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MOLECULAR SENSORS

20170370917 · 2017-12-28

    Inventors

    Cpc classification

    International classification

    Abstract

    The invention relates to a sensor molecule for detecting a target molecule comprising: (a) a rod-like molecule L and a rod-like molecule R connected to each other by a joint molecule C to form a hinge; (b) a target binding molecule A bonded to the end of rod-like molecule L opposite to the joint molecule C; (c) a binding molecule A′ bonded to the end of rod-like molecule R opposite the joint molecule C; wherein the target binding molecule A is arranged to bind to the target molecule to be detected, and binding molecule A′ is arranged to bind to: i) the same target molecule as target binding molecule A; or ii) a complex of the target binding molecule A and the target; and wherein the hinge is biased into an open position, such that target binding molecule A and binding molecule A′ are biased apart by the hinge.

    Claims

    1. A sensor molecule for detecting a target molecule comprising: (a) a rod-like molecule L and a rod-like molecule R connected to each other by a joint molecule C to form a hinge; (b) a target binding molecule A bonded to the end of rod-like molecule L opposite to the joint molecule C; (c) a binding molecule A′ bonded to the end of rod-like molecule R opposite the joint molecule C; wherein the target binding molecule A is arranged to bind to the target molecule to be detected, and binding molecule A′ is arranged to bind to: i) the same target molecule as target binding molecule A; or ii) a complex of the target binding molecule A and the target; and wherein the hinge is biased into an open position, such that target binding molecule A and binding molecule A′ are biased apart by the hinge.

    2. The sensor molecule according to claim 1, wherein the presence and binding of a target molecule by target binding molecule A, and the binding of binding molecule A′ to either i) the target molecule or ii) the complex of the target binding molecule and target molecule, is arranged to bias the hinge into a closed position in opposition to the force of the hinge, which is biased to an open position.

    3. The sensor molecule according to claim 1 or claim 2, wherein the sensor molecule comprises a state denoted as the ON state wherein A is attracted towards A′ and the hinge is arranged to repeatedly open and close; and a state denoted as the OFF state wherein the hinge is in an open position and A is not attracted towards A′.

    4. The sensor molecule according to claim 3, wherein the ON state is detectable.

    5. The sensor molecule according to any preceding claim, wherein the target binding molecule A and/or the binding molecule A′ are capable of emitting a signal for detection when they are in proximity to each other, or bound to each other.

    6. The sensor molecule according to any preceding claim, wherein the sensor further comprises a signal molecule B and a signal molecule B′.

    7. The sensor molecule according to claim 6, wherein the detectable ON state signal is provided by the pair of signal molecules B and B′ being brought into sufficient proximity to cause a detectable ON state signal to be emitted.

    8. The sensor molecule according to claim 6 or claim 7, wherein the signal molecule B and/or B′ comprises a chromophore, fluorophore or bioluminescent molecule; and/or the target binding molecule A and/or binding molecule A′ comprises a chromophore, fluorophore or bioluminescent molecule.

    9. The sensor molecule according to any of claims 4 to 8, wherein the detectable ON state signal is provided by resonance energy transfer (RET) between target binding molecule A and binding molecule A′.

    10. The sensor molecule according to any of claims 6 to 9, wherein the detectable ON state signal is provided by resonance energy transfer (RET) between signal molecule B and signal molecule B′.

    11. The sensor molecule according to claim 9 or 10, wherein the resonance energy transfer (RET) is Förster resonance energy transfer (FRET) or bioluminescent resonance energy transfer (BRET).

    12. The sensor molecule according to any of claims 6 to 11, wherein the signal molecule B is bound to ligand binding molecule A and the signal molecule B′ is bound to binding molecule A′ or vice versa.

    13. The sensor molecule according to any of claims 6 to 12, where B and B′ are respectively bound to A and A′, or vice versa, the binding is via a spacer molecule.

    14. The sensor molecule according to any of claims 6 to 13, wherein the sensor molecules B and B′ each comprise a part of a split molecule.

    15. The sensor molecule according to claim 14, wherein the split molecule comprises a split fluorescent protein.

    16. The sensor molecule according to claim 14, wherein the split molecule comprises a biological active molecule that can be split into two or more parts, such that when the parts are brought back together in the presence of a target molecule of the sensor molecule, the function of the biological active molecule is restored.

    17. The sensor molecule according to claim 16, wherein the biological active molecule comprises an active drug, a pro-drug, an enzyme, or a co-factor.

    18. The sensor molecule according to any of claims 14 to 17, wherein the split molecule comprises a toxin.

    19. The sensor molecule according to claim 18, wherein the toxin is the A and B components of an AB protein toxin.

    20. The sensor molecule according to any preceding claim, wherein the aspect ratio of the rod-like molecules L and R is about 6-10:1 (length to width).

    21. The sensor molecule according to any preceding claim, wherein the rod-like molecules L and R are each at least 40 Ångströms in length.

    22. The sensor molecule according to any preceding claim, wherein the rod-like molecules L and R are substantially rigid.

    23. The sensor molecule according to any preceding claim, wherein the rod-like molecule L and/or rod-like molecule R comprise or consist of polypeptide.

    24. The sensor molecule according to any preceding claim, wherein the rod-like molecule L and/or rod-like molecule R comprise or consist of an alpha-helical polypeptide.

    25. The sensor molecule according to any preceding claim, wherein the rod-like molecule L comprises a number N of constituent molecules q1, q2, . . . , qN; and the rod-like molecule R comprises a number N′ of constituent molecules q′1, q′2, . . . , q′N′; wherein q1, q2, . . . , qN, q′1, q′2, . . . , q′N′ are selected to be charged amino acids, or hydrophilic or hydrophobic amino acids, or a combination thereof.

    26. The sensor molecule according to any preceding claim, wherein the rod-like molecules L and R of the sensor molecule are symmetrical in sequence.

    27. The sensor molecule according to any preceding claim, wherein the binding energy of A and A′ are substantially similar to the opposing bias energy of the hinge.

    28. The sensor molecule according to any preceding claim, wherein the rod-like molecules L and R comprise an alpha helix of the following repeat residues [EAAAK(SEQ ID NO: 106)].sup.m and [KAAAE(SEQ ID NO: 107)].sup.m respectively, where m is the number of repeats ranging from 6 to 12, and E and K are positively charged at physiological pH condition; or wherein the rod-like molecules L and R comprise an alpha helix of the following repeat residues and [EAAAAK (SEQ ID NO: 108)].sup.m and [KAAAAE(SEQ ID NO: 109)].sup.m respectively, where m is the number of repeats ranging from 6 to 12, and E and K are positively charged at physiological pH condition; or wherein the the rod-like molecules L and R comprise an alpha helix of the following repeat residues [EAAAAAK(SEQ ID NO: 110)].sup.m and [KAAAAAE(SEQ ID NO: 111)].sup.m respectively, where m is the number of repeats ranging from 6 to 12, and E and K are positively charged at physiological pH condition.

    29. The sensor molecule according to any preceding claim, wherein the joint molecule C is flanked according to the following sequence [EAAAAAK(SEQ ID NO: 110)].sup.4 EAAKAAKA(SEQ ID NO: 112)-[Joint Molecule C]-AKAAKAAE(SEQ ID NO: 113) [KAAAAAE(SEQ ID NO: 111)].sup.4.

    30. The sensor molecule according to any preceding claim, wherein the rod-like molecule L and R together with the joint molecule C comprise the sequence [EAAAAAK(SEQ ID NO: 110)].sup.4 EAAKAAKA(SEQ ID NO: 112) S G S AKAAKAAE(SEQ ID NO: 113) [KAAAAAE(SEQ ID NO: 111)].sup.4.

    31. The sensor molecule according to any preceding claim, wherein the sensor molecule is a fusion protein.

    32. The sensor molecule according to any preceding claim, wherein the sensor molecule comprises or consists of the protein sequence FP1-A[GSG].sup.m1 A (SEQ ID NO: 114)-TBM-A[GSG].sup.m2 A L (SEQ ID NO: 114)-[hinge]-R A[GSG].sup.m3 A (SEQ ID NO: 114)-BM-A[GSG].sup.m4 A (SEQ ID NO: 114)-FP2, wherein FP1 and FP2 are a signal molecule B and B′ respectively; TBM and BM are the target binding molecule A and binding molecule A′ respectively; L and R denote the Left and Right alpha helices of the hinge; A, S, and G denote the amino acids Alanine, Glycine and Serine; and m1, m2, m3 and m4 are appropriately selected number of repeats to ensure that the sensor is functional according to the invention.

    33. The sensor molecule according to any preceding claim, wherein the rod-like molecules L and R and joint molecule C (the hinge) are composed of residue sequences such as: [EAAAK(SEQ ID NO: 106)].sup.n A[joint molecule C].sup.m A [KAAAE(SEQ ID NO: 107)].sup.n; or [EAAAK(SEQ ID NO: 106)].sup.n A[joint molecule C].sup.m A [KAAAE(SEQ ID NO: 107)].sup.n, wherein E, A, G, S, and K are the single letter codes for amino acids and n and m are non-zero positive integers.

    34. The sensor molecule according to any preceding claim, wherein the joint molecule C is flexible.

    35. The sensor molecule according to any preceding claim, wherein the joint molecule C comprises or consists of amino acids.

    36. The sensor molecule according to any preceding claim, wherein the joint molecule C comprises or consists of the amino acid glycine.

    37. The sensor molecule according to any preceding claim, wherein the joint molecule C comprises the amino acid sequence SGS or GS.

    38. The sensor molecule according to any preceding claim, wherein the target binding molecule A and/or binding molecule A′ comprises any one of an antibody, antibody fragment or mimic thereof; an antigen, for example a protein or peptide, which is capable of being bound by an antibody; a receptor protein, which comprises a ligand binding site; a ligand, which is capable of being bound by a receptor molecule; or nucleic acid.

    39. The sensor molecule according to any preceding claim, wherein the sensor molecule is bound to another sensor molecule according to any preceding claim.

    40. A nucleic acid encoding the sensor molecule according to any preceding claim.

    41. The nucleic acid according to claim 40, wherein the entire sensor molecule is encoded as a fusion protein.

    42. The nucleic acid according to claim 40 or 41, wherein the nucleic acid comprises or consists of a vector.

    43. A host cell comprising the nucleic acid according to any of claims 40 to 42; and/or comprising the sensor molecule according to any of claims 1 to 39.

    44. A composition comprising the sensor molecule according to any of claims 1 to 39, or the nucleic acid according to any of claims 40 to 42; optionally, wherein the composition is a pharmaceutically acceptable composition.

    45. An assay method for the detection of a target molecule in sample comprising: providing the sample; providing the sensor molecule according to any of claims 1 to 39 in the sample; detecting the presence or absence of a signal from the sensor molecule; wherein an ON signal confirms the presence of the target molecule in the sample.

    46. An assay method for the detection of a target molecule in vivo comprising: providing the sensor molecule according to any of claims 1 to 39 in vivo; detecting the presence or absence of a signal from the sensor molecule; wherein an ON signal confirms the presence of the target molecule in vivo.

    47. Use of the sensor molecule according to any of claims 1 to 39 to visualise or monitor any of the following: (a) the structure and conformation of proteins; (b) the spatial distribution and assembly of protein complexes; (c) protein receptor/ligand interactions including the local concentrations of analytes; (d) the interactions of single molecules; (e) the structure and conformations of nucleic acids; (f) the distributions and transport of lipids; (g) membrane potential sensing; (h) monitoring fluorogenic protease substrates; (i) local cellular concentrations of cyclic AMP and calcium.

    48. Use of the sensor molecule according to any of claims 1 to 39 in the detection of a target analyte, and optionally its concentration, in assays or living cells; or as a drug or drug delivery vehicle to, or within, biological cells, fluids or tissue; or to provide or catalyse a chemical reaction in the vicinity or within biological cells, organic materials, fluids or tissue; or to deliver heat in the vicinity or within biological cells, fluids, tissue or organic materials; or in photodynamic therapy in the vicinity or within biological cells, fluids, tissue, or organic materials; or to perform assays for analytes including titration measure using microtiters or vials, with and without specialised equipment; or to detect analytes in suitable continuous flow chambers.

    49. Use of the sensor molecule according to any of claims 1 to 39 for cell killing, wherein the sensor molecule comprises a split molecule that is an active toxin once the parts of the split molecule are brought together in the presence of a target molecule.

    50. A method of providing a biological active only in the presence of a target molecule comprising: providing the sensor molecule according to any of claims 1 to 39, wherein the sensor molecule comprises a split molecule, wherein the split molecule is a biological active.

    51. A method of treatment for a disease in a subject comprising the administration of the sensor molecule according to any of claims 1 to 39 or the composition according to claim 43, wherein the sensor molecule comprises a biological active in the form of a split molecule, which is capable of becoming an active suitable for treatment of the disease.

    52. The sensor molecule according to any of claims 1 to 39 or the composition according to claim 44, for use as medicament; optionally for use to treat cancer in a subject.

    53. The sensor molecule, use, method or composition substantially as described herein; optionally with reference to the accompanying drawings (except for FIG. 1).

    Description

    BRIEF DESCRIPTION OF DRAWINGS

    [0234] FIG. 1 Schematic description of FRET unimolecular sensor using a flexible linker as described in reference PT1 by Matsuda et al. The OFF state of the sensor corresponding to the absence of the ligand or analyte is displayed in the left panel, where ligand binding domain and sensor domain are on average far apart, and as a consequence the RET signal intensity is low. The donor and acceptor fluorophore proteins are depicted as cylinders. The ON state of the sensor corresponding to the presence of the ligand or analyte is displayed in the right panel, where ligand binding domain and sensor domain are on average in close contact, and as a consequence the RET signal intensity is high.

    [0235] FIG. 2 Schematic description of a multistate dynamical unimolecular hinge sensor of the present invention in the OFF state corresponding to the absence of the ligand.

    [0236] FIG. 3 Schematic description of a multistate dynamical unimolecular hinge sensor of the present invention in the ON state sensor corresponding to the presence of the ligand or analyte.

    [0237] FIG. 4 Test results for prototype 1 of a multistate dynamical unimolecular hinge sensor. The resonance energy transfer signal intensity I at physiological temperature is plotted as a function of binding energy Δε for different values of the depth of the bias D.sub.e: =0; .square-solid.=1Kcal/mol; .diamond-solid.=3 Kcal;/mol; .box-tangle-solidup.=5 Kcal/mol.

    [0238] FIG. 5 Test results for prototype 1 of a multistate dynamical unimolecular hinge sensor. The resonance energy transfer signal to noise ratio (I−I.sub.0)/I.sub.0 at physiological temperature is plotted as a function of binding energy Δε for different values of the depth of the bias D.sub.e: =0; .square-solid.=1Kcal/mol; .diamond-solid.=3 Kcal/mol; .box-tangle-solidup.=5 Kcal/mol.

    [0239] FIG. 6 Test results for prototype 1 of a multistate dynamical unimolecular hinge sensor, where the bias depth D.sub.e=4 Kcal/mol. The probability distribution P(r) of inter macromolecule distances r at physiological temperature is plotted for different values of the binding energies Δε =1 Kcal/mol; .square-solid.=3 Kcal/mol; .box-tangle-solidup.=5 Kcal/mol.

    [0240] FIG. 7 Test results for prototype 1 of a multistate dynamical unimolecular hinge sensor, where the bias depth D.sub.e=4 Kcal/mol. The probability P.sup.+(r) of inter macromolecule distances r at physiological temperature is plotted for different values of the binding energies Δε =1 Kcal/mol; .square-solid.=3 Kcal/mol; .diamond-solid.=5 Kcal/mol. Note that P.sup.+(r) is simply the integral from 0 to r of P(r) and is otherwise known as the cumulative probability. Here it gives the probability that two spheres of the model are within a distance r of each other.

    [0241] FIG. 8 Test results for prototype 2 of a multistate dynamical unimolecular hinge sensor. The resonance energy transfer signal intensity I at physiological temperature is plotted as a function of binding energy Δε for different values of the depth of the bias D.sub.e: =0; .square-solid.=1 Kcal/mol; .diamond-solid.=3 Kcal;/mol; .box-tangle-solidup.=5 Kcal/mol.

    [0242] FIG. 9 Test results for prototype 2 of a multistate dynamical unimolecular hinge sensor. The resonance energy transfer signal to noise ratio (I−I.sub.0)/I.sub.0 at physiological temperature is plotted as a function of binding energy Δε for different values of the depth of the bias D.sub.e:=0; .square-solid.=1 Kcal/mol; .diamond-solid.=3 Kcal/mol; .box-tangle-solidup.=5 Kcal/mol.

    [0243] FIG. 10 Test results for prototype 2 of a multistate dynamical unimolecular hinge sensor, where the bias depth D.sub.e=4 Kcal/mol. The probability distribution P(r) of inter macromolecule distances r at physiological temperature is plotted for different values of the binding energies Δε =1 Kcal/mol; .square-solid.=3 Kcal/mol; .diamond-solid.=5 Kcal/mol.

    [0244] FIG. 11 Test results for prototype 2 of a multistate dynamical unimolecular hinge sensor, where the bias depth D.sub.e=4 Kcal/mol. The probability P.sup.+ (r) of inter macromolecule distances r at physiological temperature is plotted for different values of the binding energies Δε =1 Kcal/mol; .square-solid.=3 Kcal/mol; .diamond-solid.=5 Kcal/mol.

    [0245] FIG. 12 Test results for prototype 3 of a multistate dynamical unimolecular hinge sensor. The resonance energy transfer signal intensity I at physiological temperature is plotted as a function of binding energy Δε for different values of the depth of the bias D.sub.e: =0; .square-solid.=1 Kcal/mol; .diamond-solid.=3 Kcal;/mol; .diamond-solid.=5 Kcal/mol.

    [0246] FIG. 13 Test results for prototype 3 of a multistate dynamical unimolecular hinge sensor. The resonance energy transfer signal to noise ratio (I−I.sub.0)/I.sub.0 at physiological temperature is plotted as a function of binding energy Δε for different values of the depth of the bias D.sub.e: =0; .square-solid.=1 Kcal/mol; .diamond-solid.=3 Kcal/mol; .box-tangle-solidup.=5 Kcal/mol.

    [0247] FIG. 14 Test results for prototype 3 of a multistate dynamical unimolecular hinge sensor, where the bias depth D.sub.e=4 Kcal/mol. The probability distribution P(r) of inter macromolecule distances r at physiological temperature is plotted for different values of the binding energies Δε =1 Kcal/mol; .square-solid.=3 Kcal/mol; .box-tangle-solidup.=5Kcal/mol.

    [0248] FIG. 15 Test results for prototype 3 of a multistate dynamical unimolecular hinge sensor, where the bias depth D.sub.e=4 Kcal/mol. The probability P.sup.+(r) of inter macromolecule distances r at physiological temperature is plotted for different values of the binding energies Δε =1 Kcal/mol; .square-solid.=3 Kcal/mol; .diamond-solid.=5 Kcal/mol.

    [0249] FIG. 16 Schematic description of a primary antibody showing the heavy and light chains, the Fab and Fc regions, as well as the location of disulphide bonds and carbohydrates.

    [0250] FIG. 17 Schematic description showing different fragments of a primary antibody realizable in experiments.

    [0251] FIG. 18 Schematic description of Tuneable Multistate Dynamical Unimolecular Hinge Sensors, when A and A′ are primary antibodies, where the analyte is an antigen, and the system is free floating in the solution, and B and B′ are fluorophores or the corresponding moieties of a single split fluorophore or split bioluminescent molecule. The antigen is represented as a cone purely for illustrative reasons, but need not be of such a shape.

    [0252] FIG. 19 Schematic description of Tuneable Multistate Dynamical Unimolecular Hinge Sensors, when A and A′ are primary and corresponding secondary antibodies, where the antigen acts like an analyte, and the system is free floating in the solution, and B and B′ are fluorophores or the corresponding moieties of a single split fluorophore or split bioluminescent molecule. The antigen is represented as a cone purely for illustrative reasons, but need not be of such a shape.

    [0253] FIG. 20 Schematic description of Tuneable Multistate Dynamical Unimolecular Hinge Sensors, when A and A′ are the antigen and a secondary antibodies, where the primary antibody acts like an analyte, and the system is free floating in the solution, and B and B′ are fluorophores or the corresponding moieties of a single split fluorophore or split bioluminescent molecule. The antigen is represented as a cone purely for illustrative reasons, but need not be of such a shape.

    [0254] FIG. 21 Schematic description of Tuneable Multistate Dynamical Unimolecular Hinge Sensors similar to FIG. 18 except that the sensor is not feely floating but instead is attached via one of the primary antibodies to a suitable surface.

    [0255] FIG. 22 Schematic description of Tuneable Multistate Dynamical Unimolecular Hinge Sensors similar to FIG. 19 except that the sensor is not feely floating but instead is attached via the primary antibody or the secondary antibody to a suitable surface.

    [0256] FIG. 23 Schematic description of Tuneable Multistate Dynamical Unimolecular Hinge Sensors similar to FIG. 20 except that the sensor is not feely floating but instead is attached via the antigen or the secondary antibody to a suitable surface.

    [0257] FIG. 24 A publically available alignment code known as modeller, the visualiser code known as vmd, and the molecules simulation code known as GROMACS to relax the system in a box with water using periodic conditions to emulate bulk conditions under one atmosphere and physiological temperature and pressure. Experimental methods to build these sensors, once the protein sequence of the hinge and interconnecting flexible linkers are defined are in the literature and outlined in the prior art descriptions.

    [0258] Snap shots of the two examples after a few nanoseconds simulation in water under normal temperature and pressure are attached (the water is not shown in order to visualise the sensor protein system). The first system comprises the fhal and pka substrate sensor and ligand binding domains combined with two fluorescent proteins interconnected by the biased hinge. The second system comprises the same fhal and pka substrate sensor and ligand binding domains combined with the two moieties of a split yellow fluorescent protein interconnected by the biased hinge, the latter having a smaller and more effective joint connecting the alpha helix proteins.

    [0259] A few non-standard amino acids from the protein sequences were removed to facilitate full scale simulation using the CHARMM 27 and 36 force fields directly, but this does not affect the structure of the system.

    [0260] First system (fhal-fpl-hinge-fp2-pka substrate, note that the “-” represent short flexible linkers A[GSG].sup.n A (SEQ ID NO: 114) and these can be modified to optimize the functioning of the system). The protein sequence is SEQ ID NO: 1 herein.

    [0261] Second system is hal-splitYFP1 -hinge-splitYFP2-pka substrate, note that the “-” represent short flexible linkers A[GSG].sup.3A (SEQ ID NO: 115) and these can be modified to optimize the functioning of the system). Here splitYFP1 and splitYFP2 denote the two moieties of yellow fluorescent protein. The protein sequence is SEQ ID NO: 2 herein.

    [0262] FIG. 25—Using the simulation methods described in the prior art and disclosed in P34, one can predict the bias energy associated with the hinge to be open to be 10-12 kcal/mol for the protein sequence above, and similar structures. For instance, [EAAAK (SEQ ID NO: 106)].sup.6A[GS].sup.2A [KAAAE(SEQ ID NO: 107)].sup.6 illustrated in FIG. 25A which has the free energy as a function of distance between the end of the hinge displayed in FIG. 25B.

    [0263] FIG. 26 Free energy as a function of distance (Angstrom) between the two end of the hinge [EAAAK(SEQ ID NO: 106)].sup.6 SGS [KAAAE(SEQ ID NO: 107)].sup.6.

    [0264] FIG. 27 (FRET) Forkhead-associated fhal and protein kinase A substrate (PKA substrate with a pair of fluorescent proteins and the biased hinge).

    [0265] FIG. 28 Sensor combining split YFP moieties with forkhead fhal and protein kinase A substrate and the hinge hinge [EAAAK(SEQ ID NO: 106)].sup.6 SGS [KAAAE(SEQ ID NO: 107)].sup.6.

    [0266] FIG. 29 Sensor combining a pair of single domain camedloid antibodies with the moieties of a fluorescent protein and the biased hinge.

    [0267] FIG. 30 Split AB toxin with a pair of single domain camedloid antibodies and the biased hinge. The AB toxin is split into two moieties: AB.sub.1 and B.sub.2. The particular division of B is made because B.sub.2 is believed to be responsible for the binding of the toxin to the cell membrane preceding penetration of the toxin into the cell.

    [0268] FIG. 31 Provides Examples 1 to 5 of sequences of the full sensor system. These examples serve to teach how the sensors are built. Also it is clear to the skilled person that the spacers would in general be adjusted to fit specific problems, reflecting steric effects of the constituent components, binding affinities, the bias specific to a given hinge, and pH and salt conditions. The hinge sequences, spacer molecules, and other molecules depicted in examples 1-5 may each be individually selected as a potential component of the sensor molecule of the invention, or combinations thereof may be selected.

    SUMMARY

    [0269] Resonance Energy Transfer (RET) based probes are widely used to understand spatio-temporal dynamics of protein pairs both in-vivo and in-vitro. It is well known that the choice of molecular linker connecting FP's in such probes can have a very strong effect on its overall performance. The approach taken here is to invent a radically new type of sensor by focusing on the structural properties required of the biased hinge mechanism to complement any give pair of sensor and ligand binding domain and associated pair of FP's and ligand of interest, thereby facilitating real time tracking of biochemical events, combined with strong signal and signal to noise characteristics. Our linker design is different in several key aspects from those devised hitherto, including flexible linkers of Matsuda et al.

    [0270] The mechanism can be understood as a radical change of the basic model of Komatsu et al, realizable when the flexible linker connecting the sensor and ligand binding domain is replaced by a biased hinge. The biased hinge in the latter context is designed to be in an open conformation (where the FP's are far apart and the FRET signal is low or negligible) in the OFF state see FIG. 2, and in the ON state oscillating between an open and closed conformation frequently enough to allow local concentration of analytes to remain close to endogenous levels see FIG. 3. In the rest of this document the sensor that results from this replacement will be referred to as a multistate dynamical unimolecular hinge sensor, or simply as the sensor in contexts when what is intended is clear.

    [0271] An example of the unimolecular FRET or BRET sensor that is realizable using this biased hinge linker is drawn schematically in FIG. 2. The spheres represent macromolecules, interconnecting straight-lines denote rigid peptides labelled L and R respectively, while the curly element C denotes a highly flexible connected linker. The points labelled q1 and q2 denote charged or hydrophilic or hydrophobic residues (note the number 2 of such residues on L and on R is illustrative, there can be more and the number per peptide need not be the same). Not shown are short peptides acting as spacers between the macromolecules and linkers, or possible additional genetic sequences used for expression of the sensor in target organelles. The residues q1, q2, . . . and their locations are selected so that the probability of the hinge being open (i.e. the angle BCB′ approximately equals 180 degrees) in the ON state is approximately the same as being closed (i.e. the angle BCB' approximately 0 degrees). In one arrangement, the FP pair are depicted as macromolecules A and A′, and the ligand binding domain and sensor domain are depicted as macromolecules B and B′. In another, different arrangement, the FP pair can be depicted as macromolecules B and B′, and the ligand binding domain and sensor domain are depicted as macromolecules A and A′.

    [0272] In the case that q1, q2, . . . are charged residues, their corresponding charges can be positive (arginine, histidine, lysine) or negative (asparatic and glutamic acid), for instance at a physiological pH corresponding to the selection of amino acid sequence.

    [0273] The alpha-helical propensity of these molecules vary with arginine (0.21), histidine (0.61), lysine (0.26), asparatic Acid (0.69) and glutamic Acid (0.40) making histidine and asparatic acids possible choices (see reference NP14).

    [0274] Test results obtained through Metropolis Monte Carlo simulation of an example of a unimolecular RET sensor (referred to as prototype 1) at physiological temperature (36° C.) are given in FIGS. 4-7 where the RET signal intensity, signal to noise ratio, and related probability distributions are displayed. The numerical model of prototype 1 consists of a potential V(r.sub.1, r.sub.2)=V.sub.s(r.sub.1, r.sub.2)+V.sub.1(r.sub.1, r.sub.2) where V.sub.s(r.sub.1, r.sub.2) represents a switchable interaction between the ligand binding domain and sensor domain, and (r.sub.1, r.sub.2) are the position vectors of the idealized spheres modelling the ligand binding domain and donor FP, and the sensor domain and acceptor FP respectively, each of diameter G. In the OFF state, i.e. in the absence of the ligand or analyte V.sub.s(r.sub.1,r.sub.2) ensures that the two spheres cannot overlap, which mathematically is implemented by the constraint that the distance r between the spheres is never less than σ, r>σ; in the ON state, it ensures that the two spheres do not overlap, but also experience a uniform attractive interaction of depth ε for σ<r≦σ+δ. The potential V.sub.1(r.sub.1, r.sub.2)=D.sub.e (1−exp(α(θ−180))).sup.2 models a biased hinge, using a Morse potential of depth D.sub.e and inverse-width α, where θ is the angle between r.sub.1 and r.sub.2. The values of the parameters ε, σ, δ are generally selected to be close to the values of the real system of interest, for instance typically E is between 2 and 10 Kcal/mol, σ is ˜2.4 nm, δ is ˜1.5 nm to and a reasonable choice for D.sub.e˜ε, and α−3.141/60.

    [0275] Prototype 2 is similar to prototype 1, except that in the ON state the attractive interaction of depth ε is replaced by a Lennard-Jones potential V.sub.s (r.sub.1, r.sub.2)=4ε([σ/r].sup.12−[σ/r].sup.6). The test results are given in FIGS. 8-11 where the RET signal intensity, signal to noise ratio, and related probability distributions are displayed.

    [0276] Prototype 3 is similar to prototype 1, except that in the OFF state V.sub.s (r.sub.1, r.sub.2)=0.004 ([σ/r].sup.12−[σ/r].sup.6); and in the ON state 1 V.sub.s (r.sub.1, r.sub.2)=4ε([σ/r].sup.12−[σ/r].sup.6). Qualitatively, the main difference between prototype 1 and prototype 3 is the use of a soft (continuous and differentiable interaction rather than a “hard core” interaction. The test results are given in FIGS. 12-15 where the RET signal intensity, signal to noise ratio, and related probability distributions are displayed. The test results are given in FIGS. 12-15 where the RET signal intensity, signal to noise ratio, and related probability distributions are displayed.

    [0277] Unimolecular sensors having the structural and dynamical features depicted in FIGS. 2 and 3, can be readily generated. First, residue sequences of amino acids giving rise to stiff rod like peptides such as L or R are well known, and are widely available in the literature in the form of long alpha helical proteins such as Basic Leucine Zipper Domain (bZIP domain) found in many DNA binding proteins of almost eukaryotes. One example of bZIP, is a domain found in Maf transcription factor proteins NP15. Other long alpha helical structural motifs include coiled coils, examples include the muscle protein tropomyosin and oncoproteins c-Fos and c-jun (see reference NP16). Shorter alpha-helical motifs include the widely studied villin headpiece (see reference NP17).

    [0278] Second, short very flexible peptides connecting the rods such as peptide C are also well known. Third, as mentioned above charged amino acids are also well known.

    [0279] Fourth, the proteins which comprise the ligand binding domain, and sensor domains, and FP's can be taken from the literature (see reference NP1). Where estimates of the binding energy between particular ligand binding domains and sensor domains in the ON state in the presence of the ligand are not available, they can be estimated experimentally (see reference NP18), or computed via molecular simulation, using publically available standard force-fields developed for biology such as CHARMM (see reference NP19) or AMBER (see reference NP20), open source and publically available simulation engines such as NAMD (see reference NP21) or GROMACS (see reference NP22) and biased sampling methods such as those available in the open software package PLUMED, (see reference NP23) as well as commercial packages.

    [0280] Once the binding energy is known (or estimated), the residues q1, q2, .. . in FIGS. 2 and 3 and their locations in the residue sequence defining the biased hinge can be optimised, via molecular simulation so that in the ON state the probability of the biased hinge sensor being open is slightly higher or equal to the probability of it being closed.

    [0281] Another example of a biased hinge type sensor can be constructed where the curly element C in FIGS. 2 and 3 denotes a combination of rigid and flexible peptides rather than only a highly flexible peptide as the interconnecting linker. This example is by design more adaptable to chemical constraints associated with charged residues, and steric effects.

    [0282] Having determined the full residue sequence of the full biosensor, the sensor can be generated using “off the shelf” biotechnology kits for example those made by: PURExpress® In Vitro Protein Synthesis Kit; Mammalian expression kits such as Jump In™ T-REx™ HEK 293 Kit; Cell-Free Expression Kits such as Expressway™ Maxi Cell-Free E. coli Expression System; and Bacterial expression kits such as Champion™ pET160 Directional TOPO® Expression Kit with LumioTM Technology.

    [0283] Thus the linker can be tailor-made to match essentially any ligand binding domain and sensor domain, ligand and FP pair.

    [0284] The ligand binding domain can be designed using various method such as Monoclonal Antibody, Polyclonal antibody or Genomic antibody technologies.

    [0285] Macromolecules (for example FPs, ligand binding domains, sensor domains and even full unimolecular sensors) can be attached to specific sites of proteins of interest using chemical labelling for example covalent bonding amine labelling, thiol labelling etc. (see reference NP24), enzymatic labelling (labelling catalysed by post-translational enzyme modification, labelling with self-modified enzymes such as cutinase or interin, see reference NP25) and non-covalent tagging (tetracysteine/ biarsenical tag, histidine tag, see reference NP26). Other tags can be genetic based which include SNAP and CLIP tags (see reference NP27).

    [0286] A practical issue in analytical chemistry, biochemistry, related sciences and industry is the perturbative effect of chemical sensors/indicators used to measure the concentrations of analytes of interest. If the sensor is not very sensitive to the target analyte, large volumes of probe may be required. Another frequent situation is that the design of the probe is such that it has a disruptive effect on the system it is designed to monitor, which complicates fine scale measurements, including the tracking of temporal and spatial variations of analyte concentrations. The present invention resolves both of these difficulties.

    [0287] In parallel with developments of RET sensors using single donor and acceptor FPs, a method using a single FP donor but multiple FP acceptors (of different colours) has been reported, for instance by Sun et al, (see reference NP13). The latter method can be combined with the present invention, where for example the additional acceptor FPs are attached to sites of the protein of interest.

    [0288] The present invention resolves many of the difficulties in performing immunoassays through the application of Tuneable Multistate Dynamical Unimolecular Hinge Sensors. In the context of immunoassays, the sensors have several novel capabilities, not possible or very difficult to implement with available methods. These include the facility to track in time the local concentrations of target analytes, to turn on pharmaceutically active molecules or toxins, and do not require the complex set of washing steps typically used with conventional immunoassays described above in the background art.

    [0289] In this invention the detection of analytes such as antigens or antibodies (see FIG. 16) is made by selecting macromolecules A and A′ of the Tuneable Multistate Dynamical Unimolecular Hinge Sensors (see FIG. 2) to be primary antibodies or suitable antibody fragments (see FIG. 17). Macromolecules B and B′ can be a variety of different types of macromolecules interconnected through a biased hinge. In one version of this invention, A and A′ are selected to target different epitopes on the analyte, typically an antigen (see FIGS. 18 and 21). Alternatively, for cases where a second antibody binds to a corresponding primary antibody on the presence of the corresponding antigen, A is said primary antibody and A′ is the secondary antibody (see FIGS. 19 and 22). In another variant of this invention, A is an antigen, A′ is a secondary antibody, and the analyte is a corresponding primary antibody (see FIGS. 20 and 23. The sensor can be tuned so that in the OFF state (i.e. in the absence of target analytes close to A or A′), the arms of the hinge are open, and in the ON state (i.e. in the presence of target analytes close to A or A′), the arms of the hinge oscillate between open and closed configurations.

    [0290] When A and A′ are primary antibodies (or primary antibody fragments containing accessible carbohydrates such as Fc or F(abc)) targeting different epitopes on the same antigen (which could be epitopes of the same type but at different locations), using the background art described above B can be conjugated with A through primary amines in the antibody, or through carbohydrates in the F.sub.C region (see FIG. 16), and similarly B′ can be conjugated with A′ (with the use of blocking reagents as described above where required) as shown in FIG. 1.

    [0291] When A and A′ are fragments of primary antibodies (e.g. F(ab′).sub.2, Fab′, Fab, Fv or F(abc)) targeting different epitopes on the same antigen (which could be epitopes of the same type but at different locations), A can be conjugated with B and A′ can be conjugated with B′ respectively using the sulfhydryl groups.

    [0292] When A is a primary antibody (or primary antibody fragments containing accessible carbohydrates such as Fc or F(abc)), and A′ is a corresponding secondary antibody (or secondary antibody fragments containing accessible carbohydrates such as Fc or F(abc)), using the background art described above B can be conjugated with A through primary amines in the antibody, or through carbohydrates in the F.sub.C region, and similarly B′ can be conjugated with A′ (with the use of blocking reagents as described above where required). It is also possible to conjugate A with B and A′ with B′ using the sulfhydryl groups described in the background art.

    [0293] When A is an antigen and A′ is a secondary antibody (or fragment thereof), both targeting the same primary antibody or fragment thereof, appropriate labelling methods include the following. B can be conjugated with A through primary amines in the antibody, or through carbohydrates in the F.sub.C region, and similarly B′ can be conjugated with A″(with the use of blocking reagents as described above where required). It is also possible to conjugate A with B and A′ with B′ using the sulfhydryl groups described in the background art.

    [0294] In variant 1, and other aspects and embodiments, of this invention, macromolecules B and B′ are suitable fluorescent proteins or bioluminescent molecules, which can undergo resonance energy transfer when brought together by the action of A and A′ on the sensing of a target analyte, such as an antigen or antibody as described above.

    [0295] In variant 2, and other aspects and embodiments, of this invention, B is a fluorescent protein or bioluminescent molecule or dye molecule capable of absorbing light from an external field and B′ to be a quencher macromolecule, for instance made from a metal such as gold, such that when A and A′ come together in their ON state, B and B′ to so that the energy absorbed by B is transferred non-radiatively to B′ and released locally in the form of heat on or close to the target analyte within or close to a cell or cellular compartment.

    [0296] In variant 3, and other aspects and embodiments, of this invention, B or B′ are macromolecules which are the reactants of a chemical reaction such that when B and B′ are far apart the reaction cannot take place (in OFF state of A and A′), and when A and A′ come together in their ON state, the reaction can take place to produce products which may be pharmaceutical active or chemo-toxic. It is also possible that B consists of reactants which require a catalyst to react, and B′ consists of the corresponding catalyst, such that when A and A′ come together in their ON state, the resulting the products of the catalysed reaction are active drugs or chemo-toxic on or close to the target analyte within or close to a cell or cellular compartment.

    [0297] In variant 4, and other aspects and embodiments, of this invention, B and B′ are each a moiety of a split single fluorophore (or split bioluminescent molecule) such that in the OFF state they are far apart and do not fluoresce (or bio-luminesce), and in the ON when A and A′ can come together, B and B′ are also brought together and fluoresce when illuminated at appropriate wavelengths (or bio-luminesce) on or close to the target analyte within or close to a cell or cellular compartment.

    [0298] In variant 5, and other aspects and embodiments, of this invention, B and B′ are each the moieties of a split macromolecule which is pharmaceutically inactive (or non-chemo-toxic or non-photosensitizing) when they are far apart (in the OFF state of A and A′), and when A and A′ come together in their ON state due to the presence of the target analyte, B and B′ are brought together so that the complex is pharmaceutical active (or chemo-toxic or photosensitizing).

    [0299] Variant 6 of this invention is a combination of variant 4 and variant 5. B consists of a moiety of a split fluorescent protein or split bioluminescent molecule and the moiety of a split drug or split chemo-toxic or split photosensitizing molecule macromolecule, and B′ consists of the other moiety of the split fluorescent protein or split bioluminescent molecule and the other moiety of a split drug or split chemo-toxic molecule, such that when B and B′ are apart the complex is inactive, and when B and B′ are brought together due to the presence of target analyte by A and A′, the complex becomes pharmaceutically active or chemo-toxic or photosensitizing and fluorescent or bioluminescent.

    [0300] In variant 7, and other aspects and embodiments, of this invention, B consists of the moieties of a split fluorescent protein or split bioluminescent molecule and a quencher molecule, and B′ consist of the other moieties of the split fluorescent protein or split bioluminescent molecule and a quencher molecule, such that B and B′ are apart they cannot become easily optically excited, for instance by an external field, but when B and B′ are brought together due to the recognition of a target analyte by A and A′, the resulting complex both easily absorbs energy from an externally applied source and transfers it non-radiatively through the quencher molecule, thereby heating the local cellular or sub-cellular region wherein the analyte is located. It is also possible to divide the quencher into two moieties, with one quencher moiety and one fluorescent moiety in B, and one quencher moiety and one fluorescent moiety in B′.

    [0301] Variant 8, and other aspects and embodiments, of this invention is a combination of variants 3 and 4. B consists of the moieties of a split fluorescent protein or split bioluminescent molecule and some of the reactants of a chemical reaction and B′ consist of the other moieties of the split fluorescent protein or split bioluminescent molecule and the rest of the reactants required for a chemical reaction. It is also possible that B consists of reactants which require a catalyst to react, and and B′ consists of the corresponding catalyst. When B and B′ are apart the they cannot easily fluoresce or bioluminesce, and the chemical reaction cannot easily take place, but when B and B′ are brought together due to the recognition of a target analyte by A and A′, the resulting complex can fluoresce or bioluminesce, and the chemical reaction can take place at or close to the local cellular or sub-cellular region wherein the analyte is located.

    [0302] As well as sensors targeting single analytes in the above variants of the invention, multiple sensors targeting different analytes, each using corresponding macromolecules (A,A′,B and B′) can be used at the same time within a living sample or microtiter testing well or vial. When appropriate, different acceptor fluorophores (or moieties thereof) emitting at different wavelengths can be used so as to allow simultaneous use and/or measurement (using microscopy) of each type of possible analyte present in the sample.

    [0303] Variants 1, 4, 6 and 8, and other aspects and embodiments, of this invention can be used for performing immunoassays to identify the presence of analytes including antibodies in samples using microscopy and suitable light sources for the selected fluorescent proteins, or no external light sources if the donor fluorescent protein is bioluminescent or chemiluminescent.

    [0304] Variants 1, 4, 6 and 8, and other aspects and embodiments, of this invention, when combined with confocal scanning microscopy described in the background art, can be used to identify the time dependent concentration and location of analytes in a sample.

    [0305] This includes the capacity to generate three dimensional spatial images of the concentration of analytes and track their position over time, including in living cells. The invention can also be used to monitor changes in real time in such analyte concentration through the use of appropriate flow chambers, or in living cells.

    [0306] Desktop scanners or an ordinary CCD camera, and either a single LED of a single wavelength or a combination of Red-Green-Blue LEDs can be combined with variants 1, 4, 6 and 8, and other aspects and embodiments, of this invention to determine the concentration of the analyte in a sample, including its time dependence, to produce multi-dimensional images tracking over time the concentration of the analyte.

    [0307] Variants 1, 4, 6 and 8, and other aspects and embodiments, of this invention can be combined with either a single LED of a single wavelength or a combination of Red-Green-Blue LEDs and photomultiplier diode chips to measure the photo emission of the sensor, and thereby determine the concentration of the analyte in a sample, including its time dependence, for instance when a three or two dimensional image is not required. This can be used for taking and analysing immunoassays in the field, as well as in specialised laboratories.

    [0308] When A or A′ is bonded to a suitable surface of a flow chamber, variants 1,4,6 and 8, and other aspects and embodiments, of this invention can be used to measure the time dependent concentration of analytes, including their detection in continuous sample measurement (see FIGS. 21,22, and 23).

    [0309] When A or A′ is bonded to a suitable surface of a suitable titration vessel, the invention can be used for measuring the concentration of one or multiple analytes during a titration.

    [0310] When macromolecules B and B′ are the moieties of a split bio-luminescent or chemiluminescent molecule, the invention can be combined with low cost microtiter and vials described in the background art, to perform and analyse immunoassays in the field i.e. in non-laboratory conditions and without the use of specialised equipment.

    [0311] When macromolecules B and B′ are the moieties of a split bio-luminescent or chemiluminescent molecule, the invention can be used to deliver light close to the location of analytes, which can be close to or within living cells or cellular compartments, for the purpose of targeted electromagnetic radiation treatment.

    [0312] When macromolecules B and B′ are pairs of chemical reactants which react spontaneously when brought together, and such that the chemical product is either a drug or a chemo-toxin or a chemo-toxin or photosensitizing, the invention can be used as a therapeutic for diseased living cells or sub-cellular compartments, and such that the drug or chemo-toxin becomes activated on the presence of a target analyte. If B also includes either a donor FP or bioluminescent molecule or a moiety of a split FP or a moiety of a split bioluminescent molecule and B′ includes a corresponding acceptor FP, or corresponding moiety of a split FP or corresponding moiety of a split bioluminescent, the activation of a drug or chemo-toxin or photosensitizing agent can be marked by the emission of light. When a donor fluorophore is used an external light source is required. When either a non-split fluorophore or non-split bioluminescent molecule is used as a donor, the emission signalling recognition of the analyte is due to resonance energy transfer.

    [0313] When B is a fluorophore or a chromophore and B′ is a quencher, and given a light source, variants 2 and 7, and other aspects and embodiments, of the invention can be used to deliver non-radiative energy in the form of heat at or close to the location of target analytes in living cells.

    Discussion

    [0314] It is well known that the choice of molecular linker connecting FP's in the probe can have a very strong effect on its overall performance. The approach taken here is to invent a radically new type of sensor by focusing on the structural properties required of the linker mechanism to complement any given pair of sensor and ligand binding domain and associated pair of FP's and ligand of interest, thereby facilitating real time tracking of biochemical events, combined with strong signal and signal to noise characteristics. Our biased hinge design and the resulting sensor is different in several key aspects from the one described in references PT1 and NP11.

    [0315] The highly tuneable multistate dynamical hinge sensor is designed (i.e. biased) to be normally fully open in OFF state (i.e. the absence of the target ligand) so as to ensure the FP pair are far apart and the corresponding RET rate is very low. The sensor is tuned so that when combined with a ligand binding domain and sensor domain and associated FP's, it can open and close frequently in the ON state, but in such a way that it is can be selected to be on average open more often than closed.

    [0316] This intrinsic fluctuating feature in the ON state accounts for the high signal and signal to noise properties, while allowing concentrations levels of target analyte to be maintained close to endogenous levels. This design feature of choosing the ON state to be fluctuating between two conformations (open and closed) rather than simply closed, is completely counter intuitive, and novel.

    [0317] The sensor described above need not be protein based, its components can be organic or inorganic or a mixture thereof.

    [0318] The present invention in the context of immunoassays also creates a series of novel capabilities not possible or very difficult to implement with available methods. In this invention the detection of analytes such as antigens is made through macromolecules A and A′ of the Tuneable Multistate Dynamical Unimolecular Hinge Sensors (see FIG. 2). Macromolecules A and A′ can be selected to each be primary antibodies targeting different epitopes on the analyte, which may the same type of epitope but at different locations (see FIGS. 18 and 21). Alternatively, for cases where a second antibody binds to a corresponding primary antibody only the presence of the corresponding antigen, A is said primary antibody and A′ is the secondary antibody (see FIGS. 19 and 22). In another variant of this invention, A is an antigen, A′ is a secondary antibody, and the analyte is a corresponding primary antibody (see FIGS. 20 and 23). The sensor can be tuned so that in the OFF state (i.e. in the absence of target analytes close to A or A′), the arms of the hinge are open, and in the ON state (i.e. in the presence of target analytes close to A or A′), the arms of the hinge oscillate between open and closed configurations. Macromolecules B and B′ can each consist of one or more selected molecules or moieties of split molecules, which when brought close together due to the presence of target analytes can produce a variety of selected effects: (a) resonance energy transfer in the presence of a suitable electro-magnetic radiation field; (b) fluorescence in the presence of a suitable electro-magnetic radiation field; (c) bioluminescence; (d) activated drug; (e) activated chemo toxin; (f) chemical reaction; (g) catalysed chemical reaction; (h) in the presence of a suitable electro-magnetic radiation field, the release of heat through quenching; (i) in the presence of a suitable electro-magnetic radiation field either of an external or endogenous source, the production of reactive oxygen. In addition several of these effects can be combined in the same sensor, including: {C1 (a-c), C2 (a-d), C3 (a-e), C4 (a-f), C5 (a-g), C6 (a-h); C7 (a-i), C8 (b-d), C9 (b-e), C10 (b-f), C11 (b-g), C12 (b-h), C13 (b-i), C14 (c-d), C15 (c-e), C16 (c-f), C17 (c-g), C17 (c-h), C18 (c-i)}. These effects take place in the vicinity on the sensor, which can be close to or within cells, cellular compartments, or in vitro.

    [0319] Variants of the invention can be used to visualise and track in time analytes in vivo and in vitro in assays to create multi-dimensional images of said analytes using confocal scanning microscopy, desktop scanners, a variety of suitable LEDS and photo cascade chips. In addition variants of the invention can be deployed in the field using microtiters or vials, without specialised equipment, and at low cost for several applications including environmental, health, and food safety. Multiple sensors targeting different analytes can be deployed to measure a single sample simultaneously, and can be used to measure the time dependent concentration of analytes in suitable flow chambers or through titration.

    [0320] Variants of the invention can be used to deliver payloads to regions close to and within cellular environments which can be specified if required by genetic targeting, and such that the payloads become activated on the presence of target analytes, and are inactive in their absence. The payloads can include drugs, chemo-toxins, chemicals, catalysts, heat through the localised absorption of external electro-magnetic or chemical fields, and hydrogen radicals using photosensitizers coupled with electro-magnetic fields of external or endogenous origin.

    [0321] The hinge of the highly tuneable multistate dynamical unimolecular hinge sensor is designed (i.e., biased) to be normally fully open in OFF state (i.e. the absence of the target ligand) so as to ensure the FP pair are far apart and the corresponding RET rate is very low. The biased hinge is tuned so that when combined with a ligand binding domain and sensor domain and associated FP's, it can open and close frequently in the ON state, but in such a way that it is can be selected to be on the average open more often than closed. This intrinsic fluctuating feature in the ON state accounts for the high signal and signal to noise properties, while allowing concentrations levels of target analyte to be maintained close to endogenous levels. This design feature of choosing the ON state to be fluctuating between two configurations (open and closed) rather than simply closed, is completely counter intuitive, and novel. A version of this sensor including inorganic components can be used in electronic, semi-conducting and quantum computing industries as electronic sensors, memory devices and nano-actuators. The present invention in the context of immunoassays also creates a series of novel capabilities not possible or very difficult to implement with available methods. Variants of the invention can be used to deliver payloads to regions close to and within cellular environments which can be specified if required by genetic targeting, and such that the payloads become activated on the presence of target analytes, and are inactive in their absence. The payloads can include drugs, chemo-toxins, chemicals, catalysts, heat through the localised absorption of external optical or chemical fields, and oxygen radicals as used in photodynamic therapy.

    EXAMPLE SEQUENCES

    [0322]

    TABLE-US-00001 fha1-fp1-hinge-fp2-pka substrate (SEQ ID NO: 1) The protein sequence (single letter) is GENITQPTQQSTQATQRFLIEKFSQEQIGENIVCRVICTTGQIPIRDLSA DISQVLKEKRSIKKVWTFGRNPACDYHLGNISRLSNKHFQILLGEDGNLL LNDISTNGTWLNGQKVEKNSNQLLSQGDEITVGVGVESDILSLVIFINDK FKQCLEQNKVDRIRAGSGGSGGSGAMSKGEELFTGVVPILVELDGDVNGH KFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLVQCFSRYPDHM KRHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGI DFKEDGNILGHKLEYNYISHNVYITADKQKNGIKANFKIRHNIEDGSVQL ADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAG IAGSGGSGGSGAEAAAKEAAAKEAAAKEAAAKEAAAKEAAAKAGSGAKAA AEKAAAEKAAAEKAAAEKAAAEKAAAEAGSGGSGGSGAMSKGEELFTGVV PILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVT TFLQCFARYPDHMKRHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFE GDTLVNRIELKGIDFKEDGNILGHKLEYNYNSQNVYIMADKQKNGIKVNF KIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKR DHMVLLEFVTAAGIAGSGASAGKPGSGEGSTKGLRRATVLDGGTGGSEL fha1-splitYFP1 -hinge-splitYFP2-pka substrate (SEQ ID NO: 2) The protein sequence (single letter) is GENITQPTQQSTQATQRFLIEKFSQEQIGENIVCRVICTTGQIPIRDLSA DISQVLKEKRSIKKVWTFGRNPACDYHLGNISRLSNKHFQILLGEDGNLL LNDISTNGTWLNGQKVEKNSNQLLSQGDEITVGVGVESDILSLVIFINDK FKQCLEQNKVDRIRAGSGGSGGSGAMSKGEELFTGVVPILVELDGDVNGH KFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTFGXGLQCFARYP DHMKRHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIEL KGIDFKEDGNILGHKAGSGGSGGSGAEAAAKEAAAKEAAAKEAAAKEAAA KEAAAKSGSKAAAEKAAAEKAAAEKAAAEKAAAEKAAAEAGSGGSGGSGA LEYNYNSQNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGD GPVLLPDNHYLSYQSALSKDPNEKRDHMVLLEFVTAAGITHGMDELYKAG SGASAGKPGSGEGSTKGLRRATVLDGGTGGSEL

    Antibody Binding Affinities

    [0323]

    TABLE-US-00002 TABLE 1 K.sub.D (the equilibrium dissociation constant between the antibody and its antigen), of 840 Rabbit Monoclonal Antibodies (RabbitMAbs) and 88 MouseAbs expressed as percentage distribution at a given binding affinity from micromolar to femtomolar range. K.sub.D Value RabbitAbs MouseAbs >10.sup.−7  6% 10.sup.−7  11% 10.sup.−8  19% 1% 10.sup.−9  39% 1% 10.sup.−10 10% 35% 10.sup.−11 13% 54% 10.sup.−12 2% 8% 10.sup.−13 1%

    [0324] K.sub.D values for 88 MouseAbs were derived from published literature. The K.sub.D measurement values for the 863 RabbitMAbs were all from the OI-RD measurements. RabbitMAbs appear to be on average 1-2 order of magnitude higher affinity. Origin of data—http://www.abcam.com/index.html?pageconfig=resource&rid=15749

    TABLE-US-00003 K.sub.D value Molar concentration (sensitivity) 10.sup.−4 to 10.sup.−6 Micromolar (uM) 10.sup.−7 to 10.sup.−9 Nanomolar (nM) 10.sup.−10 to 10.sup.−12 Picomolar (pM) 10.sup.−13 to 10.sup.−15 Femtomolar (fM)

    REFERENCES

    [0325] References discussed herein are incorporated by reference.

    [0326] Patents and Published Patent Applications

    [0327] PT1 EP 2623514 A (KYOTO UNIVERSITY) 26.09.2011

    [0328] PT2 Wang. X. Optical systems for microarray scanning. U.S. Pat. No. 7,706,419 B2. (Apr. 7, 2010)

    Non-Patent Literature

    [0329] NP1 NEWMAN, Robert, et al. Genetically Encodable Fluorescent Biosensors for Tracking Signaling. Chemical Reviews. 2011, vol. 111, no. 5, p 3614-3666.

    [0330] NP2 MIYAWAKI, Atsushi. Visualization of Spatial and Temporal Dynamics on Intracellular Signaling. Developmental Cell. 2003, vol. 4, n. 3, p. 295-305.

    [0331] NP3 MIYAWAKI, Atsushi. Cellular Functions Using Fluorescent Proteins and Fluorescence Resonance Energy Transfer. Annual Review of Biochemistry. 2011, vol. 80, p. 357-373.

    [0332] NP4 FRITZ, Rafael, et al. A Versatile Toolkit to Produce Sensitive FRET Biosensors to Visualize Signaling in Time and Space. Science 2013, vol 6, no. 285, p. 1-13.

    [0333] NP5 LAKOWICZ, Joseph. Principles of Fluorescent Spectroscopy. USA: Springer, 2010. ISBN 0387312781. p. 954.

    [0334] NP6 MIYAWAKI, Atsushi, et al. Fluorescent indicators for Ca2+ based on green fluorescent proteins and calmodulin. Nature. 1997, vol. 388, no. 6645, p 882-7.

    [0335] NP7 ALLEN, Michael, et al. Subcellular dynamics of protein kinase activity visualized by FRET-based reporters, 2006, Biochemical and bio-physical research communications. 2006, vol. 348, no. 2, p. 716-721.

    [0336] NP8 ZHANG, Jin, et al. FRET-based biosensors for protein kinases: illuminating the kinome. Mol. BioSyst. 2007, vol. 3, no. 11, p. 759-765.

    [0337] NP9 LISSANDRON, Valentina, et al. Improvement of a fret-based indicator for camp by linker design and stabilization of donor-acceptor interaction. J. Mol. Biol. 2005, vol 354, no. 3, p. 546-55.

    [0338] NP10 PERTZ, Oliver, et al. Spatiotemporal dynamics of rhoa activity in migrating cells. Nature. 2006, vol. 440, p. 1069-1072.

    [0339] NP11 KOMATSU, Naoki, et al. A Development of an optimized backbone of FRET biosensors for kinases and GTPases. Molecular Biology of the Cell. 2011, vol. 22, p. 4647-4658.

    [0340] NP12 Palmer, Amy, et al. Ca2+ indicators based on computationally redesigned calmodulin-peptide pairs. Chem. Biol. 2006, vol. 13, p. 521.

    [0341] NP13 SUN, Yuansheng, et al. Three-Color Spectral FRET Microscopy Localizes Three Interacting Proteins in Living. Biophys J. 2010, vol. 99, no. 4, p. 1274-1283.

    [0342] NP14 PACE, C, et al. A Helix Propensity Scale Based on Experimental Studies of Peptides and Proteins. Biophys J. 2010, vol. 75, no. 1, p. 422-427.

    [0343] NP15 KUSUNOKI, Hideki, et al. Solution structure of the DNA-binding domain of MafG. Nat. Struct. Biol. 2010, vol. 9, no. 4, p. 252-256.

    [0344] NP16 Mason, Jody, et al. Coiled coil domains: stability, specificity, and biological implications. Chembiochem. 2004, vol. 5, no. 2, p. 170-176.

    [0345] NP17 RIPOLL, Daniel, et al. Folding of the villin headpiece subdomain from random structures. Analysis of the charge distribution as a function of pH. J Mol Biol. 2004, vol. 339, no. 4, p. 915-925.

    [0346] NP18 VREVEN, Thom, et al. Prediction of protein-protein binding free energies. Protein Sci. 2012, vol. 21, no. 3, p. 396-404.

    [0347] NP19 MACKERELL, Alex, et al. All-atom empirical potential for molecular modeling and dynamics studies of proteins. Journal of Physical Chemistry B. 1998, vol. 102, p. 3586-3616.

    [0348] NP20 LINDORFF-LARSEN, Kresten, et al. Improved side-chain torsion potentials for the Amber ff99SB protein force field. Protein Sci. 2010, vol. 78, no. 8, p. 1950-1958.

    [0349] NP21 PHILLIPS, James, et al. Spatiotemporal dynamics of rhoa activity in migrating cells. Journal of Computational Chemistry. 2005, vol. 26, p. 1781-1802.

    [0350] NP22 PRONK, Sander, et al. GROMACS 4.5: a high-throughput and highly parallel open source molecular simulation toolkit. Bioinformatcs. 2012, vol. 29, no. 7, p. 845-854.

    [0351] NP23 Bonomi, Massimiliano, et al. PLUMED: A portable plugin for free-energy calculations with molecular dynamics. Comp. Phys. Comm. 2009, vol. 180, no 10, p. 1961-1972.

    [0352] NP24 LOMANT, A, et al. Chemical probes of extended biological structures: Synthesis and properties of the cleavable protein cross-linking reagent [35S] dithiobis (succinimidyl propionate) J. Mol. Biol. 1976, vol. 104, no. 1, p. 243-261.

    [0353] NP25 X U Ming-qun, et al. In vitro protein splicing of purified precursor and the identification of a branched intermediate. Cell. 1993, vol. 75, no. 7, p. 1371-1377.

    [0354] NP26 OJIDA, Akido, et al. Oligo-Asp Tag/Zn(II) Complex Probe as a New Pair for Labelling and Fluorescence Imaging of Proteins. J. Am. Chem. Soc., 2006, vol. 128, no. 32, p. 10452-10459.

    [0355] NP27 GAUTIER, Arnaud, et al. Selective Cross-Linking of Interacting Proteins Using Self-Labelling Tags. J. Am. Chem. Soc., 2009, vol. 131, no. 49, p. 17954-17962.

    [0356] NP28 LEQUIN, Rudolf. Enzyme Immunoassay (EIA)/Enzyme-Linked Immunosorbent Assay (ELISA). Clinical Chemistry, 2005, vol. 51, 2415-2418.

    [0357] NP29 ODELL, Ian, et al. Immunofluorescence techniques J. Invest. Dermatology, 2013, vol. 133, e4.

    [0358] NP30 MAHMOOD, Tahrin, et al. Western Blot: Technique, Theory, and Trouble Shooting. North American Journal of Medical Sciences, 2012, vol. 4, 429-434.

    [0359] NP31 HERMANSON, Greg. Bioconjugate Techniques. USA: Academic Press, 26 Jul 2010.

    [0360] NP32 HUDSON, Philipp, et al. Engineered antibody fragments and the rise of single domains. Nat. Biotechnol., 2005, vol. 23, 1126-1136.

    [0361] NP33 CABANTOUS, Stephanie, et al. Protein tagging and detection with engineered self-assembling fragments of green fluorescent protein. Nat. Biotechnol., 2005, vol. 23, 102-107.

    [0362] NP34 LUKER, Kathryn, et al. Split Gaussia Luciferase for Imaging Ligand-Receptor Binding in Bioluminescent Imaging. Methods in Molecular Biology, 2013, vol. 1098, 59-69.

    [0363] NP35 BUCHWALOW, Igor, et al. Immunochemistry: Basics and Methods. Springer: ISBN: 978-3-642-04608-7.

    [0364] NP36 PALMER, J.L., et al. Reduction and re-oxidation of a critical disulphide bond in rabit antibidy molecule. J. Biol. Chem., 1963, vol. 238, no.7, p 2369-2398.

    [0365] NP37 GE, Hui. UPA, a universal protein array system for quantitative detection of protein-protein, protein-DNA, protein-RNA and protein-ligand interactions. Nucleic Acids Res. 2000, vol. 28, e3.

    [0366] NP38 ZHU, Heng, et al. Analysis of yeast protein kinases using protein chips. Nat. Genet., 2000, vol. 26, 283-289.

    [0367] NP39 COHEN, Claudia, et al. A microchip-based enzyme assay for protein kinase A. Anal. Biochem., 1999, vol. 273, 89.

    [0368] NP40 CHENG, Chao-Min, et al. Paper-Based ELISA. Angew. Chem., 2010, vol. 49, 4771-4774.

    [0369] NP41 DULKEITH, E., et al. Fluorescence Quenching of Dye Molecules near Gold Nanoparticles: Radiative and Non-radiative Effects. Phys. Rev. Lett., 2002, vol. 89, 203002.

    [0370] NP42 SPERLING, Ralph, et al. Biological applications of gold nanoparticles. Chem. Soc. Rev., 2008, vol. 37, 1896-19

    [0371] NP43 DOLMANS, Dennis, et al. Photodynamic therapy for cancer. Nature reviews cancer, 2003, vol. 3, 380-387.

    [0372] NP44 HAMBLIN, Michael, et al. Photodynamic therapy: a new antimicrobial approach to infectious disease? Photochem. Photobiol. Sci., 2004, vol. 3, 436-450.

    Aspects and Embodiments of the Invention

    [0373] Aspects and embodiments of the invention may also be described in accordance with the following numbered paragraphs.

    [0374] 1. A multi-state dynamical hinge sensor comprising: [0375] (a) a rod L and a rod R connected to each other by a joint C; [0376] (b) the joint C comprises one or more elements connected end to end, where each element is selected to be rigid or flexible and is made of atoms or molecules;

    [0377] (b) an atom or molecule or macromolecule B bonded to the end of L opposite to the joint;

    [0378] (c) an atom or molecule or macromolecule B′ is bonded to the end of R opposite the joint;

    [0379] (d) a state denoted as the ON state where B is attracted to B′ ;

    [0380] (e) a state denoted as the OFF state where B is not attracted to B′;

    [0381] (f) spacer atoms or molecules in various locations within the hinge;

    [0382] (g) a number N constituent atoms or molecules q1, q2, . . . , qN of L and a number N′ constituent atoms or molecules q′1, q′2, . . . , q′N′ of R selected so that in the OFF state the hinge is open and in the ON state the hinge repeatedly opens and closes.

    [0383] 2. A multi-state dynamical hinge sensor as described in paragraph 1 to which is added: a fluorophore A or bioluminescent molecule and a fluorophore A′, wherein A is coupled directly to B or indirectly via the spacer atoms, and wherein A′ is coupled directly to B′ or indirectly via the spacer atoms, and wherein q1, q2, . . . , qN of L and q′1, q′2, . . . , q′N′ of R are adjustable so that in the OFF state the hinge is open and in the ON state the hinge repeatedly opens and closes.

    [0384] 3. The multi-state dynamical hinge sensor as described in paragraph 2 wherein A and A′ undergo measurable resonance energy transfer when sufficiently close to each other when B and B′ are in the ON state.

    [0385] 4. The multi-state dynamical hinge sensors described in paragraph 2 or 3 wherein one or both fluorophores or the bioluminescent molecule are photo-activatable or photo-convertible or photo-switchable or fluorescent protein timers or phosphorescent.

    [0386] 5. A multi-state dynamical hinge sensors described in paragraph 4 wherein the roles of (A and A′) and (B and B′) are exchanged.

    [0387] 6. The multi-state dynamical hinge sensors as described in paragraphs 4 to 5 wherein the constituents A,A′,B,B′,L,R, and spacers are comprised of either single amino acids, peptides or proteins and wherein q1, q2, . . . , qN, q′1, q′2, . . . , q′N′ are selected to be charged amino acids, or hydrophilic or hydrophobic amino acids, or a combination thereof.

    [0388] 7. The multi-state dynamical hinge sensors as described in paragraph 6 bonded directly or indirectly through A or A′ to any target protein or proteins of interest.

    [0389] 8. The multi-state dynamical hinge sensors as described in paragraph 7 wherein additional acceptor fluorophores absorb or emit light of different colours.

    [0390] 9. Copies of multi-state dynamical hinge sensors as described in paragraphs 7 or 8 and their insertions into sub-cellular locales described using plasmid and genetic-vector technology.

    [0391] 10. The use of multi-state dynamical hinge sensors described in paragraphs 7 to 9 to: (a) to visualise or monitor any of the following: (a) the structure and conformation of proteins; (b) the spatial distribution and assembly of protein complexes; (c) protein receptor/ligand interactions including the local concentrations of analytes; (d) the interactions of single molecules; (e) the structure and conformations of nucleic acids; (f) the distributions and transport of lipids; (g) membrane potential sensing; (h) monitoring fluorogenic protease substrates; (i) local cellular concentrations of cyclic AMP and calcium.

    [0392] 11. Use of the multi-state dynamical hinge sensors described in paragraphs 7 to 9 including within any of the following applications: biomolecular/molecular medicine and drug discovery; functional food and food for health research; development of ligands targeted at taste and olfactory receptors; cosmetics and perfume industries;

    [0393] immunoassays and membrane fusion assays in biopharmaceuticals and pharmaceuticals industries.

    [0394] 12. Use of the multi-state dynamical hinge sensors described in paragraphs 7 to 9 for applications including medical diagnostics and forensic industries for automated DNA sequencing, real-time PCR assays, SNP detection, and detection of nucleic acid hybridization.

    [0395] 13. The use of the multi-state dynamical hinge sensors described in paragraphs 7 to 9 as light/ligand activated actuators or active agents in the targeted delivery of drugs;

    [0396] and the manipulation and control of biological processes, and signalling networks.

    [0397] 14. The use of multi-state dynamical hinge sensors as described in paragraphs 2-5 in chemical, electronic, semi-conducting and quantum computing industries such as analytical indicators, electronic sensors, memory devices and nano-actuators.

    [0398] 15. Methods wherein multi-state dynamical hinge sensors described in paragraphs 7 to 9 are used including: biomolecular/molecular medicine and drug discovery; functional food and food for health research; development of ligands targeted at taste and olfactory receptors; cosmetics and perfume discovery; immunoassays and membrane fusion assays; medical diagnostics and forensic industries for automated DNA sequencing, real-time PCR assays, SNP detection, and detection of nucleic acid hybridization; targeted delivery of drugs; manipulation and control of biological processes, and signalling networks.

    [0399] 16. Methods wherein multi-state dynamical hinge sensors described in paragraphs 7 to 9 are used including: biomolecular/molecular medicine and drug discovery; functional food and food for health research; development of ligands targeted at taste and olfactory receptors; cosmetics and perfume discovery; immunoassays and membrane fusion assays; medical diagnostics and forensic industries for automated DNA sequencing, real-time PCR assays, SNP detection, and detection of nucleic acid hybridization; targeted delivery of drugs; manipulation and control of biological processes, and signalling networks.

    [0400] 17. Methods wherein multi-state dynamical hinge sensors described in paragraphs 7 to 9 are used to estimate the binding energies of ligands to receptors, and the ranking of their efficacy as agonists or antagonists in bimolecular/molecular medicine and drug discovery; functional food and food for health research; development of ligands targeted at taste and olfactory receptors; cosmetics and perfume discovery.

    [0401] 18. The multi-state dynamical hinge sensors as described in paragraph 6 and paragraph 7 where macromolecules A and A′ are selected to each be primary antibodies targeting different epitopes on the same analyte or antigen.

    [0402] 19. The multi-state dynamical hinge sensors as described in paragraph 6 and paragraph 7 where the macromolecule A is a primary antibody targeting an analyte or antigen, and and A′ is the corresponding secondary antibody.

    [0403] 20. The multi-state dynamical hinge sensors as described in paragraph 6 and paragraph 7 where the macromolecule A is an antigen targeting a primary antibody, and A′ is the corresponding secondary antibody.

    [0404] 21. The multi-state dynamical hinge sensors as described in paragraphs 6, 7, 17, 18, 19 where macromolecules B and B′ each comprise one or more selected molecules or moieties of split molecules, which, when brought close together due to the close presence of target analytes, undergo resonance energy transfer in the presence of a suitable electro-magnetic field of external or endogenous origin.

    [0405] 22. The multi-state dynamical hinge sensors as described in paragraphs 6, 7, 17, 18, 19 where macromolecules B and B′ each comprise one or more selected molecules or moieties of split molecules, which, when brought close together due to the close presence of target analytes, bioluminesce.

    [0406] 23. The multi-state dynamical hinge sensors as described in paragraphs 6, 7, 17, 18, 19 where macromolecules B and B′ each comprise one or more selected molecules or moieties of split molecules, which, when brought close together due to the close presence of target analytes, become an activated drug.

    [0407] 24. The multi-state dynamical hinge sensors as described in paragraphs 6, 7, 17, 18, 19 where macromolecules B and B′ each comprise one or more selected molecules or moieties of split molecules, which, when brought close together due to the close presence of target analytes, become an activated chemo-toxin.

    [0408] 25. The multi-state dynamical hinge sensors as described in paragraphs 6, 7, 17, 18, 19 where macromolecules B and B′ each comprise one or more selected molecules or moieties of split molecules, which, when brought close together due to the close presence of target analytes, become a chemical reaction or a catalysed chemical reaction.

    [0409] 26. The multi-state dynamical hinge sensors as described in paragraphs 6, 7, 17, 18, 19 where macromolecules B and B′ each comprise one or more selected molecules or moieties of split molecules in the presence of a suitable electro-magnetic field, which, when brought close together due to the presence of the target analytes, releases heat through quenching.

    [0410] 27. The multi-state dynamical hinge sensors as described in paragraphs 6, 7, 17, 18, 19 where macromolecules B and B′ each comprise one or more selected molecules or moieties of split molecules in the presence of a suitable electro-magnetic field, which, when brought close together due to the close presence of target analytes, become an activated photosensitizer complex producing oxygen radicals.

    [0411] 28. Multi-state dynamical hinge sensors with any combination of the effects described in paragraphs 20 to paragraph 26 in the same sensor.

    [0412] 29. The use of multi-state dynamical hinge sensors described in paragraphs 20, 21, 22 and 28 in the detection of analytes and their concentrations in assays and in living cells and their tracking over time.

    [0413] 30. The use of multi-state dynamical hinge sensors described in paragraphs 23 and 28 as a drug in the vicinity or within biological cells, fluids and tissue, and in vitro.

    [0414] 31. The use of multi-state dynamical hinge sensors described in paragraphs 24 and 28 as a chemotoxin in the vicinity or within biological cells, fluids and tissue, in vitro, and in organic materials.

    [0415] 32. The use of the multi-state dynamical hinge sensors described in paragraphs 25 and 28 as chemical reaction or a catalysed chemical reaction in the vicinity or within biological cells, fluids and tissue, in vitro, and in organic materials.

    [0416] 33. The use of the multi-state dynamical hinge sensors described in paragraphs 26 and 28 to deliver heat in the vicinity or within biological cells, fluids and tissue, in vitro, and in organic materials.

    [0417] 34. The use of the multi-state dynamical hinge sensors described in paragraphs 27 and 28 in photodynamic therapy in the vicinity or within biological cells, fluids and tissue, in vitro, and in organic materials.

    [0418] 35. Methods of use described in paragraph 28 wherein multi-state dynamical hinge sensors are used to perform assays for analytes including titration measure using microtiters or vials, with and without specialised equipment, for multiple applications including environmental, health, food safety, and security.

    [0419] 36. Methods of use described in paragraph 28 wherein multi-state dynamical hinge sensors is used to detect analytes in suitable continuous flow chambers for several applications including environmental, health, food safety, and security.