Disposable thermal in-vitro diagnostic apparatus and method of conducting an in-vitro diagnostic test

A portable, disposable in-vitro diagnostic apparatus and method of performing an in-vitro diagnostic test is provided. The apparatus includes a body configured to be hand held. The body has a reaction medium supply chamber configured in selective fluid communication with a reaction chamber via a fluid conveying channel. The reaction chamber is located beneath a sample reaction chamber. The reaction medium supply chamber contains a reaction fluid therein and the reaction chamber contains a thermal reaction medium therein. The reaction fluid is selectively reactive with the thermal reaction medium to produce one of an endothermic or exothermic reaction beneath the sample reaction chamber.

BACKGROUND

1. Technical Field

This invention relates generally to in-vitro diagnostics, and more particularly to apparatus and methods for conducting thermally controlled in-vitro diagnostics.

2. Related Art

Biological diagnostic tests are a fundamental component in the process of determining the state or condition of a biological environment. These environments include, but are not limited to, human healthcare, agriculture, live stock management, municipal systems management, and national defense. Molecular tests that utilize nucleic acid detection provide an incredibly competitive level of specificity, sensitivity, and rapid timing from sampling to result. Nearly all nucleic acid detection approaches require signal amplification, such as Polymerase Chain Reaction (PCR), to generate detectable amounts of the targeted nucleic acid segment. Traditional mechanisms used in nucleic acid detection tests requiring PCR utilize high powered, immobile, non-disposable equipment to achieve large temperature gradients with high resolution. Although, these mechanism prove useful to obtain the test results desired, they are costly and are limited to use in fixed locations, given they require large, immobile equipment.

An assay is a sequence of steps or procedures used measure the presence or absence of a substance in a sample, the amount of a substance in a sample, or the characteristics of a sample. An example of a common point of care assay, or an assay conducted by a layperson is a blood glucose test. In this test, the blood is mixed with glucose oxidase, which reacts with the glucose in the sample, creating gluconic acid, gluconic acid in turn, reacts with a chemical in the assay called ferricyanide, producing ferrocyanide. Current is passed through the ferrocyanide and the impedance reflects the amount of glucose present.

Thermal cycling is a common method of accelerating a chemical reaction or promoting a biological event. Thermal cycling is a used to amplify segments of nucleic acid by via PCR. As shown inFIG. 1, in an example of a thermal cycling process, high temperature thermal cycling is used to physically separate two stands of a double helix DNA. This process is commonly referred to as denaturing, wherein the linked strands of the DNA are separated into two single strands. Temperatures maintained during denaturing are typically in the range of 94° to 96° C. The two separated strands from the denatured DNA are used as templates to logarithmically replicate identical copies of the targeted segment of DNA. Upon reducing the temperature to approximately 52° C., synthetically designed primers bind to, or “anneal” to the template DNA strands such that they flank both sides of a target segment of denatured strands of DNA. DNA Polymerase and other cofactors then cause the primer to extend fully along the denatured strands of DNA and thus, a new double stranded piece of DNA is generated, wherein a lower controlled temperature in the range of 70° to 80° C. is maintained.

The thermal cycling discussed above during denaturing and DNA replication is typically controlled in a laboratory machine. The machine includes electrical heating and cooling elements configured in electrical communication with thermal sensors in a closed loop control scheme. These machines are relatively large, immobile and expensive.

SUMMARY OF THE INVENTION

A portable, disposable, low-powered thermal cycling in-vitro diagnostic apparatus is provided in accordance with one aspect of the invention. The apparatus is economical and it provides a quick, reliable and economical method for performing a thermally activated in-vitro diagnostic test on a selected specimen, such as DNA, for example. Further, the apparatus automatically provides a predetermined thermal cycle over a predetermined time to allow a desired analysis of the specimen contained within the apparatus to be performed without need of human intervention. The apparatus produces exothermic and endothermic thermal energy, in a balanced and controlled environment via a chemical reaction between reactants contained within the apparatus. The reactants are provided and automatically combined in a predetermined manner to provide the desired thermal cycle needed to analyze the particular specimen. Accordingly, the apparatus in accordance with one aspect of the invention is wholly self-contained, and thus, is fully functional to perform the desired analysis without need of external apparatus.

In accordance with another aspect of the invention, the apparatus can be configured for operable attachment to an external source of power. The external source of power can be provided as a hand held device that is configured for attachment to the in-vitro diagnostic apparatus. The separate source of power can be re-used, while the apparatus remains disposable.

In accordance with another aspect of the invention, the external energy source can be configured to produce the desired energy profile within the apparatus. A simple circuit may provide for intermittent, or cycling of the energy source, resulting in a thermal cycling profile in the reaction chamber of the apparatus.

In accordance with another aspect of the invention, a portable, disposable in-vitro diagnostic apparatus includes a body configured to be hand held. The body has a reaction medium supply chamber configured in selective fluid communication with a reaction chamber via a fluid conveying channel. The reaction chamber is located beneath a sample reaction chamber. The reaction medium supply chamber contains a reaction fluid therein and the reaction chamber contains a thermal reaction medium therein. The reaction fluid is selectively reactive with the thermal reaction medium to produce one of an endothermic or exothermic reaction beneath the sample reaction chamber.

In accordance with another aspect of the invention, a conductive barrier separates the sample reaction chamber from the reaction chamber.

In accordance with another aspect of the invention, the portable, disposable in-vitro diagnostic apparatus includes an overflow chamber downstream from the reaction chamber.

In accordance with another aspect of the invention, a rupturable membrane selectively closes off the fluid conveying channel from the reaction medium supply chamber.

In accordance with another aspect of the invention, a self-actuatable valve is disposed between the fluid conveying channel and the reaction chamber. The self-actuatable valve is movable between a closed position to close off the fluid conveying channel from the reaction chamber and an open position to allow fluid to flow from the fluid conveying channel into the reaction chamber.

In accordance with another aspect of the invention, a method of conducting an in-vitro diagnostic test is provided. The method includes providing a body configured to be hand held having a reaction medium supply chamber with a reaction fluid contained therein in selective fluid communication with a thermal reaction medium contained in a reaction chamber via a fluid conveying channel wherein the reaction chamber is beneath a sample reaction chamber; disposing a sample in the sample reaction chamber; and dispensing a reaction fluid from the reaction medium supply chamber into the reaction chamber and producing one of an endothermic or exothermic reaction within the reaction chamber beneath the sample reaction chamber.

In accordance with another aspect of the invention, the method further includes depressing a bulb and causing the reaction fluid to rupture a membrane and flow into the reaction chamber.

In accordance with another aspect of the invention, the method further includes causing a self-actuable valve to move between open and closed positions to respectively allow and prevent the flow of the reaction fluid into the reaction chamber in response to the endothermic or exothermic reaction.

In accordance with another aspect of the invention, the method further includes causing the self-actuable valve to move between the open and closed positions by buffering a portion of the thermal reaction medium.

In accordance with another aspect of the invention, the method further includes reacting water with CuSO4 to produce an exothermic reaction.

In accordance with another aspect of the invention, the method further includes reacting oxygen with iron to produce an exothermic reaction.

In accordance with another aspect of the invention, the method further includes reacting citric acid with sodium bicarbonate to produce an endothermic reaction.

DETAILED DESCRIPTION OF PRESENTLY PREFERRED EMBODIMENTS

Referring in more detail to the drawings, disposable in-vitro diagnostic apparatus, referred to hereafter as apparatus10, constructed in accordance with various presently preferred embodiments of the invention are illustrated, by way of example and without limitation. The apparatus10provide a quick, reliable and economical method for performing a thermally activated in-vitro diagnostic test on a selected specimen. The apparatus10is both economical in manufacture and in use, is readily portable, such that it is sized to be hand held for single use, whereupon the apparatus10is disposable after use, particularly given the low cost associated with its manufacture. The apparatus10can be provided as an all inclusive device, including an integral exothermic reaction heat producing or endothermic heat reducing and regulating mechanism, or it can be configured for operable electrical connection to a separate energy source to power a thermal reaction within the apparatus (FIGS. 12A-12B). If configured for operable attachment to a separate energy source, the energy source and/or the apparatus10can be configured to regulate the thermodynamics within the apparatus10, as discussed further below.

The heat production via the exothermic chemical reaction or heat reduction via the endothermic reaction may be achieved by combining two or more elements or chemical substances, known as reactants, provided and contained entirely and integrally within the apparatus10. The combination of the reactants produces a product and a release of energy or a reduction of energy from the surrounding environment. The change in enthalpy, (thermodynamic potential) for an exothermic reaction is less than zero (<0), and thus, a larger value of energy released in the reaction is subtracted from a smaller value of energy used to initiate the reaction, the opposite being true for an endothermic reaction.

The exothermic reactants may be provided individually as, or as a combination of, solids, liquids and gasses. Some examples include:

Combining anhydrous copper (II) sulfate with water (Solid+Liquid):
CuSO4+5H2O→CuS4·5H2O+HEAT; or

Combining oxygen with iron (Gas+Liquid):
4Fe+3O2→2Fe2O3+HEAT.

The endothermic reactants may be provided individually as, or as a combination of, solids, liquids and gasses. Some examples include:

Additionally, as shown inFIG. 6, heat or cooling can be generated remotely from the sample being heated or cooled. Remote transport of thermal energy is provided by conduction through a material of high thermal conductivity, expressed in units of power per distance multiplied by temperature; W/m·K or W/m ° C., Btu/(hr ° F. ft2/ft). Remote heat transfer provides distribution of the thermal energy throughout the disposable device to locations desired, and also provides a source of varying temperature gradient to one or more points.

In accordance with one aspect of the invention, as shown inFIGS. 7-9, the apparatus10can include a thermal cycling mechanism12to regulate and vary the heat or subtraction of energy transferred to or from to the sample. The thermal cycling mechanism12can be configured via a multi-layered composite, producing a thermal cycle based on the rate of reaction, quantity of reactants present and desired thermal cycling frequency. As the exothermal or endothermal reaction progresses, the heat or cooling produced by the chemical reaction may be regulated by the thermal regulating mechanism12, which can include a valve member13, such as a bimetallic member, located between the thermal source and the sample being heated or cooled. The bimetallic member13is designed to move between an actuated and non-actuated position at predetermined temperatures, thus, providing an automated temperature regulation and a thermal cycling profile. This function can be applied to the above described remote thermal transport element (FIG. 6) and to an electrical thermal energy control by acting as an electric switch (FIGS. 12 and 13).

As shown inFIGS. 9A-9C, thermal cycling within the apparatus10can be provided via a bimetallic, thermally actuated valve14, which, at a predetermined temperature, changes from concave (FIG. 9A) to convex (FIG. 9B) with respect to a valve port16. Upon heating or cooling to the predetermined actuation temperature, the valve14moves to a closed convex configuration (FIG. 9B), thus closing off flow of the reactant through the valve port16, and upon cooling the valve14moves to an open concave configuration (FIGS. 9A and 9C), thus restoring the flow of the reactant through the valve port16. The mechanical threshold of actuation results in a “snapping action” upon the valve member13“crossing over center”.

As shown inFIGS. 12A-13B, in addition to, or in lieu of producing heat via a chemical reaction mechanism within the apparatus10, heat may be generated by placement of an electrical heating element18proximal to the sample being heated. The heating element18may be actuated by an energy source20, such as a DC battery, by way of example, located integrally within the disposable apparatus10(FIG. 13A-13B), or from a separate energy source20external to the apparatus10, such as a DC battery, by way of example, which is configured to interface electrically with the disposable device (FIGS. 12A-12B).

An electrical switch can be provided by a manual switch or by a low-level resistive or capacitance switch via contact with the sample fluid and coupled with a transistor on the apparatus10. Furthermore, a temperature reactive bimetallic switch may be employed, proximal to the fluid sample, such as discussed with regard toFIGS. 7A-7C, to regulate the temperature, or to produce a thermal cycling profile.

As shown inFIGS. 11A-11B, the fluid reactant channeled to promote the thermal reaction may be introduced to the reaction chamber by a wick member22, and/or a controlled capillary channel. This mechanism and method of channeling the reactant can be configured to regulate the rate of fluid transfer as desired, thereby allowing the heat or cooling generated to be controlled, as desired. One or more fluid blisters24containing the fluid reactant F, or other sources of reactant, are provided to initiate the thermal reaction cycle. Additionally, fluid reactants having different reactivity can be provided via the different sources of reactant, which provide a mechanism and method for varying thermal profiles. The sequential introduction of the fluid reactants at prescribed time intervals further facilitate regulating the thermal profile and timing thereof.

InFIG. 2, an exothermic multi-layered composite medium, shown as a disk stack26of alternating exothermic values, is illustrated in accordance with one aspect of the invention. As the reaction progresses, the quantitative level of exothermic energy produced alternates in accordance to the desired thermal output level, thus, achieving the desired thermal cycle. The outer peripheral sides of the stack26can be shielded from the chemical reactant, such as by an inert coating, not subject to dissolution upon exposure to the reactive fluid. The exothermal reactant chemical discussed above, CuSO4 is provided as an example, and thus, it should be recognized that additional endothermic and exothermic reactive solids could be used.

InFIG. 3A, an exothermal or endothermal composite medium, shown as a bead or sphere28, is illustrated in accordance with another aspect of the invention, which is a derivation of the exothermic composite disk stack26, with the primary difference of surface area and the number of individual points of reaction. A plurality of the composite spheres28provides an increased outer reactive surface area, and thus, provides a more intense reaction. The plurality of reactive spheres28can be provided in a predetermined configuration and quantity to produce the desired thermal cycling effect.

InFIG. 3B-3C, exothermal or endothermal composite beads or spheres30are illustrated in accordance with another aspect of the invention. The exothermal or endothermal spheres30are similar to the previously discussed composite beads or spheres28ofFIG. 3A, however, they are buffered with a coating32. The buffered coating32provides a timed-release of the active agent34in the exothermic reaction. Reactive spheres30having differing buffers32and thicknesses of buffer32(FIG. 3Bbeing thicker thanFIG. 3C) result in a staged thermal cycle, as desired. A further difference with the buffered exothermal or endothermal composite spheres30over the spheres28ofFIG. 3Ais that the buffered spheres30ofFIGS. 3B-3Ceach consist of only one reactive element34internal to the buffered coating32.

InFIGS. 4A-4B, an apparatus10, constructed in accordance with one aspect of the invention, includes a unitized housing or body11sized to be hand held, and thus, the body11is readily carried in a palm of a hand. The body11can be constructed of any suitable materials, preferably relatively inexpensive moldable polymeric materials. The body11carries and provides the components of the apparatus10as a unitized, portable and disposable assembly. In accordance with one aspect of the invention, the body11carries an exothermal or endothermal composite series of plates or disks26, also referred to as a “reaction” stack, such as discussed and shown inFIG. 2, residing under a sample reaction chamber36. The chamber36is separated from the thermal reaction stack26by a thermally conductive barrier38, thus isolating the byproducts of the thermal reaction from the sample40being heated or cooled. A self-contained reactive fluid F is encapsulated and contained in a flexible and sealed elastic bulb or blister, also referred to as a reaction fluid supply chamber24, shown as a “blister pack”, that is in selective fluid communication with the thermal reaction medium or stack26contained in a reaction chamber50via a fluid conveying channel42, and to a distal (downstream of the sample40) waste/overflow chamber44, which is vented to atmosphere or another chamber. The sample chamber36may be covered and seal off by an optically clear cover window46to permit visual or optical analysis of the reaction within the chamber36.

As best shown inFIGS. 5A-5B, the blister24containing the reactive fluid F is depressed via an externally applied force, such as by manually depressing the blister24with a thumb or finger, for example, thereby causing the reactive fluid to selectively rupture a membrane48closing off the channel42. Upon the membrane48being ruptured, the fluid passes through the channel42to the reaction chamber50containing the thermal reaction stack26. The stack26reacts with the reactive fluid F, thereby producing an endothermic or exothermic release of energy. The thermally conductive barrier38conducts or removes the thermal energy to or from the sample40being heated or cooled. Excess fluid travels to the waste/overflow chamber44, and the gases produced by the reaction are vented via a hydrophobic membrane52, thus balancing the pressures present within the disposable apparatus10. FIG.5B is similar toFIG. 5A, however, it incorporates at least one or more types of the exothermal or endothermal reactive beads or spheres28,30discussed above and shown inFIGS. 3A-3C. As withFIG. 5A, the reaction is vented, thus balancing the pressures present within the disposable apparatus10.

FIG. 6depicts the use of a thermal conductor54to transfer the thermal energy to or from the point of an assay reaction. This allows remote generation of energy, thus, multiple sources of energy may be directed to a single point or location. The figure discussed above represent only a single location of energy.

FIG. 7Adepicts the thermally cycling mechanism in the form of a deformable bimetallic or shape memory alloy disk12. The objective of this component is to regulate the level of thermal energy extended to or from the assay chamber36. The thermal barriers38shown inFIGS. 4A-4Band5A-5B can be provided as such, such as via a bimetallic, shape memory alloy or other thermally deformable material, thus allowing for the thermal barrier12to automatically change its configuration at a prescribed temperature. The deformable thermal barrier12is in contact with the thermally conductive member38prior to the thermal reaction. The bias shape ofFIG. 7Bprovides positive physical abutment of the thermal barrier12with the thermally conductive member38upon assembly of the thermal barrier12and prior to heating or cooling. It should be recognized that the thermal barrier12can be constructed having any desired shape.

FIG. 7Cdepicts the thermally deformable, or shape memory alloy barrier12, deflected at a prescribed temperature, lifting off of the thermally conductive member38, thus limiting the conduction of energy from the thermal reaction chamber50via an insulation gap G. The natural resonate frequency governing the period between deflection and non-deflection is a function of design and temperature. This frequency results in the desired thermal cycling between the configurations shown inFIGS. 7B and 7C.

FIG. 8Aillustrates an apparatus10including the thermally deformable barrier12ofFIGS. 7A-7C, in the un-actuated state. While, in its un-actuated state, thermal energy resulting from a combination of the fluid reactant F and the exothermal or endothermal composite medium is conducted through the thermally conductive barrier38and through the thermally deformable barrier12.

FIG. 8Bdepicts the thermally deformable barrier12deflected (actuated) at a prescribed temperature to provide the gap G, thus limiting the conduction of energy from the thermal reaction chamber50to the sample chamber36. Upon reaching a designed target temperature, the thermally deformable barrier12looses physical conduct with the thermally conductive barrier38, thus eliminating conduction of heat or cooling from the reaction chamber50to the sample chamber36. The thermally deformed material12returns to its physically conductive position (FIG. 8A) upon cooling. The cooling process occurs due to the lack of physical contact with the heated or cooled conduction surface38.

FIGS. 9A-9Bdepict the valve14comprised of a thermally deformable or shape memory alloy device13, capable of deflecting at a prescribed temperature, thus regulating the flow of reaction fluid F to the reaction chamber50. InFIG. 9Athe valve14is shown in the open position, wherein the fluid F is able to flow to the reaction chamber50. A thermal conductor54, as described and shown inFIG. 6, may be adapted to this design to allow control of the valve14from a remote location. This valve14would also produce a thermal cycling, upon opening and closing, to regulate a thermal reaction.

FIG. 9Bdepicts the valve14in the closed position, thereby preventing the flow of the fluid reactant F to the reaction chamber50. As such, the thermal reaction “down-stream” of the valve14is impeded, thus reducing the thermal energy the valve14is exposed to, resulting in a reopening of the valve14.

FIG. 9Cdepicts a thermally deformable shape memory valve14with electrical contacts56for electrical actuation. Nitinol, a common shape memory alloy undergoes elastic deformation upon thermal or electrical exposure. The valve14deflects upon passing current through the valve14.

FIG. 10Aillustrates an apparatus10incorporating the valve14inFIGS. 9A-9C, and also the thermally deformable member12described inFIGS. 7A-8B. The valve14is shown in the open position, permitting flow of the liquid reaction agent F to the solid reactant.FIG. 10Bdepicts the integrated thermal valve14in the closed position, thus inhibiting the flow of the liquid reaction agent into fluid contact with the solid reactant26.

FIG. 11Adepicts a device for introducing the reaction fluid into the reaction chamber remotely via the wick member22. The wick22regulates the flow of the reaction fluid into the reaction chamber50, thus regulating the rate of the reaction. The wick22can be provided having any desired cross-sectional shape along its length, and further, can be formed from any desired wicking material, thereby allowing the flow rate of wicking of the reaction fluid to be precisely controlled.

FIG. 11Bis a cross-section of an apparatus10incorporating the remote fluid wicking device22ofFIG. 11A.

FIG. 12Adepicts a disposable apparatus10constructed in accordance with another aspect of the invention including the embedded thermal element18and electrical contacts58for interface with the electrical energy source20. The thermal element18includes a resistive element60embedded in the reaction chamber50. The external energy source20provides the current to heat the resistive elements60.

FIG. 12Bshows the apparatus10inFIG. 12Ainterfacing with an external energy source20. The energy source20is capable of producing an energy profile, which in turn, produces the desired thermal profile. A simple circuit may provide for intermittent, or cycling of the energy source20, resulting in a thermal cycling profile in the reaction chamber50.

FIG. 13Ais a schematic of a “on-device”, battery powered heating element, with a simple transistor relay. The transistor receives its signal from a pair of contacts embedded in the reaction chamber50which are “connected” upon contact with the fluid sample being heated. The fluid sample provides and electrical path between the contacts, thus completing the circuit. The addition of a thermal switch would provide a thermal cycling profile.

FIG. 13Bdepicts an embodiment of the schematic inFIG. 13Ain a disposable apparatus10. The switching transistor provides the means to turn on and of the heating element, and when coupled with one of the thermally cycling devices describe prior, may also yield the thermal cycling profile.