried, but before the elution buffer has reached the membrane 78 and before the elution blister 44 is empty. By closing the waste sump vent 102, the stem seal 164 will redirect the flow of fluids coming through the membrane 78 into the reaction chamber pathway 100. As indicated above, a Laplace valve 101 may be located in fluid communication between the membrane 78 and the waste sump 80 which is larger and has a lower valve-opening pressure than a second Laplace valve connected to the reaction chamber pathway 100. Once the waste sump vent 102 is closed, however, no further fluids may enter the waste sump 80, and the pressure in the system will increase to the point where the valve to the reaction chamber pathway 100 opens. As the elution blister 44 continues to empty, the elution buffer will flow across the membrane 78; into the reaction chamber pathway 100; into the reconstitution chambers 48b, 48c; and into the reaction chambers 24b, 24c in a manner consistent with the disclosure above. In an alternative embodiment not shown in the drawings, the reaction chamber vents will be positioned such that a further stem seal can close the reaction chamber vents after the reaction chambers are full. In such an embodiment, the plungers 152 emptying the elution blister 44 and the negative control blister 46 continue to depress after the reaction chambers 24 are full. To enable this, Laplace valves could be employed with an activating pressure higher than the reaction chamber Laplace valves and connected to overflow chambers, which could be inflatable and without vents. Accordingly, all vents of the device would be sealed, and any potentially hazardous chemicals therein would be unable to escape.
In
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The Laplace valves shown in the schematic of
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While Calcein and such stimulating lights are a method of detecting an amplification reaction, one of ordinary skill would appreciate based on the teachings herein that Calcein is only one such indicating chemical, and any other indicators which are known or may become known could be used, in conjunction with techniques which are known or which may become known to identify the results of amplification reactions. It is generally preferred that a colored dye be used to ensure simplicity of the test, in that an untrained observer will understand the change in color, but other methods of detecting amplification reactions may also be used.
As may be recognized by those of ordinary skill in the pertinent art based on the teachings herein, numerous changes, improvements, modifications, additions and deletions may be made to the above-described and other embodiments of the present invention without departing from the scope of the invention. For example, the components of the device may take any of numerous different configurations and may be made of any of numerous materials that are currently known or later become known, and features may be added to or removed therefrom, without departing the from the scope of the invention. Accordingly, this detailed description of embodiments is to be taken in an illustrative as opposed to a limiting sense.
This patent application claims priority under 35 U.S.C. § 119(e) to U.S. provisional patent application Ser. No. 63/243,005, filed Sep. 10, 2021, entitled “Device and Method for Detecting Nucleic Acids in Biological Samples,” which is hereby incorporated by reference in its entirety as part of the present disclosure. This patent application also includes subject matter related to co-pending U.S. patent application Ser. No. 17/647,828, filed Jan. 12, 2022, entitled “Device and Method for Detecting Nucleic Acids in Biological Samples,” which is assigned to the assignee of the present invention and is hereby incorporated by reference in its entirety as part of the present disclosure.
This invention was made with government support under FA864921P09 awarded by the U.S. Air Force. The government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 63243005 | Sep 2021 | US |
