WO2019204873A1

WO2019204873A1 - Light-emitting biosensors

Info

Publication number: WO2019204873A1
Application number: PCT/AU2019/050367
Authority: WO
Inventors: Kirill Alexandrov; Zhong Guo
Original assignee: The University Of Queensland
Priority date: 2018-04-24
Filing date: 2019-04-24
Publication date: 2019-10-31

Abstract

Provided herein is a molecular biosensor that includes one or more sensors and one or more proteins or protein fragments, such as an enzyme or a fluorescent protein, that are capable of emitting light, or facilitating the emission of light, in response to the sensor. Detection devices including the molecular biosensor are further described herein. Also provided are methods of detecting a target molecule and disease diagnosis that utilise the molecular biosensor.

Description

LIGHT-EMITTING BIOSENSORS

TECHNICAL FIELD

THIS INVENTION relates to biosensors. More particularly, this invention relates to light-emitting biosensors that are suitable for detection of one or more target molecules in a sample. The biosensor molecule may also relate to the field of synthetic biology such as for constructing artificial cellular signalling networks.

BACKGROUND

Detection of target molecules or analytes in biological samples is central to diagnostic monitoring of health and disease. Key requirements of analyte detection are specificity and sensitivity, particularly when the target molecule or analyte is in a limiting amount or concentration in a biological sample.

Typically, specificity is provided by monoclonal antibodies which specifically bind the analyte. Sensitivity is typically provided by a label bound to the specific antibody, or to a secondary antibody which assists detection of relatively low levels of analyte. This type of diagnostic approach has become well known and widely used in the enzyme-linked immunosorbent sandwich assay (ELISA) format. In some cases, enzyme amplification can even further improve sensitivity such as by using a product of a proenzyme cleavage reaction catalyzing the same reaction. Some examples of such “autocatalytic” enzymes are trypsinogen, pepsinogen, or the blood coagulation factor XII. However, in relation to specificity antibodies are relatively expensive and can be difficult to produce with sufficient specificity for some analytes. Polyclonal antibodies also suffer from the same shortcomings and are even more difficult to produce and purify on a large scale.

Current methods to detect specific target molecules and analytes for either prognostic or diagnostic purposes suffer from a number of limitations which significantly restrict their widespread application in clinical, peri-operative and point-of- care settings. Most importantly, the vast majority of diagnostic assays require a significant level of technical expertise and a panel of expensive and specific reagents (most notably monoclonal antibodies) along with elaborate biomedical infrastructures which are rarely available outside specialized laboratory environments. For instance, ELISAs - the gold standard for detecting specific analytes in complex biological samples - rely on the selective capture of a target analyte on a solid surface which in turn is detected with a second affinity reagent that is specific for the target analyte. ELISAs also feature extensive incubation and washing steps which are generally time consuming and difficult to standardize as the number of successive steps frequently introduces significant variation across different procedures, operators and laboratories making quantitative comparisons difficult.

SUMMARY

The present invention addresses a need to develop quantitative, relatively inexpensive and easily produced molecular biosensors that readily detect the presence and/or the activity of target molecules (e.g., analytes) on short time scales that are compatible with treatment regimes. Such biosensors can either be applied singly or in multiplex to validate and/or diagnose molecular phenotypes with high specificity and great statistical confidence irrespective of the genetic background and natural variations in unrelated physiological processes. Such molecular biosensors may be used in other testing procedures such as where the target molecule or analyte is an illicit drug or performance-enhancing substance and/or in screening assays. Other applications of the biosensors may include the screening of molecules that promote or inhibit a binding interaction between proteins or between proteins and other biological molecules such as lipids, carbohydrates, metabolites, ions and nucleic acids.

More particularly, the present invention provides a molecular biosensor that is particularly suited to incorporation into devices such as laboratory or point-of-care devices for analysis and transmission of diagnostic results.

It is an object of the invention to provide a molecular biosensor which has specificity for a target molecule and which can produce an emitted light response to detection of the target molecule.

One broad form of the invention relates to a molecular biosensor comprising a sensor and an enzyme that facilitates the emission of light upon binding a target molecule by the sensor.

In one aspect of this form, the invention relates to a molecular biosensor comprising at least one amino acid sequence of an enzyme that is capable of reacting with a substrate molecule to produce light and one or more sensors that can bind or interact with a target molecule, and/or with each other, to thereby facilitate the enzyme reacting with the substrate molecule to produce light.

In one embodiment, the amino acid sequence of the enzyme is circularly permuted. Preferably the circularly permuted enzyme comprises respective amino acid sequences connected by a linker amino acid sequence. In one embodiment, the linker amino acid sequence comprises a light-emitting molecule.

In one particular embodiment the light-emitting molecule is a dye molecule.

In another particular embodiment, the light-emitting molecule is a fluorescent protein or fragment thereof.

According to this embodiment, light is emitted at a first wavelength in response to detecting, binding or interacting with a target molecule, which then triggers or activates emission of light at a second wavelength by the light-emitting molecule dye or fluorescent protein or fragment thereof.

In one embodiment, the enzyme is a bioluminescent enzyme. In one particular embodiment, the enzyme is obtainable or derived from Oplophagus gracilirostris. According to this embodiment, a preferred substrate is fumarazine.

Another form of the invention relates to a molecular biosensor comprising: a first molecular component comprising at least one amino acid sequence of an enzyme that is capable of reacting with a substrate molecule to produce light and one or more first sensors; and a second molecular component comprising one or more second sensors, whereby the first and second sensors can bind a target molecule, and/or with each other, to thereby facilitate the enzyme reacting with the substrate molecule to produce light.

Preferably, the first and second sensors can co-operatively bind a target molecule to thereby facilitate the enzyme reacting with the substrate molecule to produce light.

In one embodiment, the first molecular component and the second molecular component comprise a switch that facilitates activating enzyme catalytic activity following an interaction between the first molecular component and the second molecular component upon binding a target molecule. In one form of this embodiment, the first molecular component comprises an amino acid sequence of calmodulin or a variant thereof that is capable of non-covalently and reversibly binding or interacting with a ligand of the second molecular component such as a calmodulin-binding peptide or variant thereof, whereby binding of the peptide by calmodulin allosterically switches the enzyme into a more catalytic ally active state.

In one embodiment, the enzyme is a bioluminescent enzyme. In one particular embodiment, the enzyme is obtainable or derived from Oplophagus gracilirostris. According to this embodiment, a preferred substrate is fumarazine. Another broad form of the invention relates to a biosensor comprising a sensor and a fluorescent protein that facilitates the emission of light upon binding a target molecule by the sensor.

In one aspect, this form the invention relates to a molecular biosensor comprising at least one amino acid sequence of one or more fragments of a fluorescent protein and one or more sensors that can bind or interact with a target molecule, and/or with each other, to thereby facilitate the respective fragments of the fluorescent protein co-operatively emitting light.

In one embodiment, the amino acid sequence of the fluorescent protein is circularly permuted, whereby the sensor interconnects the first and second fragments of the fluorescent protein.

In one embodiment, the biosensor comprises first and second fragments of the fluorescent protein and a stabilizer that facilitates or stabilizes an interaction between the first and second fragments upon binding a target molecule. In one form of this embodiment, the stabilizer comprises an amino acid sequence of an SH3 domain that is capable of non-covalently and reversibly binding or interacting with a peptide of the second molecular component, such as an SH3-binding peptide.

In an embodiment, the molecular biosensor comprises a switch that facilitates an increase in fluorescence emission by the fluorescent protein.

In one particular form of the switch, the molecular biosensor comprises an amino acid sequence of calmodulin or a variant thereof that is capable of non-covalently and reversibly binding or interacting with a ligand such as a calmodulin-binding peptide or variant thereof. Suitably, binding between calmodulin or variant and the ligand facilitates allosteric switching of the fluorescent protein to thereby increase emission of light by the fluorescent protein.

In another aspect, this form of the invention relates to a molecular biosensor comprising: a first molecular component comprising first and second fragments of a fluorescent protein and a first sensor; and a second molecular component comprising a second sensor, whereby the first and second sensors can bind a target molecule, and/or with each other, to thereby facilitate the respective fragments of the fluorescent protein co-operatively emitting light.

In an embodiment, the first and second sensors can co-operatively bind a target molecule to thereby facilitate the first and second fluorescent protein fragments co operatively emitting light. In another embodiment, the first molecular component comprises a stabilizer that facilitates or stabilizes an interaction between the first and second fragments of the fluorescent protein.

In one form of this embodiment, the stabilizer comprises an amino acid sequence of an SH3 domain that is capable of non-covalently and reversibly binding or interacting with a peptide of the second molecular component, such as an SH3 -binding peptide.

The molecular biosensor may comprise another stabilizer, wherein the first molecular component comprises an amino acid sequence of calmodulin that is capable of non-covalently and reversibly binding or interacting with a ligand such as a calmodulin-binding peptide of the second molecular component upon binding a target molecule.

A further aspect of the invention provides a molecular biosensor comprising: a first molecular component comprising a first fragment of a fluorescent protein and a first sensor; and a second molecular component comprising a first fragment of a fluorescent protein and a second sensor, whereby the first and second sensors can bind a target molecule, and/or with each other, to thereby facilitate the respective fragments of the fluorescent protein co-operatively emitting light.

In an embodiment, the first and second sensors can co-operatively bind a target molecule to thereby facilitate the first and second fluorescent protein fragments co operatively emitting light.

In another embodiment, the molecular biosensor comprises a stabilizer that facilitates or stabilizes an interaction between the first and second molecular components of the biosensor.

Preferably, the stabilizer comprises an amino acid sequence of calmodulin that is capable of non-covalently and reversibly binding or interacting with a ligand such as a calmodulin-binding peptide of the second molecular component upon binding a target molecule.

A related aspect of the invention provides a method of producing an allosterically switchable protein that includes producing one or more chimeric proteins that comprise an amino acid sequence of at least a fragment of a protein of interest and an amino acid sequence of calmodulin, or a variant or fragment thereof. This aspect also includes an allosterically switchable protein produced according to the method and/or a molecular biosensor comprising one or more of the allosterically switchable proteins.

A further aspect of the invention provides a biosensor device comprising one or more biosensors according to the aforementioned aspects immobilized or affixed to a support that is transparent to light emitted by the biosensor.

A yet further aspect of the invention provides a method of detecting a target molecule, said method including the step of contacting the biosensor or biosensor device of any of the aforementioned aspects with a sample to thereby determine the presence or absence of the target molecule in the sample.

Another yet further aspect of the invention provides a method of screening or identifying an inhibitory target molecule, said method including the step of contacting the biosensor or biosensor device of any of the aforementioned aspects with a sample to thereby determine the presence or absence an inhibitory target molecule that at least partly inhibits binding by the sensor(s).

According to this aspect, in one embodiment the sensors may bind each other directly, which is inhibited by the inhibitory target molecule.

In another embodiment, the inhibitory target molecule inhibits a binding interaction between the sensors and a target molecule.

Suitably, inhibition is detected or measured as decrease in light emission.

A still yet further aspect of the invention provides a method of diagnosis of a disease or condition in an organism, said method including the step of contacting the biosensor or biosensor device of any of the aforementioned aspects with a biological sample obtained from the organism to thereby determine the presence or absence of a target molecule in the biological sample, determination of the presence or absence of the target molecule facilitating diagnosis of the disease or condition.

The organism may include plants and animals inclusive of fish, avians and mammals such as humans.

Another still yet further aspect of the invention provides a detection device that comprises a cell or chamber that comprises the biosensor or biosensor device of any of the aforementioned aspects.

Suitably, a sample may be introduced into the cell or chamber to thereby facilitate detection of a target molecule. In certain embodiments, the detection device is capable of providing an electrochemical, acoustic and/or optical signal that indicates the presence of the target molecule.

The detection device may further provide a disease diagnosis from a diagnostic target result by comprising:

a processor; and

a memory coupled to the processor, the memory including computer readable program code components that, when executed by the

processor, perform a set of functions including:

analysing a diagnostic test result and providing a diagnosis of

the disease or condition.

The detection device may further provide for communicating a diagnostic test result by comprising:

a processor; and

a memory coupled to the processor, the memory including computer readable program code components that, when executed by the processor, perform a set of functions including:

transmitting a diagnostic result to a receiving device; and

optionally receiving a diagnosis of the disease or condition from the or another receiving device.

A related aspect of the invention provides an isolated nucleic acid encoding the biosensor of any of the aforementioned aspects..

Another related aspect of the invention provides a genetic construct comprising the isolated nucleic acid of the aforementioned aspect.

A further related aspect of the invention provides a host cell comprising the genetic construct of the aforementioned aspect.

As used herein, unless the context requires otherwise, the words“comprise” , “ comprises” and“ comprising” will be understood to mean the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.

The indefinite articles ‘a’ and ‘ an’ are used here to refer to or encompass singular or plural elements or features and should not be taken as meaning or defining “one” or a“single” element or feature. As generally used herein“about” refers to a tolerance or variation in a stated value or amount that does not appreciably or substantially affect function, activity or efficacy. Typically, the tolerance or variation is no more than 10%, 5%, 3%, 2%, or 1% above or below a stated value or amount.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1. Introduction to NanoLuc. The enzyme was isolated from a deep-water shrimp Oplophagus gracilirostris and has peak luminescence at around 460nm. Bioluminescence from the NanoLuc (NLuc) system occurs when the optimized substrate called furimazine reacts with NLuc in the presence of molecular oxygen. This reaction yields furimamide and luminescence output.

Figure 2. Development of an allosteric switch module based on Calmodulin (CaM)-NanoLuc chimera activated by calmodulin binding protein (CaM-BP). The right panel represents titration of lOnM of CaM-NanoLuc chimeric protein with increasing concentrations of CaM-BP. The data was fitted to a quadratic equation leading to a Kd of 17hM. The panel below shows wells of a 96 well plate containing 100 qL of ImM CaM-NanoLuc and 10m1 furimazine solution (from Promega) in 20mM Tris-HCl pH 7.2, 20mM NaCl and lmM CaCl₂. The right well was supplemented with 5mM of CaM- BP.

Figure 3. A two component biosensor based on CaM-NanoLuc allosterically switchable chimera. (A) A schematic representation of the two component biosensor. The ligand (shown as a black star) induces dimerization of biosensor components and activation of NanoLuc activity. (B) Titration of 200m1 10hM FKBP-CaM-NanoLuc biosensor mixed with 30nM FRB-CaM-BP component and 0.25m1 furimazine solution in buffer containing 20mM Tris-HCl pH 7.2, 20mM NaCl and 0.5mM CaCF. The luminescence values were plotted against the concentration of the drug and fitted to a quadratic equation leading to a K_d of lOnM. (C) as in (B) but using a tacrolimus biosensor composed of lOnM FKBP-CaM-NanoLuc and 30nM CalA/B-CaM-BP. The fit of the data led to a K_d of 6nM.

Figure 4. Engineering of circular permutated NanoLuc and construction thereon based biosensors. The bottom panel represents molecular representations of wt (left) and the circular permutated NanoLuc. Figure 5. A single component rapamycin biosensor based on the circular permutated Nano Luc. (A) Graphic representation of the biosensor where the NanoLuc component , FKBP and FRB are shown in ribbon representation. (B) Titration of 200m1 solution of InM Rapamycin biosensor supplemented with 0.25 mΐ furimazine stock solution in buffer containing 20mM Tris-HCl pH 7.2, 20mM NaCl with the increasing concentrations of the drug. The data was fitted to a K_d of 0.4nM.

Figure 6. A single component alpha- amylase biosensor based on the circular permutated NanoLuc. (A) Graphic representation of the biosensor where the NanoLuc component is shown in ribbon representation while VHH domains attached to the N and C terminus are shown as geometric shapes. (B) Titration of 200m1 solution of lnM alpha- amylase biosensor supplemented with 0.25m1 furimazine stock solution in buffer containing 20mM Tris-HCl pH 7.2, 20mM NaCl with the increasing concentrations of alpha- amylase. The data was fitted to a K_d of 0.4nM.

Figure 7. Comparison of the structures of FKBP:rapamycin:FRB complex with FKBP:tacrolimus:Calcineurin A/B complex. Due to the large size of the complex and its non-covalent nature the subunits of Calcineurin A/B were fused to form a single subunit entity that is more amenable to engineering.

Figure 8. Structure and performance of Tacrolimus one component biosensor (A) A model of a single component tacrolimus biosensor composed of circular permutated NanoLuc flanked with FKBP (displayed as ribbon) and a fusion of Calcineurin A and Calcineurin B proteins (displayed as a molecular surface). (B) Titration of 200m1 solution of InM Tacroli us biosensor supplemented with 0.25ml furimazine stock solution in buffer containing 20mM Tris-HCl pH 7.2, 20mM NaCl with the increasing concentrations of the drug in the presence or absence of 50% serum. The data was fitted to a K_d of 0.4nM. (C) same as in (B) but in the presence or absence of 50% saliva. The data was fitted to a K_d of 0.4nM. (D) Analysis of the sensitivity of the tacrolimus biosensor to rapamycin. In the experiment lnM solution of tacrolimus biosensor either alone of with 200nM rapamycin or lmM cyclosporine A was titrated with increasing concentrations of tacrolimus.

Figure 9. Assessing the suitability of a single component tacrolimus biosensor for PoC applications. (A) Assessing the time dependence of biosensor activation. The stock reaction containing InM Tacrolimus biosensor supplemented with 0.25m1/200m1 furimazine stock solution were mixed with the indicated concentrations of tacrolimus and incubated for indicated periods of time in the 96 well plate. The luminescence of the samples was then measured and the data was plotted against the concentration of Tacrolimus. (B) The comparison of the luminescence yield of InM solution of the wild type recombinant NanoLuc, CaM-NanoLuc in the presence of lOOnM of CaM-BP and NanoLuc-based tacrolimus biosensor in the presence of 50nM of tacrolimus. (C) Assessment of the ability of tacrolimus biosensor to withstand de- and rehydration. In the experiment 5m1 of 40nM NanoLuc-based tacrolimus biosensor in assay buffer was dried in the wells of 96 well plate and then resuspended in the 200m1 of reaction buffer. The reactions was initiated by the addition of the 0.25m1 furimazine and the indicated concentrations of tacrolimus and the samples were measured after 20 minutes incubation. The in the control reactions the stock solution of the biosensors was diluted to the final concentration of lnM with the reaction buffer, furimazine and the indicated concentrations of tacrolimus. (D) Assessment of Tacrolimus biosensor performance in whole and lysed blood. The experiments were performed using as in C but in the presence of 50% of whole or hypotonically lysed blood.

Figure 10. Construction of NanoLuc biosensors with red-shifted emission (A) The design sequence of converting the wild type NanoLuc into a BRET sensor. The circular permutation of NanoLuc followed by the insertion of a fluorescent protein domain (in this case EGFP) between de novo created N and C- terminus. The subsequent addition of the binding domains creates a biosensor that when activated by a ligand results in photon emission at 460nm that leads to fluorescent excitation of the fused EGFP that subsequently emits light with the emission maximum of 510nM. (B) a fluorescent scan of InM solution of rapamycin biosensor constructed as shown in (A). The red trace represents the emission of the biosensor in the absence of rapamycin while the green trace represents emission of the biosensor solution supplemented with 20nM of rapamycin. The maximum emission of NanoLuc is indicated by an arrow. (C) Titration of InM solution of rapamycin biosensor from A and B with increasing concentration of rapamycin. The luminescence (wavelength over 480nm) was recorded by applying filter BLUE1 (TECAN plate reader). And the determined values were plotted against the concentrations of rapamycin. The data was fitted to the quadratic equation leading to a Kd value of 0.5nM which is very close to the values obtained for the parental biosensor. Figure 11. Approaches for shifting the emission of the NanoLuc biosensors to the red part of the spectmm. (A) A biosensor based on the NanoLuc where a red fluorescent protein such as Cherry is inserted between the N and C -terminal parts of the circular permutated NanoLuc. The protein serves as a linker and is used to convert the emission of the NanoLuc from blue (460nm) into the red emission (>600nm ). (B) Same as in (A) but instead of red fluorescent protein a red organic dye is used as a luminescence acceptor and the fluorophore.

Figure 12. Further approaches for extending the emission wavelength of the NanoLuc-based biosensors. The derivatives of furimazine that can serve as substrates of NanoLuc and result in emission at higher wavelength². The bottom plot shows the emission scans of NanoLuc luminescence exposed to different compounds. The last two scans represent BRET from the compound F25 to the far-red small molecule organic dyes.

Figure 13. An alternative approach for measuring the luminescence of NanoLuc- based biosensors in the presence of lysed blood. Here the biosensor is dried in a film on the transparent surface of a substrate such as a glass slide. The drying matrix includes substances with high viscosity that restrict free diffusion. The lysed blood sample containing the analyte and the NanoLuc substrate are added on top of the film rehydrating it and resulting in diffusion of the substrate and analyte to the biosensor. The emission was collected through the transparent side of the slide.

Figure 14. Rapamycin“two-component” biosensor based on cpGFP-CalM Ca. In this example, the calmodulin-calmodulin binding interaction acts as a stabilizer.

Figure 15. Peptide“single component” biosensor based on cpGFP-CalM Ca. In this example, the calmodulin-calmodulin binding peptide interaction acts as an allosteric switch.

Figure 16. Rapamycin“two-component” biosensor based on the biosensor in FIG. 15. As in FIG. 15, the calmodulin-calmodulin binding peptide interaction acts as an allosteric switch.

Figure 17. Rapamycin“single component” biosensor based on FKBP-cpGFP-FRB.

Figure 18. Improving the dynamic ranges of biosensors based on fragment displacement.

Figure 19. Generic approach for construction of protein switches. The gene coding for a protein of interest (Pol) is used to create a library of calmodulin (CaM) insertion mutants.

DETAILED DESCRIPTION The present invention provides a biosensor which is capable of producing or generating light in response to detecting, binding or interacting with a target molecule. In one form, the biosensor comprises an enzyme or enzyme fragment switchable between catalytically“inactive” and catalytically“active” states to thereby react with a substrate molecule to produce light. More particularly, the enzyme or enzyme fragment is a bioluminescent enzyme which has been engineered to enable switching between catalytically“inactive” and catalytically“active” states. In one particular form the enzyme is circularly permuted. In one particular embodiment of this form, the circularly permuted enzyme comprises a linker amino acid sequence that may comprise a light- emitting molecule such as a dye or a fluorescent protein or fragment thereof. According to this embodiment, light is emitted at a first wavelength in response to detecting, binding or interacting with a target molecule, which then triggers or activates emission of light at a second wavelength by the light-emitting molecule dye or fluorescent protein or fragment thereof. In another form, the biosensor comprises respective fragments of a fluorescent protein, wherein binding of a target molecule causes the fragments to co operatively emit light. The biosensor molecule(s) disclosed herein may have efficacy in molecular diagnostics wherein the“target molecule” is an analyte or other molecule of diagnostic value or importance. In particular, the sensitivity of the biosensors disclosed herein may have particular efficacy in screening assays to detect target molecules that promote a binding interaction or those that inhibit a binding interaction. Another application of the biosensor disclosed herein may be in synthetic biology applications for constructing multi-component artificial cellular signalling networks.

For the purposes of this invention, by“ isolated’ is meant material (such as a molecule) that has been removed from its natural state or otherwise been subjected to human manipulation. Isolated material may be substantially or essentially free from components that normally accompany it in its natural state, or may be manipulated so as to be in an artificial state together with components that normally accompany it in its natural state. Isolated proteins and nucleic acids may be in native, chemical synthetic or recombinant form.

By‘ protein” is meant an amino acid polymer. The amino acids may be natural or non-natural amino acids, D- or L- amino acids as are well understood in the art.

A“ peptide” is a protein having less than fifty (50) amino acids.

A“ polypeptide” is a protein having fifty (50) or more amino acids. A“fluorescent protein” as used herein may relate to any protein or fragment thereof that is capable of emitting light of a particular wavelength upon excitation by light of a different wavelength. Fluorescent proteins may include those originally obtainable from animal and plant organisms such as of the genera Aequorea, Discosoma, Solanum, Montipora, Lobophyllia, Anemonia etc , which may include green fluorescent protein (GFP), red fluorescent protein (RFP), blue fluorescent protein (BFP), cyan fluorescent protein (CFP) etc and also synthetically engineered variants that have desired spectral properties different to their naturally-occurring counterparts, such as mStrawberry, mOrange and mTomato, although without limitation thereto.

While the terms“first” and“second” are used in the context of respective, separate or discrete molecular components of the biosensor, such as sensors, enzymes, fluorescent protein fragments and/or binding moieties, it will be appreciated that these do not relate to any particular non-arbitrary ordering or designation that cannot be reversed. Accordingly, the structure and functional properties of the first component or second component disclosed herein could be those of a second component or a first component, respectively. Likewise, the structure and functional properties of a first sensor and a second sensor disclosed herein could be those of a second sensor and a first sensor, respectively. Similarly, the structure and functional properties of a first binding moiety and the second binding moiety disclosed herein could be those of a second binding moiety and a first binding moiety, respectively. It will also be appreciated that the biosensor may further comprise one or more other, non-stated molecular components.

In this context, a“ component” or“ molecular component’ is a discrete molecule that forms a separate part, portion or component of the biosensor. In typical embodiments, each molecular component is, or comprises, a single, contiguous amino acid sequence ( i.e a fusion protein or chimera).

As used herein, a“ target molecule” may be any molecule detectable by the biosensor. The target molecule may bind, or be bound or interact with the one or more sensors of the biosensor. In one particular embodiment, the target molecule may promote or enhance a binding interaction between the sensors. In another particular embodiment, the target molecule may at least partly prevent or inhibit a binding interaction between the sensors, or between the sensors and another target molecule, referred to herein as an“ inhibitory target molecule”. A target molecule may be present in a“ sample”, which may be an isolate, specimen, extract, library or other mixture of compounds that potentially includes a target molecule, inclusive of inhibitory target molecules. These may include biological samples, dmg samples, molecular libraries of naturally-occurring molecules, synthetic libraries, combinatorial libraries and/or molecules produced from in silico libraries of molecular stmctures, although without limitation thereto.

Enzymatic biosensors

In one aspect of this form, the invention relates to a molecular biosensor comprising at least one amino acid sequence of an enzyme that is capable of reacting with a substrate molecule to produce light and one or more sensors that can bind or interact with a target molecule to thereby facilitate the enzyme reacting with the substrate molecule to produce light.

As used herein an“ enzyme” is a protein having catalytic activity towards one or more substrate molecules. Suitably, the enzyme is capable of displaying catalytic activity towards a substrate molecule to thereby produce light. Preferably, the enzyme is a bioluminescent enzyme or one or more fragments thereof. In one particular embodiment, the enzyme is a luciferase, such as obtainable or derived from Oplophagus gracilirostris. In one particular form, the luciferase is commercially available as NanoLuc^®. According to this embodiment, a preferred substrate is fumarazine, which is converted to fumarazide by the luciferase, with emission of light at about 460nm. This is shown schematically in FIG.1.

As generally used herein“catalytically active” and“catalytically active state” may refer to absolute or relative amounts of enzyme activity that can be displayed or achieved by an enzyme or a fragment or portion thereof. Typically, an enzyme is catalytically active or in a catalytically active state if it is capable of displaying specific enzyme activity towards a substrate molecule to produce light under appropriate reaction conditions. As generally used herein“ catalytically inactive” and“ catalytically inactive state” may refer to an enzyme, fragment or portion thereof that is substantially incapable of displaying specific enzyme activity towards a substrate molecule under appropriate reaction conditions. Typically, the light produced would be substantially less compared to that produced by a corresponding catalytically active enzyme, or would be entirely absent. In some embodiments relating to detection of inhibitory target molecules, the enzyme may switch from a“ catalytically active state” to a catalytically inactive state”.

A first particular aspect of the invention provides a molecular biosensor comprising at least one amino acid sequence of an enzyme capable of reacting with a substrate molecule, when in a catalytically active state to produce light; and one or more sensors that can bind or interact with a target molecule, and/or with each other, to thereby facilitate the enzyme reacting with the substrate molecule to produce light.

Suitably, the enzyme is in a catalytically inactive state before the one or more sensor amino acid sequences bind or interact with a target molecule.

Suitably, the enzyme is in a catalytically active state before a binding interaction between the one or more sensor amino acid sequences is inhibited by an inhibitory target molecule.

Suitably, said biosensor is in the form a single, contiguous amino acid sequence, such as in the form of a fusion protein or chimeric protein.

As hereinbefore described, preferably the enzyme is a bioluminescent enzyme or one or more fragments thereof. In one particular embodiment, the enzyme is a luciferase, such as obtainable or derived from Oplophagus gracilirostris. In one particular form, the luciferase is commercially available as NanoLuc^®. According to this embodiment, a preferred substrate is fumarazine, which is converted to fumarazide by the luciferase, with emission of light at about 460nm.

In one broad embodiment, the amino acid sequence of the enzyme is circularly permuted. Preferably, according to this embodiment, the circularly permuted amino acid sequence comprises an amino acid sequence that is normally at or near the C terminus of the enzyme located N-terminal of an amino acid sequence that is normally at or near the N terminus of the enzyme.

In a particular embodiment, the circularly permuted amino acid sequence comprises: (i) an amino acid sequence that is normally at or near the C terminus of the enzyme located N-terminal of an amino acid sequence that is normally at or near the N terminus of the enzyme; and (ii) a linker or spacer amino acid sequence between said amino acid sequence that is normally at or near the C terminus of the enzyme and said amino acid sequence that is normally at or near the N terminus of the enzyme. Thus, a general embodiment provides a circularly permuted enzyme according to the following general formula:

X - C- linker-N-Y

wherein:

X-C is normally the C-terminal amino acid sequence of the enzyme, or fragment thereof;

N-Y is normally the N-terminal amino acid sequence of the enzyme, or fragment thereof; and

the linker is an amino acid sequence contiguous with X-C and N-Y.

These embodiments are schematically shown in FIGS. 4-6, 10 and 11.

Circular permutation disrupts enzyme activity, which activity is then rescued by re-association of the enzyme portion, mediated by the sensor(s) detecting or binding a target molecule.

With reference to Fig. 6, a proposed molecular mechanism is that the b-strand fused to the binding moiety can be pulled out of the protein due to the solvation forces and binding of the target molecule (in this case a amylase) which leads to the forced re association of the enzyme portions leading to reconstitution of the enzyme and hence activation of the catalytic activity of the enzyme.

A practical issue that can arise in molecular diagnostics is that spectral filtering can occur whereby molecules in a biological sample can absorb light emitted by the biosensor. For example, the issue of spectral filtering by haemoglobin is a common problem.

In this context, an embodiment of the present invention utilizes light emitted at a first wavelength by the enzyme reacting with the substrate molecule to activate emission of light at a second wavelength by a light-emitting molecule. This phenomenon is termed Bioluminescence Resonance Energy Transfer or BRET. The light-emitting molecule may be referred to as a“wavelength converter”.

In one embodiment, the linker amino acid sequence comprises the light-emitting molecule.

In one particular embodiment, the light-emitting molecule is a fluorescent protein or fragment thereof, such as those hereinbefore described. Non-limiting examples include green fluorescent protein (GFP) and red fluorescent protein (RFP). In the context of haemoglobin and as shown in FIG. 10, the biosensor of this embodiment shifts the emission wavelength into the red and far-red spectrum in order to reduce haemoglobin-mediated signal filtering. It will therefore be appreciated that this principle may be applied to any light-emitting biosensor where the emitted light is of a wavelength that may be absorbed or attenuated by molecules potentially present in a biological sample.

In another particular embodiment, the light-emitting molecule is a dye molecule. The dye molecule may be an organic dye molecule. By way of example, a small organic red dye may be conjugated to the enzyme using site selective chemistry. A non-limiting example is shown in FIG. 11. This may be achieved using thiol chemistry in case cysteines are absent from the sequence of the protein biosensor (or present at positions where they can be mutated to serine without compromising the structure or function of the protein). Alternatively, codon reassignment can be used to incorporate a biorthogonal moiety, such as 4-Azidophenylalanine, that can be used for dye conjugation via click chemistry.

An alternative embodiment would include furimazine derivatives with red shifted emission maximum, as recently reported by Shakhmin l al, 2017, Org. Biomol. Chem. 15 8559. Such substrates can be used for forming BRET pairs resulting in far-red shifted emission as schematically shown in FIG.12.

Another form of the invention relates to a biosensor comprising: a first molecular component comprising at least one amino acid sequence of an enzyme that is capable of reacting with a substrate molecule to produce light and one or more first sensor amino acid sequences; and a second molecular component comprising one or more second sensor amino acid sequences, whereby the first and second sensor amino acid sequences can bind a target molecule to thereby facilitate the enzyme reacting with the substrate molecule to produce light.

Preferably, the first and second sensor amino acid sequences can co-operatively bind a target molecule to thereby facilitate the enzyme reacting with the substrate molecule to produce light. Accordingly, the binding interaction between the target molecule and the binding moieties of the sensor amino acid sequences facilitates co localization of the first and second molecular components.

In one form of this embodiment, the first molecular component comprises an amino acid sequence of calmodulin that is capable of binding or interacting with a ligand of the second molecular component such as a calmodulin-binding peptide. In one particular embodiment, the first molecular component and the second molecular component comprise a switch that facilitates activating enzyme catalytic activity following an interaction between the first molecular component and the second molecular component upon binding a target molecule. In one form of this embodiment, the first molecular component comprises an amino acid sequence of calmodulin or a variant thereof that is capable of non-covalently and reversibly binding or interacting with a ligand of the second molecular component such as a calmodulin-binding peptide or variant thereof, whereby binding of the ligand by calmodulin allosterically switches the enzyme into a more catalytic ally active state.

A non-limiting example of a “two-component” biosensor comprising a bioluminescent enzyme“switched” by this calmodulin- calmodulin peptide interaction is shown in FIG. 3.

Fluorescent protein biosensors

Another broad form of the invention relates to a biosensor comprising a sensor and a fluorescent protein that facilitates the emission of light upon binding a target molecule by the sensor.

In one aspect, this form the invention relates to a biosensor comprising at least one amino acid sequence of one or more respective fragments of a fluorescent protein and one or more sensors that can bind or interact with a target molecule to thereby facilitate the respective fragments of the fluorescent protein co-operatively emitting light.

The biosensor may further comprise one or more stabilizers.

The stabilizer may be, or comprise, any molecules that can bind or interact, such as“ complementary binding partners”. Preferably, this binding or interaction is non- covalent and reversible. These may include molecules that engage in ligand-receptor binding, calmodulin binding partners, protein-protein interaction domains (e.g SH3 domains and their proline-rich binding domains, leucine zippers and other dimerization domains, PDZ domains, LIM domains and their binding peptides) and antigen- antibody binding partners and affinity clamps although without limitation thereto. In one embodiment, the biosensor comprises first and second fragments of the fluorescent protein. In some embodiments, the sensor interconnects or is disposed between the first and second fragments of the fluorescent protein. In one embodiment, the biosensor comprises first and second fragments of the fluorescent protein and a stabilizer that facilitates or stabilizes an interaction between the first and second fragments.

A non-limiting example is shown in FIG. 15.

The biosensor may further comprise a switch that facilitates activating fluorescence emission by the fluorescent protein following binding a target molecule. In one form of this embodiment, the molecular biosensor comprises an amino acid sequence of calmodulin or a variant thereof that is capable of non-covalently and reversibly binding or interacting with a ligand such as a calmodulin-binding peptide or variant thereof, whereby binding of the ligand by calmodulin allosterically activates fluorescence emission by the fluorescent protein. In a particular embodiment, the amino acid sequence of calmodulin or a variant thereof interconnects respective fragments of the fluorescent protein.

A non-limiting example is shown in FIG. 15.

In another embodiment, the fluorescent protein is circularly permuted. Circular permutation disrupts fluorescence emission activity, which activity is then rescued by re-association of the fluorescent protein portions, mediated by the sensor(s) detecting or binding a target molecule.

A non-limiting example is shown in FIG. 17.

In another aspect, this form of the invention relates to a molecular biosensor comprising: a first molecular component comprising first and second fragments of a fluorescent protein and one or more first sensors; and a second molecular component comprising one or more second sensors, whereby the first and second sensors can bind a target molecule to thereby facilitate the respective fragments of the fluorescent protein co-operatively emitting light.

Preferably, the first and second sensors can co-operatively bind a target molecule to thereby facilitate the respective fluorescent protein fragments co operatively emitting light.

In one embodiment, the first and second fragments of the fluorescent protein molecular comprise a stabilizer that facilitates or stabilizes an interaction between the first and second fragments of the fluorescent protein upon binding a target molecule. Reference is made to “stabilizers” and “complementary binding partners” as hereinbefore described.

In one form of this embodiment, the stabilizer comprises an amino acid sequence of an SH3 domain that is capable of non-covalently and reversibly binding or interacting with a peptide of the second molecular component, such as an SH3 -binding peptide. A non- limiting example is shown in FIG. 16.

The biosensor may further comprise a switch that facilitates activating fluorescence emission by the fluorescent protein following binding a target molecule. In one form of this embodiment, the molecular biosensor comprises an amino acid sequence of calmodulin or a variant thereof that is capable of non-covalently and reversibly binding or interacting with a ligand, such as a calmodulin-binding peptide or variant thereof, whereby binding of the ligand by calmodulin allosterically activates fluorescence emission by the fluorescent protein. In a particular embodiment, the amino acid sequence of calmodulin or a variant thereof interconnects respective fragments of the fluorescent protein.

A non-limiting example is shown in FIG. 15.

In yet another aspect, this form of the invention relates to a molecular biosensor comprising: a first molecular component comprising at least one amino acid sequence of a first fragment of a fluorescent protein and one or more first sensors; and a second molecular component comprising at least one amino acid sequence of a second fragment of a fluorescent protein and one or more second sensors, whereby the first and second sensors can bind a target molecule to thereby facilitate the respective first and second fragments of the fluorescent protein co-operatively emitting light.

Preferably, the first and second sensor amino acid sequences can co-operatively bind a target molecule to thereby facilitate the first and second fluorescent protein fragments co-operatively emitting light.

In one embodiment, the molecular biosensor comprises one or more stabilizers that facilitate or stabilize an interaction between the first and second components upon binding a target molecule. Reference is made to“stabilizers” and“complementary binding partners” as hereinbefore described.

In one form of this embodiment, the stabilizer comprises a calmodulin amino acid sequence which is capable of reversible and releasably binding a ligand such as a calmodulin-binding peptide. Preferably, the first molecular component comprises the calmodulin amino acid sequence and the second molecular component comprises the ligand, such as a calmodulin-binding peptide amino acid sequence.

A non-limiting example is shown in FIG. 14.

It should also be appreciated that when reference is made to first and second fragments of a“fluorescent protein”, the first and second fragments could be of the same or different fluorescent protein.

Sensors and target molecules

According to the aforementioned aspects, molecular biosensors comprise: (i) an enzyme that can react with a substrate to produce light: or (ii) respective fragments of a fluorescent protein; and comprise one or more sensors that facilitate detection of a target molecule.

Suitably, the one or more sensors can bind or interact with a target molecule comprise one or more binding moieties that can bind or interact with the target molecule, or which can directly interact or bind in the absence of a target molecule. Preferably, the one or more binding moieties can co-operatively bind or interact with the target molecule.

As generally used herein a“ binding moiety” or“ binding moieties” refer to one or a plurality of molecules or biological or chemical components or entities that are capable of recognizing and/or binding each other, or one or more target molecules.

Binding moieties may be proteins, nucleic acids (e.g single-stranded or double- stranded DNA or RNA), sugars, oligosaccharides, polysaccharides or other carbohydrates, lipids or any combinations of these such as glycoproteins, PNA constructs etc or molecular components thereof

In one embodiment, the binding moieties comprise an amino acid sequence of at least a fragment of any protein or protein fragment or domain that can bind or interact directly, or bind to a target molecule. The binding moiety may be, or comprise a protein such as a peptide, antibody, antibody fragment or any other protein scaffold that can be suitably engineered to create or comprise a binding portion, domain or region (e.g. reviewed in Binz et al., 2005 Nature Biotechnology, 23, 1257-68.) which binds a target molecule.

By way of example only, binding moieties may be, or comprise: (i) an amino acid sequence of a ligand binding domain of a receptor responsive to binding of a target molecule such as a cognate growth factor, cytokine, a hormone (e.g. insulin), neurotransmitters etc; (ii) an amino acid sequence of an ion or metabolite transporter capable of, or responsive to, binding of a target molecule such as an ion or metabolite (e.g a Ca²⁺-binding protein such as calmodulin or calcineurin or a glucose transporter); (iii) a zinc finger amino acid sequence responsive to zinc-dependent binding a DNA target molecule; (iv) a helix-loop-helix amino acid sequence responsive to binding a DNA target molecule; (v) a pleckstrin homology domain amino acid sequence responsive to binding of a phosphoinositide target molecule; (vi) an amino acid sequence of a Src homology 2- or Src homology 3-domain responsive to a signaling protein; (vii) an amino acid sequence of an antigen responsive to binding of an antibody target molecule; or (viii) an amino acid sequence of a protein kinase or phosphatase responsive to binding of a phosphorylatable or phosphorylated target molecule; (ix) ubiquitin-binding domains; (x) proteins or protein domains that bind small molecules, drugs or antibiotics such as rapamycin-binding FKBP and FRB domains or tacrolimus- binding domains of calcineurin; (xi) single- or double-stranded DNA, RNA or PNA constructs that bind nucleic acid target molecules, such as where the DNA or RNA are coupled or cross-linked to an amino acid sequence or other protein-nucleic acid interaction; and/or (xii) an affinity clamp such as a PDZ-FH3 domain fusion; inclusive of modified or engineered versions thereof, although without limitation thereto.

In another embodiment, the binding moieties comprise one or a plurality epitopes that can be bind or be bound by an antibody target molecule.

In another embodiment, the binding moieties may be or comprise an antibody or antibody fragment, inclusive of monoclonal and polyclonal antibodies, recombinant antibodies, Fab and Fab’ 2 fragments, diabodies and single chain antibody fragments (e.g. scVs), although without limitation thereto. Suitably, the first and second binding moieties may be or comprise respective antibodies or antibody fragments that bind a target molecule.

In yet another particular embodiment, the binding moieties may be or comprise an antibody-binding molecule, wherein the antibody(ies) has specificity for a target molecule. The antibody-binding molecule preferably comprises an amino acid sequence of protein A, or a fragment thereof (e.g a ZZ domain), which binds an Fc portion of the antibody.

Typically, the respective binding moieties are capable of binding, interacting or forming a complex with the same target molecule. It will also be appreciated that the “same” target molecule can have respective, different moieties, subunits, domains, ligands or epitopes that can be bound by the respective binding moieties to thereby co localize and activate enzyme activity.

In one broad embodiment, the target molecule may be any ligand, analyte, small organic molecule, ion, epitope, domain, fragment, subunit, moiety or combination thereof, such as a protein inclusive of antibodies and antibody fragments, antigens, enzymes, phosphoproteins, glycoproteins, lipoproteins and glycoproteins, lipid, phospholipids, carbohydrates inclusive of simple sugars, disaccharides and polysaccharides, nucleic acids, nucleoprotein or any other molecule or analyte. These include drugs and other pharmaceuticals including antibiotics, banned substances, illicit drugs or drugs of addiction, chemotherapeutic agents and lead compounds in drug design and screening, molecules and analytes typically found in biological samples such as biomarkers, tumour and other antigens, receptors, DNA-binding proteins inclusive of transcription factors, hormones, neurotransmitters, growth factors, cytokines, receptors, metabolic enzymes, signaling molecules, nucleic acids such as DNA and RNA, membrane lipids and other cellular components, pathogen-derived molecules inclusive of viral, bacterial, protozoan, fungal and worm proteins, lipids, carbohydrates and nucleic acids, although without limitation thereto. As previously, described, it will be appreciated that the“same” target molecule can be bound by different, respective binding moieties. In a specific embodiment, the target molecule may be an inhibitory target molecule that at least partly prevents or inhibits a binding interaction between the sensor binding moieties.

In one particular embodiment, a first binding moiety is an FKBP amino acid sequence and a second binding moiety comprises a rapamycin-binding FRB amino acid sequence. Suitably, the biosensor of this embodiment is capable of detecting, binding or interacting with rapamycin.

In another particular embodiment, a first binding moiety comprises a rapamycin- binding FKBP amino acid sequence and a second binding moiety comprises one or more calcineurin amino acid sequences. Preferably, the one or more calcineurin amino acid sequences comprise a calcineurin A and a calcineurin B amino acid sequence preferably comprising an intervening linker amino acid sequence. Suitably, the biosensor of this embodiment is capable of detecting, binding or interacting with tacrolimus (FK-506).

With regard to sensors, binding moieties and target molecules, reference is made to International Publications WO2014/040129, WO2015/035452, WO2016/191812 and WO2016/065415 which provide several examples of sensors, binding moieties and target molecules that may be used according to the present invention and are incorporated by reference in their entirety herein.

In some embodiments, the target molecule is an enzyme such as a amylase. In such embodiments, the first and second binding moieties are, respectively the camelid antibodies VHH1 and VHH2.

In some embodiments, the target molecule is a secretory protein such as Human Serum Albumin. In such embodiments, the first and second binding moieties are, respectively the camelid antibodies VHH1 and bacterial albumin binding module.

In some embodiments, the target molecule is a small organic molecule such as rapamycin. In such embodiments, the first and second binding moieties are, respectively the FKBP and FRB.

In some embodiments, the target molecule is a small organic molecule such as FK506 or tacrolimus. In such embodiments, the first and second binding moieties are, respectively, the FKBP and a Calcineurin A/B complex.

In some embodiments, the target molecule is a small organic molecule such as cyclosporin. In such embodiments, the first and second binding moieties are, respectively, a peptidyl prolyl cis trans isomerase A and Calcineurin A/B complex.

In some embodiments, the target molecule is a small organic molecule such as Vitamin D. In such embodiments, the first and second binding moieties are, respectively, a Vitamin D binding protein that undergoes conformational transition when Vitamin D binds to it while the second moiety represents a protein domain that binds with much higher affinity to the Vitamin D binding protein: Vitamin D complex than to the apo form of the Vitamin D binding protein.

In a further embodiment, the binding moieties may be selected to identify previously unknown molecules that can bind or interact with the binding moieties. In some embodiments, the biosensor may facilitate screening for one or more molecules that enhance or enable a binding interaction between the binding moieties. By way of example, biosensors comprising FKBP amino acid sequence and FRB amino acid sequences may be used to identify new immunosuppressive molecules from a library of compounds.

In another embodiment, the binding moieties may be capable of directly binding or interacting without binding a target molecule. In some embodiments, the biosensor may facilitate screening for one or more inhibitors that prevent binding between the binding moieties.

It will be appreciated that the biosensors and the molecular components thereof described herein may be, or comprise, contiguous amino acid sequences such as in the form of chimeric proteins or fusion proteins as are well understood in the art. Optionally, respective amino acid sequences ( e.g binding moieties, enzyme amino acid sequences, protease amino acid sequences etc ) may be discrete or separate amino acid sequences linked or connected by spacers or linkers (e.g. amino acids, amino acid sequences, nucleotides, nucleotide sequences or other molecules) to optimize features or activities such as target molecule recognition, binding and enzyme activity or inhibition, although without limitation thereto. Non-limiting examples of amino acid sequences inclusive of enzyme amino acid sequences, engineered mutants, linkers, protease cleavage sites, and binding moieties are provided in SEQ ID NOS: l-14.

It will also be appreciated that the invention includes biosensor molecules that are variants of the embodiments described herein, or which comprise variants of the constituent enzyme, fluorescent protein, sensor and/or other protein and peptide ( e.g calmodulin, calmodulin-binding peptide) amino acid sequences disclosed herein. Typically, such variants have at least 80%, at least 85%, preferably at least 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98% or 99% sequence identity with any of the amino acid sequences disclosed herein, such as SEQ ID NOS: l-14 or portions thereof. By way of example only, conservative amino acid variations may be made without an appreciable or substantial change in function. For example, conservative amino acid substitutions may be tolerated where charge, hydrophilicity, hydrophobicity, side chain “bulk”, secondary and/or tertiary structure (e.g. helicity), target molecule binding, enzyme or fluorescence activity are substantially unaltered or are altered to a degree that does not appreciably or substantially compromise the function of the biosensor.

Variants of the invention (other than the engineered non- active mutants described herein) are selected to be functional and so retain or substantially retain catalytic activity, or the ability to reconstitute such catalytic activity when provided together with suitable further components of a biosensor as described above. Variants of the non-covalently associating amino acid sequences (such as first and second fragment sequences) described herein are selected to retain the ability to reconstitute a stable enzyme or fluorescent protein when provided in combination with their respective binding partner sequence. The term "sequence identity" is used herein in its broadest sense to include the number of exact amino acid matches having regard to an appropriate alignment using a standard algorithm, having regard to the extent that sequences are identical over a window of comparison. Sequence identity may be determined using computer algorithms such as GAP, BESTFIT, FASTA and the BLAST family of programs as for example disclosed by Altschul et al., 1997, Nucl. Acids Res. 25 3389. A detailed discussion of sequence analysis can be found in Unit 19.3 of CURRENT PROTOCOLS IN MOLECULAR BIOLOGY Eds. Ausubel et al. (John Wiley & Sons Inc NY, 1995- 1999).

Protein fragments may comprise up to 5%, 10%, 15%, 20%, 25%, 30%, 35%,

40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, preferably up to 80%, 85%, more preferably up to 90% or up to 95-99% of an amino acid sequence disclosed herein. In some embodiments, the protein fragment may comprise up to 5, 10, 20, 40, 50, 70, 80, 90, 100, 120, 150, 180 200, 220, 230. 250, 280, 300, 330, 350, 400 or 450 amino acids of an amino acid sequence disclosed herein, such as SEQ ID NOS: l-14.

A further aspect of the invention provides a biosensor device comprising one or more biosensors according to the aforementioned aspects immobilized or affixed to a support that is substantially transparent to light emitted by the biosensor.

By way of example, the support may be or include a slide, chip, wafer, strip, plate or other structure which is formed of a material that is substantially transparent to light emitted by the biosensor.

An example is schematically shown in FIG. 13.

In an embodiment, the biosensor is dried as a film on a substantially transparent surface of the support. The biosensor molecule may be applied in a“drying matrix” that includes substances with high viscosity that restricts free diffusion. A sample containing a target molecule and an enzyme substrate are added on top of the film, thereby rehydrating it and resulting in diffusion of the substrate and target molecule to the biosensor. The emitted light is collected via the substantially transparent side of the device.

Detection of target molecules

A further aspect of the invention provides a kit or composition comprising one or more biosensors disclosed herein, optionally in combination with one or more substrate molecules. Suitably, the kit is for detecting a target molecule is a sample to thereby determine the presence or absence of the target molecule in the sample.

In another further aspect, the invention provides a method of detecting a target molecule, said method including the step of contacting the composition of the aforementioned aspect with a sample to thereby determine the presence or absence of the target molecule in the sample.

In one embodiment, the sample is a biological sample. Biological samples may include organ samples, tissue samples, cellular samples, fluid samples or any other sample obtainable, obtained, derivable or derived from an organism or a component of the organism. The biological sample can comprise a fermentation medium, feedstock or food product such as for example, but not limited to, dairy products.

In particular embodiments, the biological sample is obtainable from a mammal, preferably a human. By way of example, the biological sample may be a fluid sample such as blood, serum, plasma, urine, saliva, tears, sweat, cerebrospinal fluid or am ni otic fluid, a tissue sample such as a tissue or organ biopsy or may be a cellular sample such as a sample comprising red blood cells, lymphocytes, tumour cells or skin cells, although without limitation thereto. A particular type of biological sample is a pathology sample.

Suitably, the enzyme activity of the biosensor is not substantially inhibited by components of the sample (e.g. serum proteins, metabolites, cells, cellular debris and components, naturally-occurring protease inhibitors etc).

In one embodiment, the biosensor and/or methods of use may be applicable to drug testing such as for detecting the use of illicit drugs of addiction (e.g cannabinoids, amphetamines, ***e, heroin etc.) and/or for the detection of performance-enhancing substances in sport and/or masking agents that are typically used to avoid detection of performance-enhancing substances. This may be applicable to the detection of banned performance -enhancing substances in humans and/or other mammals such as racehorses and greyhounds that may be subjected to illicit“doping” to enhance performance.

In one embodiment, the target molecule promotes an interaction between targeting moieties. The sample may comprise a library of compounds that may comprise at least one target molecule that increases, enhances or promotes a binding interaction between the binding moieties. By way of example,“immunosuppressant” biosensors could be used to identify alternative chemical structures that facilitate binding between the binding moieties. A further aspect provides a method of screening or identifying an inhibitory target molecule, said method including the step of contacting the biosensor or biosensor device of any of the aforementioned aspects with a sample to thereby determine the presence or absence an inhibitory target molecule that at least partly inhibits the sensor(s). In one embodiment, the method may be a screening assay to identify molecules in a library are potential inhibitors of binding between the binding moieties, in which case the biosensor may be used to measure or detect binding inhibition. In such an embodiment, light emission would decrease in the presence of the inhibitory target molecule.

In another particular embodiment, the biosensor and/or methods of use are for diagnosis of a disease or condition of a mammal, such as a human.

Accordingly, a preferred aspect of the invention provides a method of diagnosis of a disease or condition in a human, said method including the step of contacting the composition of the aforementioned aspect with a biological sample obtained from the human to thereby determine the presence or absence of a target molecule in the biological sample, wherein determination of the presence or absence of the target molecule facilitates diagnosis of the disease or condition.

The disease or condition may be anywhere detection of a target molecule assists diagnosis. Nonlimiting examples of target molecules or analytes include blood coagulation factors such as previously described, kallikreins inclusive of PSA, matrix metalloproteinases, viral and bacterial proteases, antibodies, glucose, triglycerides, lipoproteins, cholesterol, tumour antigens, lymphocyte antigens, autoantigens and autoantibodies, drugs, salts, creatinine, blood serum or plasma proteins, pesticides, uric acid, products and intermediates of human and animal metabolism and metals.

This preferred aspect of the invention may be adapted to be performed as a “point of care” method whereby determination of the presence or absence of the target molecule may occur at a patient location which is then either analysed at that location or transmitted to a remote location for diagnosis of the disease or condition.

A still yet further aspect of the invention provides a detection device that comprises a cell or chamber that comprises the biosensor of any of the aforementioned aspects.

a processor; and

processor, perform a set of functions including:

analysing a diagnostic test result and providing a diagnosis of

the disease or condition.

a processor; and

transmitting a diagnostic result to a receiving device; and

Diagnostic aspects of the invention may also be in the form of a kit comprising one or a plurality of different biosensors capable of detecting one or a plurality of different target molecules. In this regard, a kit may comprise an array of different biosensors capable of detecting a plurality of different target molecules. The kit may further comprise one or more amplifier molecules, deactivating molecules and/or labeled substrates, as hereinbefore described. The kit may also comprise additional components including reagents such as buffers and diluents, reaction vessels and instructions for use.

A further aspect of the invention provides an isolated nucleic acid which encodes an amino acid sequence of the biosensor of the invention, or a variant thereof as hereinbefore defined.

The term “nucleic acid’’ as used herein designates single-or double-stranded mRNA, RNA, cRNA, RNAi, siRNA and DNA inclusive of cDNA, mitochondrial DNA (mtDNA) and genomic DNA. A“ polynucleotide” is a nucleic acid having eighty (80) or more contiguous nucleotides, while an “ oligonucleotide” has less than eighty (80) contiguous nucleotides. A“primer” is usually a single-stranded oligonucleotide, preferably having 15-50 contiguous nucleotides, which is capable of annealing to a complementary nucleic acid“template” and being extended in a template-dependent fashion by the action of a DNA polymerase such as Taq polymerase, RNA-dependent DNA polymerase or Sequenase™. A “probe” may be a single or double-stranded oligonucleotide or polynucleotide, suitably labelled for the purpose of detecting complementary sequences in Northern or Southern blotting, for example.

The invention also provides variants and/or fragments of the isolated nucleic acids. Variants may comprise a nucleotide sequence at least 70%, at least 75%, preferably at least 80%, at least 85%, more preferably at least 90%, 91%, 93%, 94%, 95%, 96%, 97%, 98% or 99% nucleotide sequence identity with any nucleotide sequence disclosed herein. In other embodiments, nucleic acid variants may hybridize with the nucleotide sequence of with any nucleotide sequence disclosed herein, under high stringency conditions.

Fragments may comprise or consist of up to 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95-99% of the contiguous nucleotides present in any nucleotide sequence disclosed herein.

Fragments may comprise or consist of up to 20, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900 950, 1000, 1050, 1100, 1150, 1200, 1350 or 1300 contiguous nucleotides present in any nucleotide sequence disclosed herein.

The invention also provides“ genetic constructs” that comprise one or more isolated nucleic acids, variants or fragments thereof as disclosed herein operably linked to one or more additional nucleotide sequences.

As generally used herein, a "genetic construct" is an artificially created nucleic acid that incorporates, and/or facilitates use of, an isolated nucleic acid disclosed herein.

In particular embodiments, such constructs may be useful for recombinant manipulation, propagation, amplification, homologous recombination and/or expression of said isolated nucleic acid.

As used herein, a genetic construct used for recombinant protein expression is referred to as an " expression construct ", wherein the isolated nucleic acid to be expressed is operably linked or operably connected to one or more additional nucleotide sequences in an expression vector.

An “ expression vector” may be either a self-replicating extra-chromosomal vector such as a plasmid, or a vector that integrates into a host genome.

In this context, the one or more additional nucleotide sequences are regulatory nucleotide sequences.

By“ operably linked” or "operably connected" is meant that said regulatory nucleotide sequence(s) is/are positioned relative to the nucleic acid to be expressed to initiate, regulate or otherwise control expression of the nucleic acid.

Regulatory nucleotide sequences will generally be appropriate for the host cell used for expression. Numerous types of appropriate expression vectors and suitable regulatory sequences are known in the art for a variety of host cells.

One or more regulatory nucleotide sequences may include, but are not limited to, promoter sequences, leader or signal sequences, ribosomal binding sites, transcriptional start and termination sequences, translational start and termination sequences, splice donor/acceptor sequences and enhancer or activator sequences.

Constitutive or inducible promoters as known in the art may be used and include, for example, nisin-inducible, tetracycline-repressible, IPTG-inducible, alcohol- inducible, acid-inducible and/or metal-inducible promoters.

In one embodiment, the expression vector comprises a selectable marker gene. Selectable markers are useful whether for the purposes of selection of transformed bacteria (such as bla, kanR, ermB and tetR ) or transformed mammalian cells (such as hygromycin, G418 and puromycin resistance).

Suitable host cells for expression may be prokaryotic or eukaryotic, such as bacterial cells inclusive of Escherichia coli (DH5a for example), yeast cells such as S. cerivisiae or Pichia pastoris, insect cells such as SF9 cells utilized with a baculovirus expression system, or any of various mammalian or other animal host cells such as CHO, BHK or 293 cells, although without limitation thereto.

Introduction of expression constructs into suitable host cells may be by way of techniques including but not limited to electroporation, heat shock, calcium phosphate precipitation, DEAE dextran-mediated transfection, liposome-based transfection (e.g. lipofectin, lipofectamine), protoplast fusion, microinjection or microparticle bombardment, as are well known in the art. Purification of the recombinant biosensor molecule may be performed by any method known in the art. In preferred embodiments, the recombinant biosensor molecule comprises a fusion partner (preferably a C-terminal His tag) which allows purification by virtue of an appropriate affinity matrix, which in the case of a His tag would be a nickel matrix or resin.

So that the invention may be readily understood and put into practical effect, embodiments of the invention will be described with reference to the following non limiting Examples.

EXAMPLES

EXAMPLE 1

NanoLuc^®-based biosensors

The biomedical field has greatly benefited from the discovery of bioluminescent proteins. Currently, scientists employ bioluminescent systems for numerous biomedical applications, ranging from highly sensitive cellular assays to bioluminescence based molecular imaging. Traditionally, these systems are based on Firefly and Renilla lucif erases; however, the applicability of these enzymes is limited by their size, stability, and luminescence efficiency. NanoLuc, a novel bioluminescent enzyme, offers several advantages over established systems, including enhanced stability, smaller size, and > 150-fold increase in luminescence¹.

In addition, the substrate for NanoLuc displays enhanced stability and lower background activity, opening up new possibilities in the field of bioluminescence imaging and target molecule detection.

Converting NanoLuc into an allosteric peptide activated switch

The very high quantum yield of NanoLuc makes it an attractive platform for biosensor development. Based on our previous experience we first decided to test if NanoLuc could be converted into the allosteric switch module. To this end we analyzed the high resolution structure of NanoLuc and inserted a calmodulin (CaM) domain between critical structural elements. The resulting chimeric molecules was produced in recombinant form and analysed by its ability to process its substrate Furimazine at different concentrations of Calmodulin Binding Peptide (CaM-BP). As can be seen in Figure 2 the luminescence of the biosensor increased dose-dependently and saturably upon addition of CaM-BP. The overall luminescence change was close to 20 fold that is one of the largest dynamic changes observed in artificial signaling systems (Fig. 2 right panel).

The availability of the CaM-operated switch module encouraged us to apply the earlier developed concept of two component biosensors to construct sensors of rapamycin and tacrolimus. To this end the CaM-NanoLuc module was fused to FKBP and a mutant version of CaM-BP was fused to either FRB (rapamycin biosensor) or to Calcineurin A and Calcineurin B complex fusion by a linker (tacrolimus biosensor) (Fig 3; SEQ ID NO:4). To facilitate the expression of Calcineurin A and Calcineurin B complex, both subunits were fused into one single chain by a linker, in addition a solubility SUMO tag was attached to the N-terminal of the protein.

Titration of a mixture of these components with the respective drugs resulted in saturable increase of luminescence that could be fitted to K_dS of 10 and 6nM for rapamycin and tacrolimus respectively. The limit of detection detect for both biosensors was below to 0.5nM indicating that the developed tacrolimus biosensor may be suitable for the detection of the clinically relevant concentrations of tacrolimus that is in the range of l-40nM. The parental CaM-NanoLuc and the tacrolimus biosensor could operate at 1 mM and 0.5 mM Ca²⁺ suggesting that the sensors are likely to be suitable for the use under the physiological calcium concentration.

The fact that the activity of the NanoLuc could be operated by the CaM inserted between the two last b-strands prompted us to test if alternative conformational rearrangements could be used to elicit this change. To this end, we fused the native N and C-terminus of NanoLuc using a flexible linker and reintroduced them at a new position. This resulted in a formation of a circular permutated NanoLuc (cp-NanoLuc) (Lig. 4). We then fused the LRB and LKBP to the newly formed N and C terminus of the cp-NanoLuc and produced the resulting fusion protein in recombinant form. As can be seen in Ligure 5B, addition of rapamycin to the solution of the recombinant protein resulted in the dose depended increase in luminescence. Importantly, the biosensor displayed a Kd value of 0.4nM and the limit of detection in the range of 50pM. This represents nearly 10 fold improvement compared to the earlier version shown in the Ligure 3.

A further construct was produced where two VHH binders were extracted from PDB structure lbvn.pdb and lkxv.pdb and were fused to the N and C terminus of the cp-NanoLuc to construct and develop an alpha amylase biosensor (PIG. 6).

Encouraged by these results we decided to test if the same approach can be used to construct a tacrolimus biosensor. The main impediment to that is the size of tacrolimus binding ternary complex that is more than two times larger than the PRB:PKBP complex that is used to bind rapamycin (Ligure 7).

Having to fit a ternary complex of that size into the developed architecture is not a trivial task. We have solved it by first fusing the subunits of Calcineurin A and Calcineurin B through a linker into a single subunit and then subsequently using the resulting open reading frame to replace the FRB domain in the rapamycin biosensor shown in Figure 5 (The domain arrangement for Rapamycin is FKBP-cpNanoLuc-FRB; For FK506, CN-alpha/beta-cpNanoLuc-FKBP). The resulting biosensor is schematically shown Figure 8A. Titration of this biosensor with tacrolimus demonstrated dose dependent response and the overall affinity for tacrolimus of 0.4nM and the limit of detection close to 50pM. We repeated the experiments using samples spiked with 50% serum or saliva. In both cases while we observed the reduction in overall signal the response of the biosensor has not changed and the concentration of the drug in the sample could be reliably detected. Finally, we tested the effect of the rapamycin and cyclosporine A on the performance of the tacrolimus biosensor. We titrated lnM of tacrolimus biosensor either in buffer or in the presence of 200 molar excess of rapamycin (200nM) or 1000 molar excess of cyclosporine A (ImM). The data shown in Figure 8D shows that while neither rapamycin nor cyclosporin A did not lead to activation of the tacrolimus biosensor both drugs interfered with its activation. This is not entirely surprising as all biosensors share one common binding domain -FKBP between rapamycin and tacrolimus biosensors and Calcineurin A/ Calcineurin B complex between tacrolimus and cyclosporine A biosensors. Given the fact that even at 1000X drug excess the biosensor retained 7-10% of its dynamic range it is still likely that one can use the developed biosensors in the patients on both drugs. Further it is possible to that use of biosensor combinations may allow even more accurate measurement of drug concentrations in such patients. Additional experiments are needed to ascertain this assumption.

Testing the suitability of the developed biosensor to construction of the point of care tacrolimus test

First we tested the time dependence of the biosensor response by carrying out the measurements after different incubation periods. As shown in Figure 9A the amplitude of response increased with increasing incubation time coming to saturation at 20min. We subsequently quantified the luminescent yield of the wild type NanoLuc, CaM-NanoLuc and the tacrolimus biosensor. As can be seen in FIG. 9B the CaM NanoLuc retained 70% of the luminescent yield of the parental enzyme while the tacrolimus biosensor retained only 20% of the yield. Both of the findings described above may be related to the concentrations of furimazine substrate used. The Promega corporation that supplies the reagent does not provide its concentrations and provides volumetric measurements in its protocols. It is entirely possible that optimization of substrate concentration would significantly affect the obtained results. We observed that with increase of the furimazine concentration the signal increased linearly till it was too strong for the used instruments. In the future HPLC and extinction coefficient-based quantification methods can be used in order to perform quantitative experiments and re evaluate the current observations.

Next, we tested the stability of the tacrolimus biosensor under the drying conditions. Figure 9C shows the result of drying and rehydration that shows that the biosensor can be dried and rehydrated without any optimization of the conditions. We subsequently tested the compatibility of the biosensor with lysed and whole blood. We observed that 50% hypotonically lysed blood significantly diminished the biosensor’s signal (Fig. 9D). This is not surprising as the emission wavelength of the NanoLuc overlaps with the absorption wavelength of haemoglobin.

Addressing the inner filter effect of haemoglobin

The issue of spectral filtering by haemoglobin is common and can be addressed by different approaches:

a) Bleaching of hemoglobin with H2O2 with a subsequent hydrolysis of H2O2 by a peroxidase. b) Shift of the emission wavelength into the red and far-red spectrum in order to reduce hemoglobin-mediated signal filtering. c) Use of alternative NanoLuc substrates with the red shifted emission.

One way of executing option b) is to co-integrate the biosensor with a luminescent converter that absorbs light in the blue part of the spectrum and emits at a higher wavelength. This phenomenon is termed Bioluminescence Resonance Energy Transfer or BRET. We decided to test this idea by using green fluorescent protein as a linker between the domains of the rapamycin biosensor. Figure 10 shows the engineering steps taken to constmct a NanoLuc based rapamycin biosensor with a wavelength converter.

Figure 10B shows the emission scan of such rapamycin biosensor in the absence and presence of rapamycin. It can be seen that the emission of the biosensor, as expected is shifted to the maximum of 5l0nm that corresponds to the emission of EGFP. The results of titration of the red shifted biosensor with rapamycin are shown in Figure 10C and demonstrate that despite the integration of the wavelength“extender” domain the biosensor faithfully produced enhanced light emission with the maximum at 510nm. The signal change could be fitted to a K_d of 0.5nM that demonstrates that despite introduction of the BRET-forming domain the function of the biosensor remained largely unchanged.

Following the successful construction of the BRET biosensor the next step would be to construct tacrolimus biosensors with either EGFP or a red fluorescent protein such as Cherry or mTomato (FIG. 11 A). However, addition of yet another protein domain to already four domain biosensors may result in folding conflicts and low protein expression. As an alternative plan, a small organic red dye may be conjugated to the NanoLuc using site selective chemistry (FIG. 11B). This may be achieved using thiol chemistry in case cysteines are absent from the sequence of the protein biosensor (or present at positions where they can be mutated to serine without compromising the structure or function of the protein). Alternatively, codon reassignment can be used to incorporate a biorthogonal moiety such as 4-Azidophenylalanine that can be used for dye conjugation via click chemistry. An alternative approach would be to use the recently reported furimazine derivatives with red shifted emission maximum². Such substrates can be used for forming BRET pairs resulting in far-red shifted emission (FIG. 12 bottom panel). However, the exact quantum yields, stability and compatibility with the developed biosensors are not known and need to be investigated.

Using unidirectional light emission

The intention of using the developed biosensors in dry down format also creates an opportunity for exploiting a unidirectional emission (FIG. 13). The idea here borrows from the glucometer platform where the enzyme is embedded into PEG“cake”. Glucose from blood rapidly diffuses to the biosensor while the proteins and cells migrate much slower through viscous PEG layer. In the case of tacrolimus the dried down biosensor would be activated by rehydration that brings with it both the fluorogenic substrate and the drug. The emission can be recorded through the transparent support. The emission is expected to slowly decay as the biosensor is diffusing through the PEG layer into the lysed blood. However, optimization of this process may create a sufficient time window for quantitative detection of tacrolimus. EXAMPLE 2

Fluorescent protein-based biosensors

Fluorescent proteins such as GFP may be utilized in light-emitting biosensors. In an example shown in FIG. 15, a single component biosensor comprises first and second GFP fragments. An SH3 domain is N-terminal of the first GFP fragment, a calmodulin sensor interconnects the first and second GFP fragments and an SFI3 ligand peptide is C-terminal of the second GFP fragment. In the absence of calmodulin binding peptide target molecule, fluorescence emission is minimal. The first and second GFP fragments interact as a result of the calmodulin sensor binding calmodulin-binding peptide that induces a conformational change of the latter leading to reconstitution of the GFP structure and resulting in increased light emission. The SH3 domain and the SH3-binding peptide act as a stabilizer to facilitate the interaction between first and second GFP fragments.

The single-component biosensor of FIG. 15 may be incorporated into a“two- component” biosensor as shown in FIG. 16. In this example, the GFP peptide sensor (which is essentially the same as that shown in FIG. 15) of the first molecular component is fused to an FKBP binding moiety. The second molecular component comprises a low affinity derivative of M13 calmodulin binding peptide fused to FRB. Binding of rapamycin target molecule results in increase of the local concentration of both of components and association of the calmodulin binding peptide with calmodulin. The latter switches the conformational change restoring the active conformation of GFP thereby significantly increasing fluorescence emission by the GFP sensor.

An example of another two-component biosensor is shown in FIG. 14. In this embodiment the first component comprises an FKBP sensor fused to a GFP fragment with a C-terminal calmodulin amino acid sequence. The second component comprises an FRB sensor fused to a second GFP fragment with a C-terminal calmodulin-binding peptide. Upon binding rapamycin by FRB and FKBP, GFP is activated by conformation/solvation driven conformation change to thereby increase fluorescence emission. In this example, the calmodulin-peptide binding interaction acts as a stabilizer rather than as an allosteric switch.

In another example shown in FIG. 17, a single component GFP-based biosensor may be produced comprising first and second GFP fragments of a circularly-permuted GFP protein. FKBP is located N-terminally of the first GFP fragment and FRB is located C-terminally of the second GFP fragment. In the absence of target molecule (i.e rapamycin) binding, fluorescence emission is low. Upon binding of rapamycin by the FRB/FKBP sensor, light emission increases substantially in a dose-dependent manner.

The functional mechanism of this biosensor is expected to be similar to the one described for single component NanoLuc biosensor in Figure 6. Here a domain fused to a b-strand at the end of the reporter protein displaces this strand due to Brownian motion and solvation forces rendering the GFP reporter molecule non-functional. Addition of the ligand constrains the movement of the b- strand and increases the proportion of the functional GFP in the sample. Therefore the dynamic range of the biosensors can be further increased by rigidifying the linker between the protein and the last b-strand.

This model provides guidance in further improving the dynamic ranges of biosensors based on fragment displacement. As shown in Figure 18(A) this can be achieved by further increasing the rigidity of the linker connecting the fragment to the enzyme, alternatively this can be achieved by extending the linker thereby increasing the hydrodynamic radius of the fragment-binder assembly and decreasing the frequency of its association with the reporter domain (B). In another embodiment the dynamic range is increased by creating a steric barrier by fusing a disordered peptide or polymer sequence (such as PEG) to the binder thereby creating a barrier for ligand-independent reporter reconstitution. In yet another embodiment the increase in dynamic range can be achieved by introducing the interactions that stabilise the biosensors conformation in the “open” form where binding of the ligand shifts the equilibrium to the“closed” form.

EXAMPLE 3

Generic pipeline for protein-based switch module design .

The examples provided herein demonstrate the feasibility of a generic approach for construction of protein switches as outlined in FIG. 19. Here the gene coding for a protein of interest (Pol) is used to create a library of calmodulin (CaM) insertion mutants. Such library can be either computationally designed using available structural information and placing the calmodulin sequences in the loop regions or it can be constructed by transposon mediated random insertion of CaM sequence in to the sequence of Pol. The library is them expressed either in vivo (bacteria, yeast or eukaryotic cells) or in vitro using cell-free expression and appropriate activity assays are used to identify mutants that are activated or inactivated by calmodulin binding peptide. These variants can be then used to construct two component biosensors. The identified mutants are further analysed and are used to construct circular permutated variants. The identified insertion sides can be then used to construct single component biosensors by adding binding domains to the N and C terminus of the sequence. It should be appreciated that the calmodulin and calmodulin binding peptide amino acid sequences used in this example may be wild-type amino acid sequences or be variant sequences, such as created by mutagenesis.

BIOSENSOR SEQUENCES

NanoLuc-CalM peptide sensor (SEQ ID NO:l)

GSDN MVFTLEDFVG DWRQTAGYN LDQVLEQGGVSSLFQN LGVSVT PIQRIVLSG E NG LKI DIHVI I PYEG LSG DQMGQI E KI FKVVYPVDDH H FKVI LHYGTLVI DGVTPN M I DYFG RPYEG IAVFDG KKITVTGTLWNG N KI I DE RLI N PDGS LLF RVTI N TE EQIAE FKEAFSLFD KDG DGTITTK E LGTVM RSLGQN PTEAE LQD M I N EVDADG NGTI DFPE FLTM MARK M KDTDSE EEI REAFRVFDKDG NGYISAAELRHVMTN LG EKLTDEEV DE M I READI DG DGQVNYE E FVQM MTAGGSGGVTGWRLCE RI LA

KLAAALEH H H H H H

NanoLuc-CalM-FKBP (Rapamycin and FK506 sensor component 2; SEQ ID NO:2)

GSDN MVFTLEDFVG DWRQTAGYN LDQVLEQGGVSSLFQN LGVSVT PIQRIVLSG E NG LKI DIHVI I PYEG LSG DQMGQI E KI FKVVYPVDDH H FKVI LHYGTLVI DGVTPN M I DYFG RPYEG IAVFDG KKITVTGTLWNG N KI I DE RLI N PDGS LLF RVTI NGSGGTEEQIAE FKEAFSLFDKDG DGT ITTKELGTVM RSLGQN PTEAELQDM I N EVDADG NGTI DF PE FLTM M ARKM KDTDS EE E I REAF RVFDKDG NGYISAAE LRHVMTN LG E KLTD E EVDE M I READI DG DGQVNY EE FVQM MTAGGSGGVTGWRLCE RI L A

GGSGSGGGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDS

SRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAY

GATGHPGIIPPHATLVFDVELLKLE

KLAAALEH H H H H H

Substitute Sheet

(Rule 26) RO/AU FRB-CalM-BP ( NanoLuc-CalM-Rapamycin sensor component 1; SEQ ID NO:3)

AHHHHHHSSGTRVA1LWHEMWHEGLEEASRLYFGERNV KGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQ EWCRKYMKSGNVKDLTQAWDLYYHVFRRISGGSGGSGS GSGGSGGKRRWKKNFIAVAS ASA

SUMO-Cal-alpha/beta-CalM-BP (NanoLuc-CalM-FK506 sensor component 1; SEQ ID NO:4)

DV PLI PSQFAKAKSEN FDKKGSDSEVNQEAKPEVKPEVKPETH I N LK VSDGSSE I F FKI KKTTP LRRLM EAFAKRQG KE M DSLRFLYDG I RIQA DQTPEDLDM EDN DI I EAH REQIGGGSGSGGAHH H H H HSSGTSEPK

AIDPKLSTTDRVVKAVPFPPSHRLTAKEVFDN DG KPRVDILKAHLM KEG RLEESVALRIITEGASILRQEKNLLDIDAPVTVCG DIHGQFFDL MKLFEVGGSPANTRYLFLGDYVDRGYFSIECVLYLWALKILYPKTLF LLRG NHECRHLTEYFTFKQECKIKYSERVYDACMDAFDCLPLAALM NQQFLCVHGGLSPEINTLDDIRKLDRFKEPPAYGPMCDILWSDPLE DFGNEKTQEHFTHNTVRGCSYFYSYPAVCEFLQHN NLLSILRAHEA QDAGYRMYRKSQTTGFPSLITI FSAPNYLDVYNNKAAVLKYENNV MNIRQFNCSPHPYWLPNFMDVFTWSLPFVGEKVTEMLVNVLNIC SDDELGSEEDGSGSGSGGG N EASYPLEMCSH FDADE I KRLG KRFKK LDLDNSGSLSVEE FMSLPELQQN PLVQRVI DI FDTDG NG EVDFKE FI EGVSQFSVKG DKEQKLRFAFRIYDM DKDGYISNG E LFQVLKM MVG N N LKDTQLQQIVDKTI I NADKDG DG RISFEE FCAVVGG LDI H KKMV VDVGGSGGSGSGSGGSGG KRRWKKNFIAVASASA

Single component of Rapamycin sensor (FKBP-cpNanoLuc-FRB; SEQ ID NO:5)

D H H H H H H G V Q V E T I S P G D G R T F P K R G Q T C V V H Y T G M L E D G K K F D S S R D R N K P F K F M L G K Q E V I R G W E E G V A Q M S V G Q R A K L T I S P D Y A Y G A T G H P G I I P P H A T L V F D V E L L K L E GGSGGSGGV T G W R L C E R I L A GGSGSGSGSGGSGSGG S D N M V F T L E D F V G D W R Q T A G Y N L D Q V L E Q G G V S S L F Q N L G V S V T P I Q R I V L S G E N G L K I D I H V I I P Y E G L S G D Q M G Q I E K I F K V V Y P V D D H H F K V I L H Y G T L V I D G V T P N M I D Y F G R P Y E G I A V F D G K K I T V T G T L W N G N K I I D E R L I N P D G S L L F R V T I N GSGSGSGG L W H E M W H E G L E E A S R L Y F G E R N V K G M F E V L E P L H A M M E R G P Q T L K E T S F N Q A Y G R D L M E A Q E W C R K Y M K S G N V K D L T Q A W D L Y Y H V F R R I S

Substitute Sheet

(Rule 26) RO/AU Single component of FK506 sensor (Cal-alpha/beta-cpNanoLuc-FKBP; SEQ ID NO:6)

D H H H H H H

S S G T S E P K A I D P K L S T T D R V V K A V P F P P S H R L T A K E V F D N D G K P R V D I L K A H L M K E G R L E E S V A L R I I T E G A S I L R Q E K N L L D I D A P V T V C G D I H G Q F F D L M K L F E V G G S P A N T R Y L F L G D Y V D R G Y F S I E C V L Y L W A L K I L Y P K T L F L L R G N H E C R H L T E Y F T F K Q E C K I K Y S E R V Y D A C M D A F D C L P L A A L M N Q Q F L C V H G G L S P E I N T L D D I R K L D R F K E P P A Y G P M C D I L W S D P L E D F G N E K T Q E H F T H N T V R G C S Y F Y S Y P A V C E F L Q H N N L L S I L R A H E A Q D A G Y R M Y R K S Q T T G F P S L I T I F S A P N Y L D V Y N N K A A V L K Y E N N V M N I R Q F N C S P H P Y W L P N F M D V F T W S L P F V G E K V T E M L V N V L N I C S D D E L G S E E D G S G S G S G G G N E A S Y P L E M C S H F D A D E I K R L G K R F K K L D L D N S G S L S V E E F M S L P E L Q Q N P L V Q R V I D I F D T D G N G E V D F K E F I E G V S Q F S V K G D K E Q K L R F A F R I Y D M D K D G Y I S N G E L F Q V L K M M V G N N L K D T Q L Q Q I V D K T I I N A D K D G D G R I S F E E F C A V V G G L D I H K K M V V D VGGSGSGSGG V T G W R L C E R I L AGGSGSGSGSGGSGSGG S D N M V F T L E D F V G D W R Q T A G Y N L D Q V L E Q G G V S S L F Q N L G V S V T P I Q R I V L S G E N G L K I D I H V I I P Y E G L S G D Q M G Q I E K I F K V V Y P V D D H H F K V I L H Y G T L V I D G V T P N M I D Y F G R P Y E G I A V F D G K K I T V T G T L W N G N K I I D E R L I N P D G S L L F R V T I NGSGSSGSGG G V Q V E T I S P G D G R T F P K R G Q T C V V H Y T G M L E D G K K F D S S R D R N K P F K F M L G K Q E V I R G W E E G V A Q M S V G Q R A K L T I S P D Y A Y G A T G H P G I I P P H A T L V F D V E L L K L E

Single component of amylase sensor (VHHl-cpNanoLuc-VHH2; (SEQ ID NO:7)

D H H H H H H

DTTVSE PAPSCVTLYQSWRYSQADNGCAETVTVKVVYE DDTEG LCY

AVAPGQITTVG DGYIGSHG HARYLARC LGGSGGSGG V T G W R L C E R I

L AGGSGSGSGSGGSGSGG S D N M V F T L E D F V G D W R Q T A G Y N L D Q V L E Q G G V S S L F Q N L G V S V T P I Q R I V L S G E N G L K I D I H V I I P Y E G L S G D Q M G Q I E K I F K V V Y P V D D H H F K V I L H Y G T L V I D G V T P N M I D Y F G R P Y E G I A V F D G K K I T V T G T L W N G N K I I D E R L I N P D G S L L F R V T I NGSGSGSGG

QVQLV ESGGGTVPAGGSLRLSCAASG NTLCTYDMSWYRRAPG KG R

DFVSG I DN DGTTTYVDSVAG RFTISQG NAKNTAYLQM DSLKPDDTA

MYYCKPSLRYG LPGCPI I PWGQGTQVTVSS

Single component of Rapamycin BRET sensor (FKBP-cpNanoLuc-eGFP-FRB; (SEQ ID NO:8)

Substitute Sheet

(Rule 26) RO/AU

Single component GFP biosensor comprising SH3 stabilizer (SEQ ID NO:9)

(SH3L-GFP-CalM-SH3):

DGSGPPPPLPPKRRRGLENVYIKADK

GSGGTEEQIAE FKEAFSLFDKDG DGTITTKELGTVM RSLG QN PTEAE LQDM I N EVDADG NGTI DFPEFLTM MARKM KDTDSEE E IREAFRVF DKDG NGYISAAELRHVMTIN LG EKLTDEEVDEM I READI DG DG QVN YE E FVQM MTAGGSGG

KNGIKANFKIRHNIEDGGVQLAYHYQQNTPIGDGPVLLPDNHYLSVQSKLSKDP NEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSMVSKGEELFTGVVPILVELD GDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLGYGVQCFS RYPDHMKQHDFFKSAMPEGYIQERTIFFKDDGNYKTRAEVKFEGDTLVNRIEL KGIDFKEDGNILGHKLEYNLPD G

AEYVRALFDFNGNDEEDLPFKKGDILRIRDKPEEQWWNAEDSEGKRGMIPVPYVEKY

KLAAA LEH H H H H H

Substitute Sheet

(Rule 26) RO/AU Single component GFP biosensor for detecting rapamycin (SEQ ID NO: 10)

FKBP-GFP-FRB:

D H H H H H HG

VQVETISPG DG RTFPKRGQTCVVHYTG M LEDG KKFDSSRDRIMK PFKFM LG KQEVI RGWEEGVAQMSVGQRAKLTISPDYAYGATG H PG I I PPHATLVFDVELLKL

EGLENVYIKADKQKNGIKANFKIRHNIEDGGVQLAYHYQQNTPIGDGPVLLPDNHYLSVQSKLSK DPNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSMVSKGEELFTGVVPILVELDGDVNGHKF SVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLGYGVQCFSRYPDHMKQHDFFKSAMPE GYIQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNLPDGL W H E M W H EG LEEAS R LYFG ERNVKG M F EVL E P LH AM M E RG PQTLKETS F N QAYG R D LM EAQ EWCR KYM KSG NVK DLTQAWD LYY HVF R R IS

Two component GFP biosensor for detecting rapamycin (SEQ ID NO: 11 & 12)

Zgs453: FKBP12-cpGFP-CalM

FKBP12-cpGFP-CalM:

GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVA QMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLEGSGSGGKNGIKANFKIRHNIEDGG VQLAYHYQQNTPIGDGPVLLPDNHYLSVQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGG

TGGSMVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLV TTLGYGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKG IDFKEDGNILGHKLEYNLPDQLTEEQIAEFKEAFSLFDKDGDGTITTKELGTVMRSLGQNPTEAELQ DMINEVDADGDGTIDFPEFLTMMARKGSYRDTEEEIREAFGVFDKDGNGYISAAELRHVMTN LG EKLTDEEVDEMIREADIDGDGQVNYEEFVQMMTAK

Zgs466: CalM peptide -cpGFP-FRB

CalM peptide -cpGFP-FRB

GWNKTGHAVRAIGRLSSLENVYIKADKGGSGGGSGGS GGLWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLH AMMERGPQTLKETSFNQAYGRDLMEAQEWCRKYMKS GNVKDLTQAWDLYYHVFRRIS FKBP-GFP peptide sensor first component; SEQ ID NO:13):

FKBP-GFP peptide sensor(SH3L-GFP-CalM-SH3) :

DGVQVETIS PG DG RTFPKRG QTCVVHYTG M LEDG KK F

DSSR D R N KP FKFM M LG KQ EVI RGWE EGVAQ M SVG Q R

Substitute Sheet

(Rule 26) RO/AU AK LT I S P DYAYGATG H PG I I P P HATLVF DV ELL KLE GGSGGSGSGS GGPPPPLPPKRRRGLENVYIKADK

GSGGTEEQIAEFKEAFSLFDKDGDGTITTKELGTVMRSLGQNPTEAE LQDMINEVDADGNGTIDFPEFLTMMARKMKDTDSEEEIREAFRVF DKDGNGYISAAELRHVMTNLGEKLTDEEVDEMIREADIDGDGQVN YE E FVQM MTAGGSGG

AEYVRALFDFNGNDEEDLPFKKGDILRIRDKPEEQWWNAEDSEGKRGMIPVPYVEKY

KLAAA LEH H H H H H

FRB-CalM peptide second component SEQ ID NO: 14):

AHHHHHHSSGTRVAILWHEMWHEGLEEASRLYFGERNV KGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQ EWCRKYMKSGNVKDLTQAWDLYYHVFRRISGGSGGSGS GSGGSGGKRRWKKNFIAVASASA

Throughout the specification, the aim has been to describe the preferred embodiments of the invention without limiting the invention to any one embodiment or specific collection of features. Various changes and modifications may be made to the embodiments described and illustrated without departing from the present invention.

The disclosure of each patent and scientific document, computer program and algorithm referred to in this specification is incorporated by reference in its entirety.

Substitute Sheet

(Rule 26) RO/AU REFERENCES

(1) Hall, M. P., Unch, J„ Binkowski, B. F„ Valley, M. P„ Butler, B. L„ Wood, M. G., Otto, P., Zimmerman, K., Vidugiris, G., Machleidt, T., Robers, M. B., Benink, H. A., Eggers, C. T., Slater, M. R., Meisenheimer, P. L., Klaubert, D. H., Fan, F., Encell, L. P., and Wood, K. V. (2012) Engineered Luciferase Reporter from a Deep Sea Shrimp Utilizing a Novel Imidazopyrazinone Substrate. ACS Chem. Biol. 7, 1848-1857.

(2) Shakhmin, A., Hall, M. P., Machleidt, T., Walker, J. R., Wood, K. V, and Kirkland,

T. A. (2017) Coelenterazine analogues emit red-shifted bioluminescence with NanoLuc. Org. Biomol. Chem. 15, 8559-8567.

Claims

1. A molecular biosensor comprising at least one amino acid sequence of at least one protein, or at least one fragment thereof, and at least one sensor, wherein the at least one protein is capable of emitting light, or facilitating the emission of light, in response to the sensor.

2. The molecular biosensor of Claim 1, wherein the at least one protein is or comprises an enzyme or fragment thereof that facilitates the emission of light upon binding a target molecule by the at least one sensor, or a binding interaction between the sensors.

3. A molecular biosensor comprising at least one amino acid sequence of an enzyme that is capable of reacting with a substrate molecule to produce light and one or more sensors that can bind or interact with a target molecule, and/or with each other, to thereby facilitate the enzyme reacting with the substrate molecule to produce light.

4. The molecular biosensor of any preceding claim, wherein the sensor comprises an amino acid sequence of calmodulin or variant that is capable of binding or interacting with a calmodulin-binding peptide or variant.

5. The molecular biosensor of Claim 4, wherein binding between calmodulin or variant and the calmodulin-binding peptide or variant facilitates allosteric switching of the enzyme into a catalytically active state.

6. The molecular biosensor of Claim 3, Claim 4 or Claim 5, wherein the amino acid sequence of the enzyme is circularly permuted.

7. The molecular biosensor of Claim 6, which comprises a linker amino acid sequence.

8. The molecular biosensor of Claim 7, wherein the linker amino acid sequence comprises a light-emitting molecule.

9. The molecular biosensor of Claim 8, wherein the light-emitting molecule is a dye molecule or a fluorescent protein or fragment thereof.

10. A molecular biosensor comprising: a first molecular component comprising at least one amino acid sequence of an enzyme that is capable of reacting with a substrate molecule to produce light and one or more first sensors; and a second molecular component comprising one or more second sensors, whereby the first and second sensors can bind a target molecule, and/or each other, to thereby facilitate the enzyme reacting with the substrate molecule to produce light.

11. The molecular biosensor of Claim 10, wherein the first and second sensors can co-operatively bind a target molecule to thereby facilitate the enzyme reacting with the substrate molecule to produce light.

12. The molecular biosensor of Claim 10 or Claim 11, wherein the first molecular component and the second molecular component comprise a stabilizer that facilitates or stabilizes an interaction between the first molecular component and the second molecular component upon binding a target molecule.

13. The molecular biosensor of any one of Claims 2-12, wherein the enzyme is a bioluminescent enzyme.

14. The molecular biosensor of Claim 1, the at least one protein is or comprises a fluorescent protein that facilitates the emission of light upon binding a target molecule by the sensor.

15. A molecular biosensor comprising at least one amino acid sequence of one or more fragments of a fluorescent protein and one or more sensors that can bind or interact with a target molecule to thereby facilitate the respective fragments of the fluorescent protein co-operatively emitting light.

16. The molecular biosensor of Claim 15, wherein the biosensor comprises first and second fragments of the fluorescent protein and a stabilizer that facilitates or stabilizes an interaction between the first and second fragments upon binding a target molecule.

17. The molecular biosensor of Claim 16, wherein, the stabilizer comprises an amino acid sequence of an SH3 domain that is capable of non-covalently and reversibly binding or interacting with a peptide of the second molecular component.

18. The molecular biosensor of any one of Claims 15-17, which comprises an allosteric switch that facilitates the increase in fluorescence emission by the fluorescent protein.

19. The molecular biosensor of Claim 18, wherein the allosteric switch comprises an amino acid sequence of calmodulin or a variant thereof that is capable of non- covalently and reversibly binding or interacting with a calmodulin-binding peptide or variant thereof of the second molecular component.

20. The molecular biosensor of Claim 15, wherein the molecular biosensor comprises first and second fragments of the fluorescent protein that are circularly permuted.

21. A molecular biosensor comprising: a first molecular component comprising first and second fragments of a fluorescent protein and a first sensor; and a second molecular component comprising a second sensor, whereby the first and second sensors can bind a target molecule, or each other, to thereby facilitate the respective fragments of the fluorescent protein co-operatively emitting light.

22. The molecular biosensor of Claim 21, wherein, the first and second sensors can co-operatively bind a target molecule to thereby facilitate the first and second fluorescent protein fragments co-operatively emitting light.

23. The molecular biosensor of Claim 21 or Claim 22, wherein the first molecular component comprises a stabilizer that facilitates or stabilizes an interaction between the first and second fragments of the fluorescent protein.

24. The molecular biosensor of any one of Claims 20-23, wherein, the stabilizer comprises an amino acid sequence of an SH3 domain that is capable of non- covalently and reversibly binding or interacting with a peptide of the second molecular component.

25. The molecular biosensor of any one of Claims 20-24, further comprising an allosteric switch, wherein the first molecular component comprises an amino acid sequence of calmodulin or a variant that is capable of non-covalently and reversibly binding or interacting with a calmodulin-binding peptide or variant of the second molecular component upon binding a target molecule.

26. A molecular biosensor comprising: a first molecular component comprising a first fragment of a fluorescent protein and a first sensor; and a second molecular component comprising a first fragment of a fluorescent protein and a second sensor, whereby the first and second sensors can bind a target molecule, or each other, to thereby facilitate the respective fragments of the fluorescent protein co operatively emitting light.

27. The molecular biosensor of Claim 26, wherein the first molecular component and the second molecular component comprise a stabilizer that facilitates or stabilizes an interaction between the first and second molecular components.

28. The molecular biosensor of Claim 27, wherein, the first molecular component comprises an amino acid sequence of calmodulin or a variant thereof that is capable of non-covalently and reversibly binding or interacting with a calmodulin-binding peptide or variant thereof of the second molecular component.

29. The molecular biosensor of Claim 28, wherein binding between calmodulin or variant and the calmodulin-binding peptide or variant facilitates allosteric switching of the fluorescent protein to thereby increase emission of light by the fluorescent protein.

30. The molecular biosensor of any preceding claim, which comprises an amino acid sequence set forth in any one of SEQ ID NOS:l-14, or a fragment or variant thereof.

31. A method of producing an allosterically switchable protein that includes producing one or more chimeric proteins that comprise an amino acid sequence of at least a fragment of a protein of interest and an amino acid sequence of calmodulin, or a variant or fragment thereof.

32. An allosterically switchable protein produced according to the method of Claim 31.

33. A molecular biosensor comprising one or more of the allosterically switchable proteins of Claim 32.

34. A biosensor device comprising one or more molecular biosensors according to any one of Claims 1-30 or 33 that is immobilized or affixed to a support that is substantially transparent to light emitted by the biosensor.

35. A method of detecting a target molecule, said method including the step of contacting the molecular biosensor of any one of Claim 1-30 or 33 or the biosensor device of Claim 34 with a sample to thereby determine the presence or absence of the target molecule in the sample.

36. A method of screening or identifying an inhibitory target molecule, said method including the step of contacting the biosensor or biosensor device of any of the aforementioned aspects with a sample to thereby determine the presence or absence an inhibitory target molecule that at least partly inhibits a binding interaction between the sensor(s).

37. The method of Claim 36, wherein the presence of the inhibitory target molecule is indicated by a decrease in light emission by the biosensor.

38. A method of diagnosis of a disease or condition in an organism, said method including the step of contacting the molecular biosensor of any one of Claim 1- 30 or 33 or the biosensor device of Claim 34 with a biological sample obtained from the organism to thereby determine the presence or absence of a target molecule in the biological sample, wherein determination of the presence or absence of the target molecule facilitates diagnosis of the disease or condition.

39. A detection device that comprises a cell or chamber that comprises the molecular biosensor of any one of Claims 1-30 or 33 or the biosensor device of Claim 34.

40. An isolated protein which comprises an amino acid sequence set forth in any one of SEQ ID NOS:l-l4, or a fragment or variant thereof.

41. An isolated nucleic acid encoding the molecular biosensor of any one of Claims

1-30 or 33, or a component thereof, or the isolated protein, fragment or variant of Claim 40.

42. A genetic construct comprising the isolated nucleic acid of Claim 41.

43. A host cell comprising the genetic construct of Claim 42.

44. A method of producing a recombinant protein biosensor or a component thereof as defined in any one of Claims 1-30 or 34, or a recombinant protein, fragment or variant according to Claim 40, the method including at least one step selected from: expressing the molecule in a host cell; and isolating the recombinant biosensor or protein from the host cell.

45. A composition or kit comprising the molecular biosensor, the biosensor device, the detection device, the isolated protein, the isolated nucleic acid, the genetic constmct and/or the host cell as defined in any of the preceding claims.