Patent Publication Number: US-2022219169-A1

Title: Device and method for detecting nucleic acids in biological samples

Description:
CROSS-REFERENCE TO PRIORITY PROVISIONAL APPLICATIONS 
     This patent application claims priority to provisional patent application No. 63/136,435, filed Jan. 12, 2021, entitled “Device and Method for Detecting Nucleic Acids in Biological Samples,” provisional patent application no. 63/154,217, filed Feb. 26, 2021, entitled “Device and Method for Detecting Nucleic Acids in Biological Samples,” and provisional application no. 63/243,005, filed Sep. 10, 2021, entitled “Device and Method for Detecting Nucleic Acids in Biological Samples,” each of which is hereby incorporated by reference in its entirety as part of the present disclosure. 
    
    
     FIELD OF THE INVENTION 
     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. 
     BACKGROUND INFORMATION 
     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 &#39;176 patent”). The &#39;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 FIG. 11 of the &#39;176 patent, a Flanders Technologies Associates or FTA™ membrane plug is mounted at the base of the reaction chamber, and pouches 1, 2 and 3 are connected to the reaction chamber above the FTA membrane. The total volume of the reaction chamber is about 20 μl. Sample material is added to the reaction chamber. Lysis buffer in pouch 1 is added to the reaction chamber by compressing pouch 1. The lysate mixture is incubated for a prescribed time, with optional stirring by magnetic rods that may be turned by a rotating magnet. Next, an absorbent sink pad is contacted to the FTA membrane, which wicks in lysed sample to the absorption sink pad. Nucleic acid is adsorbed on the FTA membrane plug. Next, pouch 2 is compressed to add wash buffer to the reaction chamber. Then, the absorbent pad is again contacted to the FTA membrane to wick the wash buffer through the membrane. Next pouch 3 is compressed to fill the chamber with molecular or de-ionized water. The chamber is then heated by an external heating element or by chemical heating (exothermic reaction). The heating releases pre-stored, encapsulated reagents for isothermal nucleic acid amplification. This can be achieved by encapsulating the dry reagents with low melting point paraffin, which melts upon heating the reaction chamber to the desired incubation temperature (e.g., 60° C.) and releases the reagents for amplification. The amplification step proceeds at elevated temperatures for about 20-60 minutes. After amplification, a lateral flow strip is contacted to a porous membrane plug of the reaction chamber. This is made of a material that has low nucleic acid binding. The strip is loaded with amplification product, which is functionalized with antibody or antigen to capture the labeled amplicon. The LF strip loading pad contains reporter particles to enhance detection of product captured on the strip. 
     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 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 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 and/or accurate 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. 
     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. 
     SUMMARY OF THE INVENTION 
     In accordance with a first aspect, the present invention is directed to a device for amplifying nucleic acids in a biological sample, such as saliva, to allow for detection of such amplified nucleic acids. The device comprises a sample port for receiving therein the biological sample, a lysis chamber including a lysis agent therein, a mixing chamber for mixing the biological sample and lysis agent into a sample-lysis mixture, one or more wash stations including a wash solution therein, and an elution station including an eluent therein. A solid-state membrane is located downstream of the mixing chamber, wash station and elution station, and is configured to capture nucleic acids in the biological sample passed across the membrane. A waste chamber is located downstream of the solid-state membrane, and one or more reaction chambers are also located downstream of the solid-state membrane. The sample port, lysis chamber and mixing chamber 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 across the solid-state membrane to purify nucleic acids captured therein, and receive the wash solution from the solid-state membrane 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 the reaction chamber for amplifying the captured nucleic acids. The amplified nucleic acids may be detected by visibly observing the reaction chamber, such as through a transparent window or other portion of the reaction chamber, and detecting a color change or other surrogate marker indicative of the amplified nucleic acids. Alternatively, the amplified nucleic acids may be passed through or outside of the reaction chamber for detection, such as into a viewing window or chamber in fluid communication with the reaction chamber. 
     In some embodiments of the present invention, the device further comprises a sample conduit defining the mixing chamber therein. The sample conduit is in fluid communication between each of the sample port, the lysis chamber and the wash station or wash stations, and the solid-state membrane. If desired, a lyophilized reconstitution chamber may be provided in fluid communication between the solid-state membrane and each reaction chamber. The sample port, lysis station and sample conduit are configured to mix the sample and lysis agent to form the 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 the sample conduit following the sample-lysis mixture, pass the wash solution across the solid-state membrane to purify nucleic acids captured therein, and receive the wash solution from the solid-state membrane in the waste chamber. The elution station is configured to pass the eluent across the solid-state membrane and elute captured nucleic acids from the solid-state membrane. If one or more lyophilized reconstitution chambers are provided, the captured nucleic acids pass through the lyophilized agent reconstitution chamber and into the reaction chamber for amplifying the captured nucleic acids. Alternatively, the captured nucleic acids may pass into a combined reaction and reconstitution chamber where the lyophilized agent is added to the reaction chamber, reconstituted with the eluted nucleic acids, and subsequently amplified. In some such embodiments, the mixing chamber is defined by a mixer, such as a static mixer, located within the sample conduit in fluid communication between a sample-lysis junction and the solid-state membrane, that mixes the sample and lysis agent and forms the sample-lysis mixture prior to passage across the solid-state membrane. In one exemplary embodiment, the static mixer is defined by a plurality of axially-spaced recesses or grooves formed in the sample conduit. 
     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, the wash leg is in fluid communication with the sample conduit at a sample-wash junction located adjacent to the sample port and is 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. Also in such embodiments, the lysis leg is in fluid communication with the sample conduit at a sample-lysis junction located downstream of the sample-wash junction and is 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 second wash station in fluid communication with the sample conduit and including 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 passed across the solid-state membrane also is received in the waste chamber. In some such embodiments, the second wash station includes a sealed second wash chamber containing the second wash solution. A second wash leg extends 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, and across the solid-state membrane to purify nucleic acids captured therein. 
     In some embodiments of the present invention, the elution station includes a sealed eluent chamber containing the eluent and an elution leg extending in fluid communication between the elution station and the solid-state membrane. The eluent chamber is configured to release the eluent from the eluent chamber through the elution leg and across the solid-state membrane, in order to elute captured nucleic acids from the solid-state membrane and pass the captured nucleic acids into the reaction chamber. 
     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 membrane 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. Some embodiments of the present invention further 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. 
     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, and (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 such embodiments, closure of a waste chamber vent, or movement of a waste chamber vent seal into a 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 and not into 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 with the lysis agent. 
     Some embodiments of the present invention further comprise a body where the solid-state membrane and/or the body is movable relative to the other. In some such embodiments, at least the solid-state membrane is movable relative to the body from (i) a sample position where the solid-state membrane is in fluid communication with the sample port for receiving across the solid-state membrane the biological sample and capturing nucleic acids in the biological sample therein, (ii) to a wash position where the solid-state membrane is in fluid communication with the wash station for the passage of the wash solution across the solid-state membrane to purify nucleic acids captured therein, and (iii) to a reaction position where the solid-state membrane is in fluid communication with the reaction chamber for eluting captured nucleic acids from the solid-state membrane into the reaction chamber and amplifying captured nucleic acids. Some such embodiments comprise a plurality of wash stations. In such embodiments, the solid-state membrane and/or the body is movable relative to the other from the sample position to a plurality of successive wash positions. In each wash position, the solid-state membrane is in fluid communication with a respective one of the plurality of wash stations for the passage of a respective wash solution across the solid-state membrane to purify nucleic acids captured therein. 
     In some embodiments of the present invention, the body further includes an absorbent waste pad in fluid communication with the waste chamber and engageable with the solid-state membrane in the sample position for absorbing therein fluid passed through the solid-state membrane in the sample position and/or engageable with the solid-state membrane in the wash position(s) for absorbing therein the wash solution passed through the solid-state membrane in the wash position(s). Some such embodiments further comprise a waste pad support movably mounted on the body and including the waste pad mounted thereon. The waste pad is movably engageable with an underside of the solid-state membrane to facilitate engagement of the solid-state membrane and waste pad. Some embodiments of the present invention further comprise a membrane support including the solid-state membrane mounted thereon, a microfluidic chip defining a microfluidic reaction chamber, and a microfluidic chip support movably mounted on the body and including the microfluidic chip mounted thereon. The microfluidic chip is engageable with the solid-state membrane and/or membrane support upon movement of the solid-state membrane into the reaction position, to facilitate fluid communication between the solid-state membrane and the microfluidic reaction chamber. In some embodiments of the present invention, the membrane support and/or body is movable relative to the other from the sample position to the wash position, and from the wash position to the reaction position. In some such embodiments, the membrane support includes a manually-engageable portion, such as a knob or button, that is manually engageable to move the membrane support and membrane thereon from the sample position to the wash position, and from the wash position to the reaction position. 
     In some embodiments of the present invention, the lysis station, wash station and/or elution station includes a sealed chamber containing a lysis agent, wash solution or eluent, and an actuator, such as a button actuator or plunger, movable between a non-actuated position and an actuated position. In the actuated position, the lysis agent, wash solution or eluent is released from the sealed chamber. In some such embodiments, the actuator is manually engageable and moveable from the non-actuated position to the actuated position. The sealed chamber includes a frangible or breakable wall, such as formed by a blister or foil, that is breakable by the actuator in the actuated position to release the lysis agent, wash solution or eluent from the sealed chamber. 
     In some embodiments of the present invention, the membrane support and/or body is movable relative to the other from the sample position to the wash position, and from the wash position to the reaction position. The actuator includes a locking member movable between a locked position preventing actuation of the actuator, and an unlocked position allowing the actuator to be moved from the non-actuated position to the actuated position. The locking member is engageable with the membrane support upon relative movement into the wash position or reaction position, to thereby move the locking member from the locked position to the unlocked position. In some such embodiments, the locking member is pivotally or rotatably mounted on the body and is engageable with the membrane support. Upon movement of the membrane support into the wash positon or reaction position, the membrane support causes the locking member to rotate from the locked position to the unlocked position, and thereby allow actuation in the respective position. 
     Some embodiments of the present invention further comprising (i) a disposable cartridge containing the sample port, wash station, elution station, solid-state membrane, waste chamber and reaction chamber, and (ii) a base station configured to receive the disposable cartridge therein and including a heat source for facilitating a reaction in the microfluidic reaction chamber. 
     Some embodiments of the present invention further comprise a sample receptacle including therein a lysis fluid and configured to receive therein the biological sample for mixture with the lysis fluid. The sample receptacle includes an outlet port connectable in fluid communication with the sample port for releasing a lysis fluid and biological sample mixture into the sample port and onto the solid-state membrane. The sample receptacle includes a sealed chamber containing the lysis fluid, and a frangible or breakable wall configured to be ruptured after receiving the biological sample therein, to allow mixture of the lysis fluid and biological sample. In some of such embodiments, the sample receptacle includes a closure movable between an open position for allowing introduction of the biological sample into the sample receptacle, and a closed position sealing the biological sample and lysis fluid within the receptacle. One or more protuberances are engageable with the frangible or breakable wall when the closure is in the closed position to break the frangible or breakable wall and thereby mix the lysis fluid with the biological sample. The sample receptacle may further include a pump that is manually engageable to pump the lysis fluid and sample mixture through the outlet port and into the sample port. 
     In accordance with another aspect, the present invention is directed to a device comprising (i) first means for receiving therein a biological sample; (ii) second means for sealing a lysis agent therein and releasing the lysis agent therefrom for mixture with the biological sample; (iii) third means for mixing the biological sample and lysis agent into a sample-lysis mixture; (iv) fourth means for sealing a wash solution therein and releasing the wash solution therefrom; (v) fifth means for sealing an eluent therein and releasing the eluent therefrom; (vi) sixth means in fluid communication with the third means for receiving the sample-lysis mixture and capturing nucleic acids in the biological sample therein, in fluid communication with the fourth means for receiving the wash solution following the sample-lysis mixture and passing the wash solution across the sixth means for purifying nucleic acids captured therein, and in fluid communication with the fifth means for receiving the eluent across the sixth means for eluting captured nucleic acids from the sixth means; (vii) seventh means located downstream of the sixth means for receiving the remainder of the sample-lysis mixture that passes through the sixth means and the wash solution that passes through the sixth means as waste and storing the waste therein; and (viii) eighth means located downstream of the sixth means for receiving the eluted captured nucleic acids from the sixth means and for amplifying the captured nucleic acids therein. 
     In some such embodiments, (i) the first means is sample port; (ii) the second means is a sealed lysis chamber containing a lysis agent therein and including a frangible or breakable wall that is breakable to release the lysis agent therefrom; (iii) the third means is a sample vial connectable in fluid communication with the sample port, or a sample conduit in fluid communication with the sample port and including a static mixer therein; (iv) the fourth means is a sealed wash solution chamber containing a wash solution therein and including a frangible or breakable wall that is breakable to release the wash solution therefrom; (v) the fifth means is a sealed eluent chamber containing an eluent therein and including a frangible or breakable wall that is breakable to release the eluent therefrom; (vi) the sixth means is a solid-state membrane; (vii) the seventh means is a waste chamber in fluid communication with the solid-state membrane for receiving the remainder of the sample-lysis mixture that passes through the solid-state membrane and the wash solution that passes through the solid-state membrane as waste and storing the waste therein; and (viii) the eighth means is a microfluidic reaction chamber. 
     Some embodiments of the present invention further comprise means for closing the seventh means after receiving the lysis agent and wash solution therein, and for opening the eighth means for directing the captured nucleic acids from the sixth means therein. 
     In accordance with another aspect, the present invention is directed to a method of capturing nucleic acids in a biological sample and amplifying the captured nucleic acids therein in a reaction chamber. The method comprises the following steps:
         (i) passing a biological sample and lysis fluid mixture across a solid-state membrane and capturing nucleic acids in the biological sample in the solid-state membrane;   (ii) preventing the flow of the sample-lysis mixture that passes across the solid-state membrane into the reaction chamber, and receiving the remainder of the sample-lysis mixture that passes across the solid-state membrane in a waste chamber;   (iii) passing a wash solution across the solid-state membrane and purifying nucleic acids capturing therein;   (iv) preventing the flow of the wash solution that passes across the solid-state membrane into the reaction chamber, and receiving the remainder of wash solution that passes across the solid-state membrane in the waste chamber; and   (v) passing an eluent across the solid-state membrane and eluting captured nucleic acids from the solid state membrane, directing the eluted captured nucleic acids from the solid-state membrane into the reaction chamber and not into the waste chamber, and amplifying captured nucleic acids therein in the reaction chamber.       

     In some embodiments of the present invention, step (i) includes introducing a lysing agent into a 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. In some embodiments of the present invention, steps (iii) and (iv) include introducing a wash solution into the sample conduit 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. In some embodiments of the present invention, step (v) includes introducing an eluent across the solid-state membrane, 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 reaction chamber, and amplifying the captured nucleic acids in the reaction 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 for directing the captured nucleic acids from the solid-state membrane therein. 
     In some embodiments of the present invention, step (iii) includes moving the solid-state membrane and/or a first washing station relative to the other into a first wash position, step (v) includes moving the solid-state membrane and/or a microfluidic reaction chamber relative to the other into a reaction position, eluting captured nucleic acids from the solid-state membrane into the microfluidic reaction chamber, and amplifying captured nucleic acids therein. Some embodiments further comprise moving the solid-state membrane, and/or a support for the solid-state membrane, into a reaction position. Then, upon moving the solid-state membrane or support therefor into the reaction position, moving a microfluidic chip containing the microfluidic reaction chamber into engagement with an underside of the solid-state membrane and/or the support therefor, and placing the microfluidic reaction chamber in fluid communication with the solid-state membrane. 
     Some embodiments of the present invention further comprise introducing a cartridge containing the biological sample and lysis fluid, solid-state membrane, wash solution, eluent and chamber, into a base station, performing at least step (v) with the cartridge located in the base station, and disposing of the cartridge after use. 
     One advantage of the present invention, and/or of embodiments thereof, is that the lysis mixture and sample is passed across the solid-state membrane, and the wash solution is passed across the solid state membrane, all prior to connecting the solid-state membrane in fluid-communication with the microfluidic reaction chamber. Accordingly, the volume of the biological sample, and the volume of lysing agent and/or washing solution(s) passed across the solid-state membrane, are not limited by the relatively small volume of the microfluidic reaction chamber and/or capacity of a single adsorbent pad, as encountered in the above-described prior art. As a result, the device and method of the present invention allow for larger volumes of biological samples and provide for a correspondingly greater capture of targeted nucleic acids. This, in turn, allows for a more sensitive detection of targeted nucleic acids and more accurate testing for such nucleic acids. Yet another advantage is that the device and method of the invention, and/or of the embodiments thereof, allow for even significantly greater amounts of biological samples, lysis fluids and/or wash solutions as compared to prior art devices and methods, particularly as compared to prior art hand-held or mobile devices or methods. Yet another advantage is that the components of the device and method can be provided in a cartridge that can be mounted in a base station to heat the microfluidic reaction chamber and/or otherwise facilitate the amplification and detection of the captured nucleic acids. A still further advantage is that each cartridge can be disposed of after use and the base station can be reused with additional cartridges to conduct additional tests. A further advantage is that the use of paraffin or like sealants to prevent premature hydrolyzation and the loss of reagent before the reaction as required by the above-described prior art can be avoided. 
     Other objects and advantages of the present invention, and/or of embodiments thereof, will become more readily apparent in view of the following detailed description of embodiments and accompanying drawings. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1  includes a perspective view of a device embodying the present invention including a disposable cartridge received within a base station for amplifying and detecting nucleic acids in a biological sample; 
         FIGS. 2A through 2C  include (i) in  FIG. 2A , a perspective view of a sample receptacle or vial for biological sample collection and lysis, including a left-hand view showing the vial open, and a right-hand view showing the vial closed, (ii) in  FIG. 2B , a perspective view of a disposable cartridge for amplifying and detecting nucleic acids in a biological sample, and the closed sample vial suspended above the cartridge and ready to supply a sample and lysis mixture to the cartridge for testing, and (iii) in  FIG. 2C , a perspective view of the cartridge including the sample vial with the outlet port thereof connected in fluid communication with the sample port of the cartridge, and the cartridge received within the base station for capturing, concentrating, purifying, amplifying and detecting targeted nucleic acids in the biological sample; 
         FIGS. 3A through 3I  are a series of perspective views of the cartridge and sample vial of  FIGS. 1 and 2  illustrating the following procedural steps: (i) in  FIGS. 3A and 3B , loading a saliva sample from the sample vial into the sample receptacle of the cartridge, (ii) in  FIGS. 3C  and  3 D, washing the sample, (iii) in  FIGS. 3E and 3F , washing the sample again, (iv) in  FIGS. 3G and 3H , eluting the DNA/RNA into the reaction chamber, and (v) in  FIG. 3I , incubating the reaction chamber, e.g., at about 62° C. for about 20 minutes, and then comparing the color of the reaction chamber fluid to a test chip to determine if targeted nucleic acids are detected; 
         FIGS. 4A through 4E  include a series of perspective, cut-away views of the cartridge and sample vial of  FIGS. 1 and 2  illustrating the following procedural steps: (1) in  FIG. 4A , the sample vial received in the sample port of the cartridge, and in  FIG. 4B , the sample waste sump/waste pad engaged with the underside of the solid-state membrane slider for capturing nucleic acids from the sample in the solid-state membrane; (2) in  FIG. 4C , manual advancement of the membrane slider toward the first wash station, and disengagement of the sample waste sump/waste pad therefrom; (3) in  FIG. 4D , rotation of the blister plunger in the first wash station as the membrane slider is manually moved therein and engagement of the first wash waste pad with the underside of the membrane slider when received in the first wash station; and (4) in  FIG. 4E , unlocking of the blister plunger in the first wash station to, in turn, allow the first blister plunger to be manually depressed to break the blister and wash the captured nucleic acids in the solid-state membrane in the first wash station; 
         FIGS. 5A through 5E  include a series of perspective, cut-away views of the cartridge and sample vial of  FIGS. 1 and 2  illustrating the following additional procedural steps: (5) in  FIG. 5A , the sample vial received in the sample port of the cartridge, and in  FIG. 5B , manual movement of the membrane slider from the first wash station toward the second wash station, and disengagement of the first wash waste pad from the underside of the membrane slider; (6) in  FIG. 5C , manual advancement of the membrane slider to the second wash station and alignment of the membrane slider with the second wash station blister and waste sump/pad; (7) in  FIG. 5D , unlocking of the second blister plunger in the second wash station to, in turn, allow the second blister plunger to be manually depressed to break the blister and wash the captured nucleic acids in the solid-state membrane in the second wash station; and (8) in  FIG. 5E , disengagement of the second wash waste pad from the underside of the membrane slider and manual movement of the membrane slider from the second wash station toward the reaction position in fluid communication with the reaction chamber where the third blister plunger is depressed; 
         FIG. 6A  is a perspective, cut-away view of the cartridge of  FIGS. 1 and 2 ,  FIG. 6B  is a separate perspective view of the microfluidic chip and microfluidic chip carrier,  FIG. 6C  is an exemplary perspective, cross-sectional view of the blister plunger and blister assembly in each wash station, and  FIG. 6D  is another perspective view of the cartridge and sample vial of  FIGS. 1 and 2 ; 
         FIGS. 7A through 7G  are a series of perspective, partial cross-sectional views of the cartridge of  FIGS. 1 and 2  showing (1) in  FIG. 7A , the membrane slider in fluid communication with the sample port, (2) in  FIG. 7B , movement of the membrane slider into the first wash station, (3) in  FIG. 7C , unlocking of the blister plunger in the first wash station and engagement of the first waste pad with the underside of the membrane slider for absorbing the wash solution in the first wash station, (4) in  FIG. 7D , disengagement of the first wash waste pad and movement of the membrane slider from the first wash station into the second wash station, (5) in  FIG. 7E , unlocking of the blister plunger in the second wash station and engagement of the second wash waste pad with the underside of the membrane slider for absorbing the wash solution in the second wash station, (6) in  FIG. 7F , disengagement of the second wash waste pad and movement of the membrane slider from the second wash station into the reaction position, (7) in  FIG. 7G , unlocking of the blister plunger in the reaction position and engagement of the microfluidic reaction chamber with the underside of the membrane slider for placing the microfluidic reaction chamber in fluid communication with the solid-state membrane in the reaction position; 
         FIGS. 8A and 8B  are top plan views of an exemplary front face of a cartridge of the type shown in  FIGS. 1 and 2  with arrows indicating the sample port and rotational direction of the blister plungers, and showing in  FIG. 8A  the test windows of the cartridge indicating a negative test result, and in  FIG. 8B  the test windows of the cartridge indicating a positive test result; 
         FIG. 9  is a flow chart illustrating a clinical workflow when performing a test, such as a test for Covid-19, with the device of  FIGS. 1 and 2 ; 
         FIGS. 10A through 10G  are a series of the following views: (1)  FIGS. 10A and 10B  are a perspective view and a perspective cross-sectional view, respectively, of the sample vial as received and ready for receiving a sample, (2)  FIG. 10C  is a perspective, cross-sectional view of the sample vial after receiving a sample therein, such as a saliva sample, and its closure in an open position, (3)  FIGS. 10D and 10E  are a perspective view and a perspective cross-sectional view, respectively, of the sample vial with its closure in the closed position; (4)  FIG. 10F  is a cross-sectional view of the sample vial after the protuberances or piercing members of the closure break the sealed chamber of the vial containing lysis fluid, and gentle agitation thereof to mix the sample and lysis fluid within the vial, and (5)  FIG. 10G  is a perspective, cross-sectional view illustrating insertion of the outlet port and valve of the sample vial into the sample port of the cartridge of  FIGS. 1 and 2  so that the dome-shaped pump of the closure may be depressed to, in turn, pump the sample-lysis fluid mixture into the sample port and solid-state membrane in fluid communication therewith; 
         FIGS. 11A and 11B  are two perspective views of the cartridge and base station of  FIGS. 1 and 2 , including in  FIG. 11A  the cartridge prior to insertion into the base station, and in  FIG. 11B  the cartridge inserted into the base station; 
         FIGS. 12A through 12C  include perspective views of another embodiment of a cartridge and base station for amplifying and detecting nucleic acids in a biological sample wherein the cartridge includes the solid-state membrane near the distal end thereof, and the base station includes the wash station(s) and microfluidic reaction chamber(s) (not shown), and illustrating in  FIG. 12A  the cartridge prior to insertion into the base station, in  FIG. 12B  the cartridge itself, and in  FIG. 12C  the cartridge inserted into the base station; 
         FIG. 13A  is a perspective view of the membrane slider of the device of  FIGS. 1-7  and including the solid-state membrane mounted therein,  FIG. 13B  is a cross-sectional view of the membrane slider and solid-state membrane of  FIG. 13A , and  FIG. 13C  is a perspective, cross-sectional view of the membrane slider and solid-state membrane of  FIG. 13A ; 
         FIGS. 14A and 14B  include upper and lower perspective views, respectively, of a device embodying the present invention including a disposable cartridge that is received within a base station for amplifying and detecting nucleic acids in a biological sample; 
         FIGS. 15A and 15B  include upper and lower perspective views, respectively, of the device of  FIGS. 14A and 14B , and  FIG. 15C  is a partial, perspective view of a saliva collection syringe-type device receivable within the sample port of the device of the device of  FIGS. 14A and 14B  for introducing a saliva sample therein; 
         FIG. 16  is a somewhat schematic illustration of the device of  FIGS. 14A and 14B  illustrating the introduction of a saliva sample through a sample port, where the device includes a first wash station, a lysis station, a mixing chamber, a second wash station, an elution station, a solid-state membrane, a waste chamber and microfluidic reaction chambers, wherein the lysis and wash solutions are received within the waste chamber which, in turn, creates a sufficient back pressure to open a one-way valve between the solid-state membrane and the microfluidic reaction chambers to allow the elution fluid and targeted nucleic acids to flow through the one-way valve and into the microfluidic reaction chambers; 
         FIGS. 17A and 17B  are perspective views of the capillary tube LAMP reaction chip of the device of  FIGS. 14A and 14B  where the microfluidic reaction chip includes three reaction chambers; and 
         FIG. 18  is a perspective view of an alternative embodiment of a capillary tube LAMP reaction chip of the device of  FIGS. 14A and 14B  where the microfluidic reaction chip includes five reaction chambers. 
     
    
    
     DETAILED DESCRIPTION 
     In  FIG. 1 , a device embodying the present invention for amplifying and detecting nucleic acids in a biological sample is indicted generally by the reference numeral  10 . The device  10  can be used to identify target nucleic acid sequences corresponding to target pathogen or host genetic sequences including any genetic target in a biological or environmental sample. Identification is done through the isolation, concentration, isothermal amplification, and detection of nucleic acids. Multiple targets can be identified simultaneously. Examples of use include without limitation: (i) the detection of infectious agents such as SARS-CoV-2, in saliva, swab, urine, or stool; (ii) the detection of specific mutations in blood samples; and (iii) the detection of infectious agents from a surface swab. The device  10  in  FIG. 1  comprises a body or test cartridge  12 , a base station  14 , and a sample receptacle or vial  16 . A biological sample will be placed into the sample vial  16  and combined with a lysis chemistry facilitating cell lysis, releasing any nucleic acids from cells in the biological sample. The sample vial  16  is inserted into a sample port  18  of the test cartridge  12 . The sample and lysis chemistry mixture is extruded from the sample vial  16  into the sample port  18  of the test cartridge  12 . 
       FIG. 2  shows the sample vial  16  and test cartridge  12  in greater detail. The sample vial  16  comprises a vial body  20  and a vial closure or cap  22 . When the vial cap  22  is inserted onto the vial body  20 , the vial cap  22  seals the sample vial  16 . The vial cap  22  of the sample vial  16  comprises a dome-shaped pump  24  which can be used to pump the sample mixture through an outlet port  26  in the sample vial  16 . When the sample vial  16  is inserted into a sample port  18  of the test cartridge  12 , the sample will move through an outlet port  26  of the sample vial  16  to enter the test cartridge  12 . The test cartridge  12  is inserted into the base station  14  for isolation, concentration, and amplification of any targeted nucleic acids in the sample. 
       FIGS. 3-7  show the mechanism and operation of the exemplary device  10  in greater detail. Once the sample vial  16  is inserted into the sample port  18  of the test cartridge  12 , the outlet port  26  of the sample vial  16  will be placed in fluid communication with the top of a solid-state membrane  28  (shown in  FIG. 4 ) and a waste pad  30  will be engaged with the underside of the solid-state membrane  28 . The outlet port  26  is in fluid communication with the solid-state membrane  28  and the solid-state membrane  28  is in fluid communication with the waste pad  30 . Fluids can pass through the solid-state membrane  28 , such as by capillary action, and therefore the outlet port  26  is in fluid communication with the waste pad  30 . The dome-shaped pump  24  is used to pump the sample-lysis mixture into the test cartridge  12  and across the solid-state membrane  28 . Portions of the sample-lysis mixture will be adsorbed onto the solid-state membrane  28  and isolated from the remainder of the mixture, which will be absorbed by the waste pad  30 . The solid-state membrane  28  is made of a capture material that binds nucleic acids, resulting in the adsorption of nucleic acids in the biological sample. In the illustrated embodiment, the membrane is defined by a solid-state structure, i.e., it does not include moving parts, and includes without limitation a solid-state filter matrix. The solid-state filter matrix may be formed of silica, glass fibers, cellulose or other materials that are currently known, or that later become known for such purpose. An exemplary capture material is the Pall A/E Borosilicate Glass Membrane without binders from Pall Corporation. Such a membrane is made of or includes borosilicate glass fibers that bind nucleic acids. However, as may be recognized by those of ordinary skill in the pertinent art based on the teachings herein, the solid-state membrane  28  may be made of any of numerous different materials and may take any of numerous different configurations that are currently known, and/or that later become known. 
     The solid-state membrane  28  is contained within a membrane support  31  which is, in turn, mounted on a membrane slider  32  movably mounted on the test cartridge  12 . As shown in  FIGS. 1-7 and 14 , the membrane slider  32  is manually engageable and movable through an axially-elongated slot  33  formed in the body  12  in a direction parallel to the elongated sides of the test cartridge  12 . As may be recognized by those of ordinary skill in the pertinent art based on the teachings herein, the solid-state membrane  28  may be mounted and movable within the test cartridge, or the test cartridge may be movable relative to the solid-state membrane, in accordance with any of numerous different mechanisms or configurations that are currently known, or that later become known. For example, the solid-state membrane could be mounted on a support that is rotatably mounted on the cartridge to allow the membrane to be rotated from one station or position to the next. Alternatively, as described below, the solid-state membrane may be fixedly mounted on the test cartridge or device, i.e., not movable on the device or movably mounted. 
     As shown in  FIGS. 4-6 , the membrane slider  32  begins in a sample position  34 , is moved to each of a plurality of wash positions  36  and ends at a reaction position  38 . The membrane slider  32  comprises a manually-engageable portion or knob  40  which is manually engageable and movable through the axially-elongated slot  33  in the test cartridge  12  to move the membrane slider  32  and membrane  28  thereon from the sample position  34  to a first wash position  36 , from the first wash position to a second wash position  36 , and from the second wash position  36  to the reaction position  38 . A plurality of waste pad supports  42 ,  42  are pivotably mounted on the test cartridge  12  to support waste pads  30 ,  30  at the sample position  34  and each of the wash positions  36 ,  36 . When the membrane slider  32  is located at the sample position  34 , or any of the wash positions  36 , the waste pad support  42  located at the respective position including a waste pad  30  pivotably mounted thereon pivots to engage with the underside of the solid-state membrane  28 . Engagement of each waste pad  30  with the solid-state membrane  28  facilitates effective capillary action of the waste pad  30  on the solid-state membrane  28 . As the slider  32  advances away from the sample position  34  or any of the wash positions  36 , the respective waste pad support  42  and waste pad  30  located at the position disengage from the solid-state membrane  28 . As may be recognized by those of ordinary skill in the pertinent art based on the teachings herein, the waste pads  30 ,  30  and waste pad supports  42 ,  42  may take any of numerous different configurations that are currently known, or that later become known, and the device may include any desired number of waste pads and/or waste pad supports. For example, while the exemplary implementation includes separate waste pads  30  and waste pad supports  42  for each of the sample position  34  and wash positions  36 ,  36 , the device could include waste pads and waste pad supports spanning multiple positions, multiple waste pads at a single position, multiple waste pads on a single waste pad support, a single waste pad supported by multiple waste pad supports, or any combination thereof. For example, a single waste pad and waste pad support spanning the sample position and both wash positions could be employed. Alternatively, as described below, the device may include a waste chamber or receptacle without any waste pad or pads. 
     As the membrane slider  32  reaches each wash position  36 ,  36 , the solid-state membrane  28  will complete a wash station  44  at that position. As shown in  FIGS. 4-7 , each wash station  44  comprises the solid-state membrane  28 , an interlock  55  located above the solid-state membrane  28 , a sealed chamber or blister  46  mounted within the interlock  55  and containing a wash solution, an actuator or blister plunger  48  mounted on the interlock  55  and movable between a non-actuated position  50  and an actuated position  52 , an upper seal or sealing pad  54 A ( FIG. 6C ) extending about the blister  46  and sealing the blister within the interlock  55 , and a waste pad  30  supported by and pivotably mounted on a waste pad support  42  located on the underside of the solid-state membrane. As can be seen, the actuator or blister plunger  48  located at each wash station  36  is manually engageable and depressible from the non-actuated position  50  into the actuated position  52 . When the actuator or blister plunger  48  is moved to the actuated position  52 , the lower end of the actuator or blister plunger  48  engages and breaks the sealed chamber of the blister  46  to release the wash solution therein. The force exerted by the blister plunger  48  pushes the wash solution into the solid-state membrane  28  and capillary action of the waste pad  30  pulls the wash solution into the waste pad  30  to thereby purify and isolate the nucleic acids adsorbed on the solid-state membrane  28  by washing away unbound matter from the solid-state membrane. 
     As shown in  FIG. 6C , the sealed chamber or blister  46  includes a frangible or breakable wall  56  that is configured to break as the actuator or blister plunger  48  moves into the actuated position  52 , releasing the wash solution from the sealed chamber  46  and allowing it to pass across the solid-state membrane  28  to purify the nucleic acids captured therein. Movement of the plunger  48  from the non-actuated position  50  to the actuated position  52  causes the actuator or blister plunger  48  to exert pressure on the sealed chamber or blister  46 . Blister rupture pins (not shown) are located below the frangible wall  56  and come in contact with and burst the frangible wall as the blister actuator  48  is compressed to release the wash solution onto the solid-state membrane  28 . The upper sealing pad  54 A is impermeable to the wash solution and sufficiently compressible to ensure a fluid-tight seal between the interlock  55  and the membrane slider  32 . In some configurations, the upper sealing pad  54 A is sufficiently compressible to facilitate a substantially complete emptying of the blister  46  upon depressing the blister plunger  48  into the fully-actuated position  52 . One of ordinary skill will recognize that while the upper sealing pad  54 A is attached to an actuator in  FIG. 6C , it could provide the same functional benefits if attached to the membrane slider  32  or other structure. 
     In the exemplary implementation shown in  FIGS. 4 and 5 , each of the actuators  48 ,  48  includes a locking member  58  movable between a locked position  60  preventing actuation of the actuator  48 , and an unlocked position  62  allowing the actuator  48  to be moved from the non-actuated position  50  to the actuated position  52 . Each locking member  58  starts or is normally located in the locked position  60 . When the membrane slider  32  moves into a respective wash position  36 ,  36  or the reaction position  38 , the corresponding locking member  58  is engaged by the membrane slider  32  to move the locking member  58  from the locked position  60  to the unlocked position  62 . The locking member  58  is pivotably or rotatably mounted on the test cartridge  12  and engageable with the membrane slider  32  such that the membrane slider  32  causes the locking member  58  to rotate from the locked position  60  to the unlocked position  62 . While in this exemplary implementation each rotating locking member  58  is part of the respective actuator  48 , one of ordinary skill in the relevant art will recognize that alternative locking configurations may be used including, for example, a configuration where the locking member and actuator are separate components, or where no locking member is employed at all. 
     Each waste pad  30 ,  30  is made of absorbent materials of a type known to those of ordinary skill in the pertinent art to pull the sample, washes, and other liquids across and/or through the solid-state membrane  28 . Such materials allow the fluid flow to be driven by capillary forces. The action of depressing the sample vial pump  24  and/or depressing an actuator  48 ,  48  to the actuated position  52  may not itself generate sufficient thrust to push liquids through or across the solid-state membrane  28 . Accordingly, the waste pads  30 ,  30  induce capillary forces to pull the liquids across the solid-state membrane  28 . The membrane  28  otherwise (without contact with an absorption pad  30 ) may resist liquid flow, and the liquid may not fully or sufficiently pass across the solid-state membrane  28  without the absorbing waste pad  30 . In the exemplary implementation shown in  FIGS. 6 and 7 , each waste pad  30  is lowered or dropped out of engagement with the membrane support  40  and/or solid-state membrane  28  upon movement thereof out of the respective wash station  30 . 
     While the exemplary implementation shown allows for manual movement of the manually-engageable portion  40  of the slider and manual engagement of each actuator by the operator of the device, one of ordinary skill in the pertinent art would recognize that the device could be made with any number of manually or automatically operated mechanisms to accomplish the same or essentially the same function, including a drive mechanism, such as a screw or gear drive, that may or may not be connected or connectible to an electric motor. 
       FIG. 6  shows an exemplary microfluidic chip  64  pivotably mounted on a chip carrier  66  and comprising a plurality of microfluidic reaction chambers  68 ,  68 . The microfluidic chip  64  may include a plurality of reaction chambers  68 ,  68 , preferably about three to seven reaction chambers, and in the illustrated embodiment, includes three reaction chambers  68 ,  68 . The chip carrier  66  is pivotably mounted to the body  12  so that when the membrane slider  32  moves into the reaction position  38 , the microfluidic chip  64  engages the underside of the solid-state membrane  28 . Similar to the wash stations  44 ,  44 , a reaction station  69  (shown in  FIG. 7 ) at the reaction position  38  is completed by movement of the solid-state membrane  28  into the reaction position  38 . The reaction station  69  includes the solid-state membrane  28 , the microfluidic chip  64  mounted on the chip carrier  66  underneath the solid-state membrane  28 , an interlock  55  located above the solid-state membrane  28 , a sealed chamber or blister  46  mounted within the interlock  55  and containing an elution buffer, an actuator or blister plunger  48  mounted on the interlock  55  and movable between a non-actuated position  50  and an actuated position  52 , and the upper seal or sealing pad  54 A extending about the sealed chamber or blister  46  and sealing the blister  46  within the interlock  55 . As the actuator or blister plunger  48  is moved to the actuated position  52 , the elution buffer is released when the lower end of the plunger engages the sealed container or blister  46 , breaks the blister and thereby releases the elution buffer therein. The pressure created by depressing the blister plunger  48  and the capillary action induced by the microfluidic chip  64  cause the elution buffer to pass across the solid-state membrane  28  and into the reaction chambers of the microfluidic chip for amplification and identification. The elution buffer releases the adsorbed, purified nucleic acids from the solid-state membrane  28  as it passes through, eluting the nucleic acids into the elution buffer and carrying the nucleic acids into the microfluidic reaction chambers  68 ,  68 . 
     As shown in  FIG. 6C , the sealed chamber or blister  46  includes a frangible or breakable wall  56  that is configured to break when the actuator  48  moves into the actuated position  52 , releasing the elution buffer from the sealed chamber  46  and allowing it to pass across the solid-state membrane  28  and carry with it the nucleic acids captured therein. Movement of the blister plunger  48  from the non-actuated position  50  to the actuated position  52  causes the plunger to exert pressure on the blister  46 . Blister rupture pins (not shown) are located below the frangible wall  56  and come in contact with and burst the frangible wall as the blister actuator  48  is compressed to release the elution buffer across the solid-state membrane  28 . The upper sealing pad  54 A is impermeable to the elution buffer and sufficiently compressible to ensure a fluid-tight seal between the interlock  55  and the membrane slider  32 . In some configurations, the upper sealing pad  54 A is sufficiently compressible to facilitate a substantially complete emptying of the sealed chamber  46 . 
     Nucleic acid amplification reagents, including primers and enzymes, are provided in the reaction chambers  68 ,  68  in the form of dry and stable reagents, such as lyophilized reagents, to be hydrolized when the elution buffer enters the microfluidic chip  64  and flows into the reaction chambers  68 ,  68 . The illustrated embodiment of the device  10  employs a loop-mediated isothermal amplification (“LAMP”) method of amplification. However, as may be recognized by those of ordinary skill in the pertinent art based on the teachings herein, other methods of amplification that are currently known or that later become known may be employed. 
     As indicated in  FIGS. 11A and 11B , the device  10  may be used to detect SARS-COV-2 using a heating element (not shown) in the base station  14  to ensure that the reaction chambers  68 ,  68  are maintained at a substantially constant temperature for LAMP reaction at a desired temperature, such as about 63° C., when the cartridge is inserted into the base station. The base station  14  maintains the LAMP reaction temperature for a set period of time, which in the exemplary embodiment is approximately 28 minutes for the LAMP implementation detection of SARS-COV-2. As shown in  FIG. 8 , at the end of this period, the results of the test will be visible through a results window  70 . The results window  70  contains a negative control  72 , a positive control  74 , and a sample indicator  76 . If the color of the sample indicator  76  matches the color of the negative control  72 , the test indicates a negative result. If the color of the sample indicator  76  matches the color of the positive control  74 , the test indicates a positive result. The detection method is a colorimetric or fluorescent dye that can be easily seen by the user of the device  10 . As may be recognized by those of ordinary skill in the pertinent art based on the teachings herein, different reaction solutions, amplification reactions, and detection methods may have different or non-isothermal ideal reaction temperatures. Accordingly, the base station  14  may be adapted to provide different reaction temperatures that may be warmer or cooler than ambient or room temperature, including cyclic variation of warmer and cooler temperatures as required for polymerase chain reaction (“PCR”) amplification or other temperature settings, which are currently known or that later become known for performing such functions. 
       FIGS. 10A through 10G  show in greater detail an embodiment of the sample receptacle or vial  16 . The sample vial  16  includes a vial body  20  containing therein a lysis fluid  78  and configured to receive therein the biological sample  80  for mixture with the lysis fluid  78 . An outlet port  26  of the sample receptacle  16  is configured to be inserted into the sample port  18  of the test cartridge  12  for releasing the lysis fluid and biological sample mixture  82  into the sample port  18  and across the solid-state membrane  28  in the sample position  34 . The sample receptacle  16  includes a sealed chamber  84  containing the lysis fluid  78 , and a frangible or breakable wall  86  configured to be ruptured after receiving the biological sample  80  therein to allow mixture of the lysis fluid  78  and biological sample  80 . The sample receptacle  16  includes a cap or closure  22  movable between an open position  90  for allowing introduction of the biological sample  80  into the sample receptacle  20 , and a closed position  92  sealing the biological sample  80  and lysis fluid  78  within the receptacle. One or more protuberances or piercing members  94  are engageable with the frangible or breakable wall  86  when the closure  22  is in the closed position  92  to break the frangible or breakable wall  86  and thereby mix the lysis fluid  78  with the biological sample  80 . Manual agitation may be required to ensure adequate mixing of the sample  80  and lysis fluid  78 . The cap or closure  22  includes an elastomeric, dome-shaped pump  24  and the piercing members  94  mounted thereon below the pump. As can be seen, the cap or closure  22  includes four piercing members  94 ,  94  equally spaced relative to each other and extending radially inwardly. The end of each piercing member includes a pointed protuberance or tip that projects downwardly. When the closure  22  is moved into the closed position, the tips of the piercing members  94 ,  94  engage the sealed chamber and break the frangible wall thereof to place the lysis fluid in fluid communication with the sample and allow mixing thereof in the sealed vial  16 . The pump  24  is manually engageable to pump the mixture  82  through the outlet port  26  and into the sample port  18 . As can be seen, the pump  24  includes an approximately dome-shaped or hemispherical shaped, flexible wall, which is manually engageable and depressible to pump the lysis fluid and biological sample mixture  82 . One of ordinary skill in the pertinent art will recognize that alternative pump configurations can accomplish the same or similar results, and that the frangible or breakable wall  86  of the sealed chamber  84  may be broken using alternative mechanisms or configurations. The sample receptacle  16  includes an outlet valve (not shown) in fluid communication with the vial body  20 . The outlet valve defines a normally-closed position preventing release of fluid from the interior of the receptacle through the outlet port, and an open position allowing fluid from the interior of the receptacle to flow through the outlet port. The outlet valve may include a valve member movable between a closed position and an open positon. The sample port  18  may include a valve-engaging member that engages the valve member when the outlet port  26  is in fluid communication with the sample port  18  to move the valve member from the closed position to the open position. 
     As shown in  FIGS. 13A through 13C , the membrane slider  32  comprises the membrane support  31  including the solid-state membrane  28  mounted therein. As can be seen, in the illustrated embodiment, the solid-state membrane  28  includes two layers  28 A,  28 B of the glass material received within an aperture  98  in the membrane support  31 . A porous support  100  is mounted on the membrane support  31  below, and underlies the layers  28 A,  28 B of membrane material to support the membrane  28  within the membrane support  31 . In the illustrated embodiment, the porous support  100  is made of a relatively rigid plastic to support the overlying layers  28 A,  28 B of membrane material, but is sufficiently porous to allow any fluids flowing across the membrane to pass therethrough. The porous support  100  engages the respective waste pads  30 ,  30  and microfluidic chip  64  in the sample position  34 , wash positions  36 ,  36  and reaction position  38  to facilitate the flow of fluids across the membrane  28  by capillary action through the porous support and into the waste pads  30 ,  30  or microfluidic chip  64 . As may be recognized by those of ordinary skill in the pertinent art based on the teachings herein, the solid-state membrane  28  can include any desired number of layers, and the layers may take any of numerous different thicknesses, and the porous support  100  may take the form of any of numerous different frits, sieves, grates or like structures, that are currently known, or that later become known, for performing the functions of these components. The upper peripheral seal  54 A is mounted on the upper side of the membrane support  31  and surrounds the solid-state membrane  28 , and a lower peripheral seal  54 B is mounted on the underside of the membrane support and surrounds the underside of the membrane and porous support. As described above, the upper peripheral seal  54 A sealingly engages the outlet port  26  of the sample vial  16  in the sample position  34 , and extends about the blister  46  and seals the blister  46  within the interlock  55  in each wash position  36 ,  36  and reaction position  38 . The lower peripheral seal  54 B sealingly engages the waste pads  30 ,  30  and/or waste pad supports  42 ,  42  in each of the sample position  34  and wash positions  36 ,  36 , and sealingly engages the microfluidic chip  64  and/or chip carrier  66  in the reaction position  38 . The construction and characteristics of the lower peripheral seal  54 B may be the same as or substantially similar the construction and characteristics of the upper peripheral seal  54 A, as described above. In the illustrated embodiment, each peripheral seal  54 A,  54 B is a rubber gasket. However, as may be recognized by those of ordinary skill in the pertinent art based on the teachings herein, the seals may take any of numerous different configurations and may made of any of numerous different materials, and the device any include any desired number of seals, that are currently known or that later become known. In addition, rather than mount the upper and lower seals on the membrane support, they could be mounted on the other components of the device that engage the membrane support, such as the actuator/blister assemblies, the waste pads, the waste pad supports, the microfluidic chip and/or the chip carrier. 
     An exemplary operation of the device  10  includes the following procedural steps:
         1) A biological sample  80  of about 100 μl to about 460 μl is collected in the sample receptacle  16 . Samples can be saliva, urine, blood, or a swab in a buffered solution.   2) The sample receptacle cap  22  is closed and sealed causing the biological sample  80  to mix with a cell lysis solution  78 . The cell lysis solution  78  may be any of numerous different lysis buffers that are currently known, or that later become known for performing this function, including detergent, salt solutions, chaotropic agents, or hypertonic solutions.   3) The sample receptacle  16  is inserted in the test cartridge  14  and the substantially complete contents  82  of the sample receptacle  16  are dispensed from the receptacle  16  into the test cartridge  14  using the manually operated pump  24  in the closure or cap  22  of the receptacle.   4) When the sample mixture  82  is dispensed into the test cartridge  14 , it passes across the solid-state membrane  28  which adsorbs nucleic acids from the sample  80  as the solution  82  passes across the membrane and into the waste pad  30  contained in the test cartridge  14 . The solid-state membrane  28  is then moved, in succession, to two wash positions  36 ,  36  where wash solutions are used to purify the captured nucleic acids.   5) The solid-state membrane  28  is then moved to a final reaction position  38  where a hypotonic solution is used to elute the captured nucleic acids adsorbed to the membrane  28 . The solution and nucleic acids flow into the microfluidic reaction chambers  68 ,  68 . The elution volume is in the range of about 15 μl to about 100 μl.   6) The movement of the elution solution into the reaction chambers  68 ,  68  hydrolyzes reagents stored dry in the reaction chambers.   7) The device  10  is then heated to a pre-determined temperature using a general or customized external heating device (not shown) in the base station  14 . The temperature may be optimized for any number of isothermal amplification chemistries.   8) The amplified target (or the lack thereof) is determined by the amplified target nucleic acid binding to or interacting with colorimetric or fluorescent dyes.   9) The resulting color change or lack thereof allows the user to determine the presence of absence of the target in the original sample  80 .       

     In  FIG. 9 , an exemplary clinical workflow for collecting and assaying a biological sample  80  with the device  10  is illustrated in further detail. First, in step  9 - 1 , a sample  80  is collected using a cotton swab or equivalent device inserted into the mouth of the test subject and rubbed against surfaces containing saliva thereon. In step  9 - 2 , once the swab is sufficiently saturated, the swab is agitated in the lysis buffer  78  of the vial  16  to break down cellular walls in the sample and release the nucleic acids contained therein. Next, in step  9 - 3 , the swab is placed into the sample port  18  of the test cartridge  12  and onto the solid-state membrane  28  positioned in the sample position  34  to transfer the lysed sample  82  onto the solid-state membrane  28 . In step  9 - 4 , the knob  40  of the membrane slider  32  is manipulated to move the membrane slider  32  into a first wash position  36  and move the membrane  28  into a first wash station  44 . Then, also in step  9 - 4 , with the locking member  58  of the first actuator  48  moved to the unlocked position  62 , the first actuator or plunger  48  is manually depressed, releasing the wash solution from the blister  46  at the first wash station to wash the solid-state membrane  28  at the first wash position  36 . Next, in step  9 - 5 , the slider knob  40  of the membrane slider  32  is used to push the membrane slider into the second wash position  36  and move the membrane  28  into the second wash station  44 . Then, also in step  9 - 5 , with the locking member  58  of the second plunger  48  moved to its unlocked position  62 , the second plunger  48  is depressed, releasing the wash solution from the second blister  46  to wash the solid-state membrane  32  at the second wash position  36 . In step  9 - 6 , the slider knob  40  is then used to push the membrane slider  32  into the reaction position  38  and accordingly move the membrane  28  into the reaction station  69 . Then, also in step  9 - 6 , with the locking member  58  of a third plunger  48  moved to its unlocked position  62 , the third plunger  48  is depressed, breaking the blister  46  and releasing its elution buffer onto the solid-state membrane  28  to elute the sample nucleic acids at the reaction position  36  into the microfluidic reaction chambers  66 ,  66  by capillary action. Then, in step  9 - 7 , the test cartridge  12  is inserted into the base station  14 , activating the heating element in the base station to provide the appropriate temperature for the amplification reaction. In step  9 - 8 , the reaction chamber is heated a set period of time, e.g., about 20 minutes, for a LAMP amplification reaction. Then, in step  9 - 9 , the test cartridge may be removed from the base station, and the results of the test are visible through the results window  70 . In step  9 - 10 , if the color of the sample indicator  76  matches the color of the negative control  72 , the test indicates a negative result. If the color of the sample indicator  76  matches the color of the positive control  74 , the test indicates a positive result. 
     Table 1 below includes in the first column the step number, in the second column the procedural steps for assaying a biological sample with the device of  FIGS. 1 and 2 , in the third column the primary function(s) performed at each such step, and in the fourth column variable(s) to be controlled at each such step. 
     
       
         
           
               
               
               
               
             
               
                 TABLE 1 
               
               
                   
               
               
                 Step 
                   
                   
                   
               
               
                 No. 
                 Assay Step 
                 Primary Function(s) 
                 Variables to be Controlled 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
            
               
                 1 
                 Sample Collection 
                 Collect saliva sample from patient 
                 0.1 ml +− 0.02 ml 
               
               
                   
                   
                 Absorb saliva sample 
               
               
                 2 
                 Sample preparation 
                 Chaotropic agent to lysis 
                 0.6 ml lysis chemistry + 0.1 
               
               
                   
                 (use kit) 
                 RNA/DNA from cell nucleus 
                 sample = 0.7 ml total 
               
               
                   
                   
                 Dilute sample to target viscosity 
                 Time for diffusion of 
               
               
                   
                   
                   
                 sample into diluent 
               
               
                   
                   
                   
                 Time for chaotropic 
               
               
                   
                   
                   
                 chemistry to complete 
               
               
                   
                   
                   
                 Perform medium agitation 
               
               
                   
                   
                   
                 (20 sec) 
               
               
                 3 
                 Position membrane 
                 Exposes membrane to open 
               
               
                   
                 and slider at sample 
                 sample port in this position as 
               
               
                   
                 load location 
                 received 
               
               
                 4 
                 Insert sample 
                 Dispense sample into sample port 
                 0.7 ml lysed saliva 
               
               
                   
                 preparation vial 
                 Flow sample onto membrane 
                 Whatman FTA ® membrane 
               
               
                   
                 into test cartridge 
                 surface and through membrane 
                 size/volume 
               
               
                   
                 port and depress 
                 bulk 
                 Absorbent foam in contact 
               
               
                   
                 pump 
                 Absorb into waste pad 
                 with back side of membrane 
               
               
                   
                   
                   
                 Foam capillarity 
               
               
                   
                   
                   
                 Other “glass” membranes 
               
               
                   
                   
                   
                 may be equivalent 
               
               
                 5 
                 Move slider to first 
                 Locate membrane under first 
                 Force/Human Factors 
               
               
                   
                 blister location 
                 blister 
                 Detent to hold membrane in 
               
               
                   
                   
                   
                 place 
               
               
                   
                   
                   
                 Absorbent foam in contact 
               
               
                   
                   
                   
                 with back side of membrane 
               
               
                   
                   
                   
                 Foam capillarity 
               
               
                 6 
                 Actuate first blister 
                 Wash sample on membrane 
                 Qiagen AW1 buffer 0.5 ml 
               
               
                   
                 (first wash buffer) 
                 surface (wash away cell 
                 in blister 
               
               
                   
                 by compressing 
                 pieces/parts and other unbound 
                 Volume dispensed in 
               
               
                   
                 first button 
                 matter) 
                 sample membrane and 
               
               
                   
                   
                 Absorb into waste pad 
                 positive control membrane 
               
               
                 7 
                 Move slider to 
                 Locate membrane under second 
                 Force/Human Factors 
               
               
                   
                 second blister 
                 blister 
                 Detent to hold membrane in 
               
               
                   
                 location 
                   
                 place 
               
               
                   
                   
                   
                 Absorbent foam in contact 
               
               
                   
                   
                   
                 with back side of membrane 
               
               
                   
                   
                   
                 Foam capillarity 
               
               
                 8 
                 Actuate second 
                 Wash sample on membrane 
                 Qiagen AW2 buffer 0.5 ml 
               
               
                   
                 blister by 
                 surface (wash away cell pieces 
                 Volume dispensed in 
               
               
                   
                 depressing second 
                 and parts and other unbound 
                 sample membrane and 
               
               
                   
                 button 
                 matter) 
                 positive control membrane 
               
               
                   
                   
                 Absorb into waste pad 
               
               
                 9 
                 Optionally move 
                 Dry membrane 
                 Force/Human Factors 
               
               
                   
                 slider to a 
                   
                 Wait predetermined period 
               
               
                   
                 membrane drying 
                   
                 of time 
               
               
                   
                 location (this 
               
               
                   
                 operation is not 
               
               
                   
                 present in the 
               
               
                   
                 illustrated 
               
               
                   
                 embodiments) 
               
               
                 10 
                 Move slider to third 
                 Locate membrane under third 
                 Force/Human Factors 
               
               
                   
                 blister location 
                 blister 
                 Seal leakage requirement 
               
               
                   
                   
                 Engage and seal with 
               
               
                   
                   
                 microfluidics chip 
               
               
                 11 
                 Actuate third blister 
                 Dispense elution buffer above 
                 Molecular H2O 
               
               
                   
                   
                 reaction chamber 
                 0.06 ml through membrane 
               
               
                   
                   
                 Dispense molecular water into 
                 and into chip 
               
               
                   
                   
                 internal control and COVID test 
                 0.03 ml into negative 
               
               
                   
                   
                 reaction chambers to mix with 
                 control channel of chip 
               
               
                   
                   
                 dried LAMP reagents 
                 Dispense volume per 
               
               
                   
                   
                 Dispense molecular water into 
                 channel (all three channels 
               
               
                   
                   
                 negative control reaction 
                 positive, negative and 
               
               
                   
                   
                 chambers to mix with dried 
                 sample) 
               
               
                   
                   
                 LAMP reagents 
                 Channel must be filled - 
               
               
                   
                   
                   
                 maximum predetermined 
               
               
                   
                   
                   
                 percentage of air 
               
               
                   
                   
                   
                 Rehydrolization of dried 
               
               
                   
                   
                   
                 reagents 
               
               
                 12 
                 Plug inlet and 
                 Seal ports to prevent evaporation 
                 Seal leak rate 
               
               
                   
                 outlet ports 
                 and/or DNA/RNA escaping into 
                 Leak test negative pressure 
               
               
                   
                   
                 environment 
                 level 
               
               
                   
                   
                 Baseline is paraffin washer 
               
               
                   
                   
                 seal on inlet port and hydro- 
               
               
                   
                   
                 plugging membrane on exit 
               
               
                   
                   
                 port 
               
               
                   
               
            
           
         
       
     
     As set forth in Table 1, first the biological sample  80  is collected and placed in the sample vial  16 . The sample  80  is agitated with the lysis buffer  78  in the vial to break down cellular walls and release nucleic acids contained therein. Next, the sample receptacle  16  is placed into the sample port  18  of the test cartridge  14  and the pump  24  is depressed one or more times, to extrude the sample-lysis mixture  82  through the sample port  18  and across the solid-state membrane  28  positioned in the sample position  34 . The slider knob  40  is then pushed forward to move the membrane slider  32  into the first wash position  36  and accordingly move the membrane  28  into the first wash station  44 . The first actuator or plunger  48  is then depressed, releasing the wash solution from the sealed chamber or blister  46  to wash the sample or captured nucleic acids at the first wash position  36 . Next, the slider knob  40  is pushed to move the membrane slider  32  into the second wash position  36  and accordingly move the membrane  28  into the second wash station  44 . The second actuator or plunger  48  is then depressed, releasing the wash solution from the sealed chamber or blister  46  to wash the sample or captured nucleic acids at the second wash position  36 . Following the second wash, the solid-state membrane is allowed to dry. If desired, the slider knob  40  may be manipulated to move the membrane slider  32  into a drying position. For example, the drying position may be located between the second wash position  36  and the reaction position  38 . Next, the slider knob  40  is pushed to move the membrane slider  32  into the reaction position  38  and accordingly move the membrane  28  into the reaction station  69 . Then, with the locking member  58  of the third actuator or plunger  48  moved to the unlocked position  62 , the third actuator or plunger  48  is depressed, releasing the elution buffer from the sealed chamber or blister  46  to elute the sample nucleic acids at the reaction position into the microfluidic chip  64 . Finally, the test cartridge  12  is inserted into the base station  14  to begin nucleic acid amplification. When the test cartridge  12  is inserted, inlet and outlet ports of the reaction chambers may be closed by paraffin seals. The heating element (not shown) in the base station provides the appropriate temperature for the LAMP reaction, which as shown in the illustrated embodiment, is about 63° C. If necessary, this temperature is sufficient to melt paraffin seals and open respective inlet and/or outlet ports. During this step, after a set period of time, e.g., about 28 minutes, the results of the test will be visible through the results window  70 . The results window  70  contains the negative control  72 , the positive control  74 , and the sample indicator  76 . If the color of the sample indicator  76  matches color of the negative control  72 , the test indicates a negative result. If the color of the sample indicator  76  matches the color of the positive control  74 , the test indicates a positive result. 
     Turning to  FIGS. 14A and 14B , another embodiment of a device including a disposable cartridge that is received within a base station for amplifying and detecting nucleic acids in a biological sample is indicated generally by the reference number  110 . The device  110  of  FIGS. 14A-14B  differs from the cartridge described above in that it is a true microfluidic “Lab On Chip Device.” The device  110  of  FIGS. 14A-14B  does not require a moving membrane slider, waste pad or chip components. Rather, it is a passive-monolithic device that contains each assay function. As can be seen, the device  110  is a smart fluidic channel design, where blister activation sequence and passive valving control each of the assay operations as needed. Inherently, the fluid channel when activated in proper sequence will purge residual fluid from the solid-state membrane with a small burst of air before each sequential step, without an extra supply of air. Toxic lysing buffer is safely contained in a blister and is isolated from the user. As shown in  FIG. 15 , an off-the-shelf absorbent sample collection device  112  (Super-SAL) works with the integrated syringe body style sample port  114  of the device  110 . 
     As shown in  FIG. 16 , a saliva or other biological sample is inserted into a sample port  114 , such as via the sample collection device  112 , where the liquid sample enters a microfluidic conduit  113  of the microfluidic device  110 . The sample collection device  112  includes a saliva collection swab  109  for collecting saliva thereon and a syringe-type plunger  111  for compressing the saliva collection swab  109  within the sample port  114  to release the saliva therefrom and into the microfluidic conduit  113 . A lysis station  116  including a lysis buffer blister  118 , and a first wash station  120  including a wash buffer blister  122 , are then depressed simultaneously. As shown in  FIG. 16 , this pushes the sample solution and the lysis buffer from the lysis blister  118  into a static mixer  124  for mixing the sample and lysis buffer. As shown in  FIG. 16 , the sample-lysis mixture then passes through a solid-state, RNA/DNA capture membrane  126  and into a waste container  128  built into the device  110 . At this point, the target RNA or DNA is captured on the solid-state membrane  126 . The membrane  126  is then washed with the first wash solution from the first wash blister  122  of the first wash station  120 , which also passes through the membrane  126  and into the waste container  128 . The first wash solution from the first wash blister  122  and the sample/lysis mixture are separated by an air pocket generated when the first wash blister  122  is depressed. A second wash station  130  including a second wash blister  132  is then depressed and the second solution passes through the solid-state membrane  126  and into the waste container  128 . The first and second wash solutions are also separated by an air pocket. At this point, the waste container  128  is full and creates a back pressure that diverts the subsequent fluids, i.e., the eluent(s), into the reaction chambers  134 ,  134  through a one-way valve  136  in fluid communication between the solid-state membrane  126  and the reaction chambers  134 ,  134 . An eluent station  138  including an eluent blister  140  is then depressed to, in turn, cause the eluent to pass from the eluent blister  140  through the membrane  126 , and release the captured RNA or DNA into the corresponding reaction chambers  134 ,  134 . In the illustrated embodiment, the device  110  includes two reaction chambers  134 ,  134 ; however, as may be recognized by those of ordinary skill in the pertinent art based on the teaching herein, the device may include any desired number of reaction chambers, including from about one to about five reaction chambers, or any other number of chambers. In a separate fluidic path, a negative control station  142  including a negative control blister  144  is depressed to release a negative control fluid and fill the negative control chamber  146 . As may be recognized by those of ordinary skill in the pertinent art based on the teachings herein, the device  110 , or the base station for receiving the device  110 , may include an actuator or plunger associated with each blister that may be the same as or similar to those set forth above and that may be manually or otherwise engageable to depress and rupture the blisters and, in turn, release their fluids in the desired sequence as set forth herein. 
     Turning to  FIGS. 17A and 17B , the capillary tube LAMP reaction chip  148  included within the device  110  of  FIGS. 14A-14B  is illustrated. LAMP reaction lab assay kits are sensitive to thermal gradients, material interactions and incomplete filling of the reaction chamber(s). Glass capillary tubes have excellent thermal conductivity, are chemically inert, and have capillary fill properties making the structure perform better than other options when used in a LAMP reaction device. 
     The capillary tube LAMP reaction chip  148  illustrated in  FIG. 17A-17B  utilizes three glass capillary tubes  150  that are over-molded or co-molded into a polymer resin chassis  152  that, as can be seen, integrates the capillary tubes, molded fluid channels and features for ancillary components. The integrated design allows for simultaneous filling of the internal control chamber and CV- 19  or other reaction chambers  134 ,  134  with elutant, while the negative control  146  is filled from an isolated blister  144 , as shown in  FIGS. 14A-14B  and described above. 
     Included in each of the three channels  150 ,  150  is a unique reconstitution chamber  154 ,  154  where stabilized master-mix reagents are placed during assembly. In  FIG. 17B , each reconstitution chamber  154 ,  154  is shown capped or sealed to retain the master-mix reagents, such as lyophilized master mix pellets, therein. The chassis  152  defines a negative control inlet  156  in fluid communication between the negative control station  142  ( FIG. 16 ) and the negative control reaction chamber  146 , and a another inlet  158  in fluid communication between the solid state membrane  126  ( FIG. 16 ) and the internal control reaction chamber  134  and the CV- 19  or other reaction chamber  134 . As shown in  FIG. 17A , the chassis  152  defines a split fluid path  160  between the inlet  158  and the two reconstitution chambers  154 ,  154  for the reaction chambers  134 ,  134 . When in use, during elution, each of the reconstitution chambers  154 ,  154  fills and simultaneously reconstitutes each master mix lyosphere into a homogeneous mixture that is delivered directly into the reaction chambers  134 ,  134  and  146  fully mixed and ready for the LAMP reaction. As shown in  FIGS. 17A and 17B , a vent  162 ,  162  is located within the chassis  152  at the downstream end of each reaction chamber  134 ,  134  and  146  and is in fluid communication with the respective reaction chamber. Each vent  162  is filled or plugged with a material that allows air or other gas to vent therethrough as each tube  150 ,  150  is filled with eluent, but that does not allow liquid to pass therethrough, such as a hydrophobic vent membrane, in order to retain the eluent and reaction components within each respective chamber. 
     More generally, glass provides for alternative embodiments outside of capillary tubes. For example, laminated glass structure assemblies could be employed as well. These embodiments use planar (like a microscope slide) glass elements in a laminated structure where internal microfluidic channels are fabricated through common glass fabrication and bonding processes. These structures could also be glass and polymer composite assemblies. 
     In  FIG. 18 , another capillary tube LAMP reaction chip  148  that may be mounted within the device  110  of  FIGS. 14A-14B  includes five reaction chambers rather than the three reaction chambers as shown in the device of  FIGS. 17A-17B  above. As can be see, the LAMP reaction chip of  FIG. 18  includes one negative control reaction chamber  146  and four reaction chambers  134 ,  134 . In the illustrated embodiment, each pair of reaction chambers  134 ,  134  includes an internal control reaction chamber and a CV- 19  or other reaction chamber. As may be recognized by those of ordinary skill in the pertinent art based on the teachings herein, any desired number of glass capillary reaction tube chambers may be employed. One advantage of the glass reaction tubes is that they can both improve the reaction and, as can been seen, allow visualization of the colored test results therethrough. 
     As shown in  FIG. 16 , the device  110  further comprises a lysis leg  164  extending in fluid communication between the lysis station  116  and the sample conduit  113  and configured to direct the flow of the lysis agent from the lysis station/blister into the sample conduit  113 . A first wash leg  166  extends in fluid communication between the first wash station  120  and the sample conduit  113  at a point upstream relative to the lysis leg  164  and is configured to direct the flow of the first wash solution from the first wash station/blister into the sample conduit  113  behind the sample-lysis mixture. As can be seen, the first wash leg  166  is in fluid communication with the sample conduit  113  at a sample-wash junction  168  located adjacent to the sample port  114  and configured to allow a substantial portion of the sample to flow into the sample conduit  113  downstream of the sample-wash junction  168  prior to introducing the first wash solution through the first wash leg  166  and into the sample conduit  113 . The lysis leg  164  is in fluid communication with the sample conduit  113  at a sample-lysis junction  170  located downstream of the sample-wash junction  168  and is configured to allow the lysis agent to mix with the sample and form the sample-lysis mixture and the first wash solution to flow into the sample conduit behind or upstream of the sample-lysis mixture. 
     As shown in  FIG. 16 , the static mixer  124  is in fluid communication between the sample-lysis junction  170  and the solid-state membrane  126  to mix the sample and lysis agent and form a sample-lysis mixture prior to passage across the solid-state membrane  126 . As indicated schematically in  FIG. 16 , in the exemplary embodiment, the static mixer  124  defines a plurality of axially-spaced recesses or grooves  172 ,  172  formed in the sample conduit to facilitate mixing the sample and lysis therein. However, as may be recognized by those of ordinary skill in the pertinent art based on the teachings herein, the mixer may take the form of any of numerous different mixers, including static mixers, that are currently known, or that later become known, such as, for example, pulsatile flow mixers, rotational flow mixers, and combinatorial micromixers. 
     As shown in  FIG. 16 , the second wash station  130  is configured to introduce the second wash solution into the sample conduit  113  following the first wash solution and to pass the second wash solution across the solid-state membrane  126  to purify nucleic acids captured therein. The second wash solution passes across the solid-state membrane  126  and is received in the waste chamber  128 . A second wash leg  174  is in fluid communication between the second wash station  130  and the sample conduit  113  downstream of the first wash leg  166  and is configured to direct the flow of the second wash solution from the second wash station/blister into the sample conduit  113 . The second wash solution is released from the chamber of the second wash blister  132  through the second wash leg  174  and into the sample conduit  113 , is passed across the solid-state membrane  126  to purify nucleic acids captured therein, and is received in the waste chamber  128 . 
     The elution station  138  includes the elution blister  140  defining a sealed eluent chamber containing the eluent. An elution leg  176  extends in fluid communication between the elution station  138  and the solid-state membrane  126 . Upon depressing the elution blister  140 , the eluent is released from the chamber of the elution blister  140  through the elution leg  176  and across the solid-state membrane  126  to elute captured nucleic acids from the solid-state membrane and pass the captured nucleic acids into the reaction chambers  134 ,  134 . 
     As shown in  FIG. 16 , the waste chamber  128  includes a waste chamber vent  178  in fluid communication between the waste chamber and ambient atmosphere. The waste chamber vent  178  defines an open condition and a closed condition. In the open condition fluid passing across the solid-state membrane  126  is received within the waste chamber  128 . In the closed condition fluid passing across the solid-state member  126  is prevented from passing into the waste chamber  128 . During passage of the sample-lysis mixture and first and second wash solutions across the solid-state membrane  126 , the waste chamber vent  178  is in the open condition and the sample-lysis mixture and the wash solutions passing across the solid-state membrane flow into the waste chamber  128  and are prevented from flowing into the reaction chambers  134 ,  134 . As may be recognized by those of ordinary skill in the pertinent art based on the teachings herein, the waste chamber vent may take any of numerous different configurations that are currently known, or that later become known, for performing the function of the waste chamber vent as described herein. For example, the waste chamber vent may include a valve movable between open and closed positions, or could include a hydrophobic membrane, in order to create sufficient back pressure to direct the flow of eluent across the solid-state membrane  126  through the reaction chamber valve  136  and into the reaction chambers  134 ,  134 . 
     As shown in  FIG. 16 , the reaction chamber valve  136  (i) is closed to prevent fluid flow into the reaction chambers  134 ,  134  when the fluid pressure between the solid-state membrane  126  and the reaction chamber valve  136  is below a valve-opening pressure, and (ii) is open to allow fluid flow into the reaction chambers  134 ,  134  when the fluid pressure between the solid-state membrane  126  and the reaction chamber valve  136  is above the valve-opening pressure. In the illustrated embodiment, closure of the waste chamber vent  178  causes the fluid pressure between the solid-state membrane  126  and reaction chamber valve  136  to exceed the valve-opening pressure and thereby allow fluid flow from the solid-state membrane  126  into the reaction chambers  134 ,  134  and not into the waste chamber  128 . 
     As indicated above, the sequence of fluid activation/release from the blisters will purge residual fluid from the solid-state membrane  126  with a small burst of air before each sequential step. In the illustrated embodiment, the bursts of air are provided by the legs  164 ,  166 ,  174  and  176  extending between each respective station and the sample conduit  113  and/or solid state membrane  126 . In other words, each such leg is filled with air such that when the respective blister is depressed and the fluid is released through the respective leg, the air in the leg forms an air gap separating the fluid on either side of the air gap. These pockets or bursts of air also pass over the solid-state membrane and can facilitate drying or evaporating chemical constituents from the membrane, if desired. 
     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.