WO2015067302A1 - Molécules capteurs et leurs utilisations - Google Patents

Molécules capteurs et leurs utilisations Download PDF

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WO2015067302A1
WO2015067302A1 PCT/EP2013/073078 EP2013073078W WO2015067302A1 WO 2015067302 A1 WO2015067302 A1 WO 2015067302A1 EP 2013073078 W EP2013073078 W EP 2013073078W WO 2015067302 A1 WO2015067302 A1 WO 2015067302A1
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binding
sensor molecule
sensor
molecule
analyte
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PCT/EP2013/073078
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Kai Peter JOHNSSON
Alberto SCHENA
Rudolf GRISS
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Ecole Polytechnique Federale De Lausanne (Epfl)
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
    • G01N33/542—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with steric inhibition or signal modification, e.g. fluorescent quenching

Definitions

  • the invention generally relates to methods, reagents and devices for determining a concentration of an analyte in a sample, such as an analyte in a sample of bodily fluid, as well as methods and devices which can be used to support the making of such determinations. More in particular, it relates to the field of in vitro detection methods using luminescence.
  • Luminescence is a phenomenon in which energy is specifically channeled to a molecule to produce an excited state. Return to a lower energy state is accompanied by release of a photon. Luminescence includes fluorescence, phosphorescence, chemiluminescence, and bioluminescence. Luminescence can be used, among others, in the analysis of free analytes or biological interactions.
  • the inventors introduced an approach for the generation of semisynthetic protein-based biosensors for small molecule analytes.
  • the fluorescent biosensors were named SNAP-tag based Indicator protein with a Fluorescent Intramolecular Tether (SNIFIT). See Brun et al. J Am Chem Soc. 2009;131(16):5873-84 and Brun et al. J Am Chem Soc. 2011;133(40):16235-42.
  • the sensor is constituted of a fusion of a binding protein, specific for the analyte of interest, and a synthetic portion constituted of a ligand for the binding protein and two fluorophores forming a FRET-pair tethered to the protein construct via self- labeling-tag fusion.
  • the proteinaceous sensor molecule undergoes a conformational change resulting from the displacement of the tethered ligand by the analyte of interest. This causes a detectable change in FRET-efficiency between the two fluorophores.
  • a suitable binding protein and its relative tetherable ligand virtually any small metabolite can be sensed and several examples have been disclosed (Brun et al., , J Am Chem Soc 2012, 134, 7676-8; Masharina et al., J Am Chem Soc 2012, 134, 19026-34).
  • the SNIFIT sensor design suffers from severe limitations: the binding protein has to fulfil strict geometrical requirements, namely the relative spacing of the binding site and the protein terminus has to be small. When a suitable binding protein is available, an optimal ligand with suitable affinity must be found and successfully tethered. These requirements make the generation of a new sensor non-trivial and time-consuming.
  • the SNIFIT concept is limited to the change in FRET-efficiency as readout. To overcome these limitations, the inventors aimed at a substantial innovation in the SNIFIT sensor concept to allow a much broader application and to introduce a relative ease in novel sensor generation. Ideally, the new sensor concept should not be limited to resonance energy transfer as readout and facilitate a broad practical applicability, e.g. in a point-of-care device.
  • an optimized sensor with a second ligand which is specific for the analyte of interest e.g. a protein or for a binding partner of a small molecule analyte.
  • a proteinaceous sensor molecule for detecting an analyte of interest (A) comprising a signal generating moiety tethered to a synthetic regulatory molecule capable of modulating signal
  • the synthetic regulatory molecule comprises a primary ligand (LI) capable of intramolecular binding to a primary partner (BPl) on the sensor molecule; wherein (ii) the sensor molecule comprises a secondary ligand (L2) capable of binding to a secondary binding partner (BP2), BP2 being A or a binding partner thereof; and wherein (iii) binding of LI and L2 to their respective binding partners is mutually exclusive, such that binding of BP2 to L2 influences binding of LI to BPl, resulting in a conformational change within the sensor molecule such that the generated signal is modulated.
  • LI primary ligand
  • L2 secondary ligand
  • the primary ligand (LI) is intramolecularly bound to a primary binding partner (BPl ) on the sensor.
  • the sensor contains a moiety which can modulate a detectable signal depending on the conformational configuration of the sensor.
  • the configuration is controlled by the interaction between LI and its primary binding site. Since the binding of primary and secondary ligand (L2) to their respective binding sites is mutually exclusive i.e. either LI or L2 is bound, the presence of analyte influences the extent of LI binding to BP1 and hence the signal of the signal generating moiety by inducing a conformational change.
  • the binding of L2 to an analyte of interest displaces LI from its primary binding site on the sensor molecule, such that the sensor reaches an open conformation.
  • L2 is bound to a binding partner of the analyte, thus preventing binding of LI to the primary binding site such that the sensor is in an open conformation.
  • the presence of analyte displaces L2 from the binding partner, allowing LI to close the
  • the conformational change of the sensor modulates the signal generating moiety of the sensor.
  • the conformational change e.g. from an open to closed conformation can result in a modulation of the detectable signal depending on the design and nature of the signal generating moiety. For instance, if detection is based on FRET or BRET between a donor and a fluorochrome acceptor comprised in the sensor molecule, an open conformation typically results in a decrease in resonance energy transfer efficiency.
  • the binding site of LI is within the active site of an enzyme comprised in the sensor, opening of the sensor can release the inhibition and increase or induce enzymatic activity.
  • This novel concept referred to as Steric Displacement-SNIFITs (SD-
  • SNIFIT opens the way to adapt the SNIFIT sensor concept to virtually any biochemical analyte and makes it adaptable to the most diverse readout systems.
  • detection can be based on any enzyme capable of generating a detectable signal, e.g. luciferases, enzymes catalyzing chromogenic or fluorogenic reactions, redox enzymes that can be coupled to electrochemical devices, etc.
  • Detector systems that rely on steric competition are known in the art.
  • US 3,935,074 relates to immunoassays for detecting a ligand of interest, and the use thereof in medical diagnosis.
  • a reagent is provided having at least two epitopes, one of the epitopes being common with the ligand of interest, and the other epitopes being foreign to the ligand.
  • the two epitopes are positioned in the reagent so that antibody bound to one of the epitopes sterically inhibits the binding of antibody to the second epitope.
  • US 6,455,288 relates to methods for immunoassay of analytes employing mutant glucose- 6- phosphate dehydrogenase (G6PDH) enzymes as labels.
  • G6PDH glucose- 6- phosphate dehydrogenase
  • methods for determining the presence or amount of an analyte in a sample suspected of containing the analyte comprising the steps of: a) combining in an assay medium: 1) the sample, 2) a conjugate of an analyte analog and a mutant G6PDH wherein the G6PDH has at least one amino acid mutation per subunit as compared to precursor G6PDH wherein at least one of the mutations comprises the introduction of a cysteine residue proximate to an epitope recognized by an inhibitory anti- G6PDH antibody capable of simultaneously binding to two of the subunits within the same G6PDH molecule, 3) an antibody capable of binding the analyte and the analyte analog conjugate, and 4) substrates for
  • an analyte-label conjugate employing as the label mutant G6PDH enzymes having at least one mutation per subunit as compared to precursor G6PDH wherein the mutations are proximate to an epitopic site recognized by an anti-G6PDH antibody capable of inhibiting the activity of the precursor G6PDH.
  • the prior art reagents differ significantly from a sensor of the invention.
  • none of them comprises the use of tethered, intramolecular ligands that affect the conformation of the sensor.
  • both LI and L2 are part of the synthetic regulatory molecule.
  • Figure 1A for a schematic drawing of an exemplary sensor molecule wherein LI is bound to a moiety of the sensor moiety referred to as BP1 comprising a primary binding site for LI. Since LI is bound and L2 is free, the sensor has a closed conformation in the absence of analyte (in Fig. 1A referred to as BP2), e.g. a protein of interest. However, the presence of BP2, which is capable of binding to L2, displaces LI from BPl, resulting in the opening of the sensor.
  • BP2 analyte
  • Figure IB shows a schematic drawing of an exemplary sensor molecule wherein L2 is bound to BP2, wherein BP2 is also a binding partner of the analyte A. Since L2 is bound and LI is free, the sensor has an open conformation in the absence of analyte. However, the presence of analyte A displaces L2 from BP2 thus allowing LI to bind to BPl. This results in the closing of the sensor.
  • BP2 can be a separate entity or it can be part of the sensor molecule. In one embodiment, BP2 is a separate entity. In another embodiment, BP2 is part of the regulatory molecule.
  • L2 is not part of the regulatory molecule but specifically tethered to or part of the proteinaceous region of the sensor molecule that contains the primary binding site. See for instance Figure IC or ID wherein L2 is conjugated or attached to BPl.
  • Figure IC binding of analyte BP2 to L2 displaces LI from BPl, resulting in the opening of the sensor.
  • the presence of A displaces L2 from BP2 thus allowing LI to bind to BPl.
  • BP2 can but does not have to be part of the sensor molecule.
  • BPl is the active site of dihydrofolate reductase (DHFR) or a circularly permuted variant thereof, preferably in combination with trimethoprim, methotrexate, or a variant thereof as LI.
  • BPl is the active site of human carbonic anhydrase (HCA), preferably in combination with an aromatic sulfonamide or variant thereof as primary ligand.
  • HCA human carbonic anhydrase
  • a sensor molecule of the invention is characterized by a secondary ligand capable of binding to a secondary binding partner, which can be the analyte of interest itself, or a binding partner (e.g.
  • the invention provides a sensor wherein the secondary ligand (L2) is capable of binding to BP2, BP2 being the analyte, such that in the absence of free analyte L2 is free and LI is bound to BPl and wherein the binding of analyte to L2 displaces LI from BPl resulting in an open
  • Figures 1A and 1C for a schematic drawing of exemplary sensors based on normal steric displacement.
  • the secondary ligand (L2) is capable of binding to a binding partner of the analyte of interest (BP2), such that in the absence of analyte L2 is bound to BP2 and LI is free and wherein the binding of analyte to BP2 displaces BP2 from L2 allowing LI to bind to BPl resulting in a change in the conformational state of the sensor molecule such that the signal generation is modulated.
  • BP2 can be a separate entity or it can be part of the sensor molecule.
  • BP2 is not covalently bound to the sensor. See for example Figure 4.
  • BP2 is part of the proteinaceous sensor molecule and binding of L2 to BP2 is intramolecular. The presence of analyte disrupts intramolecular binding of L2 to BP2 and induces intramolecular binding of LI to BPl.
  • the relative positions of BP2 and BPl within the sensor molecule are designed in such a manner to ensure that the change in intramolecular binding alters the conformation of the sensor such that signal generation is modulated.
  • each of BP2 and BPl can be located at one of the termini of the sensor and the regulatory molecule comprising LI and L2 can be tethered to an
  • the synthetic regulatory molecule is tethered to the proteinaceous moiety in a site-specific fashion to ensure a single, homogenous product.
  • the site of attachment can be chosen among any part of the proteinaceous moiety.
  • the site of attachment of the synthetic regulatory molecule to the proteinaceous moiety is chosen such that it allows for a signal change when the sensor molecule switches between the different conformations upon analyte- induced steric displacement of either LI or L2 from its respective binding partner.
  • Site-specific attachment of the synthetic regulatory molecule can be achieved by methods known in the art.
  • an amino acid naturally or non- natural showing a unique reactivity is suitably used.
  • Suitable amino acids include cysteine and any (unnatural) amino acid that allows for a site-specific chemical conjugation reaction, such as click-chemistry, of an appropriate synthetic regulatory molecule.
  • the unnatural amino acid azidohomoalanine (AHA) can be used.
  • the synthetic regulatory molecule is site- specifically tethered to the proteinaceous moiety by means of a protein labelling tag.
  • the protein labelling tag is a self-labelling protein known in the art, such as SNAP-tag, CLIP-tag or Halo-Tag, and wherein the synthetic regulatory molecule is tethered via the appropriate reactive group.
  • the self-labeling protein tag is based on a human 0 6 -alkylguanine- DNA-alkyltransferase (hAGT) to which the synthetic regulatory molecule is tethered via a reactive group for hAGT.
  • hAGT human 0 6 -alkylguanine- DNA-alkyltransferase
  • the protein tag is a SNAP-tag or CLIP-tag.
  • the reactive group is a 0 6 -benzylguanine (BG), 04-benzyl- 2-chloro-6-aminopyrimidine (CP) or 0 2 -benzylcytosine (BC) derivative.
  • the self-labeling protein tag is based on a modified haloalkane dehalogenase to which the synthetic regulatory molecule is tethered via a chloroalkane (Halo-Tag).
  • the protein labeling tag can be a tag that is labeled with the synthetic regulatory molecule through the action of an enzyme, such as sortase (and mutants thereof), lipoic acid ligase (and mutants thereof), biotin ligase (and mutants thereof), phosphopantetheine transferase (PPTase; and mutants thereof).
  • Labeling can be achieved by directly transferring a molecule carrying the synthetic regulatory molecule to the protein tag or by a two-step procedure where in the first step a molecule comprising a bioorthogonal group is attached and in the second step the bioorthogonal group is reacted with the synthetic regulatory molecule comprising an appropriate functional group.
  • enzymatic transfer of a modified phosphopantetheine derivative carrying the synthetic regulatory molecule results in labeling of a specific serine within a certain peptide sequence derived from acyl carrier proteins and thus allows the synthetic regulatory molecule to be linked at exactly one residue present in the protein (see N. George et al. J Am Chem Soc. 2004 126, 8896).
  • ACP-tag and MCP-tag are such sequences derived from acyl carrier protein.
  • the presence of the phosphopantetheine transferase is required for the formation of a covalent link between the ACP-tag or MCP-tag and their substrates, which are derivatives of Coenzyme A (CoA).
  • the group conjugated to CoA is covalently attached to the ACP-tag or MCP-tag by the phosphopantetheine transferase.
  • An example for the two-step strategy would be a labeling in which in the first step, a mutant of lipoic acid ligase (LplA) ligates a ircmscyclooctene derivate onto a LplA acceptor peptide which is part of the sensor molecule.
  • LplA lipoic acid ligase
  • ligated ircms-cyclooctene is chemoselectively derivatized with a synthetic regulatory molecule conjugated to a tetrazine. Details of such a two step procedure are described by Liu et al. (J Am Chem Soc. 2012 Jan 18;134(2):792-5).
  • the synthetic regulatory molecule is site-specifically tethered to the proteinaceous moiety by means of intein-based labeling.
  • intein-based labeling For example, the use of so-called expressed protein ligation (T. Muir, Annu. Rev.
  • Biochem. 2003. 72:249-289) would entail expressing the proteinaceous moiety as fusion protein with a C-terminal intein and the subsequent isolation of the corresponding C-terminal thioester. This thioester is then reacted with a cysteine residue to which the synthetic regulatory molecule is attached, resulting in formation of functional sensor molecule.
  • split-intein-based protein labeling Volkmann G, Liu X-Q (2009) PLoS ONE 4(12): e8381
  • the proteinaceous part of the sensor molecule can be expressed as a fusion protein with a C- or N-terminal split intein.
  • the site of specific attachment of the synthetic regulatory molecule in the sensor molecule may be connected via a proteinaceous linker moiety to the other part of the proteinaceous moiety.
  • the linker moiety can be an artificial polypeptide sequence or a naturally occurring protein designed to ensure that analyte-induced ligand displacement causes a sufficient change in the conformational state of the sensor.
  • Poly-L-proline linkers can be used as precise molecular rulers due to their well-defined property of forming a stable and rigid helical structure (the polyproline II helix) with a pitch of 3.1 A per residue in aqueous solution.
  • the linker moiety is preferably a helical linker rich in prolines, which leads to structural rigidity and isolation of the synthetic regulatory molecule from the attached luciferase.
  • Very good results were obtained with a poly-L-Proline linker consisting of at least 15 Pro residues, for instance Prois, or Pro3o or even longer.
  • Brun et al. (2011) investigated polyproline linkers of varying length (0, 6, 9, 12, 15, 30, 60) that were inserted between SNAP- and CLIP-tag in the conventional SNIFIT-sensors. It was found that a length of 30 or 60 proline residues yielded an improved maximum ratio change of the sensor.
  • the linker moiety consists of a poly-L-Pro linker comprising at least 15, preferably at least 20, more preferably at least 30, residues.
  • a sensor molecule of the invention may contain any type of signal generating moiety which is capable of generating a detectable signal.
  • the activity of the signal generating moiety is modulated by a change in the binding ratio L1/L2 to their respective binding partners (LI to BP1 and L2 to BP2).
  • the signal generating moiety comprises an enzyme E.
  • E is selected from the group of enzymes catalyzing a chromogenic, luminescent, fluorogenic or redox reaction. Examples of E include peroxidases, luciferases, hydrolases, glucose oxidases, beta-galactosidases.
  • Enzyme modulation can be direct, for example by designing BP1 to be part of E, preferably in or close to the active site of E. Therefore, in one embodiment of the invention a proteinaceous sensor molecule for detecting an analyte of interest comprises a signal generating moiety comprising an enzyme (E), to which is tethered a synthetic regulatory molecule capable of modulating signal generation by intramolecular binding, wherein (i) the synthetic regulatory molecule comprises a primary ligand (LI) capable of intramolecular binding to a BP1 on E, preferably wherein BP1 is in the active site or an allosteric site of E, and wherein (ii) binding of LI to the active site of E leads to inhibition of the corresponding enzymatic activity of E and wherein (iii) binding of LI and L2 to their respective binding site is mutually exclusive, such that binding of BP2 to L2 influences binding of LI to the active site of E, resulting in a change of the enzymatic activity of E within the sensor
  • LI is an inhibitor of the enzyme such that binding of analyte to L2 ligand sterically displaces LI, resulting in activation of the reporter enzyme.
  • this sensor design TURNON-reporter.
  • Example 4 herein below which exemplifies a sensor using Renilla luciferase as reporter enzyme in a sensor tethered to a regulatory molecule comprising the luciferase inhibitor
  • coelenteramide as primary ligand and biotin as secondary ligand to sense the analyte protein streptavidin.
  • LI and L2 can both be part of the synthetic regulatory molecule or L2 can be specifically tethered to or part of the enzyme. Also, the sensor can be based on the normal or competitive displacement.
  • RET methods are based on the use of compatible energy donor and acceptor pairs allowing RET to take place when donor and acceptor are in close proximity ( ⁇ 10nm).
  • the signal generating moiety may comprise the donor and acceptor pair, e.g. two fluorescent proteins.
  • the donor or acceptor is contained in the signal generating moiety, the other member of the pair being part of the regulatory molecule (see Figure 2A for an exemplary design).
  • the donor and/or acceptor is suitably attached to the signal generating moiety via a self-labeling protein tag e.g.
  • both the donor and acceptor have a proteinaceous character and are comprised in the amino acid sequence of the sensor (see Figure 2B).
  • a sensor may contain two fluorescent proteins or one fluorescent protein plus a luciferase enzyme.
  • the signal generating moiety is capable of generating a fluorescence resonance energy transfer (FRET) signal.
  • FRET is a distance-dependent interaction between the electronic excited states of two dye molecules in which excitation is transferred from a donor molecule to an acceptor molecule without emission of a photon.
  • the efficiency of FRET is dependent on the inverse sixth power of the intermolecular separation, making it useful to monitor an analyte-induced conformational change in a sensor molecule of the invention.
  • FRET can be detected by the appearance of sensitized fluorescence of the acceptor or by quenching of donor fluorescence.
  • donor and acceptor molecules must be in close proximity (typically 10-100 A) and that the absorption spectrum of the acceptor must overlap the fluorescence emission spectrum of the donor.
  • donor and acceptor transition dipole orientations must be approximately parallel.
  • a sensor is provided wherein the signal generating moiety comprises a fluorescent acceptor and wherein the synthetic regulatory molecule comprises a fluorescent donor, or vice versa.
  • Example 3 herein below demonstrates that the steric displacement concept of the present invention is advantageously used to improve the FRET-ratio change of a sensor molecule. Suitable FRET
  • a sensor of the invention is advantageously based on Bioluminescence Resonance Energy Transfer (BRET).
  • BRET Bioluminescence Resonance Energy Transfer
  • the donor fluorophore of the FRET pair is replaced by a bioluminescent donor protein (BDP) which, in the presence of a substrate, excites the acceptor fluorophore through the same resonance energy transfer mechanisms as FRET.
  • BDP Bioluminescent donor protein
  • the signal generating moiety of a sensor provided herein comprises a BDP.
  • the BDP is a luciferase, for example the luciferase from Renilla reniformis).
  • Alternative BDPs that can be employed in this invention are enzymes which can act on suitable substrates to generate a luminescent signal. Specific examples of such enzymes are beta- galactosidase, alkaline phosphatase, beta-glucuronidase and beta-glucosidase. Synthetic luminescent substrates for these enzymes are well known in the art and are commercially available from companies, such as Tropix Inc. (Bedford, MA, USA).
  • the BDP has luciferase activity.
  • Luciferases, and nucleic acid constructs encoding them are available from a variety of sources or by a variety of means. Examples of bioluminescent proteins with luciferase activity may be found in U. S. Patent Nos. 5,229,285; 5,219,737; 5,843,746;
  • luciferases include NanoLuc luciferase, Renilla luciferase, firefly luciferase and Gaussia luciferase. Also encompassed are non- naturally occurring luciferases, e.g. a mutated luciferase.
  • a sensor of the invention comprises the previously described NanoLucTM Luciferase (Nluc), a 19.1 kDa, monomeric, ATP independent enzyme that utilizes a novel substrate to produce high intensity, glow- type luminescence. See WO 2012/061530 and Hall et al. ACS Chem Biol.
  • the enzyme was generated using directed evolution from a deep-sea shrimp luciferase, creating a luciferase that is much brighter than other forms of luciferase, including both firefly ⁇ Photinus pyralis) and Renilla reniformis.
  • the high intensity luminescence of the NanoLuc enzyme combined with low autoluminescence of the furimazine substrate allows the sensitive detection of low levels of luciferase.
  • the fluorescent acceptor molecule is chosen to function as BRET pair together with the BDP i.e. to accept the bioluminescence energy from the donor in the presence of an appropriate substrate. Furthermore, the fluorescent acceptor molecule is adapted to emit light after accepting the bioluminescence.
  • Suitable fluorescent acceptors to form a BRET pair include any fluorophore whose excitation spectra at least partially overlaps with the emission spectra of the respective luciferase.
  • the relative positioning of the BRET donor/acceptor pair within a sensor of the invention can vary.
  • the signal generating moiety comprises the BDP and the synthetic regulatory molecule comprises a fluorescent acceptor molecule.
  • Other useful formats include the signal generating moiety comprising both the BDP and the fluorescent acceptor, either in the form of a fluorescent protein or a synthetic fluorophore tethered either using a self-labelling tag or by other means of attachment.
  • a sensor molecule of the invention is broadly applicable for the detection, quantification and/or imaging of any analyte of interest.
  • the sensor finds its use in many different areas, ranging from clinical diagnostics, drug screening, and fundamental research.
  • a method for in vitro detecting an analyte of interest in a sample comprising the steps of: (a) contacting the sample with a sensor molecule according to the invention under conditions allowing for an analyte-induced conformational change within the sensor molecule to occur such that the signal generated by the signal generating moiety is modulated; and (b) analyzing a change in a signal generated by the signal generating moiety.
  • the analyte of interest is a substance for which the presence, absence, location and/or quantity is to be determined.
  • the analyte can have a chemical, biological, synthetic or semi- synthetic nature. It can be a small molecule or a protein.
  • the analyte of interest is a drug, a metabolite, an inhibitor, a protein, a biomarker or a nucleic acid molecule.
  • the analyte of interest has biological or pharmacological activity.
  • Biologically active analytes are often of biological origin themselves, but can also be of synthetic or semi- synthetic origin.
  • the sample is a biological sample or a fraction thereof, preferably a bodily fluid, more preferably selected from the group consisting of blood, serum, saliva, urine, spinal fluid, pus, sweat, tears, breast milk, (in text: wherein the sample absorbs light in the blue light region).
  • the sample comprises cells.
  • a sensor or method of the invention is used for drug quantification.
  • finding the balance between efficacy and toxicity requires quantification of their concentrations in the patient's blood.
  • Many immunosuppressants, anti-epileptics, antibiotics and others have unpredictable pharmacokinetics and narrow therapeutic ranges, requiring monitoring of their concentrations in the body.
  • This process known as therapeutic drug monitoring (TDM)
  • TDM therapeutic drug monitoring
  • TDM therapeutic drug monitoring
  • TDM therapeutic drug monitoring
  • the development of fast and low-cost assays would improve safety and therapeutic outcome in regions with poor infrastructure and allow personalized dosage at bedside or at home. Quantifying drug levels at bedside or at home would have obvious advantages in terms of therapeutic outcome and convenience, but current techniques require the setting of a diagnostic laboratory.
  • Moving TDM from the diagnostic lab to the patient requires tools that (i) are capable of handling minimal sample volumes down to a single drop, (ii) are quantitative, and (iii) permit readout with inexpensive devices. It was found that a bioluminescent sensor described herein permits precise measurements of drug concentrations in patient samples e.g. by spotting minimal volumes on paper and recording the signal using a simple digital camera.
  • the sensors can be readily engineered to selectively recognize a wide range of drugs, including immunosuppressants, antiepileptics, anticancer agents, and antiarrhythmics. This low-cost point-of-care method could make therapies safer, increase the convenience of doctors and patients, and make therapeutic drug monitoring available in regions with poor infrastructure.
  • Another specific aspect of the invention relates to the use ratiometric RET sensors for quantification of analytes in complex samples that absorb light at the emission wavelengths of the sensor, e.g. serum or other bodily fluids. Analysis of such samples is prone to artefacts and often leads to unreliable assay outcomes. Whereas sensors based on luciferases as an internal light source (i.e. BRET) would in theory reduce the fluorescent background problem and potentially increase sensitivity, no ratiometric BRET-based sensors have yet been introduced that are suitably used for the quantification of analytes in light-absorbing samples.
  • BRET internal light source
  • the invention therefore also provides an analytical device comprising a BRET sensor molecule as described herein above, wherein the sensor molecule is arranged in such a manner that, when the device is in use for detecting an analyte of interest in a sample, the photons that are emitted from the sensor molecule and that are collected by a detector pass through the sample for a distance shorter than 330 ⁇ .
  • the sensor molecule is immobilized or absorbed to a solid carrier, preferably a glass or transparent plastic.
  • the sensor molecule is absorbed to a paper carrier or a gel, preferably to chromatography or filter paper.
  • the sensor molecule is comprised in a thin film, or confined in a tube, capillary or (microfluidic) chamber.
  • Still further applications of the invention relate to the in vivo targeting and/or imaging of cells.
  • cancer cells expressing a cancer- specific cell surface marker like a receptor can be targeted by a sensor of the invention carrying as secondary ligand that is specific for such cancer marker.
  • the analyte of interest is not necessarily a free compound in solution.
  • Sensor molecules based on a luciferase reporter enzyme which is inhibited by LI in the absence of analyte are particularly suitable for such application in view of their negligible background emission and the established use of luciferase for in vivo imaging.
  • a sensor molecule of the invention in particular one being based on the TURNON design, is suitably used as optical probe.
  • the invention also provides a method for detecting a cell of interest, comprising the steps of contacting a sample known or suspected of comprising the cell with a sensor molecule according to the invention wherein L2 is capable of specifically binding to at least one surface marker expressed on said cell, under conditions allowing for an analyte-induced conformational change within the sensor molecule to occur such that the signal generated by the signal generating moiety is modulated; and analyzing a change in a signal generated by the signal generating moiety.
  • the cell is a cancer cell.
  • the sample can be a clinical sample, like a tissue biopsy or bodily sample , or a research sample e.g. a cell culture.
  • the invention also relates to a method for providing a sensor molecule of the invention. As is illustrated in Examples 3-7, the proteinaceous moiety and the synthetic regulatory molecule (or precursor thereof) are typically produced as separate entities, after which the synthetic molecule is tethered to the
  • the method comprises the steps of providing the proteinaceous moiety and the synthetic regulatory molecule or precursor thereof, and assembling both to yield the sensor molecule.
  • the proteinaceous moiety can be prepared using standard recombinant DNA techniques well known to those skilled in the art.
  • the nucleic acid sequence coding the proteinaceous region comprising BP1 can be genetically introduced into the multiple cloning site of a bacterial expression vector comprising a luciferase sequence such that the BP1 sequence is operatively linked to the Luc coding sequence.
  • Other proteinaceous components like a protein labeling tag and/or linker sequences, can also be incorporated using standard techniques.
  • the DNA constructs for various configurations of the proteinaceous moiety of a sensor of the invention can be transfected/transformed in suitable cell lines (eukaryotic or prokaryotic) for its production. The various configurations of the fusion proteins produced in cells, are then purified or semipurified from the
  • transfected/transformed cells A convenient procedure to purify a proteinaceous moiety is by affinity chromatography e.g. using a His- and/or Strep-tag engineered in the DNA construct. Standard biochemical techniques can be also used alone or in combination with affinity chromatography to purify to various levels the various fusion proteins. Finally, these purified fusion proteins can be also chemically or enzymatically modified before their tethering to the synthetic regulatory molecule.
  • the proteinaceous moiety is produced by a combination of in vivo and in vitro methods.
  • a fusion protein is genetically engineered and expressed in cells using recombinant techniques.
  • the fusion protein is then purified or semi-purified before being modified by chemically or enzymatically attaching a further proteinaceous element, e.g. an element which can serve as a binding protein such as an antibody. Attachment of the further element can be peptide-based or chemically-based.
  • the synthetic regulatory molecule or precursor thereof can be synthesized using methods known in the art.
  • the primary and the secondary ligand are linked in close proximity to each other and appended to the fluorophore-0 6 -benzylguanine derivative by chemical synthesis.
  • the skilled person will understand that the methods used can be selected based on the chemical nature of the fluorophore and/or the ligand(s).
  • the coupling of each element can essentially be performed according to what has been described in the art on conventional FRET-based SNIFITs.
  • the regulatory molecule or precursor thereof may contain an element which mediates tethering to the proteinaceous moiety.
  • the synthetic regulatory molecule if the synthetic regulatory molecule is to be site-specifically tethered to the proteinaceous moiety of the sensor molecule via a self-labelling protein such as SNAP-tag, CLIP-tag or Halo-Tag, the synthetic regulatory molecule must contain the appropriate reactive group such as a reactive group for hAGT, a 0 6 -benzylguanine (BG), 0 4 -benzyl-2-chloro-6-aminopyrimidine (CP), O 2 - benzylcytosine (BC) derivative, or a chloroalkane.
  • Reactive groups mediating tethering may be advantageously coupled to the fluorophore acceptor molecule via spacer comprising several polyethylene glycol (PEG) units.
  • PEG polyethylene glycol
  • spacer of 3-27 PEG units is suitably used. See for example Brun et al. J Am Chem Soc.
  • a regulatory molecule to be used in combination with cysteine or enzyme-mediated coupling can be synthesized based on the examples below, wherein the BG is exchanged with a maleimide for cysteine coupling, or with a CoA derivative for coupling via phosphopantetheine transferases.
  • FIG. 1 Pictorial description of the sensing mechanism of exemplary sensor molecules.
  • the ligands which can be synthetic or proteinaceous, are denoted LI and L2 and their corresponding binding partners are labelled BPl and BP2.
  • X represents the attachment position of the synthetic regulatory molecule.
  • A "Normal steric displacement” with both LI and L2 situated on the synthetic regulatory molecule.
  • B "Competition steric displacement” with both LI and L2 situated on the synthetic regulatory molecule.
  • BP2 can either be a separate entity or part of the sensor molecule (represented by dashed line).
  • C "Normal steric displacement" with LI being part of the synthetic regulatory molecule and L2 situated on the signal generating moiety.
  • RET-based readout with both RET partners being part of the signal generation moiety.
  • the RET partners can be synthetic fluorophores, fluorescent proteins, or a luciferase.
  • Displacement of LI from BPl leads to a change in RET efficiency.
  • C TURN-ON reporter.
  • the binding partner of LI is an enzyme (E) that is capable of generating a detectable signal.
  • LI can be an inhibitor that reduces enzymatic activity. Binding of L2 to BP2 prevents LI from inhibiting E and a signal is observed.
  • FIG. 3 Exemplary sensor molecule for detecting streptavidin based on "normal steric displacement” and BRET as readout.
  • the signal generating moiety comprises human carbonic anhydrase (HCA) as BPl, NanoLuc luciferase as BRET donor, a 30-proline linker for geometrical optimization, and SNAP-tag as attachment position for the synthetic regulatory molecule.
  • the synthetic regulatory molecule comprises BG, which is recognized by SNAP-tag, the BRET acceptor Cy3, an aromatic sulfonamide (SA) as LI, and biotin (b) as L2.
  • Figure 4 Exemplary sensor molecule for detecting methotrexate based on
  • FIG. 5 Exemplary sensor molecule for detecting acetylcholine esterase inhibitors based on "competition steric displacement" and FRET as readout.
  • A Pictorial description of the structure and sensing mechanism of the sensor molecule.
  • the signal generation moiety consists of HCA as BPl, CLIP-tag labelled with Cy3 as FRET donor, a 30-proline linker for geometrical optimization, SNAP-tag as attachment position for the synthetic regulatory molecule, and acetylcholine esterase (AChE) as BP2 - binding partner for the analyte of interest.
  • the signal generation moiety is tethered to the surface of mammalian cells.
  • the synthetic regulatory molecule consists of BG, which is recognized by SNAP-tag, the FRET acceptor Cy5, an aromatic sulfonamide (SA) as LI, and edrophonium (e / edro) as L2.
  • SA aromatic sulfonamide
  • edrophonium e / edro
  • the analyte tacrine T
  • B Structure of the synthetic regulatory molecule.
  • C Response of the sensor on the surface of mammalian cells perfused with the analyte tacrine. For details see Example 3.
  • FIG. 6 Exemplary TURN-ON reporter for detecting streptavidin based on "normal steric displacement” and bioluminescence as readout.
  • A Pictorial description of the structure and sensing mechanism of the sensor molecule.
  • the signal generation moiety consists of Renilla luciferase as enzyme, E, and SNAP-tag as attachment position for the synthetic regulatory molecule.
  • the synthetic regulatory molecule consists of BG, which is recognized by SNAP-tag,
  • coelenteramide coent
  • biotin biotin
  • Binding of the analyte streptavidin (strept) to L2 prevents coent from inhibiting the luciferase and a signal can be observed.
  • C Picture of the sensor molecule in the absence and presence of 1 ⁇ streptavidin. The change in bioluminescence intensity can be seen by eye.
  • D Quantification of the bioluminescence intensity produced by the sensor molecule in the absence and presence of 1 ⁇ streptavidin.
  • Peptide couplings were performed by activation of the respective carboxylic acid with 0-(N-Succinimidyl)-N,N,N',N'-tetramethyluronium tetrafluoroborate (TSTU) or N,N,N',N'-Tetramethyl-0-(lH-benzotriazol-l- yl)uronium hexafluorophosphate (HBTU) in the presence of diisopropylethylamine (DIEA) as base in anhydrous dimethylsulfoxide (DMSO) at room temperature.
  • DIEA diisopropylethylamine
  • DMSO dimethylsulfoxide
  • the basic principle underlying the Steric Displacement- SNIFITs is the mutually exclusive binding of two tethered ligands due to steric hindrance between the two protein targets.
  • the two ligands, the primary and the secondary ligand are linked in close proximity to each other and appended to the fluorophore-O 6 - benzylguanine derivative by chemical synthesis.
  • the BG -fluorophore- double ligand molecule can be used to label an existing sensor protein of an optimized SNIFIT, retaining its original optimized FRET-ratio change, but making it sensitive to the analyte protein of interest. In absence of the analyte protein, the primary ligand is bound to the receptor protein and strong resonance energy transfer occurs.
  • the analyte protein at a sufficient concentration can bind to the secondary ligand, but the steric hindrance between the analyte protein and the receptor protein leads to unbinding of the primary ligand from the receptor protein leading to a detectable decrease in RET-efficiency.
  • This allows to adapt a previously optimized probe to virtually any different analyte, with minimal synthetic effort.
  • SD-SNIFITs are not anymore limited to sense small molecules and we were able to extend the field of application to whole protein analytes.
  • a BRET sensor capable of sensing streptavidin concentrations was constructed by introducing biotin as a secondary ligand into a human carbonic anhydrase II (HCA) -based sensor molecule (see Figure 3A).
  • HCA human carbonic anhydrase II
  • Scheme 1 Schematic representation of the synthesis of the molecule BG-Cy3-SA- biot.
  • A-l 4-carboxybenzene sulfonamide
  • A-2 N-alpha-Boc-(L)- 2,3-diaminopropanoic acid
  • A-4 was deprotected by stirring at room temperature in trifluoroacetic acid (TFA) to obtain A-4.
  • TFA trifluoroacetic acid
  • the product was then coupled to biotin to give the bifunctional ligand A-5. that was subsequently tethered to a short PEG2 tether by peptide coupling to l-N-Boc-3,6-dioxa-l,8- diaminooctane (A-6) to afford A-7.
  • Deprotection was performed by stirring in TFA at room temperature to give A-8.
  • BG-EG11-NH2 (A-10) and Cy3 (A-9) were prepared as previously described (Mujumdar et al. Bioconjugate Chemistry 1993, 4, 105-111 Masharina et al. J Am Chem Soc. 2012;134(46):19026-34) and the three building blocks were coupled together with A-8 to give the polyfunctional molecule BG-Cy3-SA-Biot (A-ll).
  • a fusion protein of SNAP-tag, a 30-proline linker, NanoLuc luciferase (Promega, Fitchburg, WI) and HCA was constructed by replacing the coding sequence of CLIP-tag in the previously described sensor SNAP-PP30-CLIP-HCA (Brun et al. J Am Chem Soc. 2011;133(40):16235-42) by the coding sequence of NanoLuc luciferase using standard cloning techniques.
  • the fusion protein was expressed in the E. coli strain Rosetta-gami and purified using a C-terminal His- tag as well as an N-terminal Strep-tag.
  • the sensor molecule was assembled by labeling SNAP-tag with the synthetic molecule BG-Cy3-SA-biot ( Figure 3B).
  • the purified protein was diluted to a concentration of 1 ⁇ in HEPES buffer (50mM HEPES, 50mM NaCl, pH 7.2) and incubated with a 4-fold molar excess of the synthetic compound BG-Cy3-SA-biot for 1 hour at room temperature. Excess of the synthetic compound was removed using centrifugal filter devices (Amicon Ultra-0.5, Merck Millipore, Billerica, MA).
  • the assembled sensor molecule was diluted to a concentration of 10 nM in 100
  • HEPES buffer containing defined concentrations of streptavidin in white non- binding 96-well plates (Greiner Bio-One, Kremsmiinster, Austria). The solutions were incubated at room temperature for at least 10 minutes to ensure that the sensor had reached equilibrium.
  • Figures 3C and 3D show the response of the sensor to different streptavidin concentrations. At low concentrations, the sensor is in its closed conformation, permitting efficient resonance energy transfer from NanoLuc to Cy3 and leading to a low NanoLuc / Cy3 emission ratio. At high streptavidin
  • HCA-binding molecules such as sulfonamide drugs can in principle interfere with the streptavidin quantification and this should be taken into account for some application, but the presence of both the compounds is rather uncommon.
  • biotin concentration can thus be calculated from the shifting observed in the titration curve compared to a blank titration performed in the absence of biotin derivative. This approach has interesting clinical applications.
  • Biotin is a coenzyme for five carboxylases in the human body (propionyl-CoA carboxylase,
  • biotin is essential for amino acid catabolism
  • Biotin is also necessary for gene stability because it is covalently attached to histones. Biotinylated histones play a role in repression of transposable elements and some genes. Normally, the amount of biotin in the body is regulated by dietary intake, biotin transporters
  • biotin deficiency could occur. Since biotin is in many foods at low concentrations, deficiency is rare except in locations where malnourishment is very common. Pregnancy, however, alters biotin catabolism and despite a regular biotin intake, half of the pregnant women in the U.S. are marginally biotin deficient. If biotin metabolism is defective, all four carboxylases will be deficient. Biotin is covalently linked to a key lysine residue in each carboxylase by action of
  • holocarboxylase synthetase When the carboxylase proteins are degraded, biotinoyl-lysine is subsequently cleaved by biotinidase releasing free biotin that can be reutilized.
  • the two defects in biotin metabolism associated with Multiple Carboxylase Deficiency are caused by deficient activity of holocarboxylase synthetase and biotinidase.
  • the disorders tend to present clinically at different ages, with holocarboxylase synthetase deficiency being known as early-onset (neonatal) multiple carboxylase deficiency and biotinidase deficiency referred to as late-onset multiple carboxylase deficiency. Both respond to biotin supplementation.
  • a sensor molecule of the present invention is advantageously used in support of diagnosis, treatment, and other clinical decision- making.
  • a BRET sensor capable of sensing methotrexate concentrations was constructed by introducing the dihydrofolate reductase (DHFR) inhibitor trimethoprim as a secondary ligand into a human carbonic anhydrase II (HCA)- based sensor molecule.
  • DHFR binds to this secondary ligand, keeping the sensor in its open state.
  • Methotrexate competes for binding to DHFR and can thus be detected by competition steric displacement (see Figure 4A).
  • BG-EG11-TMR-COOH (B-7) was prepared as previously described (Brun et al. J Am Chem Soc. 2009;131(16):5873-84; Kvach et al. Bioconjug Chem. 2009, 20(8), 1673-82) was subsequently coupled to B-6 to give the polyfunctional molecule BG-TMR-SA-tmp (B-8).
  • a fusion protein of SNAP-tag, a 30-proline linker, NanoLuc luciferase (Promega, Fitchburg, WI) and HCA was constructed by replacing the coding sequence of CLIP-tag in the previously described sensor SNAP-PP30-CLIP-HCA (Brun et al. J Am Chem Soc. 2011;133(40):16235-42) by the coding sequence of NanoLuc luciferase using standard cloning techniques.
  • the fusion protein was expressed in the E. coli strain Rosetta-gami and purified using a C-terminal His- tag as well as an N-terminal Strep-tag.
  • the sensor molecule was assembled by labeling SNAP-tag with the synthetic molecule BG-TMR-SA-tmp ( Figure 4B).
  • the purified protein was diluted to a concentration of 1 ⁇ in HEPES buffer (50mM HEPES, 50mM NaCl, pH 7.2) and incubated with a 4-fold molar excess of the synthetic compound BG-TMR-SA- tmp for 1 hour at room temperature. Excess of the synthetic compound was removed using centrifugal filter devices (Amicon Ultra-0.5, Merck Millipore, Billerica, MA).
  • the assembled sensor molecule was diluted to a concentration of 10 nM in 100 HEPES buffer supplemented with 100 ⁇
  • NADPH NADPH
  • DHFR a defined concentration of DHFR
  • methotrexate a defined concentration of methotrexate in white non-binding 96-well plates
  • a FRET sensor capable of sensing the concentration of acetylcholine as well as acetylcholine esterase inhibitors was constructed by introducing
  • HCA human carbonic anhydrase II
  • a synthetic regulatory molecule containing an 0 6 -benzylguanine (BG) group for SNAP-tag labeling, the fluorophore Cy5, a sulfonamide as primary ligand, and the acetylcholine esterase inhibitor edrophonium as secondary ligand was synthesized according to scheme 3.
  • the synthetic procedure involved three convergent steps.
  • the edrophonium derivative was prepared by reacting Phloroglucinol C-l with dimethylamine to obtain C-2 that was alkylated in DMF with 10-azido-l- bromodecane in the presence of potassium carbonate to afford C-3.
  • Treatment with ethyl iodide in acetonitrile gave the cationic C-4 that was coupled to
  • BG-EG11-NH2 (A-10) and Cy5 (C-ll) were prepared as previously described (Mujumdar et al. Bioconjugate Chemistry 1993, 4, 105-111 Masharina et al. J Am Chem Soc. 2012;134(46):19026-34) and were coupled together also with 6- aminohexanoic acid C-12 to give C-13.
  • C-13 and C-10 were coupled using TSTU to obtain the polyfunctional molecule BG-Cy5-SA-edro (C-14).
  • Scheme 3 Schematic representation of the synthesis of the regulatory molecule BG-Cy5-SA-edro.
  • a fusion protein of acetylcholine esterase, SNAP-tag, a 30-proline linker, CLIP-tag and HCA was constructed by inserting the coding sequence of acetylcholine esterase into the previously described sensor SNAP-PP30-CLIP-HCA in pDisplay (Brun et al. J Am Chem Soc. 2011;133(40):16235-42) using standard cloning techniques.
  • the plasmid encoding for the senor was transfected into HEK293 cells using Lipofectamine 2000 (Life Technologies, Carlsbad, CA) following the supplier's recommendations.
  • the sensor molecule was assembled on the cell surface by labeling SNAP-tag with the synthetic molecule BG-Cy5-SA-deca ( Figure 5B) as well as CLIP-tag with the fluorophore CLIP- Surface 547 (New England Biolabs, Ipswich, MA).
  • the response of the sensor to inhibitors of acetylcholine esterase was then investigated by live-cell microscopy. The methods for labelling and imaging have been previously described (Brun et al. J Am Chem Soc. 2011;133(40):16235-42).
  • Figure 5C shows the response of the sensor to perfusion of the cells with tacrine.
  • the secondary ligand In the absence of tacrine, the secondary ligand is bound to acetylcholine esterase. In this state, resonance energy transfer from CLIP- Surface 547 to Cy5 is inefficient, leading to a high FRET ratio.
  • the secondary ligand In the presence of tacrine, the secondary ligand is displaced making it possible for the primary ligand to bind HCA, bringing the two fluorophores closely together. In this state, resonance energy transfer is efficient, leading to a high FRET ratio.
  • the steric displacement approach of the present invention can allow the quantification of metabolites or proteins by any detection means.
  • a reporter enzyme is used as signal generating moiety and tethered as primary ligand an inhibitor of such reporter enzyme.
  • secondary ligand we chose a binding partner of the analyte protein to be detected.
  • the reporter enzyme is in its off-state, because the primary ligand inhibits it.
  • the binding of the analyte protein to the secondary ligand sterically displaces the primary ligand, resulting in activation of the reporter enzyme.
  • this sensor design TURN-ON reporter.
  • D-l 2-Amino-3-azido-propionic acid
  • D-2 2-Amino-3-azido-propionic acid
  • BG-EGn- NH2 A-10
  • D-2 was coupled to D-2 to obtain the azido-modified 0 6 -benzylguanine- biotinylated derivative D-3.
  • coelenteramide D-4 was O-alkylated with propargyl bromide with sodium carbonate in dimethylformamide to give the O- propargyl-coelenteramide D-5.
  • Scheme 4 Schematic representation of the synthesis of the regulatory molecule BG-coel-biot.
  • a fusion protein of SNAP-tag and humanized Renilla luciferase was constructed by replacing the coding sequence of the 30-proline linker, CLIP-tag, and HCA in the previously described sensor SNAP-PP30-CLIP-HCA (Brun et at. J Am Chem Soc. 2011;133(40):16235-42) by the coding sequence of humanized Renilla luciferase using standard cloning techniques.
  • the fusion protein was expressed in the E. coli strain Rosetta-gami and purified using a C-terminal His- tag as well as an N-terminal Strep-tag.
  • the sensor molecule was assembled by labeling SNAP-tag with the synthetic molecule BG-coel-biot ( Figure 6B).
  • the purified protein was diluted to a concentration of 1 ⁇ in HEPES buffer (50mM HEPES, 50mM NaCl, pH 7.2) and incubated with a 4-fold molar excess of the synthetic compound BG-coel-biot for 1 hour at room temperature. Excess of the synthetic compound was removed using centrifugal filter devices (Amicon Ultra-0.5, Merck Millipore, Billerica, MA).
  • the assembled sensor molecule was diluted to a concentration of 50 nM in 100 HEPES buffer containing different
  • Figure 6D shows the response of the sensor to streptavidin.
  • the primary ligand In the absence of streptavidin, the primary ligand is bound to the luciferase and inhibits the enzyme.
  • streptavidin When streptavidin is added, it binds to biotin and displaces the primary ligand from the luciferase, bringing it into an active state and leading to a 17-fold increase in luminescence intensity. This change is also clearly visible by naked eye; a picture taken with a digital camera is shown in Figure 6C.
  • the TURN-ON luciferase sensor shows negligible background emission and the application of luciferases for in vivo imaging has been widely demonstrated (Roda et. al. Trac-Trend Anal Chem 2009;28:307). This allows its application for targeting and visualizing specific cells in living animals, e.g. cancer cells expressing a specific receptor and tethering a secondary ligand specific for such a receptor. Moreover, the TURN-ON reporter concept is not limiting to
  • An enzyme used for colorimetric assay can be used in place of the luciferase with one of its inhibitors as primary ligand, to provide a colorimetric assay to determine the concentration or the activity of any analyte protein of interest, by tethering one of its binders as secondary ligand.
  • a redox sensor is envisioned analogously.

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Abstract

La présente invention concerne de manière générale des procédés, des réactifs et des dispositifs qui permettent de déterminer la concentration d'une substance à analyser dans un échantillon. La présente invention porte également sur une molécule capteur protéique pour la détection d'une substance à analyser d'intérêt (A), comportant une fraction de génération de signal fixée à une molécule régulatrice synthétique pouvant moduler une génération de signal par liaison intramoléculaire, (i) la molécule régulatrice synthétique comportant un ligand primaire (L1) apte à une liaison intramoléculaire à un partenaire primaire (BP1) sur la molécule capteur ; (ii) la molécule capteur comportant un ligand secondaire (L2) pouvant se lier à un partenaire de liaison secondaire (BP2) qui est A ou un partenaire de liaison de ce dernier ; (iii) la liaison de L1 et L2 à leurs partenaires de liaison respectifs étant mutuellement exclusives, de telle sorte que la liaison de BP2 à L2 influence la liaison de L1 à BP1, menant à un changement de conformation dans la molécule capteur de telle sorte que le signal généré est modulé.
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WO2019164402A1 (fr) * 2018-02-26 2019-08-29 Technische Universiteit Eindhoven Biocapteur bioluminescent pour la détection et la quantification de biomolécules
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CN115141128A (zh) * 2022-06-24 2022-10-04 沈阳药科大学 3-芳基-3-(磺胺苯甲酰胺基)丙(烯)酸衍生物及其制备方法及应用
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