APPARATUS, SYSTEM AND METHOD

Abstract

A DNA nano device (2) and a method of producing a DNA nano device (2) for detecting a biomarker are provided. The DNA nano device (2) includes an ensemble of DNA formations. The formations include a detector (4) adapted to accept a target biomarker and thereby to release a trigger DNA sequence; an amplifier (6) adapted for hybridisation with the trigger DNA sequence which thereby releases a key DNA sequence and the trigger DNA sequence or a further trigger DNA sequence; and a responder (8) adapted for hybridisation with the key DNA sequence and thereby to produce a signal. The DNA nano device (2) thereby produces a signal detectable to an observer in response to the detection of the target biomarker.

Claims

1. An ensemble of DNA formations for detecting a biomarker, the formations including: a detector adapted to accept a target biomarker and thereby to release a trigger DNA sequence; an amplifier adapted for hybridisation with the trigger DNA sequence and thereby to release a key DNA sequence and the trigger DNA sequence or a further trigger DNA sequence; and a responder adapted for hybridisation with the key DNA sequence and thereby to cause a signal detectable to an observer to be produced.

2. An ensemble of DNA formations according to claim 1, wherein two or more of the formations are attached to a common body.

3. An ensemble of DNA formations according to claim 2, wherein the detector, at least part of the amplifier, and the responder are attached to the common body.

4. An ensemble of DNA formations according to claim 2, wherein the common body is a DNA formation, a nanoparticle or a substrate.

5. An ensemble of DNA formations according to claim 1, wherein two or more of the formations are attached to the common body in a predetermined arrangement.

6. An ensemble of DNA formations according to claim 1, wherein the detector is adapted to accept a target DNA sequence and thereby to release a trigger DNA sequence.

7. An ensemble of DNA formations according to claim 6, wherein the target DNA sequence is circulating tumour DNA.

8. An ensemble of DNA formations according to claim 1, wherein the detector is adapted for hybridisation with a plurality of biomarkers and thereby to release one or more trigger DNA sequences.

9. An ensemble of DNA formations according to claim 1, wherein the detector comprises one or more probe DNA sequences attached to a DNA body and adapted to hybridise with a target DNA sequence and release a trigger DNA sequence.

10. (canceled)

11. An ensemble of DNA formations according to claim 1, further comprising a blocker adapted to hybridise an interfering DNA sequence.

12. An ensemble of DNA formations according to claim 11, wherein the interfering DNA sequence is cell free DNA.

13. An ensemble of DNA formations according to claim 1, wherein the amplifier comprises a plurality of amplifier subunits, each amplifier subunit adapted to hybridise a trigger DNA sequence and to release a key DNA sequence and the trigger DNA sequence or a further trigger DNA sequence.

14. An ensemble of DNA formations according to claim 1, wherein the amplifier comprises one or more instances of one or more amplifier DNA sequences adapted to hybridise with a trigger DNA sequence; and one or more instances of a key holder DNA sequence adapted to release a key DNA sequence.

15. An ensemble of DNA formations according to claim 14, wherein a first amplifier DNA sequence is adapted to change conformation upon hybridisation with the trigger DNA sequence.

16. An ensemble of DNA formations according to claim 15, wherein a second amplifier DNA sequence is adapted to change conformation upon hybridisation with the first DNA sequence and thereby to release the trigger DNA sequence.

17. (canceled)

18. An ensemble of DNA formations according to claim 1, wherein the amplifier comprises a plurality of amplification pathways for release of different species of key DNA sequences.

19. An ensemble of DNA formations according to claim 1, wherein the responder comprises a DNA capture formation adapted at least partially to enclose a reporter in a first configuration, and to release the reporter in a second configuration.

20. (canceled)

21. (canceled)

22. A method of detecting a target biomarker comprising introducing an ensemble of DNA formations according to claim 1 to a sample of bodily fluid.

23. A method of detecting a target biomarker, comprising: hybridising a target biomarker to a probe and thereby releasing a trigger DNA sequence; hybridising the trigger DNA sequence to an amplifier DNA sequence and thereby causing one or more conformational changes of the amplifier DNA sequence, releasing a key DNA sequence, and releasing a trigger DNA sequence; and hybridising the key DNA sequence to a lock DNA sequence portion of a DNA capture formation and thereby cause a signal detectable to an observer to be produced, preferably by changing configuration of the DNA capture formation to release a reporter.

24. (canceled)

25. A computer program product comprising software code adapted to generate, when executed, a simulation of the ensemble of DNA formations according to claim 1.

Description

[0039] These and other aspects of the present invention will become apparent from the following exemplary embodiments that are described with reference to the following figures in which:

[0040] FIG. 1 is a schematic of a DNA nano device;

[0041] FIGS. 2a-c are diagrams of a detector portion of a DNA nano device in different states;

[0042] FIGS. 3a-d are diagrams of an amplifier portion of a DNA nano device in different states;

[0043] FIGS. 4a and 4b are diagrams of a responder portion of a DNA nano device in different states;

[0044] FIGS. 5a-d are diagrams of an alternative amplifier portion of a DNA nano device in different states;

[0045] FIG. 6 is a diagram of another alternative amplifier portion of a DNA nano device;

[0046] FIG. 7 is a diagram of a DNA nano device with a blocking portion;

[0047] FIG. 8 is a diagram of a blocking portion of a DNA nano device; and

[0048] FIG. 9 illustrates an example of a method of generating a DNA nano device.

[0049] FIG. 1 shows a DNA nano device 2 (also referred to as a nanorobot). The DNA nano device 2 is designed to allow early detection of cancer using a blood sample. The DNA nano device is sensitive enough to distinguish between circulating tumour DNA (ctDNA) 18 and cell-free DNA (cfDNA) which are both present in blood; the ctDNA target fragment 18 may merely carry a single point mutation 19 compared to the cfDNA. When ctDNA is detected the DNA nano device causes a signal that is observable to a user (e.g. a fluorescent signal), thus giving a rapid response to a clinician for diagnosis. Because the DNA nano device identifies specific genetic mutations which originate in tumour cells, it can provide insight into type and stage of the disease.

[0050] The DNA nano device 2 is formed of oligonucleotides—short, single-stranded molecules of DNA—through a self-assembly process, as is well known in the field.

[0051] The DNA nano device 2 includes a number of functional DNA formations that are anchored to a larger common DNA structure, in the illustrated example a rectangular body 3. The functional DNA formations can be between 10 and 90 base pairs long in some examples, but they may be longer (or shorter) as well—e.g. 100 base pairs long, 250 base pairs long, 300 base pairs long, 400 base pairs long, or more.

[0052] In the illustrations DNA strands that make up functional DNA formations are indicated by lines, with the 3′ ends of DNA strands indicated with hooks, and anchor points of DNA formations to the body 3 are indicated with a symbol as indicated in the legend of FIG. 1. The position of a point mutation on the target cfDNA strand is indicated with a symbol as indicated in the legend of FIG. 1. In figures illustrating DNA strands in more detail groups of DNA bases, also referred to as domains, are represented as lower case letters, such as a, b, c, etc. Starred letters indicate complimentary domains—e.g. domain a* is complementary to domain a. Hybridisation of complementary domains is indicated in the illustration by an array of connecting lines between the complementary domains. Domains are a construct to aid in the understanding of the behaviour and interaction of DNA strands. The sequence of DNA bases in a domain may be given (e.g. for a specific target) or it may be selected to engineer the behaviour of a DNA strand (e.g. propensity to form a hairpin loop). In an example a domain can be between 5 and 50 base pairs long, for example 10 base pairs, 15 base pairs, 20 base pairs, 25 base pairs, 30 base pairs, or 40 base pairs long. Different domains on the same strand may have different lengths.

[0053] A wide variety of two- and three-dimensional shapes and forms can be used for the body 3 of the nano device 2, for example a tubular shape, an irregular shape or as illustrated a DNA sheet. The DNA nano device 2 includes a number of functional formations that are designed to interact with the environment (including the sample) and with each other. The functional formations are anchored to the body 3 at suitable positions. This can enable collocation of functional features that are intended to interact with each other, and facilitate their interaction. DNA is a particularly advantageous material for the body 3, as it can permit highly controllable anchoring of functional features at specific positions relative to each other. The functional formations include portions that can be released, exchanged, or accept other portions. This can enable interaction of functional features with the environment and each other. By virtue of the collocation and the ability to incorporate formations performing different functions, the molecular events resulting in an observable signal can be engineered to optimise performance of the DNA nano device 2.

[0054] The DNA nano device 2 illustrated in FIG. 1 includes three functional portions or modules: [0055] detector 4, [0056] amplifier 6 and [0057] responder 8.

[0058] Each module performs a specific function, and by interacting they perform the intended detection and signalling.

[0059] The functional DNA formations interact according to the principles of competitive hybridisation to accept and release one another spontaneously, notably by way of toehold-mediated strand displacement and toehold exchange. Some of the functional DNA formations include portions with bistable configurations and can change configuration in response to interactions with one another (this is also referred to as molecular logic).

[0060] The detector module 4 and the amplifier module 6 are based on DNA computing principles such as toehold-mediated strand displacement, toehold exchange and molecular logic gates (molecules that perform a logical operation based on one or more physical or chemical inputs and a single output).

[0061] The responder 8 is based on dynamic DNA nanotechnology principles (DNA origami) and a cell-free expression system for producing a fluorescent signal.

[0062] The individual modules and their interactions are now described in more detail.

[0063] FIGS. 2a to 2c show the detector 4 in more detail in different states of operation. FIGS. 2a and 2b show a detector 4 before and after exposure to a target T, and FIG. 2c shows the DNA strands of the detector 4 in more detail.

[0064] The detector 4 comprises a DNA strand that acts as probe P. The probe P is anchored to the body 3 of the DNA nano device 2 at an anchor point 12 (the illustrations generally show anchor points with a symbol as indicated in the legend of FIG. 1). The probe P in FIG. 2a is shown before exposure to the target T (the ctDNA sequence to be detected). In the initial configuration an output strand O, also referred to as a ‘trigger’ DNA strand or DNA sequence is (partially) hybridised to the probe strand P.

[0065] In FIG. 2b the target T has displaced the output O by way of toehold-mediated strand displacement. The target T is hybridised to the probe P and the output O is released.

[0066] In use, the target DNA strand T attaches to the toehold region of the probe P and outcompetes the output strand O to displace it from the probe P.

[0067] The probe P and the output O are designed such that the probe P can distinguish between the target T ctDNA (carrying cancer mutations) and cfDNA. The probe P is complementary to the target T, whereas the output O has higher affinity to the probe than cfDNA, but has lower complementarity than the target T.

[0068] The output O triggers the downstream module, the amplifier 6.

[0069] FIGS. 3a, 3b, 3c and 3d show a subunit of an amplifier 6 in more detail in different states of operation.

[0070] The illustrated amplifier 6 subunit includes three DNA strands: two strands that act as amplifiers A1 and A2 and a third strand that acts as a key holder H. Both the amplifiers A1, A2 and the key holder H are tethered to the body 3 of the DNA nano device 2 by way of tethers 13 that are attached to the body 3 at anchor points 12.

[0071] In FIG. 3a the amplifier 6 subunit is shown in its initial configuration before exposure to the output O. In the initial configuration a key DNA strand K is hybridised to the key holder H. The two amplifier strands A1, A2 each include a molecular logic gate; that is, each has a hairpin loop metastable configuration that can be opened or closed depending on the circumstances.

[0072] In FIG. 3b the amplifier 6 subunit is shown after exposure to the output O (from the detector module 4). The trigger strand O facilitates the conformational change of the logic gate of the first amplifier strand A1, that is O hybridises to A1, thereby changing the configuration (opening the hairpin loop) of A1.

[0073] In FIG. 3c the amplifier 6 subunit is shown at a further stage of the amplification procedure. The exposed portion of A1 then hybridises to A2, during which the hairpin loop of A2 opens and A2 displaces O. Subsequently or concurrently, H hybridises to A1 and K is thereby released.

[0074] FIG. 3d shows the amplifier 6 subunit where the second amplifier strand A2 and the key holder strand H are both hybridised to the first amplifier strand A1, and the key K and the trigger strand O have been released.

[0075] As a result of the operation of the amplifier 6 subunit two strands are released: a single key strand K and the original trigger strand O. The trigger strand O is then recycled to another similar amplifier 6 subunit. The released output O continues to a further amplifier subunit in the initial condition as shown in FIG. 3a and by the analogous sequence of steps at that subunit a further key K 28 is released as well as the output strand O.

[0076] The keys K activate the downstream module, the responder 8.

[0077] In a catalytic fashion a single input O can be reused to release multiple key strands K from multiple amplifier subunits. Each amplifier 6 subunit takes an output O, and outputs a key K as well as an output O for further consumption. In the example illustrated in FIG. 1 three amplifier 6 subunits are shown (each comprising two amplifier strands and a key holder), but any number of subunits may be included in the amplifier 6. The number of amplifier subunits can be varied to vary the amplification desired, that is the number of key strands released for each activating strand (output O from the detector). By inclusion of n amplifier subunits, n keys K are released. An initial single output strand O is amplified to n key strands K. This allows for detection of low concentrations of the target ctDNA by providing an amplified response in the form of n key strands K for a single target strand T.

[0078] Other specific amplification processes (generally known as hairpin DNA cascade amplification) may be used to produce multiple key strands catalytically from a single trigger strand (output strand O from the detector).

[0079] FIGS. 4a and 4b show a responder 8 in more detail in different states of operation. FIG. 4a shows a responder 8 before exposure to a key K.

[0080] In the initial configuration shown in FIG. 4a the illustrated responder 8 includes a capturing box C and a reporter gene R captured by the capturing box C.

[0081] The capturing box C includes a number of DNA strands which can form an enclosure that constrains the reporter gene R and prevents exposure of the enclosed portion. The enclosed portion of the reporter gene is a promoter sequence, so expression of the reporter gene is prevented until such time as the reporter gene R is released from the capturing box C and the promoter portion is exposed. The reporter gene R encodes a fluorescent protein such as green fluorescent protein, which can be produced by cell-free protein expression as is known in the art.

[0082] The capturing box C is a bistable DNA formation with a closed and an open configuration. The responder 8 includes a lock DNA strand L. Hybridisation of a key strand K to the lock strand L causes the capturing box C to undergo a change from the closed configuration into the open configuration.

[0083] In FIG. 4b the key strand K is hybridised to the lock strand L, the capturing box C is in the open configuration and the reporter gene R is released.

[0084] The released reporter gene is expressed, and fluorescence of the expressed protein can indicate to an observer the presence of the target ctDNA in the sample.

[0085] The responder may include a number of instances of the capturing box C, each with reporter gene R. Each capturing box C can be opened by one of the key sequences K and thereby produce an observable fluorescent signal for a trace amount of analyte.

[0086] FIGS. 5a to 5d illustrate another amplifier 6 subunit where each amplification subunit includes an amplifier strand A and a key holder strand H, both anchored to the DNA body 3 at anchor points 12. FIGS. 5a-5c show the amplifier 6 subunit in different states of operation, and FIG. 5d shows the DNA strands of the amplifier 6 subunit in more detail.

[0087] FIG. 5a shows the initial configuration where two DNA strands are (partially) hybridised to the amplifier A: a further copy of the output strand O, and an intermediate DNA strand I. In the illustrated example the output strand O is in a hairpin loop metastable configuration.

[0088] In FIG. 5b the output O has become hybridised to the amplifier A by way of toehold-mediated strand displacement. The output O attaches to the toehold region of the amplifier A and outcompetes the intermediate Ito displace it from the amplifier A. The {intermediate I+output O} hybrid is released.

[0089] In FIG. 5c the intermediate I has become hybridised to the key holder H by way of toehold-mediated strand displacement. The output O is released, as is the key K.

[0090] In the next step the released output O continues to a further amplifier subunit in the initial condition as shown in FIG. 4a, and the output strand O is hybridised to a further amplifier A, thus displacing a further {intermediate I+output O} hybrid and by the analogous sequence of steps a further key K is released.

[0091] FIG. 6 shows a variant of the amplifier subunit as described above with reference to FIGS. 3a to 3d. In this variant, the responder requires two keys, K1, K2 to operate (that is, change configuration from closed to open to release the reporter). The amplifier includes two pathways: a right-hand pathway and left-hand pathway (corresponding to the initial ctDNA being detected); for each pathway a probe PR, PL provides the appropriate trigger OR, OL. Because the responder requires two keys in this example high stability of the responder in the initial configuration can be enabled, and spontaneous activation of the responder can be prevented, thus enabling a low rate of false positives. In a related variant a single probe P carries two triggers, OR and OL, and both are released upon hybridisation of the target T to the probe P.

[0092] In a further variant both pathways (right and left) are adapted to amplify each other, creating a feed-forward loop. In this case the detection of a single target ctDNA molecule can cause a chain reaction, with the effect of high sensitivity and the response time of the DNA nano device being very short.

[0093] FIG. 7 shows a variant of the DNA nano device described above. A DNA nano device 50 includes the detector 4, the amplifier module 6 and the responder 8 as described above. In addition the DNA nano device 50 includes a blocking portion (or module) 40 for capturing cfDNA (naturally present in blood) and releasing strands which are non-interacting with the rest of the device. The blocking module 40 includes a number of blocking probes B. The blocking probes B are similar to the probes P of the detector 4 described above, except that they hybridise best with interfering cfDNA strand X whereas the probe P hybridises best with the target ctDNA strand T.

[0094] FIG. 8 shows a probe P, as described above with reference to the detector 8, and a blocking probe B in more detail. The blocking probe B is a DNA strand that is anchored to the body 3 of the DNA nano device 50 at an anchor point. In the initial configuration a non-interacting strand N is (partially) hybridised to the blocking probe B. An interfering strand X, such as a cfDNA strand, is hybridised to the blocking probe B and displaces a non-interacting strand N by way of toehold-mediated strand displacement. The non-interacting strand N released from the blocking probe B does not interact with the rest of the DNA nano device 50. By capturing interfering strands X, such as cfDNA, the blocking probes 40 can prevent non-target DNA strands X from causing unintended release of trigger DNA sequences O, and thereby reduce or prevent cross-talk and increase the specificity of the detector.

[0095] The blocking portion 40 may include a number of instances of the same species of blocking probe as illustrated in FIG. 7, or the blocking portion 40 may include a number of different species of blocking probe (with one or more instances of each species of blocking probe).

[0096] In the examples illustrated above, the functional DNA strands that participate in the designed hybridisations are either anchored directly to the main DNA body 3 at an anchor point 12 (illustrated for example in FIG. 2a), or they are anchored by way of a tether 13 connecting the functional DNA strands to the main body 3 at an anchor point 12 (as illustrated for example in FIG. 3a). In a variant some functional DNA strands are anchored directly, some by way of a tether. Optionally some (or all) functional DNA strands may be provided untethered (e.g. the second amplifier strand A2 of an amplifier subunit), in free solution surrounding the DNA nano device. Direct anchoring gives good control over location and collocation different species of functional DNA strands in order to facilitate their interaction. Tethering gives less control over location, but it permits greater freedom of movement and change of conformation and interaction (hybridisation) with other functional DNA strands, and may provide better exposure of the functional DNA strands. Free solution provision may require relatively high concentrations to ensure sufficient availability. The tether may be a DNA sequence that is selected to discourage interaction with other parts of the system and to discourage formation of secondary structures such as hairpin loops. The tether may be of a material other than DNA such as a biopolymer (e.g. actin, lignin, tubulin). The tether length may be selected to give the functional DNA strand described above a certain range of movement.

[0097] In a variant the body to which the functional units (DNA strands) are attached is not a DNA structure as described above, but another material. For example the body may be a nanoparticle (organic or inorganic), a carbon nanotube, a micelle or a liposome. In a variant the body is a substrate (e.g. glass, silicon, polymer, ceramic) that may form part of a larger device such as a microfluidic device, a flow cell, a well plate, a cuvette, a test strip or other assay devices. Patterning of different functional units to a substrate may be with microfabrication techniques such as photolithography or soft lithography. Covalent bonding, for example, can anchor or tether the functional units to the body.

[0098] For designing a DNA nano device as described above a computer program product is used. The design of the DNA nano device is a multivariate problem and the exact sequence of DNA strands needed to build up a suitable DNA nano device depends on many factors. These include but are not limited to: [0099] The desired target DNA sequence, which is determined by the genetic mutations or biomarkers of interest for medical diagnostics or other purposes. [0100] The chemical stability of the nanorobot as a whole in the sample environment. [0101] The reaction rates [0102] The structure of the individual modules, i.e. the number of probes and amplifiers required for the desired sensitivity and the desired mechanism of transfer of output/inputs between them. [0103] Crosstalk minimisation: e.g. the specificity of the detector portion, how many and what structure of blocking probes, crosstalk between functional portions and units of the same DNA nano device, and also cross talk between different pathways (whether on a common DNA nano device or on separate DNA nano devices) intended to detect different targets. For example, a trigger of the type X (released by detecting ctDNA of type X) should only interact with amplifiers of type X. It should not interact with amplifiers of type Y and Z which indicate other type of mutations and possibly other types of cancer.

[0104] Performance of the DNA nano device can be optimised over these factors. A machine learning system is provided to perform such a multivariate optimisation and design the DNA nano device according to the desired characteristics and given a pool of cancer mutations to be detected.

[0105] The training data for the AI can be provided by simulation data, experimental data or both.

[0106] FIG. 8 shows an example of a set of inputs, outputs, and the resulting DNA nano device 2. The software 62 simulates 60 the performance of the DNA nano device 2 in dependence on the inputs. For the optimum simulated DNA nano device 2 the software 50 outputs DNA sequences to form the DNA nano device, and potentially a feature layout and/or further manufacture information.

[0107] A DNA nano device 2 is generated as specified by the outputs. Properties of the DNA nano device 2 are tested and fed back to the software 50 to validate the simulation 60.

[0108] In the examples described above the DNA nano device is for in vitro analysis of a blood sample; it will be understood that the blood sample may undergo preparation, for example separation of the plasma component, DNA amplification, addition of reagents, or other processing. In a variant the DNA nano device can similarly be used with another bodily fluid or excretion such as lymph fluid, cerebrospinal fluid, pancreatic juice, bile juice, urine, saliva, sputum, stool, pleural fluid, amniotic fluid, tear fluid, or sweat. In a variant the DNA nano device can be used for in vivo detection.

[0109] In the examples described above the DNA nano device is for detection of ctDNA; in a variant the DNA nano device is adapted for detecting circulating tumour RNA.

[0110] In other examples the DNA nano device is adapted for detecting a circulating tumour protein, or other biomarkers such as alpha-feto protein, ion channel proteins or inflammatory markers. In this example the probe described above may be adapted to release a trigger DNA sequence on exposure to the biomarker, and the trigger DNA sequence causes the amplification and response to proceed as described above. In an example the detector described above with reference to FIG. 1 includes a probe DNA sequence that is adapted (by selection of a suitable sequence) to form an aptamer capable of binding a target molecule such as a biomarker. An aptamer is a single-stranded oligonucleotide with a unique 3-dimensional structure and capable of binding targets (typically ligands) with high selectivity and specificity. The aptamer is selected such that binding to a target molecule causes a conformational change of the aptamer (for example a change in the secondary structure of the aptamer). The conformational change of the aptamer then causes release of a trigger DNA sequence, and the trigger DNA sequence causes the amplification and response to proceed as described above. In another variant the detector module of the DNA nano device includes, in addition to the probe DNA sequence P and trigger DNA sequence O hybrid as described with reference to FIG. 1, an aptamer (optionally tethered to the DNA body) adapted to bind a target biomarker. In the initial configuration the aptamer does not bind to the probe P. The aptamer is adapted to change conformation upon binding the target biomarker, thus exposing a region that then hybridises to the probe P and thereby cause release of the trigger DNA sequence O from the probe P; the trigger DNA sequence then causes the amplification and response to proceed as described above.

[0111] In another example the DNA nano device is for detection of other trace DNA, RNA or other biomarkers, for example in forensic analysis.

[0112] While the examples described above refer to a DNA nano device for detecting a single ctDNA target species, in a variant a DNA nano device carries a library of probes, each for a different ctDNA species. The probes may all use the same pathway, or each probe may trigger a distinct pathway accommodated in the DNA nano device and may produce an observable signal specific to a probe type.

[0113] In the examples described above the DNA nano devices respond by way of a reporter gene for cell-free expression of a fluorescent protein. In variants other means of signalling may be implemented, for example by way of: [0114] fluorescence where a key K output at the amplification stage is adapted to displace a quencher from a suitable fluorescent tag; [0115] fluorescence resonance energy transfer (FRET) where a key K output at the amplification stage is adapted to include a FRET donor or acceptor molecule, and a reporter strand includes a complementary FRET acceptor/donor and is adapted to receive a key K strand; [0116] radioactive labelling, magnetic bead decoration, surface-enhanced Raman spectroscopy (SERS), gold or silver nanoparticle decoration

[0117] It will be understood that the present invention has been described above purely by way of example, and modifications of detail can be made within the scope of the invention.

[0118] Reference numerals appearing in the claims are by way of illustration only and shall have no limiting effect on the scope of the claims.

[0119] The term ‘comprising’ as used in this specification and claims preferably means ‘consisting at least in part of’.