Apparatus for Addressing Wells Within a Microarray Plate

Abstract

An apparatus, including at least one piezoelectric chip having a working surface, and an opposing at least substantially parallel transducer surface; and at least one interdigital transducer applied to the transducer surface of the chip for generating acoustic energy within the chip in response to an application of an electrical signal to the interdigital transducer; wherein the working surface of the chip is, when in use, in contact with a fluid receptacle to thereby acoustically actuate fluid accommodated within said fluid receptacle, the chip being directly in contact with the receptacle or in contact with a fluid coupling medium that is in contact with the receptacle.

Claims

1. An apparatus, including: a plurality of piezoelectric chips, each chip having a working surface, and an opposing transducer surface at least substantially parallel to the working surface; and at least one interdigital transducer applied to the transducer surface of each chip for generating acoustic energy within each chip in response to an application of an electrical signal to the interdigital transducer; wherein the working surface of each chip is, when in use, in direct or indirect contact with a fluid receptacle to thereby respectively acoustically actuate fluid accommodated within said fluid receptacle, each chip being directly in contact with the receptacle or in contact with a fluid coupling medium that is in contact with the receptacle.

2. The apparatus according to claim 1, wherein said fluid receptacles are a microarray plate including a plurality of wells for respectively accommodating fluid therein.

3. The apparatus according to claim 2, wherein the chips are dimensioned to facilitate acoustic actuation and/or transfer of fluid within a single said well.

4. The apparatus according to claim 3, wherein the chips are located in a grid and/or arbitrary pattern to match the position of individual said wells of the microarray plate.

5. The apparatus according to claim 1, wherein at least one of the chips is supported on a printed circuit board having a conductive circuit layout for providing said electrical signal to the interdigital transducer of the at least one chip.

6. The apparatus according to claim 1, wherein the generated acoustic energy includes surface reflected bulk waves (SRBW).

7. The apparatus according to claim 6, wherein the acoustic energy includes surface acoustic waves (SAW).

8. The apparatus according to claim 6, wherein the acoustic energy includes bulk acoustic waves.

9. The apparatus according to claim 1, wherein the generated acoustic energy includes hybrid surface acoustic waves and surface reflected bulk waves (SRBW).

10. The apparatus according to claim 1, wherein the acoustic actuation of the fluid includes any one or more of manipulation, vibration, mixing, pre-concentration, jetting, nebulisation, particle/cell patterning, centrifugation, fluid or particle or cell transport, drop transport, streaming, and atomisation.

11. The apparatus according to claim 1, wherein a thickness of the chip and a wavelength of the generated acoustic energy are determined by a width and gap of the interdigital transducer patterns.

12. (canceled)

13. A method of acoustically actuating fluid accommodated within one or more wells of a microarray plate including: providing a plurality of piezoelectric chips, each chip having a working surface, and an opposing at least substantially parallel transducer surface; providing at least one interdigital transducer applied to the transducer surface of each chip; and generating acoustic energy within the chip in response to an application of an electrical signal to the interdigital transducer; wherein the working surface of each chip is, in use, in contact with said microarray plate.

14. The method according to claim 13, wherein each chip is dimensioned to facilitate acoustic actuation of fluid within a single said well.

15. The method according to claim 13, and further comprising locating the chips in a grid pattern to match the position of the wells in the microarray plate.

16. The method according to claim 13, and further comprising supporting each chip on a circuit board having a conductive circuit layout for providing said electrical signal to the interdigital transducer of each chip.

17. The method according claim 13, wherein the generated acoustic energy includes surface reflected bulk waves (SRBW).

18. The method according to claim 17, wherein the acoustic energy includes surface acoustic waves.

19. The method according to claim 17, wherein the acoustic energy includes bulk acoustic waves.

20. The method according to claim 13, wherein the acoustic actuation of the fluid includes any one or more of manipulation, vibration, mixing, pre-concentration, jetting, nebulisation, particle/cell patterning, centrifugation, fluid or particle or cell transport, drop transport, streaming, and atomisation.

21. The apparatus according to claim 1, wherein each chip is in contact with the fluid coupling medium that is in contact with the receptacle, wherein the fluid coupling medium is an acoustic fluid, gel or tape couplant such as, but not limited to, a thin layer of water or silicone oil.

22. An apparatus, including: a plurality of piezoelectric chips, each chip having a working surface, and an opposing transducer surface at least substantially parallel to the working surface; and at least one interdigital transducer applied to the transducer surface of each chip for generating acoustic energy within the chip in response to an application of an electrical signal to the interdigital transducer; wherein the working surface of each chip is, when in use, in direct contact with a fluid droplet to be acoustically actuated.

23. The apparatus according to claim 22, wherein the chips are dimensioned to facilitate acoustic actuation and/or transfer of fluid onto the working surface of each chip.

24. The apparatus according to claim 23, wherein the chips are located in a grid.

25. The apparatus according to claim 22 wherein each chip is supported on a printed circuit board having a conductive circuit layout for providing said electrical signal to the interdigital transducer of each chip.

26. The apparatus according to claim 22, wherein the generated acoustic energy includes surface reflected bulk waves (SRBW).

27. The apparatus according to claim 26, wherein the acoustic energy includes surface acoustic waves (SAW).

28. The apparatus according to claim 26, wherein the acoustic energy includes bulk acoustic waves.

29. The apparatus according to claim 22 wherein the generated acoustic energy includes hybrid surface acoustic waves and surface reflected bulk waves (SRBW).

30. The apparatus according to claim 22, wherein the acoustic actuation of the fluid includes any one or more of manipulation, vibration, mixing, pre-concentration, jetting, nebulisation, particle/cell patterning, centrifugation, fluid or particle or cell transport, drop transport, streaming, and atomisation.

31. The apparatus according to claim 22, wherein a thickness of the chip and a wavelength of the generated acoustic energy are determined by a width and gap of the interdigital transducer patterns.

32. (canceled)

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0033] It will be convenient to further describe the invention with reference to the accompanying drawings which illustrate a preferred embodiment of the apparatus according to the present invention. Other embodiments are possible, and consequently, the particularity of the accompanying drawings is not to be understood as superseding the generality of the preceding description of the invention.

[0034] In the drawings:

[0035] FIG. 1(a) respectively shows schematic side and top views of a commercial prior art apparatus which fails to address individual wells;

[0036] FIG. 1(b) respectively shows schematic side and top views of an apparatus according to the present invention which enables individual wells to be addressed in the absence of cross-talk;

[0037] FIG. 1(c) is an photographic image showing a top view of a partially assembled apparatus according to the present invention;

[0038] FIG. 2(a) are photographic images respectively showing different top views of a prior art SAW apparatus;

[0039] FIG. 2(b) are photographic images respectively showing further different top views of the apparatus of the present invention;

[0040] FIG. 3(a) is a photographic image showing simultaneous mixing within wells of a 96-well microarray plate driven using the apparatus according to the present invention;

[0041] FIG. 3(b) are photographic images respectively showing sequential mixing within wells of a 96-well microarray plate driven using the apparatus according to the present invention;

[0042] FIG. 3(c) is a graph showing the normalised mixing index in three different wells of a microarray plate over time using an apparatus according to the present invention;

[0043] FIGS. 4(a) to (d) are respectively photographic images and graphs showing the rapid concentration of a suspension of particles and cells within a well of a microarray plate using an apparatus according to the present invention; and

[0044] FIGS. 5(a) and (b) are respectively photographic images showing the formation of a liquid jet in a well of a microarray plate using the apparatus according to the present invention.

[0045] FIG. 6(a) demonstrates droplet ejection from one piezoelectric chip, while in (b) multiple droplet ejection from multiple piezoelectric chips. The timing of each droplet generation can be programmed individually and independently.

[0046] FIG. 7 respectively shows schematic side and top views of an apparatus according to the present invention which enables individual droplets to be addressed in the absence of cross-talk.

[0047] While the above-identified figures set forth one or more embodiments of the present invention, other embodiments are also contemplated, as noted in the discussion. In all cases, this disclosure presents the invention by way of representation and not limitation. It should be understood that numerous other modifications and embodiments can be devised by those skilled in the art, which fall within the scope and spirit of the principles of the invention. The figures may not be drawn to scale, and applications and embodiments of the present invention may include features, steps and/or components not specifically shown in the drawings.

DETAILED DESCRIPTION OF THE INVENTION

[0048] Referring initially to FIG. 1(a), there is shown a prior art apparatus 1 for addressing wells 7 of a microarray plate 5. That apparatus 1 utilises a piezoelectric substrate 9, typically made from Lithium Niobate (LN). That substrate 9 has a transducer surface 12, upon which are applied two interdigital transducers (IDT) 11. Application of an electric signal to each IDT 11 results in surface acoustic waves (SAW) 15 being generated along the transducer surface 12. The microarray plate 5 is located in contact with the transducer surface 12 so that fluid 3 held within the wells 7 can be acoustically actuated by the SAW 15.

[0049] FIG. 1(b) shows an apparatus 2 according to the present invention for addressing wells 7 of a microarray plate 5. The apparatus 2 includes a plurality of piezoelectric chips 17, for example made from LN, that are respectively dimensioned to address a single well 7 of the microarray plate 5. Each chip 17 has a bottom transducer surface 19 upon which is applied an IDT 21, and an opposing top working surface 23. The transducer surface 19 is at least substantially parallel to the working surface 23. The piezoelectric chips 17 are supported on a printed circuit board PCB) 22 vic pins 29 which supports the chip modules 17 in a grid pattern matching the positions of the wells 7 of a standard microarray plate 5. The PCB 22 includes a conductive circuit layout for enabling an electrical signal to be applied to each IDT 21. The microarray plate 5 is in contact with the top working surfaces 23 of each chip 17. Alternatively, the microarray plate 5 is in contact with the top working surfaces 23 of each chip 17 through a coupling layer 30. The fluid coupling medium is an acoustic fluid, gel or tape couplant such as, but not limited to, a thin layer of water or silicone oil.

[0050] Application of an electrical signal to each IDT 21 results in acoustic energy being generated within each chip 17. The acoustic energy is primarily in the form of surface reflected bulk waves (SRBW) 25 which propagate though the chip 17 to the working surface 23. The Applicant's International publication no. WO2016/179664 describes in more detail how a SRBW is generated. It is in particular noted that SRBW is generated as a result of SAW being propagated along the transducer surface 19 of each chip 17. This in turn generates SRBW 25 that is reflected between the transducer and working surfaces 19, 21 of each chip 17. The generation of SRBW is optimised by having the thickness of each chip 17 at or around the wavelength of the SAW propagated in the transducer surface 19. The acoustic energy generated within the chip 17 can have a hybrid wave configuration due to the combining of the SFBW with the SAW and any other bulk acoustic waves generated within the chip 17. In some embodiments, the chip thickness is matched to the wavelength, set by the width and gap of the IDT patterns, which, in turn, specifies the resonant frequency at which the IDT is excited. In one embodiment, the chip thickness h500 m and the resonate frequency at which the IDT is excited is 10 MHz.

[0051] The apparatus 2 according to the present invention provides a modular and reconfigurable platform that utilises individual chips 17 whose dimensions completely match the well dimensions, so that each well 7 can be directly and individually, or even simultaneously, addressed on demand without incurring crosstalk of the signal with neighbouring wells.

[0052] FIGS. 1(a) and (b) respectively depicts two different principles by which (a) SAWs, and, (b) SRBWs in the present invention, can be coupled from a piezoelectric lithium niobate (LN) substrate 9, 17 to individually address a target well 8 (shown in red) in a microarray plate. FIG. 1(a) shows a commercially available system similar to the Advalytix PlateBooster system, where liquid manipulation in the target well 8 can be driven by exciting two orthogonal SAWs 15 with the aid of a pair of IDTs 11 whose transmission paths intersect beneath the well 8. In this commercial system however, it can be clearly seen that addressability of a single well is not possible since entire rows of wells 7, 8 in the transmission pathway of the SAWs 15 are also concurrently excited. Conversely, in the apparatus 2 in accordance with the present invention, as shown in FIG. 1(b), addressability of a single target well 7, 8 is achieved by mounting standalone LN chips 17 beneath each well 7, 8 that have IDTs 19 on their underside which are electrically connected by plugging the chip modules 18 supporting each chip 17 onto the PCB 22 as shown in FIG. 1(c). The SRBW 25 that is generated on the underside transducer surface 19 of the chip 17 where the IDTs 21 are patterned propagate through the thickness of the chip 17 to the top working surface 23 where they are transmitted into the wells 7, 8. Not only can each well or multiple wells be addressed in this manner sequentially or simultaneously, the modules can also be arbitrarily arranged to flexibly support any desired well or microarray plate configuration, as shown in FIG. 1(c). It is further envisaged that other embodiments, without a fluid receptacle, may be configured to provide addressability of a single droplet, as detailed in FIG. 7.

[0053] FIG. 2 (a) show top view images of a SAW device 27 (left) interfacing with a well 7 in a 24-well microarray plate 5 (centre). The magnified view on the right clearly shows the interference of the acoustic wave generated by the IDTs with neighbouring wells, thus highlighting the inability of the device to provide individual addressability of all the wells on the plate, and the limitation encountered when attempting further size reduction beyond the 24-well plate format. FIG. 2(b) shows top view images of the much smaller chip modules 18, each accommodating a chip 17 (left). The modules 18 are imaged flipped to show the IDTs 21 on the underside of the chip 17. Each of the modules 18 can be mounted beneath every single well 7 on a 96-well plate 5 (centre) and electrically connected to a PCB 22 from beneath (for clarity, only one module 18 has been plugged into the PCB 22). The magnified view on the right shows the possibility for individual addressability of each well 7 or even simultaneous addressability of multiple wells 7 on demand since the chips 17 are not only matched in dimension so that they only transmit acoustic energy into the well that is directly above them, but are also isolated from neighbouring chips 17 by a 3D printed housing that encases them to form the chip module 18. The scale bars denote a length of 10 mm.

[0054] The miniaturisation of the chip dimensions without loss in efficiency is therefore made possible by patterning the IDTs 21 on the underside of the chip 17 and employing SRBWs generated within the chip 17, where the chip thickness (h500 m) is matched to the wavelength, set by the width and gap of the IDT patterns. This in turn specifies the resonant frequencyhere at, 10 MHzat which the IDT 21 is excited. Unlike SAWs, which are only generated and propagate on the bottom transducer surface 19 of the chip 17 on which the IDTs 21 are patterned, these hybrid surface and bulk waves are generated on the IDTs 21 but propagate through the thickness of the chip 17 to the top working surface 23, where they interface with and are transmitted into each well 7 (FIG. 1(b)). In this way, it is possible to reduce the chip dimensions to that comparable with the IDT dimensions since no additional surface area for the transmission of the acoustic wave is required on the transducer surface 19. This then allows the dimensions to be exactly matched to, for example, a 96-well plate as shown in FIG. 2(b). It is also envisaged that the chip dimensions can be further scaled down to accommodate the additional wells in a 384-well plate given that the IDT dimensions are fundamentally limited only by the acoustic wavelength. In still other embodiments, it is envisaged that droplets may be placed directly onto a piezoelectric chip, in the absence of any fluid receptacle, allowing direct interaction between the acoustic waves and the droplets, as further outlined in FIGS. 6 and 7.

[0055] Moreover, the placement of the IDTs 21 on the underside surface 19 allows circumvention of the limited space available for electrical connections that have plagued preceding technologies. This is because it is possible to directly access the IDTs 21 from below by snap fitting each chip 17, mounted in a 3D printed housing 10, onto each of the 96 protruding connection pin pairs 26 soldered on the custom-designed printed circuit board (PCB) platform 22 shown in FIG. 1(c). Traces for the electrical excitation of each individual well 7 are linked to edge connectors 24 at the periphery of the PCB 22 (FIG. 2(b)). These can then be manually or digitally triggered by switches controlled by an Arduino board. Further, the modular nature of the present invention allows flexible reconfiguration of the apparatus to accommodate widely different formats beyond the standard microarray plate, as exemplified in FIG. 1(c).

[0056] The present invention has the capability for on-demand addressability of individual wells to carry out a number of typical liquid handling processes required in the microarray workflow, such as sequential mixing, particle/cell concentration, and single droplet ejection from single or multiple wells via liquid jettingsuch a capacity to carry out a combination of these modes on the same platform is an advance over many current technologies, which are limited to carrying out only a single operation. FIG. 3 shows the possibility of driving on-demand mixing of a small quantity of blue dye which was deposited with the same quantity into each of the 96 wells on the microarray plate that initially contained a pink-dyed solution. Prior to the excitation of the SRBW under select wells, each well contained the same amount of pink-dyed solution (100 l) into which an equal amount of blue dye (1 l) was placed. The ability for individual addressability can both be seen in FIG. 3(a), which shows the mixing to be arbitrarily actuated only in wells that were excited with the SRBW, whereas FIGS. 3(b) and 3(c) shows the possibility of sequentially addressing these individual wells. That negligible mixing is apparent in unexcited wells adjacent to those that were excited suggests minimal crosstalk of the acoustic wave between neighbouring devices as well as crosstalk of the vibrational signal between neighbouring wellsa problem which besets the setup shown in FIGS. 1(a) and 2(a) where the piezoelectric substrate spans neighbouring wells or even many wells. FIG. 3(c) shows where the acoustic excitation beneath wells 1, 2 and 3 were triggered at 0, 3 and 6 s, respectively, as shown by the vertical dashed lines. In FIG. 3(c), a value of 1 denotes the completely unmixed state and a value of 0 denotes a completely mixed state. The scale bars represent a length of 10 mm.

[0057] FIG. 4, on the other hand, shows the possibility for inducing microcentrifugation and hence particle/cell concentration in individual wells on demand. It can be seen from FIG. 4(a), which shows a top view image showing the rapid concentration of a suspension of 11 m fluorescently-labelled particles, that the suspension of polystyrene particles housed in the central well is rapidly aggregated into a tight cluster within 5 s upon excitation of the SRBW beneath that well. The mechanism by which the azimuthal microcentrifugation flow arises, which, in turn, drives the particles to concentrate has been previously described in the Applicant's U.S. Pat. No. 8,998,483. That the liquid in all of the neighbouring wells, which contained the exact same suspension and which were not excited, remained quiescent and that the particles remained dispersed, again testifies to the ability for on-demand single-well addressability without crosstalk with neighbouring wells. The ability of the present invention to rapidly drive the clustering of cells, which has been shown to be a pre-cursor step for spheroid formation, within a specified well is shown in the image of FIG. 4(b). That image shows the rapid concentration of HeLa cells in a select well on a 96-well plate with the present invention within 4 s and 3 s, respectively. It was verified that the cells remained unaffected by the exposure to the acoustic excitation, as can be seen from the results of the short and long term cell viability and proliferation tests in FIGS. 4(c) and 4(d), respectively. FIG. 4(c) shows viability, as measured using a trypan blue assay, and, FIG. 4(d) proliferation, as measured using a MTT assay, of the HeLa cells, immediately and after 24 and 48 hours following their concentration under the acoustic excitation in the central well compared to cells in the neighbouring well which were not acoustically excited. In FIG. 4(d), the proliferation of the cells is quantified by the absorbance at 540 nm of dissolved formazan crystals converted from the MTT reagent by actively proliferating cells.

[0058] The possibility of extracting a small volume of liquid from individual wells at will is shown in FIG. 5, this ability for external sample transfer being useful for sampling individual wells for further separation or analysis. FIG. 5(a) shows the formation, elongation and subsequent pinch-off of a liquid jet within a well when subjected to an acoustic wave pulse from beneath to form a single droplet, which, in turn, is ejected from the well. Successive droplet ejection from different select wells can also be effected by sequentially triggering SRBW pulses under each well, as shown in FIG. 5(b). Sequential single droplet ejection from the different select wells is shown in the inset by successively triggering a SRBW pulse under each well at 0.1 s intervals. Each ejected droplet consisted of approximately the same volume (70050 nl). The scale bars denote lengths of approximately 2 mm. Given the modular setup, the flexibility to simultaneously drive these events from multiple wells or sequentially from the same well can also be easily envisaged, which constitutes a far more attractive alternative compared to the slow and complex process of mechanically moving a single ultrasonic transducer to actuate different wells in the LabCyte Echo system.

[0059] In still other embodiments, as shown in FIGS. 6 and 7, droplets may be placed directly onto a piezoelectric chip, in the absence of any fluid receptacle, allowing direct interaction between the acoustic waves and the droplets. An array of piezoelectric chips may be configured in any format (i.e. one or two dimensional array) and droplets can be placed onto the chips using pipette(s), pump(s), wicking conduit(s) or directly contacting the surface of the chip with another plate containing droplets. It is envisaged that embodiments without a receptacle may be utilised with emerging technology, such as DNA microarray (also referred to a DNA chip); that is, a collection of DNA spots on a solid surface. It is further envisaged that such an embodiment may be used with a droplet volume in the nanolitre scale (10.sup.9 litres), preferably a droplet volume in the picolitre scale (10.sup.12 litres). The capability for on-demand addressability of individual droplets to carry out a number of typical liquid handling processes required in, for example, DNA workflow, such as sequential mixing and single droplet ejection from single or multiple droplets via liquid jetting, represents a substantial advance over existing technology.

[0060] FIG. 7 shows an apparatus 2A according to the present invention for addressing droplets 32, 34 on the working surface. Like apparatus 2, apparatus 2A includes a plurality of piezoelectric chips 17, respectively dimensioned to address a single droplet 32 on the working surface 23. Each chip 17 has a bottom transducer surface 19 upon which is applied an IDT 21, and an opposing top working surface 23. The transducer surface 19 is at least substantially parallel to the working surface 23. The piezoelectric chips 17 are supported on a printed circuit board PCB) 22 vic pins 29 which supports the chip modules 17 in a grid pattern matching the positions of the droplets 32 or 34. The PCB 22 includes a conductive circuit layout for enabling an electrical signal to be applied to each IDT 21. The droplets 32 or 34 are in direct contact with the top working surfaces 23 of each chip 17.

[0061] For example, in the absence of a fluid receptacle, single or multiple droplets may be ejected from the piezoelectric chip array, where there is no acoustic cross-talk (i.e. interference) since each chip is fed with an independent electric wave and each chip is mechanically isolated from the neighbouring ones with a 3D printed case. The 3D printed casing may also provide the structure for which the electrical pins protrude from the printed circuit board (PCB) to contact piezoelectric chips. The independent electrical signals can therefore be programmed in any configuration to locally address each chip to jet, eject droplet or nebulise them. This represents a distinct advantage over existing technologies wherein an entire row of droplets or wells must be actuated or alternatively a single PZT is placed under a target well/droplet and then mechanically moved to a subsequent well/droplets. Instead, the present invention provides a solid-state format which can achieve precise, accurate single drop addressability without interference and furthermore without the need for a mechanically manipulated/moving PZT.

[0062] In summary, a versatile modular plugandactuate concept has been demonstrated that is truly compatible with the ubiquitous microarray titre plate and emerging technologies such as DNA microarrays on a picomolar scale. The present invention is capable of efficiently driving a range of microfluidic actuation processes from mixing, sample preconcentration and external liquid transferall of which comprise critical steps in the drug discovery workflowon demand, with the possibility of addressing individual, multiple or all wells/droplets on the plate sequentially or simultaneously, thus constituting a significant step towards improving the functionality associated with existing laboratory protocols and processes.

[0063] The present invention therefore provides for true sequential or simultaneous single- and multi-well or droplet addressability in a microarray plate using a reconfigurable modular platform from which MHz-order hybrid surface acoustic waves and surface reflected bulk waves can be coupled to drive a variety of microfluidic modes including mixing, sample pre-concentration and droplet jetting/ejection in individual or multiple wells/droplets on demand, thus constituting a highly versatile yet simple setup capable of improving the functionality of existing laboratory protocols and processes.

[0064] The apparatus and method according to the present invention has a number of benefits: [0065] a) contamination is minimised and reduced when compared to conventional liquid handling technologies such as robotically actuating micro-pipetting; [0066] b) while robotically actuating micro-pipetting have volume limitations, this is not an issue for the present invention; [0067] c) robotically actuating micro-pipetting is prone to mechanical failure, which is also not an issue for the present invention; [0068] d) the LabCyte (liquid handling system) device is limited to one drop at a time and only to liquid dispensing not to other sample manipulations such as mixing and/or pre-concentration; [0069] e) conventional SAW devices cannot target individual cells easily. The only way to target individual cells is to include a chip for each cell, however, this is too expensive and too much space on the substrate would be required to house all the individual chips; and [0070] f) this technology can be retrofitted to existing devices.

[0071] The present invention provides a solid-state solution to fluid actuation within multiple wells/droplets, unlike other technologies that would require the transducers to slide beneath fluid wells to target them individually.

[0072] Although the present invention has been described with reference to preferred embodiments, workers skilled in the art will recognize that changes may be made in form and detail without departing from the spirit and scope of the invention. For example, modifications and variations as would be deemed obvious to the person skilled in the art are included within the ambit of the present invention as claimed in the appended claims