EP3377610A2 - Ratiometrische biosensoren und nichtgeometrisch modulierter fret - Google Patents

Ratiometrische biosensoren und nichtgeometrisch modulierter fret

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Publication number
EP3377610A2
EP3377610A2 EP16867307.7A EP16867307A EP3377610A2 EP 3377610 A2 EP3377610 A2 EP 3377610A2 EP 16867307 A EP16867307 A EP 16867307A EP 3377610 A2 EP3377610 A2 EP 3377610A2
Authority
EP
European Patent Office
Prior art keywords
fluorophore
ligand
biosensor
binding protein
protein
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP16867307.7A
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English (en)
French (fr)
Other versions
EP3377610A4 (de
Inventor
Homme W. Hellinga
Malin J. Allert
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Duke University
Original Assignee
Duke University
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Filing date
Publication date
Application filed by Duke University filed Critical Duke University
Publication of EP3377610A2 publication Critical patent/EP3377610A2/de
Publication of EP3377610A4 publication Critical patent/EP3377610A4/de
Withdrawn legal-status Critical Current

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Classifications

    • 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
    • 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
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/533—Production of labelled immunochemicals with fluorescent label

Definitions

  • the present invention relates to compositions and methods for detecting compounds and determining the concentration thereof.
  • Fluorescent chemosensors have wide-ranging applications in cell biology and analytical chemistry.
  • the present subject matter provides methods for converting monochromatic responses into dichromatic responses that enable ratiometric sensing. If the fluorescence emission spectrum changes shape in response to analyte binding such that the ratio of emission intensities at two appropriately chosen wavelengths reports on analyte concentration
  • ratiometric measurements can be used to monitor analyte concentrations.
  • these methods are based on establishing non-geometrically modulated Forster resonance energy transfer (ngmFRET) between a monochromatic, chemoresponsive fluorophore (a directly responsive partner), and a second fluorophore that neither interacts directly with the ligand, nor is sensitive to ligand-mediated changes in its environment (an indirectly responsive partner).
  • ngmFRET non-geometrically modulated Forster resonance energy transfer
  • Biosensors that undergo ngmFRET (or altered ngmFRET) upon ligand binding are also provided herein, as well as compositions and devices comprising such biosensors.
  • a biosensor that exhibits a monochromatic response (which does not produce a ratiometric signal) to ligand binding may be converted into a biosensor that produces a
  • ratiometric biosensors dichromatic/ratiometric signal.
  • fluorophores that typically do not show a dichromatic response to ligand binding such as fluorescein and derivatives thereof
  • an additional reporter group such as another fluorophore
  • methods, compounds, and compositions relating to biosensors with multiple reporter groups that have improved ratiometric signals compared to other ratiometric biosensors (e.g., ratiometric biosensors having a single reporter group).
  • tgmFRET is a physical phenomenon that was first described over 50 years ago.
  • the transfer of excited state energy from a donor fluorophore to an acceptor fluorophore i.e. energy transfer
  • tgmFRET is modulated by a ligand-binding event through changes in the distance and/or angle between the donor and acceptor fluorophores.
  • tgmFRET is manifested by opposing changes in the fluorescence emission intensities of the donor and acceptor fluorophores, respectively, in response to ligand binding. For instance, a decrease in distance results in a decrease of the donor fluorescence emission intensity and an increase in the acceptor fluorescence intensity, as energy is transferred from the former to the latter.
  • a ligand-mediated increase in the distance between the partners has the opposite effect (the fluorescence emission intensity of the donor increases, whereas that of the acceptor decreases).
  • ligand-mediated modulation of fluorescence intensity arises from global changes in the entire system, and can occur only if both partners are present.
  • ligand-mediated modulation of fluorescence intensity arises from changes that are localized to the photophysics of the directly responsive fluorophore.
  • ligand-mediated changes in fluorescence therefore occur also if only the directly responsive partner is present in isolation by itself.
  • the entire ngmFRET system comprising two partners is not required for evincing ligand-mediated changes in fluorescence emission intensity, the response of such a system is qualitatively changed or quantitatively enhanced over the responses of the isolated directly responsive partner (e.g. converting a monochromatic into a dichromatic response, thereby enabling ratiometry).
  • the pattern of fluorescence intensity changes manifested by ligand binding in ngmFRET systems are not limited to opposing changes only. Instead, in ngmFRET almost all combinations of emission intensity changes are possible: opposing changes in the two partners, both partners increase, both decrease, one partner remains unchanged whereas the other increases or decreases.
  • the ligand-mediated alteration of the photophysics of the directly responsive partner includes changes to its spectral properties such as the shape of the excitation or emission spectra, and the ratio of radiative to non-radiative emission rates.
  • the fluorescence emission intensity of the indirectly responsive partner in isolation does not change in response to ligand binding; its intensity changes only in the presence of a directly responsive partner in the complete ngmFRET system.
  • quenching has often been used loosely to refer to a decrease fluorescence emission intensity. However, as used herein, the term “quenching” strictly means a "change in the ratio of radiative to non-radiative emission rates" of a fluorophore.
  • ngmFRET occurs between two or more reporter groups (e.g., a donor fluorophore and an acceptor fluorophore) of the biosensor.
  • ngmFRET may change (e.g., increase or decrease) when ligand is bound to the biosensor and a donor fluorophore is contacted with radiation within its excitation wavelength. Effects from tgmFRET and ngmFRET may occur together and be combined into an overall ligand-mediated change in fluorescence emission intensity. In preferred embodiments, less than half or none of the change in overall ligand-mediated change in fluorescence emission intensity is due to tgmFRET.
  • most of the overall ligand-mediated change in fluorescence emission intensity change is not due to a change in the distance between the donor and acceptor fluorophore or as a result of a change in the orientation between the donor and acceptor fluorophore. In non-limiting examples, less than about 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, or 0.5% of the change in overall ligand-mediated change in fluorescence emission intensity is due to tgmFRET.
  • the change in overall ligand-mediated change in fluorescence emission intensity comprises a spectral change (e.g., in the excitation or emission spectrum) and/or a change in the ratio of the radiative to non-radiative decay rates of one of the fluorophores (by itself and regardless of the presence of any other fluorophore/partner) upon ligand binding.
  • ligand binding mediates spectral shifts in the absorption or emission spectrum of the directly responsive partner.
  • such changes are due at least in part to a switch between different excited states in the ligand-free and ligand-bound biosensor.
  • the two excited states are associated with different transition dipoles.
  • This class of changes is termed "dipole switching" herein.
  • biosensors that show dipole sensing include ttGGBP 17C Badan-pZif Alexa532 and ttGGBP 182C Acrylodan-pZif Alexa532.
  • the reporter groups include a directly responsive partner (which may be a donor fluorophore or an acceptor fluorophore) and an indirectly responsive partner (which may be a donor fluorophore or an acceptor fluorophore).
  • a "directly responsive" partner is a fluorophore that responds to (i) ligand-induced protein conformational changes upon ligand binding to a ligand-binding protein; or (ii) ligand binding to the directly responsive partner itself.
  • the directly responsive partner comprises a fluorophore (i.e., it is a directly responsive fluorophore).
  • the directly responsive fluorophore exhibits a monochromatic or dichromatic spectral change, and/or a change in the ratio of radiative to non-radiative emission rates, upon ligand binding.
  • the directly responsive partner may be a fluorophore such as a fluorescent protein or a small molecule fluorescent compound.
  • An "indirectly responsive" partner is a fluorophore for which no change in emission spectra, excitation spectra, or change in the ratio of radiative to non-radiative emission rates is caused by ligand binding in the absence of a directly responsive partner.
  • the indirectly responsive partner comprises a fluorophore (i.e., it is an indirectly responsive fluorophore).
  • the emission fluorescence intensity of the indirectly responsive partner changes due to a change in energy flow in the ngmFRET pathway upon ligand binding. See, e.g., FIG. 28. ngmFRET Biosensors
  • compositions, biosensors, and devices comprising multiple reporter groups, e.g. a directly responsive fluorophore and an indirectly responsive fluorophore, between which ngmFRET occurs.
  • multiple reporter groups e.g. a directly responsive fluorophore and an indirectly responsive fluorophore, between which ngmFRET occurs.
  • aspects include a method of detecting a ligand in a sample, comprising contacting a biosensor with a ligand.
  • the biosensor comprises a ligand-binding protein, a directly responsive fluorophore and an indirectly responsive fluorophore.
  • the directly responsive and the indirectly responsive fluorophores are located at two distinct sites of the ligand-binding protein.
  • the directly responsive fluorophore is a donor fluorophore and the indirectly responsive fluorophore is an acceptor fluorophore.
  • the directly responsive fluorophore is an acceptor fluorophore and the indirectly responsive fluorophore is a donor fluorophore.
  • the method includes contacting the biosensor with radiation comprising a wavelength within the excitation spectrum of the donor fluorophore.
  • a fluorescence property of the directly responsive fluorophore changes in response to ligand binding. This change in fluorescent property is independent of the indirectly responsive fluorophore, and occurs regardless of whether the indirectly responsive fluorophore is absent or present.
  • the fluorescence properties of the indirectly responsive fluorophore do not change in response to ligand binding in the absence of the directly responsive fluorophore.
  • the method further comprises measuring fluorescent light that is emitted from the directly responsive fluorophore and the indirectly responsive fluorophore, and calculating a ratiometric signal to detect the ligand in the sample.
  • the ratiometric signal (i- 1;2 ) comprises a quotient of two intensities, I ⁇ and I%2, measured at two independent wavelengths, ⁇ ] and ⁇ 2 and is calculated according to the following equation:
  • the change in the fluorescent property of the directly responsive fluorophore comprises (i) a bathochromic or hypsochromic shift in the emission or excitation spectrum thereof; and/or (ii) a change in the ratio of radiative to non-radiative emission rates thereof.
  • the directly responsive fluorophore is Badan and emission intensity is measured at a wavelength or range of wavelengths between about 400 nm and 1 OOOnm (e.g., including a wavelength of about 450, 451 , 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, or 475 nm), and wherein the indirectly responsive fluorophore is 5-iodoacetamidofluorescein (5-IAF) and emission intensity is measured at a wavelength or range of wavelengths between about 400 nm and lOOOnm (e.g., including a wavelength of about 510, 511, 512, 513, 514, 515, 516,517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529
  • the directly responsive fluorophore is Badan and emission intensity is measured at a wavelength or range of wavelengths between about 400 nm and 1 OOOnm (e.g., including a wavelength of about 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, or 475 nm), and wherein the indirectly responsive fluorophore is Alexa532 and emission intensity is measured at a wavelength or range of wavelengths between about 400 nm and 1 OOOnm (e.g., including a wavelength of about 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, or 570 nm).
  • the directly responsive fluorophore is Pacific Blue and emission intensity is measured at a wavelength or range of wavelengths between about 400 nm and lOOOnm (e.g., including a wavelength of about 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, or 465 nm), and wherein the indirectly responsive fluorophore is 5-IAF and emission intensity is measured at a wavelength or range of wavelengths between about 400 nm and lOOOnm (e.g., including a wavelength of about 510, 511, 512, 513, 514, 515, 516,517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, or 530 nm).
  • the directly responsive fluorophore is Acrylodan and emission intensity is measured at a wavelength or range of wavelengths between about 400 nm and lOOOnm (e.g., including a wavelength of about 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, or 475 nm), and wherein the indirectly responsive fluorophore is 5-IAF and emission intensity is measured at a wavelength or range of wavelengths between about 400 nm and lOOOnm (e.g., including a wavelength of about 510, 511, 512, 513, 514, 515, 516,517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, or 530 nm).
  • the directly responsive fluorophore is Acrylodan and emission intensity is measured at a wavelength or range of wavelengths between about 400 nm and 1 OOOnm (e.g., including a wavelength of about 470, 471 , 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490 nm), and wherein the indirectly responsive fluorophore is Alexa532 and emission intensity is measured at a wavelength or range of wavelengths between about 400 nm and lOOOnm (e.g., including a wavelength of about 540, 541, 542,543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, or 560 nm).
  • the directly responsive fluorophore is 5-IAF and emission intensity is measured at a wavelength or range of wavelengths between about 400 nm and lOOOnm (e.g., including a wavelength of about 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, or 465 nm), and wherein the indirectly responsive fluorophore is Pacific Blue and emission intensity is measured at a wavelength or range of wavelengths between about 400 nm and lOOOnm (e.g., including a wavelength of about 510, 511, 512, 513, 514, 515, 516,517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, or 530 nm).
  • the directly responsive fluorophore is Oregon Green and emission intensity is measured at a wavelength or range of wavelengths between about 400 nm and lOOOnm (e.g., including a wavelength of about 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, or 465 nm), and wherein the indirectly responsive fluorophore is Pacific Blue and emission intensity is measured at a wavelength or range of wavelengths between about 400 nm and lOOOnm (e.g., including a wavelength of about 510, 511, 512, 513, 514, 515, 516,517, 518, 519, 520, 521, 522, 523,
  • the directly responsive fluorophore is N-(Iodoacetaminoethyl)- l-naphthylamine-5-sulfonic acid (IAEDANS) and emission intensity is measured at a wavelength or range of wavelengths between about 400 nm and lOOOnm (e.g., including a wavelength of about 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, or 475 nm), and wherein the indirectly responsive fluorophore is 5-IAF and emission intensity is measured at a wavelength or range of wavelengths between about 400 nm and lOOOnm (e.g., including a wavelength of about 510, 511, 512, 513, 514, 515, 516,517, 518, 519, 520, 521, 5
  • the directly responsive fluorophore is Alexa532 and emission intensity is measured at a wavelength or range of wavelengths between about 400 nm and 1000 nm (e.g. including a wavelength of about 530, 531, 532, 534, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, or 570 nm), and wherein the indirectly responsive fluorophore is Acrylodan and emission intensity is measured at a wavelength or range of wavelengths between about 400 nm and 1000 nm (e.g.
  • the directly responsive fluorophore is a yellow fluorescent protein and emission intensity is measured at a wavelength or range of wavelengths between about 400 nm and lOOOnm (e.g., including a wavelength of about 520, 521, 522, 523, 524,
  • the indirectly responsive fluorophore is Acrylodan and emission intensity is measured at a wavelength or range of wavelengths between about 400 nm and lOOOnm (e.g., including a wavelength of about 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, or 510 nm.
  • the directly responsive fluorophore is a yellow fluorescent protein and emission intensity is measured at a wavelength or range of wavelengths between about 400 nm and lOOOnm (e.g., including a wavelength of about 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, or 540 nm), and wherein the indirectly responsive fluorophore is Pacific Blue and emission intensity is measured at a wavelength or range of wavelengths between about 400 nm and lOOOnm (e.g., including a wavelength of about 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, or 465 nm).
  • lOOOnm e.g., including a wavelength of about 520, 521,
  • the directly responsive fluorophore comprises a donor fluorophore and the indirectly responsive fluorophore comprises an acceptor fluorophore.
  • the emission intensity of the donor fluorophore decreases and the emission intensity of the acceptor fluorophore increases upon ligand binding to the ligand-binding protein when the donor fluorophore is contacted with radiation within the excitation spectrum of the donor fluorophore.
  • the emission intensity of the donor fluorophore increases and the emission intensity of the acceptor fluorophore decreases upon ligand binding to the ligand-binding protein when the donor fluorophore is contacted with radiation within the excitation spectrum of the donor fluorophore.
  • the emission intensities of the donor fluorophore and the acceptor fluorophore both decrease upon ligand binding to the ligand-binding protein when the donor fluorophore is contacted with radiation within the excitation spectrum of the donor fluorophore.
  • the emission intensity of the donor fluorophore decreases and the emission intensity of the acceptor fluorophore increases, decreases, or remains about the same upon ligand binding to the ligand-binding protein when the donor fluorophore is contacted with radiation within the excitation spectrum of the donor fluorophore. In some embodiments, the emission intensity of the donor fluorophore increases, decreases, or remains about the same and the emission intensity of the acceptor fluorophore decreases upon ligand binding to the ligand-binding protein when the donor fluorophore is contacted with radiation within the excitation spectrum of the donor fluorophore.
  • the emission intensities of the donor fluorophore and the acceptor fluorophore both increase upon ligand binding to the ligand-binding protein when the donor fluorophore is contacted with radiation within the excitation spectrum of the donor fluorophore. In some embodiments, the emission intensity of the donor fluorophore increases, decreases, or remains about the same and the emission intensity of the acceptor fluorophore increases upon ligand binding to the ligand-binding protein when the donor fluorophore is contacted with radiation within the excitation spectrum of the donor fluorophore.
  • the emission intensity of the donor fluorophore increases and the emission intensity of the acceptor fluorophore increases, decreases, or remains about the same upon ligand binding to the ligand-binding protein when the donor fluorophore is contacted with radiation within the excitation spectrum of the donor fluorophore.
  • the directly responsive fluorophore comprises an acceptor fluorophore and the indirectly responsive fluorophore comprises a donor fluorophore.
  • the emission intensity of the donor fluorophore decreases and the emission intensity of the acceptor fluorophore increases, decreases, or remains about the same upon ligand binding to the ligand-binding protein when the donor fluorophore is contacted with radiation within the excitation spectrum of the donor fluorophore.
  • the emission intensity of the donor fluorophore increases and the emission intensity of the acceptor fluorophore increases, decreases, or remains about the same upon ligand binding to the ligand-binding protein when the donor fluorophore is contacted with radiation within the excitation spectrum of the donor fluorophore. In some embodiments, the emission intensity of the donor fluorophore remains about the same and the emission intensity of the acceptor fluorophore decreases upon ligand binding to the ligand-binding protein when the donor fluorophore is contacted with radiation within the excitation spectrum of the donor fluorophore.
  • the emission intensity of the donor fluorophore decreases and the emission intensity of the acceptor fluorophore increases, decreases, or remains about the same upon ligand binding to the ligand-binding protein when the donor fluorophore is contacted with radiation within the excitation spectrum of the donor fluorophore. In some embodiments, the emission intensity of the donor fluorophore increases and the emission intensity of the acceptor fluorophore increases, decreases, or remains about the same upon ligand binding to the ligand-binding protein when the donor fluorophore is contacted with radiation within the excitation spectrum of the donor fluorophore.
  • the emission intensity of the donor fluorophore remains about the same and the emission intensity of the acceptor fluorophore increases upon ligand binding to the ligand- binding protein when the donor fluorophore is contacted with radiation within the excitation spectrum of the donor fluorophore. In some embodiments, the emission intensity of the donor fluorophore decreases and the emission intensity of the acceptor fluorophore increases upon ligand binding to the ligand-binding protein when the donor fluorophore is contacted with radiation within the excitation spectrum of the donor fluorophore. In some
  • the emission intensity of the donor fluorophore increases and the emission intensity of the acceptor fluorophore remains about the same, increases, or decreases upon ligand binding to the ligand-binding protein when the donor fluorophore is contacted with radiation within the excitation spectrum of the donor fluorophore.
  • the increase may be, e.g., between about 0.1% to 10%, 10% to 50%, or 50% to 100%, or at least about 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 50%, 75%, 100%, 2-fold, 3-fold, 4-fold, 5-fold, 6- fold, 7-fold, 8-fold, 9-fold, or 10-fold.
  • the decrease may be, e.g., a decrease of between about at least about 0.1% to 10%, 10% to 50%, or 50% to 100%, or at least about 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90%.
  • the increases are not equal.
  • both the emission intensity of the donor fluorophore and the acceptor fluorophore decreases then the decreases are not equal.
  • the ligand-binding protein comprises the directly responsive fluorophore.
  • the directly responsive fluorophore is formed by an autocatalytic cyclization of an oligopeptide within the ligand-binding protein.
  • the oligopeptide is located within an interior a helix.
  • the oligopeptide comprises 2, 3, 4, 5, 6, 7, 8, 9, or 10 consecutive residues.
  • the directly responsive fluorophore is formed by an autocatalytic cyclization of a tripeptide located in an interior a helix of the ligand-binding protein.
  • ligand-binding protein comprises a yellow fluorescent protein (YFP), i.e. the YFP binds to ligand such as a halide anion.
  • YFP yellow fluorescent protein
  • ligand binding causes a change in signaling by the directly responsive fluorophore.
  • the directly responsive and the indirectly responsive fluorophores are located at two distinct sites of the amino acid or polypeptide, and the directly responsive fluorophore is chemoresponsive.
  • the method may further comprise contacting the biosensor with radiation comprising a wavelength within the excitation spectrum of the donor fluorophore, wherein (i) a fluorescence property of the directly responsive fluorophore changes in response to ligand binding in the absence or presence of the indirectly responsive fluorophore; (ii) a fluorescence property of the indirectly responsive fluorophore does not change in response to ligand binding in the absence of the directly responsive fluorophore; (iii) ngmFRET occurs between the directly responsive fluorophore and the indirectly responsive fluorophore; (iv) fluorescent light is emitted from the biosensor, wherein the light emitted from the biosensor comprises a combination of light emitted from the directly responsive fluorophore and light emitted from the indirectly responsive fluorophore; and (v) the ratio of the fluorescence emission intensity emitted from the biosensor at each of two distinct wavelengths changes in response to ligand binding.
  • the method may also include measuring fluorescent light that is emitted from the directly responsive fluorophore and the indirectly responsive fluorophore and calculating a ratiometric signal, to detect the ligand in the sample.
  • the ratiometric signal (i? 1;2 ) comprises a quotient of two intensities, I ⁇ and 1 ⁇ 2, measured at two independent wavelengths, ⁇ ] and ⁇ 2 and is calculated according to the following equation:
  • chemoresponsive fluorophore is a fluorophore to which ligand binds, wherein ligand binding causes a change in signaling by the fluorophore.
  • signaling refers to the emission of energy (which may be referred to as a "signal") by one or more reporter groups.
  • the signal comprises electromagnetic radiation such as a light.
  • the signal is detected as a complete emission spectrum (or spectra) or a portion (or portions) thereof.
  • a signal may comprise emitted light at a particular wavelength or wavelengths, or range(s) of wavelengths.
  • a change in signaling comprises a spectral change (e.g., a spectral shift and/or change in intensity).
  • a change in signaling comprises a dichromatic shift or a monochromatic fluorescence intensity change.
  • the directly responsive fluorophore is a donor fluorophore and the indirectly responsive fluorophore is an acceptor fluorophore.
  • the directly responsive fluorophore is an acceptor fluorophore and the indirectly responsive fluorophore is a donor fluorophore.
  • the change in the fluorescent property of the directly responsive fluorophore comprises (i) a bathochromic or hypsochromic shift in the emission or excitation spectrum thereof; and/or (ii) a change in the ratio of radiative to non-radiative emission rates thereof.
  • the directly responsive fluorophore is 5-IAF and emission intensity is measured at a wavelength or range of wavelengths between about 400 nm and lOOOnm (e.g., including a wavelength of about 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, or 470 nm), and wherein the indirectly responsive fluorophore is Acrylodan and emission intensity is measured at a wavelength or range of wavelengths between about 400 nm and lOOOnm (e.g., including a wavelength of about 510, 511, 512, 513, 514, 515, 516,517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, or 530 nm).
  • the directly responsive fluorophore is 5-IAF and emission intensity is measured at a wavelength or range of wavelengths between about 400 nm and lOOOnm (e.g., including a wavelength of about 510, 511, 512, 513, 514, 515, 516,517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, or 530 nm), and wherein the indirectly responsive fluorophore is Pacific Blue and emission intensity is measured at a wavelength or range of wavelengths between about 400 nm and lOOOnm (e.g., including a wavelength of about 445, 446, 447,448, 449, 450, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, nm).
  • lOOOnm e.g., including a wavelength of about 510, 511, 512, 513, 514, 515
  • Any amino acid or polypeptide may be used to link the chemoresponsive directly responsive fluorophore with the indirectly responsive fluorophore, provided the two fluorophores are close enough for ngmFRET to occur. Suitable distances may be determined in part by the distance-dependence of the energy transfer between a given donor-acceptor pair (see, e.g, J.R. Lakowicz, 2006, Principles of Fluorescence Spectroscopy, Springer,
  • the amino acid or polypeptide comprises 1 amino acid, or a stretch of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 500, 750, or 1000 amino acids.
  • the amino acid or polypeptide comprises at least 1, 2, or 3 thiol groups; at least 1, 2, or 3 cysteines that each comprise a sulfhydryl group; at least 1, 2, or 3 primary amine groups; or at least 1, 2, or 3 lysines that each comprise a primary amine.
  • the polypeptide comprise two cysteines, and there is no disulfide bond between the two cysteines. In some embodiments there is no disulfide bond between any pair of cysteines within the amino acid sequence of the polypeptide.
  • the polypeptide comprises a stretch of at least 50, 60, 70, 80, 90, or 100 amino acids in a sequence that is at least about 85%, 90%, 95%, or 99% identical to the amino acid sequence of ecTRX (SEQ ID NO: 151).
  • the polypeptide comprises a mutant of ecTRX comprising a D3X, K4X, K19X, D27X, K37X, K53X, K58X, K70X, R74X, K83X, K91X, K97X, or K101X mutation, or any combination thereof, wherein X is any amino acid, and wherein each ecTRX amino acid position is numbered as in SEQ ID NO: 151.
  • the polypeptide comprises a mutant of ecTRX comprising a D3A, K4R, K4Q, K19R, K19Q, D27A, K37R, K53M, K53R, K58M, K70R, R74C, K83R, K91R, K97R, or K101R mutation, or any combination thereof, wherein each ecTRX amino acid position is numbered as in SEQ ID NO: 151.
  • the polypeptide comprises a mutant of ecTRX that does not comprise a lysine.
  • the polypeptide comprises amino acids in the sequence of any one of SEQ ID NOS: 69-86 or 151.
  • the polypeptide further comprises a hexahistidine tag.
  • the ligand comprises a hydrogen ion.
  • the biosensor for pH wherein the directly responsive fluorophore is pH-sensitive.
  • the fully excited emission intensity of the directly responsive fluorophore is different at a pH less than about 7.0 (e.g. 6.9, 6.8, 67, 6.6, 6.5, 6.4, 6.3, 6.2, 6.1, or 6.0), or about 4.0 to 10.0, compared to a pH of about 7.3, 7.4, 7.5, 7.6, or 7.7.
  • the directly responsive fluorophore comprises a pH-sensitive fluorophore comprising fluorescein or a derivative thereof. In embodiments, the directly responsive fluorophore transitions from a monoanion to a dianion at a pH that is less than 7.0 in an aqueous solution.
  • the indirectly responsive fluorophore is attached to the ligand-binding protein via a covalent bond.
  • a covalent bond comprises a disulfide bond, a thioester bond, a thioether bond, an ester bond, an amide bond, or a bond that has been formed by a click reaction.
  • the indirectly responsive fluorophore is attached to the ligand- binding protein via a non-covalent bond. In certain embodiments, the indirectly responsive fluorophore is attached to a cysteine or a lysine of the protein.
  • the indirectly responsive fluorophore is attached to the N- terminus or the C-terminus of the protein. In some embodiments, the indirectly responsive fluorophore is attached to the N-terminus or the C-terminus of the protein via a fluorophore attachment motif. In some embodiments, fluorophore attachment motif comprises an amino acid or polypeptide. Various embodiments may be used to link a fluorophore with a ligand-binding protein. In some embodiments, the amino acid or polypeptide comprises 1 amino acid, or a stretch of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 500, 750, or 1000 amino acids.
  • the polypeptide comprises amino acids in the sequence of ⁇ (SEQ ID NO: 42).
  • the polypeptide comprises a stretch of at least 50, 60, 70, 80, 90, or 100 amino acids in a sequence that is at least about 85%, 90%, 95%, or 99% identical to the amino acid sequence of E. coli thioredoxin (ecTRX; SEQ ID NO: 151).
  • the directly responsive fluorophore is attached to the ligand- binding protein via a covalent bond.
  • the covalent bond comprises a disulfide bond, a thioester bond, a thioether bond, an ester bond, an amide bond, or a bond that has been formed by a click reaction.
  • directly responsive fluorophore is attached to a cysteine or a lysine of the protein.
  • the acceptor fluorophore comprises palladium, platinum, ruthenium, or osmium, then the acceptor fluorophore is not attached to the amino group of the N-teiminus of the ligand-binding protein.
  • the acceptor fluorophore does not comprise j Ru(bpy) 3 ] , [Ru(Ph 2 phen)3] ⁇ , [Ru(bpy) 2 (dcbpy)] ⁇ , or [Ru(bpy) 2 (phen-ITC)] 2+ , where bpy is 2,2" ⁇ bipyridme, phen is 1,10-phenanthroline, dcbpy is 4,4'-dicarboxy ⁇ 2,2'-bipyridine, and ITC is isothiocyanate.
  • the biosensor does not comprise an E. coli glutamine-binding protein with Acrylodan attached to 179C. In some embodiiments, the biosensor does not comprise E. coli glucose-binding protein with Acrylodan attached to 255C.
  • an overlap of the emission spectrum of the donor fluorophore and the excitation spectrum of the acceptor fluorophore increases upon ligand binding.
  • the directly responsive fluorophore comprises the donor fluorophore, and the increase results from a bathochromic shift in the emission spectrum of the donor fluorophore.
  • the directly responsive fluorophore comprises the acceptor fluorophore, and the increase results from a hypsochromic shift in the excitation spectrum of the acceptor fluorophore.
  • an overlap of the emission spectrum of the donor fluorophore and the excitation spectrum of the acceptor fluorophore decreases upon ligand binding.
  • the directly responsive fluorophore comprises the donor fluorophore, and the decrease results from a hypsochromic shift in the emission spectrum of the donor fluorophore.
  • the directly responsive fluorophore comprises the acceptor fluorophore, and the decrease results from a bathochromic shift in the excitation spectrum of the acceptor fluorophore.
  • the directly responsive fluorophore has a monochromatic spectral change upon ligand binding.
  • the directly responsive fluorophore has a dichromatic spectral change upon ligand binding.
  • the emission intensity of the donor fluorophore and/or the acceptor fluorophore increases in two phases as ligand concentration increases.
  • the ratio of radiative to non-radiative emission or intensity of the directly responsive fluorophore increases by at least about 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 50%, 75%, 100%, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8- fold, 9-fold, or 10-fold upon ligand binding to the ligand-binding protein.
  • the ratio of radiative to non-radiative emission or intensity of the directly responsive fluorophore decreases by at least about 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 50%, 75%, 90%, 95%, or 99% upon ligand binding to the ligand-binding protein.
  • the directly responsive fluorophore and the indirectly responsive fluorophore are not a naphthalene derivative. In some embodiments, the directly responsive fluorophore and the indirectly responsive fluorophore are not Prodan, Acrylodan, or Badan. In certain embodiments, the directly responsive fluorophore is not a naphthalene derivative. In some embodiments, the directly responsive fluorophore is not Prodan, Acrylodan, or Badan.
  • the directly responsive fluorophore comprises xanthene, a xanthene derivative, fluorescein, a fluorescein derivative, coumarin, a coumarin derivative, cyanine, a cyanine derivative, rhodamine, a rhodamine derivative, phenoxazine, a phenoxazine derivative, squaraine, a squaraine derivative, coumarin, a coumarin derivative, oxadiazole, an oxadiazole derivative, anthracene, an anthracene derivative, a
  • boradiazaindacine (BODIPY) family fluorophore pyrene, a pyrene derivative, acridine, an acridine derivative, arylmethine, an arylmethine derivative, tetrapyrrole, or a tetrapyrrole derivative.
  • the directly responsive fluorophore comprises fluorescein or a derivative thereof.
  • the directly responsive fluorophore and/or the indirectly responsive fluorophore comprises a fluorescent protein.
  • the directly responsive fluorophore and/or the indirectly responsive fluorophore comprises an organic compound having a molecular weight less than about 2000 Da (e.g., 5- iodoacetamidofluorescein (5-IAF) or 6-iodoacetamidofluorescein (6-IAF), rhodamine, Oregon Green, eosin, Texas Red, indocarbocyanine, oxacarbocyanine, thiacarbocyanine, merocyanine, Badan, Acrylodan, IAEDANS, comprising 3-cyano-7-hydroxycoumarin, 7- hydroxycoumarin-3-carboxylic acid, 6,8-difluoro-7-hydroxy- 4-methylcoumarin, or 7-amino-
  • 5-IAF 5- iodoacetamidofluorescein
  • 6-IAF 6-iodoace
  • the donor fluorophore comprises Pacific Blue and the acceptor fluorophore comprises 5-IAF or 6-iodoacetamidofluorescein (6-IAF);
  • the donor fluorophore comprises Pacific Blue and the acceptor fluorophore comprises Oregon Green;
  • the donor fluorophore comprises IAEDANS and the acceptor fluorophore comprises 5- IAF or 6-IAF;
  • the donor fluorophore comprises acrylodan and the acceptor fluorophore comprises Alexa532;
  • the donor fluorophore comprises acrylodan and the acceptor fluorophore comprises 5-IAF or 6-IAF;
  • the donor fluorophore comprises acrylodan and the acceptor fluorophore comprises Pacific Blue or YFP;
  • the donor fluorophore comprises
  • 5- IAF or 6-IAF and the acceptor fluorophore comprises Pacific Blue; (h) the donor fluorophore comprises badan and the acceptor fluorophore comprises 5-IAF or 6-IAF; or (i) the donor fluorophore comprises badan and the acceptor fluorophore comprises Alexa532.
  • the ligand-binding protein is selected from the group consisting of a glucose-galactose binding protein (GGBP), a glucose-binding protein, a urea-binding protein (UBP), a lactate-binding protein (LacBP), a calcium-binding protein, a calcium-bicarbonate binding protein (BicarbBP), and an iron-bicarbonate binding protein (FeBP).
  • GGBP glucose-galactose binding protein
  • UBP urea-binding protein
  • LacBP lactate-binding protein
  • CacBP calcium-binding protein
  • BacarbBP calcium-bicarbonate binding protein
  • FeBP iron-bicarbonate binding protein
  • aspects include a biosensor for a ligand comprising a ligand-binding protein, a directly responsive fiuorophore and an indirectly responsive fiuorophore, the directly responsive and the indirectly responsive fluorophores being located at two distinct sites of the ligand-binding-protein, wherein (i) the directly responsive fiuorophore is a donor fluorophore and the indirectly responsive fiuorophore is an acceptor fiuorophore; or (ii) the directly responsive fiuorophore is an acceptor fiuorophore and the indirectly responsive fiuorophore is an donor fiuorophore, and wherein if the acceptor fiuorophore comprises ruthenium or osmium, then the acceptor fiuorophore is not attached to the amino group of the N-terminus of the ligand-binding protein.
  • the ligand-binding protein comprises the directly responsive fiuorophore.
  • the directly responsive fiuorophore is formed by an autocatalytic cyclization of an oligopeptide within the ligand-binding protein.
  • the ligand-binding protein comprises a Yellow Fluorescent
  • the ligand comprises a halide anion.
  • the halide anion comprises a fluoride (F ⁇ ), chloride (CF), a bromide (Br ⁇ ), an iodide ( ⁇ ), an astatide (At ⁇ ) anion, or an ununseptide (Ts ⁇ ) anion.
  • the mutant comprises a mutation that alters the interaction of the mutant with a bound halide anion compared to YFP.
  • the mutant comprises a mutation that alters the affinity and/or specificity of the mutant for a halide anion compared to YFP.
  • the ligand-binding protein comprises 1, 2, 3, 4, or 5 halide anion binding sites.
  • At least one amino acid of the YFP or the fluorescent mutant thereof has been substituted with a cysteine.
  • the cysteine is within a first ⁇ - strand ⁇ ), a second ⁇ -strand ( ⁇ 2 ), a third ⁇ -strand ( ⁇ 3 ), a fourth ⁇ -strand ( ⁇ 4 ), a fifth ⁇ -strand ( ⁇ 5 ), a sixth ⁇ -strand ( ⁇ 6 ), a seventh ⁇ -strand ( ⁇ 7 ), an eighth ⁇ -strand ( ⁇ 8 ), a ninth ⁇ -strand 9), a tenth ⁇ -strand ( ⁇ 10 ), or an eleventh ⁇ -strand ( ⁇ ) of the YFP or the fluorescent mutant thereof.
  • the ligand-binding protein comprises one or more of the following substitutions: E17X, E32X, T43X, F64X, G65X, L68X, Q69X, A72X, H77X, K79X, R80X, E95X, R109X, R122X, D133X, H148X, N149X, V163X, N164X, D173X, Y182X, Q183X, Y203X, Q204X, L221X, and H231X, wherein X is any amino acid, a conservative substitution, or a cysteine, wherein each YFP amino acid position is numbered as in SEQ ID NO: 150.
  • the ligand-binding protein comprises one or more of the following substitutions: F64L, G65T, L68V, Q69T, A72S, K79R, R80Q,
  • the ligand-binding protein comprises an R at the 96 position, a Y at the 203 position, a S at the 205 position, and an E at the 222 position, wherein each YFP amino acid position is numbered as in SEQ ID NO: 150.
  • ligand binding causes a change in signaling by the directly responsive fluorophore.
  • the ligand-binding protein comprises a mutation compared to a naturally occurring protein. For example, at least one amino acid of the ligand-binding protein has been substituted with a cysteine.
  • the ligand-binding protein comprises a mutant of a microbial ligand-binding protein. In certain embodiments, the ligand-binding protein comprises a mutant of a microbial periplasmic ligand-binding protein.
  • the ligand comprises glucose, galactose, lactose, arabinose, ribose, maltose, lactate, urea, bicarbonate, phosphate, sulfate, chloride, fluoride, iodide, astatide, ununseptide, bromide, calcium, a hydrogen ion, a dipeptide, histidine, glutamine, glutamate, aspartate, or iron.
  • the ligand-binding protein comprises a GGBP.
  • the GGBP comprises or comprises a mutant of: an Escherichia sp. GGBP; a
  • Thermoanaerobacter sp. GGBP a Clostridium sp. GGBP; a Salmonella sp. GGBP; a
  • Caldicellulosiruptor sp. GGBP Caldicellulosiruptor sp. GGBP; a Paenibacillus sp. GGBP; a Butyrivibrio sp. GGBP; a
  • Roseburia sp. GGBP a Faecalibacterium sp. GGBP; an Erysipelothrix sp. GGBP; or an
  • the ligand-binding protein comprises a UBP.
  • the UBP comprises or comprises a mutant of: an Marinomas sp. UBP; a Marinobacter sp. UBP; a Bacillus sp. UBP; a Desulfotomaculum sp. UBP; a Geobacillus sp. UBP; a Clostridium sp.
  • UBP Caldicellulosiruptor sp. UBP; a Thermocrinis sp. UBP; a Synechoccus sp UBP; a
  • Paenibacillus sp. UBP Paenibacillus sp. UBP; or a Thermosynechococcus sp UBP.
  • the ligand-binding protein comprises a GBP.
  • the GBP comprises or comprises a mutant of: an Thermus sp GBP; a Deinococcus sp. GBP; a
  • Thermotoga sp. GBP a Kosmotoga sp. GBP; a Bacillus sp. GBP; a Staphylothermus sp.
  • the ligand-binding protein comprises a LacBP.
  • the LacBP comprises or comprises a mutant of: a Thermus sp. LacBP; a Thioalkalivibrio sp. LacBP; a Roseobacter sp. LacBP; a Marinobacter sp. LacBP; a Anaeromyxobacter sp. LacBP; a Pseudomonas sp. LacBP; a Rhodobacter sp. LacBP;, a Flexistipes sp. LacBP; or a Thermanaerovibrio sp. LacBP.
  • the ligand-binding protein comprises a calcium-binding protein or a BicarbBP.
  • the ligand-binding protein comprises or comprises a mutant of: a Synechocystis sp. BicarbBP; a Thermosynechococcus sp. BicarbBP; a
  • the ligand-binding protein comprises a FeBP.
  • the ligand-binding protein comprises or comprises a mutant of: a Mannheimia sp. FeBP; an Exiguobacterium sp. FeBP; a Thermosynechococcus sp FeBP; a Candidatus Nitrospira sp. FeBP; a Thermus sp. FeBP; a Meiothermus sp. FeBP; a Salinibacter sp. FeBP; or a
  • a biosensor for a ligand comprising an amino acid or a polypeptide, a directly responsive fluorophore and an indirectly responsive fluorophore, the directly responsive and the indirectly responsive fluorophores being located at two distinct sites of the amino acid or polypeptide, wherein the directly responsive fluorophore is chemoresponsive, and wherein (i) the directly responsive fluorophore is a donor fluorophore and the indirectly responsive fluorophore is an acceptor fluorophore; or (ii) the directly responsive fluorophore is an acceptor fluorophore and the indirectly responsive fluorophore is an donor fluorophore.
  • any amino acid or polypeptide may be used to link the
  • the amino acid or polypeptide comprises 1 amino acid, or a stretch of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 500, 750, or 1000 amino acids.
  • the polypeptide comprises a stretch of at least 50, 60, 70, 80, 90, or 100 amino acids in a sequence that is at least about 85%, 90%, 95%, or 99% identical to the amino acid sequence of ecTRX (SEQ ID NO: 151).
  • the polypeptide comprises a mutant of ecTRX comprising a D3X, K4X, K19X, D27X, K37X, K53X, K58X, K70X, R74X, K83X, K91X, K97X, or K101X mutation, or any combination thereof, wherein X is any amino acid, and wherein each ecTRX amino acid position is numbered as in SEQ ID NO: 151.
  • the polypeptide comprises a mutant of ecTRX comprising a D3A, K4R, K4Q, K19R, K19Q, D27A, K37R, K53M, K53R, K58M, K70R, R74C, K83R, K91R, K97R, or K101R mutation, or any combination thereof, wherein each ecTRX amino acid position is numbered as in SEQ ID NO: 151.
  • the polypeptide comprises a mutant of ecTRX that does not comprise a lysine.
  • the biosensor comprises amino acids in the sequence of any one of SEQ ID NOS: 69-86 or 151.
  • the polypeptide further comprises a hexahistidine tag.
  • the amino acid or polypeptide comprises at least 1, 2, or 3 thiol groups; at least 1, 2, or 3 cysteines that each comprise a sulfhydryl group; at least 1, 2, or 3 primary amine groups; or at least 1, 2, or 3 lysines that each comprise a primary amine. In some embodiments, there is no disulfide bond between cysteines within the amino acid sequence of the polypeptide.
  • the ligand comprises a hydrogen ion.
  • the biosensor is a biosensor for pH, wherein the directly responsive fiuorophore is pH- sensitive.
  • the fully excited emission intensity of the directly responsive fiuorophore is different at a pH less than about 7.0 compared to a pH of 7.5.
  • the directly responsive fiuorophore comprises a pH-sensitive fiuorophore comprising fluorescein or a derivative thereof.
  • the directly responsive fiuorophore transitions from a monoanion to a dianion at a pH that is less than 7.0 in an aqueous solution.
  • the directly responsive fiuorophore is attached to the ligand- binding protein, the amino acid, or the polypeptide via a covalent bond.
  • the covalent bond comprises a disulfide bond, a thioester bond, a thioether bond, an ester bond, an amide bond, or a bond that has been formed by a click reaction.
  • the directly responsive fiuorophore is attached to a cysteine or a lysine of the protein.
  • the indirectly responsive fiuorophore is attached to the N- terminus or the C-terminus of the protein. In some embodiments, the indirectly responsive fiuorophore is attached to the N-terminus or the C-terminus of the protein via a fiuorophore attachment motif. In some embodiments, the fiuorophore attachment motif comprises an amino acid or a polypeptide. In certain embodiments, the polypeptide comprises amino acids in the sequence of ⁇ (SEQ ID NO: 42). In various embodiments, polypeptide comprises a stretch of at least 50, 60, 70, 80, 90, or 100 amino acids in a sequence that is at least about 85%, 90%, 95%, or 99% identical to the amino acid sequence of E. coli thioredoxin (ecTRX; SEQ ID NO: 151).
  • ecTRX E. coli thioredoxin
  • the indirectly responsive fluorophore is attached to the ligand-binding protein via a covalent bond.
  • the covalent bond comprises a disulfide bond, a thioester bond, a thioether bond, an ester bond, an amide bond, or a bond that has been formed by a click reaction.
  • the indirectly responsive fluorophore is attached to a cysteine or a lysine of the protein.
  • aspects of the present subject matter further provide a method for assaying the level of a ligand in a subject, comprising contacting a biosensor with a biological sample from the subject.
  • ligands include glucose, galactose, lactose, arabinose, ribose, maltose, lactate, urea, bicarbonate, phosphate, sulfate, chloride, fluoride, iodide, astatide, ununseptide, bromide, calcium, a hydrogen ion, a dipeptide, histidine, glutamine, glutamate, aspartate, and iron.
  • the subject has or is suspected of having abnormal kidney function, abnormal adrenal gland function, diabetes, hypochloremia, bromism,
  • hypothyroidism hyperthyroidism, cretinism, depression, fatigue, obesity, a low basal body temperature, a goiter, a fibrocystic breast change, lactic acidosis, septic shock, carbon monoxide poisoning, asthma, a lung disease, respiratory insufficiency, Chronic Obstructive Pulmonary Disease (COPD), regional hypoperfusion, ischemia, severe anemia, cardiac arrest, heart failure, a tissue injury, thrombosis, or a metabolic disorder, diarrhea, shock, ethylene glycol poisoning, methanol poisoning, diabetic ketoacidosis, hypertension, Cushing syndrome, liver failure, cancer, or an infection.
  • COPD Chronic Obstructive Pulmonary Disease
  • the biological sample comprises sweat, tear fluid, blood, serum, plasma, interstitial fluid, amniotic fluid, sputum, gastric lavage, skin oil, milk, fecal matter, emesis, bile, saliva, urine, mucous, semen, lymph, spinal fluid, synovial fluid, a cell lysate, venom, hemolymph, or a fluid obtained from a plant.
  • Also provided is a method for assaying the level of ligand in an environmental sample comprising contacting a biosensor with the environmental sample.
  • the environmental sample is from an environmental site that is suspected of being polluted.
  • the environmental sample has been obtained or provided from an environmental substance, fluid, or surface.
  • the environmental substance comprises (a) rock, soil, clay, sand, a meteorite, an asteroid, dust, plastic, metal, a mineral, a fossil, a sediment, or wood; (b) the environmental surface comprises the surface of a satellite, a bike, a rocket, an automobile, a truck, a motorcycle, a yacht, a bus, or a plane, a tank, an armored personnel carrier, a transport truck, a jeep, a mobile artillery unit, a mobile antiaircraft unit, a minesweeper, a Mine-Resistant Ambush Protected (MRAP) vehicle, a lightweight tactical all-terrain vehicle, a high mobility multipurpose wheeled vehicle, a mobile multiple rocket launch system, an amphibious landing vehicle, a ship, a hovercraft, a submarine, a transport plane, a fighter jet, a helicopter, a rocket, or an Unmanned Arial Vehicle, a drone, a robot, a building, furniture,
  • aspects of the present subject matter further provide a method for monitoring the level of a ligand, comprising periodically continuously detecting the level of the ligand, wherein detecting the level of the ligand comprises (a) providing or obtaining a sample; (b) contacting the sample with a biosensor for the ligand; and (c) detecting a signal produced by the biosensor.
  • the sample is provided or obtained from a subject or from a culture of microbial cells.
  • aspects of the present subject matter also provide a method for constructing a biosensor, comprising: (a) providing a ligand-binding protein; (b) identifying at least one putative allosteric, endosteric, or peristeric site of the ligand-binding based a structure of the ligand-binding protein; (c) mutating the ligand-binding protein to substitute an amino acid at the at least one putative allosteric, endosteric, or peristeric site of the second protein with a cysteine; (d) conjugating a donor fluorophore or an acceptor fluorophore to the cysteine to produce single labeled biosensor; (e) detecting whether there is a spectral shift or change in emission intensity of the single labeled biosensor upon ligand binding when the donor fluorophore or the acceptor fluorophore is fully excited; and (f) if a spectral shift or change in emission intensity is detected in (e), attaching a donor
  • the ligand-binding protein has been identified by (i) selecting a first protein having a known amino acid sequence (seed sequence), wherein the first protein is known to bind a ligand; (ii) identifying a second protein having an amino acid sequence (hit sequence) with at least 15% sequence identity to the seed sequence; (iii) aligning the seed amino acid sequence and the hit sequence, and comparing the hit sequence with the seed sequence at positions of the seed sequence that correspond to at least 5 primary complementary surface (PCS) amino acids, wherein each of the at least 5 PCS amino acids has a hydrogen bond interaction or a van der Waals interaction with ligand when ligand is bound to the first protein; and (iv) identifying the second protein to be a ligand-binding protein if the hit sequence comprises at least 5 amino acids that are consistent with the PCS.
  • seed sequence seed sequence
  • the spectral shift comprises a monochromatic fluorescence intensity change or a dichromatic spectral shift.
  • Also provided is a method of converting a biosensor that shows a monochromatic response upon ligand binding into a biosensor with a dichromatic response upon ligand binding comprising (a) selecting a biosensor that exhibits a monochromatic response upon ligand binding, wherein the biosensor comprises a ligand-binding protein and a first reporter group; and (b) attaching a second reporter group to the biosensor, wherein the second reporter group has (i) an excitation spectrum that overlaps with the emission spectrum of the first reporter group; or (ii) an emission spectrum that overlaps with the excitation spectrum of the first reporter group.
  • the present subject matter also includes method of converting a biosensor that shows a monochromatic response upon ligand binding into a biosensor with a dichromatic response upon ligand binding, the method comprising (a) selecting a biosensor that exhibits a monochromatic response upon ligand binding, wherein the biosensor comprises a ligand- binding fluorescent protein; and (b) attaching an acceptor fluorophore or a donor fluorophore to the biosensor, wherein (i) the acceptor fluorophore has an excitation spectrum that overlaps with the emission spectrum of the fluorescent protein; or (ii) the donor fluorophore has an emission spectrum that overlaps with the excitation spectrum of the fluorescent protein.
  • Also provided is a method of increasing a dichromatic response of a biosensor to ligand binding comprising (a) selecting a biosensor that exhibits a dichromatic response upon ligand binding, wherein the biosensor comprises a ligand-binding protein and a first reporter group; and (b) attaching a second reporter group to the biosensor, wherein the second reporter group has (i) an excitation spectrum that overlaps with the emission spectrum of the first reporter group; or (ii) an emission spectrum that overlaps with the excitation spectrum of the first reporter group.
  • the second reporter group is within about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 4, 6, 8, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, or 200 angstroms (A) of the first reporter group regardless of whether ligand is bound to the biosensor. Suitable distances may be determined in part by the distance-dependence of the energy transfer between a given donor-acceptor pair ⁇ see, e.g. J.R. Lakowicz, 2006,
  • the average distance between the first reporter group and the second reporter group changes by less than about 5, 4, 3, 2, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.05, or 0.01 angstroms (A) compared to when ligand is not bound to the ligand-binding protein.
  • the present subject matter further provides a method of converting a biosensor that shows a monochromatic response upon ligand binding into a biosensor with a dichromatic response upon ligand binding, the method comprising (a) selecting a biosensor that exhibits a monochromatic response upon ligand binding, wherein said biosensor comprises an amino acid or polypeptide and a first reporter group, wherein the first reporter group comprises a chemoresponsive fluorophore; and (b) attaching a second reporter group to said biosensor, wherein said second reporter group has (i) an excitation spectrum that overlaps with the emission spectrum of said first reporter group; or (ii) an emission spectrum that overlaps with the excitation spectrum of said first reporter group.
  • a method of increasing a dichromatic response of a biosensor to ligand binding comprising (a) selecting a biosensor that exhibits a dichromatic response upon ligand binding, wherein said biosensor comprises an amino acid or a polypeptide and a first reporter group, wherein the first reporter group comprises a chemoresponsive fluorophore; and (b) attaching a second reporter group to said biosensor, wherein said second reporter group has (i) an excitation spectrum that overlaps with the emission spectrum of said first reporter group; or (ii) an emission spectrum that overlaps with the excitation spectrum of said first reporter group.
  • tgmFRET may be used alternatively or in addition to ngmFRET in certain embodiments.
  • the biosensor comprises multiple reporter groups, including a first reporter group and a second reporter group.
  • the first reporter group may comprise a donor fluorophore and the second reporter group may comprise an acceptor fluorophore.
  • FRET is detectable by a change in the fluorescence of the acceptor fluorophore or by a decrease in donor fluorophore fluorescence.
  • the donor fluorophore, and/or the acceptor fluorophore is fluorescent. In some embodiments, both the donor fluorophore and the acceptor fluorophore are fluorescent.
  • the angle and/or distance between the donor fluorophore and the acceptor fluorophore changes upon ligand binding.
  • neither the donor fluorophore nor the acceptor fluorophore is directly responsive to ligand binding.
  • the donor fluorophore and/or the acceptor fluorophore is attached to the N-teiminus or the C-terminus of the ligand-binding protein (e.g., directly or via a fluorophore attachment motif).
  • the donor fluorophore and/or the acceptor fluorophore is attached to a fluorophore attachment motif.
  • the fluorophore attachment motif may be conjugated to the N-teiminus or the C-terminus of the ligand- binding protein.
  • the donor fluorophore and/or the acceptor fluorophore comprises a fluorescent protein.
  • the donor fluorophore and/or the acceptor fluorophore comprises an organic compound having a molecular weight less than about 2000 Da (e.g., 5-iodoacetamidofluorescein (5-IAF) or 6-iodoacetamidofluorescein (6- IAF), rhodamine, Oregon Green, eosin, Texas Red, indocarbocyanine, oxacarbocyanine, thiacarbocyanine, merocyanine, Badan, Acrylodan, IAEDANS, comprising 3-cyano-7- hydroxycoumarin, 7-hydroxycoumarin-3-carboxylic acid, 6,8-difluoro-7-hydroxy- 4- methylcoumarin, or 7-amino-4-methylcoumarin, pyridyloxazole, nitrobenzoxadiazole, benzoxadia
  • 5-IAF 5-
  • a biosensor that comprises a one or more reporter groups attached to a ligand-binding protein, wherein binding of a ligand to a ligand-binding domain of the ligand-binding protein causes a change in signaling by the reporter group.
  • the reporter group is attached to an endosteric site, an allosteric site, or a peristeric site of the ligand-binding protein.
  • the reporter group is covalently or noncovalently attached to the ligand-binding protein.
  • a reporter group may be referred to by a name of an unattached form of the reporter group regardless of whether the reporter group is attached to a ligand-binding protein.
  • a compound known as "Compound A” when in an unconjugated form may be referred to herein as “Compound A” when in a form that is attached to a ligand-binding protein.
  • the term "Acrylodan” is used to refer to unreacted/unconjugated Acrylodan, as well as Acrylodan that is conjugated to a ligand-binding protein.
  • a biosensor comprises a reporter group that is conjugated to a ligand-binding protein, and the reporter group is conjugated to an amino acid of the protein that is at least about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 angstroms (A) from the ligand when the ligand is bound to the protein.
  • A angstroms
  • the reporter group is conjugated to an amino acid of the protein that is about 0.1 A to about 5 A, about 5 A to about 10 A, about 10 A to about 20 A, about 20 A to about 50 A, about 50 A to about 75 A, or about 75 A to about 100 A from the ligand when the ligand is bound to the protein. In some embodiments, the reporter group is conjugated to an amino acid of the protein that is within an a-helix or a ⁇ -strand.
  • the reporter group is conjugated to an amino acid that (i) is not within an a- helix or a ⁇ -strand, but is within about 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acids of an amino acid of the protein's amino acid sequence that is within an a-helix or a ⁇ -strand. In some embodiments, the reporter group is conjugated to an amino acid that is in an inter-domain hinge amino acid region between two domains of a protein.
  • the reporter group is conjugated to an amino acid that is in an inter-domain hinge amino acid region between (i) a a-helix and a ⁇ -strand; (ii) two a-helixes; or (iii) two ⁇ -strands of a protein.
  • the reporter group is conjugated to an amino acid (e.g., a cysteine such as a cysteine added by substitution compared to a naturally corresponding polypeptide) between positions 1-25, 25-50, 50-75, 75-100, 100-125, 125-150, 150-175, 175- 200, 200-225, 225-250, 250-275, 275-350, 275-300, 275-325, 300-325, 300-350, 300-400, or 350-450 (inclusive) of a polypeptide (e.g., not including N-teiminal fusion proteins compared to the polypeptide's naturally occurring counterpart).
  • an amino acid e.g., a cysteine such as a cysteine added by substitution compared to a naturally corresponding polypeptide
  • the directly or indirectly responsive fluorophore is conjugated (directly or via a fluorophore attachment motif) to an amino acid that is no more than about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 5-15, 5-20, 5-25, 5-100, 10-15, 10-20, 10-25, 10-50, 10-100, 25-50, 25-75, or 25-100 amino acids from the N-terminus or the C-terminus of the ligand-binding protein.
  • the directly or indirectly responsive fluorophore is conjugated (directly or via a fluorophore attachment motif) to an amino acid that is at least about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 5-15, 5-20, 5-25, 5-100, 10-15, 10-20, 10-25, 10-50, 10-100, 25-50, 25-75, or 25-100 amino acids from the N-terminus or the C-terminus of the ligand-binding protein.
  • 50, 60, 70, 80, 90 100, 5-15, 5-20, 5-25, 5-100, 10-15, 10-20, 10-25, 10-50, 10-100, 25-50, 25-75, or 25-100 amino acids (including or not including the signal peptide) have been deleted (e.g. are absent) from the N- terminus of the protein compared to its naturally occurring counterpart.
  • Periplasmic binding proteins are characterized by two lobes connected by a hinge region; ligand bind at a location at the interface between the two domains.
  • Such proteins or engineered versions thereof can adopt two different conformations: a ligand-free open form and a ligand-bound closed form, which interconvert through a relatively large bending motion around the hinge (FIG. 1 A; Dwyer et al., 2004, Current Opinion in Structural Biology 12:495-504).
  • Direct signaling relationships between proteins and reporter groups are readily designed by replacing a residue known to form a ligand contact with a cysteine to which the fluorophore is attached ("endosteric" attachment site).
  • Other, indirect signaling relationships can be established in two ways. The first relies on visual inspection of the ligand complex structure, and identifying residues that are located in the vicinity of the binding site, but do not interact directly with the ligand, and that are likely to be involved in conformational changes. Typically, such "peristeric" sites are located adjacent to the residues that form direct contacts with the bound ligand. In the case of the bPBPs, such residues are located at the perimeter of the inter-domain cleft that forms the ligand binding site location.
  • the environment of these peristeric sites changes significantly upon formation of the closed state. These are examples of positions which are proximal to the ligand-binding pocket/domain.
  • the second, most general, approach identifies sites in the protein structure that are located anywhere in the protein, including locations at some distance away from the ligand-binding site (i.e., distal to the ligand-binding pocket/domain), and undergo a local conformational change in concert with ligand binding. If the structures of both the open and closed states are known, then such "allosteric" sites can be identified using a computational method that analyzes the conformational changes that accompany ligand binding (Marvin et al., Proc. Natl. Acad. Sci. USA 94:4366-4371, 1997).
  • the reporter group is attached to the ligand-binding protein via a biotin-avidin interaction.
  • the reporter group may be, e.g., conjugated to biotin and the ligand-binding protein is conjugated to avidin.
  • the avidin is bound to four biotin molecules wherein each biotin molecule is individually conjugated to a reporter group.
  • the reporter group is conjugated to avidin and the ligand-binding protein is conjugated to biotin.
  • the avidin is bound to four biotin molecules, wherein each biotin molecule is individually conjugated to a ligand-binding protein.
  • conjugated means covalently attached.
  • One compound may be directly conjugated to another compound, or indirectly conjugated, e.g., via a linker.
  • the reporter group is directly attached to the ligand-binding protein.
  • the reporter group is attached to an amino acid of the ligand-binding protein that is at least about 2, 4, 6, 8, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 angstroms (A) from the ligand when the ligand is bound to the ligand-binding protein.
  • the reporter group is conjugated to an amino acid having a position within positions 1-25, 25-50, 50-75, 75-100, 100-125, 125-150, 150-175, 175-200, 200-225, 225-250, 250-275, or 275-300 of the ligand-binding protein, wherein position 1 is the N-terminal amino acid of the ligand- binding protein.
  • the reporter group is conjugated to an amino acid of the ligand-binding protein that is (a) within an a-helix or a ⁇ -strand of the ligand-binding protein; (b) not within an a-helix; (c) not within a ⁇ -strand; (d) within about 5 or 10 amino acids of an amino acid that is within an a-helix or ⁇ -strand; (e) within a stretch of consecutive amino acids that links two domains of the ligand-binding protein; (f) within a stretch of consecutive amino acids that links an a-helix and a ⁇ -strand; (g) within a stretch of consecutive amino acids that links two a-helices; or (h) within a stretch of consecutive amino acids that links two ⁇ -strands.
  • the reporter group is directly attached to the N-terminus or the C-terminus of the ligand-binding protein.
  • the reporter group may be conjugated to the ligand-binding protein a variety of linkers or bonds, including (but not limited to) a disulfide bond, an ester bond, a thioester bond, an amide bond, or a bond that has been formed by a click reaction.
  • the click reaction is a reaction between (a) an azide and an alkyne; (b) an azide and an alkyne in the presence of Cu(I); (c) an azide and a strained cyclooctyne; (d) an azide and a dibenzylcyclooctyne, a difluorooctyne, or a biarylazacyclooctynone; (e) a diaryl- strained-cyclooctyne and a 1,3-nitrone; (f) an azide, a tetrazine, or a tetrazole and a strained alkene; (g) an azide, a tetrazine, or a tretrazole and a oxanorbornadiene, a cyclooctene, or a trans-cycloalkene; (h) a tetrazole and an alkene
  • linker refers to a molecule or sequence (such as an amino acid sequence), that attaches, as in a bridge, one molecule or sequence to another molecule or sequence. "Linked” means attached or bound by covalent bonds, or non-covalent bonds, or other bonds, such as van der Waals forces.
  • a linker comprises a chemical structure that has resulted from a reaction used to attach one molecule to another.
  • the reporter group is conjugated to a cysteine of the ligand-binding protein.
  • the cysteine may be present on a natural counterpart or version of the ligand-binding protein or added to the ligand-binding protein by a substitution mutation.
  • the cysteine is at the N-terminus or the C-terminus of the ligand-binding protein.
  • Non-limiting examples relate to the conjugation of a reporter group to a primary amine of the ligand-binding protein.
  • the primary amine is present in a lysine of the ligand-binding protein.
  • the lysine may be present on a natural counterpart or version of the ligand-binding protein or added to the ligand-binding protein by a substitution mutation.
  • the lysine is at the N-terminus or the C-terminus of the ligand-binding protein.
  • aspects of the present subject matter provide a biosensor in which the reporter group is attached to the ligand-binding protein via a linker.
  • the linker comprises an organic compound that is less than about 30, 20, 15, or 10 A long.
  • Non- limiting examples of linkers include O, S, NH, PH, and alkyl linkers.
  • Alkyl refers to the radical of saturated or unsaturated aliphatic groups, including straight-chain alkyl, alkenyl, or alkynyl groups, branched-chain alkyl, alkenyl, or alkynyl groups, cycloalkyl, cycloalkenyl, or cycloalkynyl (alicyclic) groups, alkyl substituted cycloalkyl, cycloalkenyl, or cycloalkynyl groups, and cycloalkyl substituted alkyl, alkenyl, or alkynyl groups.
  • a straight chain or branched chain alkyl has 30 or fewer carbon atoms in its backbone (e.g., Q-C30 for straight chain, C3-C30 for branched chain), more preferably 20 or fewer carbon atoms, more preferably 12 or fewer carbon atoms, and most preferably 8 or fewer carbon atoms.
  • preferred cycloalkyls have from 3-10 carbon atoms in their ring structure, and more preferably have 5, 6 or 7 carbons in the ring structure. The ranges provided above are inclusive of all values between the minimum value and the maximum value.
  • alkyl includes both "unsubstituted alkyls” and “substituted alkyls,” the latter of which refers to alkyl moieties having one or more substituents replacing a hydrogen on one or more carbons of the hydrocarbon backbone.
  • substituents include, but are not limited to, halogen, hydroxyl, carbonyl (such as a carboxyl, alkoxycarbonyl, formyl, or an acyl), thiocarbonyl (such as a thioester, a thioacetate, or a thioformate), alkoxyl, phosphoryl, phosphate, phosphonate, a phosphinate, amino, amido, amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, aralkyl, or an aromatic or heteroaromatic moiety.
  • carbonyl such as a carboxyl, alkoxycarbonyl, formyl, or an acyl
  • thiocarbonyl such as a thioester, a
  • lower alkyl as used herein means an alkyl group, as defined above, but having from one to ten carbons, more preferably from one to six carbon atoms in its backbone structure. Likewise, “lower alkenyl” and “lower alkynyl” have similar chain lengths.
  • Preferred alkyl groups are lower alkyls.
  • the alkyl groups may also contain one or more heteroatoms within the carbon backbone. Preferably the heteroatoms incorporated into the carbon backbone are oxygen, nitrogen, sulfur, and combinations thereof. In certain embodiments, the alkyl group contains between one and four heteroatoms.
  • the linker comprises a bond formed by a chemical reaction involving a reactive group such as a maleimide group.
  • the linker comprises a stretch of amino acids.
  • the linker comprises a polyglycine linker.
  • the polyglycine linker comprises 2, 3, 4, 5, or more glycines.
  • the polyglycine linker further comprises a serine.
  • the reporter group is attached to a linker via a covalent bond and the linker is attached to a ligand-binding protein via a covalent bond.
  • the covalent bond between the linker and the reporter group and/or the covalent bond between the linker and the ligand-binding protein is a disulfide bond, an ester bond, a thioester bond, an amide bond, a carbamate bond, or a bond that has been formed by a click reaction.
  • Non-limiting examples of click reactions include reactions between an azide and an alkyne; an azide and an alkyne in the presence of Cu(I); an azide and a strained cyclooctyne; an azide and a dibenzylcyclooctyne, a difluorooctyne, or a
  • biarylazacyclooctynone a diaryl-strained-cyclooctyne and a 1,3-nitrone
  • an azide, a tetrazine, or a tetrazole and a strained alkene an azide, a tetrazine, or a tretrazole and a
  • oxanorbornadiene a cyclooctene, or a trans-cycloalkene
  • a tetrazole and an alkene or a tetrazole with an amino or styryl group that is activated by ultraviolet light and an alkene.
  • the present subject matter also includes biosensors having one or more reporter groups attached to a ligand-binding protein via a fiuorophore attachment motif.
  • aspects of the present subject matter include the use of one or more fluorophore attachment motifs to attach one or more reporter groups to a ligand-binding protein.
  • a reporter group may be attached to a fluorophore attachment motif that is attached to the N-terminus or the C-terminus of the ligand-binding protein.
  • the fluorophore attachment motif comprises a polypeptide.
  • the polypeptide comprises amino acids in the ⁇ amino acid sequence (SEQ ID NO: 42).
  • the polypeptide comprises a stretch of at least 50, 60, 70, 80,
  • the polypeptide is a mutant of ecTRX comprising a D3X, K4X, K19X, D27X, K37X, K53X, K58X, K70X, R74X, K83X, K91X, K97X, or K101X mutation, or any combination thereof, wherein X is any amino acid, and wherein each ecTRX amino acid position is numbered as in SEQ ID NO: 151.
  • the polypeptide is a mutant of ecTRX comprising a D3A, K4R, K4Q, K19R, K19Q, D27A, K37R, K53M, K53R, K58M, K70R, R74C, K83R, K91R, K97R, or K101R mutation, or any combination thereof, wherein each ecTRX amino acid position is numbered as in SEQ ID NO: 151.
  • the polypeptide comprises amino acids in the sequence of any one of SEQ ID NOS: 69-86 or 151.
  • the polypeptide comprises (a) at least 1, 2, or 3 thiol groups; (b) at least 1, 2, or 3 cysteines that each comprise a sulfhydryl group; (c) at least 1, 2, or 3 primary amine groups; and/or (d) at least 1, 2, or 3 lysines that each comprise a primary amine. In some embodiments there is no disulfide bond between cysteines within the amino acid sequence of the polypeptide.
  • the polypeptide comprises a hexahistidine tag.
  • the hexahistidine tag is attached to another portion of the polypeptide via a GGS linker.
  • the reporter group may comprise a fiuorophore that produces a fluorescent signal.
  • Biosensors comprising a fiuorophore may be referred to herein as fiuorescently responsive sensors (FRSs).
  • a ratiometric signal (i- 1;2 ) is defined as the quotient of two intensities, I ⁇ and I%2, measured at two independent wavelengths, ⁇ ] and ⁇ 2 and may be calculated according to the following equation:
  • the two independent wavelengths ⁇ ] and ⁇ 2 may be from a single fiuorophore or from a combination of two or more fiuorophores (e.g., a pair of fiuorophores between which tgmFRET and/or ngmFRET occurs).
  • ⁇ ] falls within the emission spectrum of a directly responsive fluorophore and ⁇ 2 falls within the emission spectrum of an indirectly responsive fluorophore.
  • ⁇ ] falls within the emission spectrum of an indirectly responsive fluorophore and ⁇ 2 falls within the emission spectrum of a directly responsive fluorophore.
  • ⁇ ] falls within the emission spectrum of both a directly responsive fluorophore and an indirectly responsive fluorophore.
  • ⁇ 2 falls within the emission spectrum of both a directly responsive fluorophore and an indirectly responsive fluorophore.
  • intensities are, e.g., integrated, filtered, assessed, detected, or evaluated over a range of wavelengths. In some embodiments, intensities are integrated over a range of wavelengths in a recorded emission spectrum. In some embodiments, a range of wavelengths is selected using a filter. In some embodiments, ⁇ ] is the intensity over a 1 run to 60 nm interval centered between 400 and 1000 nm, and ⁇ 2 is the intensity over a 1 nm to 60 nm interval centered between 400 nm and 1000 nm.
  • intensities are integrated, filtered, assessed, detected, or evaluated over a 1 nm, 2 nm, lOnm, 15nm, 20nm, 25nm, 30nm, 35nm, 40nm, 45nm, 50nm, 55 nm, 60 nm, 75 nm, 100 nm, 10-40 nm, 10-50 nm, 20-50 nm, or 10-100 nm regions, centered between 400-1000 nm, e.g. between 420 nm and 520 nm for ⁇ ⁇ 5 and 400-lOOOnm, e.g. between 500 nm to 600 nm for ⁇ 2 .
  • intensities are recorded through a bandpass filter.
  • a non-limiting example of a bandpass filter is a 10 nm, 15 nm, 20 nm, 25 nm, 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 75 nm, 100 nm, 10-40 nm, 10-50 nm, 20-50 nm, or 10-100 nm bandpass filter, centered between 400-1000 nm, e.g. at 452 nm ⁇ and at 400-lOOOnm, e.g. at 528 nm ( ⁇ 2 ).
  • aspects of the present subject matter provide FRSs whose emission spectra change (e.g., the shape of the emission spectra change) in response to ligand binding.
  • the ratio of intensities at two chosen wavelengths of an FRS's emission spectrum changes upon ligand binding.
  • the emission spectra of two or more fluorophores contributes to ⁇ and/or 3 ⁇ 42 .
  • the emission spectrum of a directly responsive fluorophore contributes to I ⁇ and/or I %2 and the emission spectrum of an indirectly responsive fluorophore contributes to I ⁇ and/or 3 ⁇ 42 .
  • a directly responsive fluorophore contributes to I ⁇ and the emission spectrum of an indirectly responsive fluorophore contributes to 1 ⁇ 2.
  • a directly responsive fluorophore contributes to 1 ⁇ 2 and the emission spectrum of an indirectly responsive fluorophore contributes to In various embodiments, both the emission spectrum of a directly responsive fluorophore and the emission spectrum of an indirectly responsive fluorophore contributes to In some embodiments, both the emission spectrum of a directly responsive fluorophore and the emission spectrum of an indirectly responsive fluorophore contributes to
  • the emission wavelength and/or intensity of a fluorophore changes when the positions of atoms within the fluorophore change with respect to each other (e.g., due to the rotation of bound atoms with respect to each other or a change in the angle of a bond).
  • the emission wavelength and/or intensity of the fluorophore changes when (i) one portion of the fluorophore rotates around a bond axis compared to another portion of the fluorophore and/or (ii) when the angle of a bond between two atoms of the fluorophore changes.
  • the fluorophore is a prodan-derived fluorophore (e.g. , Acrylodan or Badan) and binding of ligand alters the orientation of a dimethylamino group, a naphthalene ring, and/or a carbonyl with respect to the ligand-binding protein and/or each other.
  • the degree of polarization of a dipole on the fluorophore changes in response to ligand binding.
  • the emission wavelength and/or intensity of the fluorophore changes when an atom electrostatically interacts with the fluorophore.
  • the emission wavelength and/or intensity of the fluorophore changes when the source of a positive or negative charge changes its distance with respect to the fluorophore within about 1, 2, 3, 4, 5, or 10 A of the fluorophore.
  • the fluorophore exhibits hypsochromicity or bathochromicity upon ligand binding to the ligand-binding domain of the ligand-binding protein.
  • the fluorophore has an emission spectrum comprising radiation with a wavelength (e.g., a peak emission wavelength) of about 400 nm, 410 nm, 420 nm, 430 nm, 440 nm, 450 nm, 460 nm, 470 nm, 480 nm, 490 nm, 500 nm, 510 nm, 520 nm, 530 nm, 540 nm, 550 nm, 560 nm, 570 nm, 580 nm, 590 nm, 600 nm, 610 nm, 620 nm, 630 nm, 640 nm, 650 nm, 660 nm, 670 nm, 680 nm, 690 nm, 700 nm, 710 nm, 720 nm, 730 nm, 740 nm, 750 nm, 760 nm, a wavelength (e
  • the signal comprises the emission intensity of the FRS recorded at a single wavelength or range of wavelengths.
  • the change in signal may be a shift in the single wavelength or range of wavelengths.
  • the shift in the wavelength is at least about 1 nm, at least about 2 nm, at least about 3 nm, at least about 4 nm, at least about 5 nm, at least about 6 nm, at least about 7 nm, at least about 8 nm, at least about 9 nm, at least about 10 nm, at least about 11 nm, at least about 12 nm, at least about 13 nm, at least about 14 nm, at least about 15 nm, at least about 16 nm, at least about 17 nm, at least about 18 nm, at least about 19 nm, at least about 20 nm, at least about 25 nm, at least about 30 nm, at least about 35 nm, at least about 40 nm, at least about 45 n
  • the shift in the wavelength is about 1 nm to about 20 nm, about 2 nm to about 20 nm, about 3 nm to about 20 nm, about 4 nm to about 20 nm, about 5 nm to about 20 nm, about 1 nm to about 19 nm, about 1 nm to about 18 nm, about 1 nm to about 17 nm, 1 nm to about 16 nm, about 1 nm to about 15 nm, about 1 nm to about 14 nm, about 1 nm to about 13 nm, about 1 nm to about 12 nm, about 1 nm to about 11 nm, or about 1 nm to about 10 nm.
  • the shift in the wavelength is about 1 nm to about 20 nm. In some embodiments, the shift in the wavelength is about 1 nm to about 130 nm.
  • the signal comprises the ratio or quotient of the emission intensities recorded at two distinct wavelengths or ranges of wavelengths, i.e., a ratiometric signal.
  • ligand binding may be determined by measuring the ratio of blue to green emission intensities.
  • the change in signal may be decreased emission intensity at one wavelength, and no change in emission intensity at the other wavelength.
  • the change in signal may be increased emission intensity at one wavelength, and no change in emission intensity at the other wavelength.
  • the change in signal may be increased emission intensity at one wavelength, and increased emission intensity at the other wavelength.
  • the change in signal may be decreased emission intensity at one wavelength, and decreased emission intensity at the other wavelength.
  • the change in signal may be increased emission intensity at one wavelength, and decreased emission intensity at the other wavelength.
  • the change in ratio of the emission intensities recorded at two distinct wavelengths or ranges of wavelengths may be at least about 1.1 -fold, at least about 1.2-fold, at least about 1.4-fold, at least about 1.6-fold, at least about 1.8-fold, at least about 2.0-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 4.5-fold, at least about 5-fold, at least about 5.5-fold, at least about 6-fold, at least about 6.5-fold, at least about 7-fold, at least about 7.5-fold, at least about 8-fold, at least about 8.5- fold, at least about 9-fold, at least about 9.5-fold, at least about 10-fold, at least about 12-fold, at least about 14-fold, at least about 16-fold, at least about 18-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 35-fold, at least about 40-fold, at least about 45
  • the change in ratio of the emission intensities recorded at two distinct wavelengths or ranges of wavelengths may be a decrease of at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%, or of 5-25%, 25-50%, 25-75%, 50-75%, 50-90%, or 75-99% or the reciprocal thereof.
  • the change in signal may be a change in the ratio of the two distinct wavelengths or ranges of wavelengths.
  • the change in signal may be a shift in the two distinct wavelengths or ranges of wavelengths. In some embodiments, one wavelength shifts. In some embodiments, both wavelengths shift.
  • the shift in the wavelength is at least about 1 nm, at least about 2 nm, at least about 3 nm, at least about 4 nm, at least about 5 nm, at least about 6 nm, at least about 7 nm, at least about 8 nm, at least about 9 nm, at least about 10 nm, at least about 11 nm, at least about 12 nm, at least about 13 nm, at least about 14 nm, at least about 15 nm, at least about 16 nm, at least about 17 nm, at least about 18 nm, at least about 19 nm, at least about 20 nm, at least about 25 nm, at least about 30 nm, at least about 35 nm, at least about 40 nm, at least about 45 nm, at least about 50 nm, at least about 55 nm, at least about 60 nm, at least about 65 nm, at least about 70 nm, at least about 75
  • the shift in the wavelength is about 1 nm to about 20 nm, about 2 nm to about 20 nm, about 3 nm to about 20 nm, about 4 nm to about 20 nm, about 5 nm to about 20 nm, about 1 nm to about 19 nm, about 1 nm to about 18 nm, about 1 nm to about 17 nm, 1 nm to about 16 nm, about 1 nm to about 15 nm, about 1 nm to about 14 nm, about 1 nm to about 13 nm, about 1 nm to about 12 nm, about 1 nm to about 11 nm, or about 1 nm to about 10 nm.
  • the shift in the wavelength is about 1 nm to about 20 nm. In some embodiments, the shift in the wavelength is about 1 nm to about 130 nm.
  • a fluorophore may comprise, e.g., a fluorescent protein or an organic compound having a molecular weight less than about 2000 Daltons (Da).
  • fluorophores include such as 5-iodoacetamidofluorescein (5-IAF) or 6-iodoacetamidofluorescein (6-IAF), rhodamine, Oregon Green, eosin, Texas Red, indocarbocyanine, oxacarbocyanine, thiacarbocyanine, merocyanine, Badan, Acrylodan,
  • IAEDANS comprising 3-cyano-7-hydroxycoumarin, 7-hydroxycoumarin-3-carboxylic acid, 6,8-difluoro-7-hydroxy- 4-methylcoumarin, or 7-amino-4-methylcoumarin, pyridyloxazole, nitrobenzoxadiazole, benzoxadiazole, DRAQ5, DRAQ7, or CyTRAK Orange, cascade blue, Nile red, Nile blue, cresyl violet, oxazine 170, profiavin, acridine orange, acridine yellow, auramine, crystal violet, malachite green, porphin, phthalocyanine, bilirubin, pyrene, ⁇ , ⁇ '- dimethyl-N-(iodoacetyl)-N'-(7-nitrobenz-2-ox- a-l,3-diazol-4-yl)ethylenediamide (NBD), N- ((2-(iodoacetoxy)ethyl)-
  • the reporter group was thiol- reactive prior to being conjugated to a polypeptide disclosed herein.
  • the reporter group is linked to a polypeptide disclosed herein via a disulfide bond.
  • fluorophores include fluorescent proteins such as Blue Fluorescent Protein (BFP), TagBFP, mTagBFP2, Azurite, Enhanced Blue Florescent Protein 2 (EBFP2), mKalamal, Sirius, Sapphire, T-Sapphire, Cyan Fluorescent Protein (CFP); Enhanced Cyan Fluorescent Protein (ECFP), Cerulean, SCFP3A, mTurquoise, mTurquoise2, monomeric Midoriishi-Cyan, TagCFP, mTFPl, AmCyanl, Green Fluorescent Protein (GFP), Enhanced Green Fluorescent Protein (EGFP), Emerald, Superfolder GFP, AcGFPl, ZsGreenl, Monomeric Azami Green, TagGFP2, mUKG, mWasabi, Clover, mNeonGreen, Yellow Fluorescent Protein (YFP), Enhanced Yellow Fluorescent Protein (EYFP), Citrine, Venus, Super Yellow Fluorescent Protein 2 (SYFP2),
  • BFP Blue Fluorescent
  • the fluorophore comprises xanthene, a xanthene derivative, fluorescein, a fluorescein derivative, coumarin, a coumarin derivative, cyanine, a cyanine derivative, rhodamine, a rhodamine derivative, phenoxazine, a phenoxazine derivative, squaraine, a squaraine derivative, coumarin, a coumarin derivative, oxadiazole, an oxadiazole derivative, anthracene, an anthracene derivative, a boradiazaindacine (BODIPY) family fluorophore, pyrene, a pyrene derivative, acridine, an acridine derivative, arylmethine, an arylmethine derivative, tetrapyrrole, or a tetrapyrrole derivative.
  • BODIPY boradiazaindacine
  • the fluorophore may comprise a xanthene derivative comprising fluorescein or a fluorescein derivative, rhodamine, Oregon Green, eosin, or Texas Red.
  • fluorescein derivatives include 5-fluorescein, 6-carboxyfluorescein, 3'6-carboxyfluorescein, 5(6)- carboxyfluorescein, 6-hexachlorofluorescein, 6-tetrachlorofluorescein, or isothiocyanate.
  • the fluorophore comprises a cyanine derivative comprising
  • the fluorophore comprises a squaraine derivative comprising a ring-substituted squaraine.
  • the fluorophore comprises a naphthalene derivative comprising a dansyl or prodan naphthalene derivative.
  • the fluorophore comprises prodan or a derivative thereof.
  • the fluorophore comprises Badan, Acrylodan, or N-(Iodoacetaminoethyl)-l-naphthylamine-5- sulfonic acid (IAEDANS).
  • the fluorophore comprises a coumarin derivative such as 3-cyano-7-hydroxycoumarin, 7-hydroxycoumarin-3-carboxylic acid, 6,8- difluoro-7-hydroxy- 4-methylcoumarin (DiFMU), or 7-amino-4-methylcoumarin.
  • the fluorophore comprises an oxadiazole derivative such as pyridyloxazole, nitrobenzoxadiazole, or benzoxadiazole.
  • the fluorophore comprises an anthracene derivative comprising an anthraquinone such as DRAQ5, DRAQ7, or CyTRAK Orange.
  • the fluorophore comprises a pyrene derivative comprising cascade blue.
  • the fluorophore comprises an oxazine derivative such as Nile red, Nile blue, cresyl violet, or oxazine 170.
  • the fluorophore comprises an acridine derivative such as proflavin, acridine orange, or acridine yellow.
  • the fluorophore comprises an arylmethine derivative such as auramine, crystal violet, or malachite green.
  • the fluorophore comprises a tetrapyrrole derivative comprising porphin, phthalocyanine, or bilirubin.
  • a fluorophore may comprise a sulfhydryl group prior to attachment to a ligand- binding protein that is reacted with a moiety of the ligand-binding protein to attach the fluorophore to the ligand-binding protein.
  • the fluorophore comprised a thiol group prior to attachment to the ligand-binding protein.
  • the fluorophore was thiol reactive prior to attachment to the ligand-binding protein.
  • Non-limiting examples of fluorophores that may readily be attached to ligand-binding proteins using thiol reactions include fluorescein, pyrene, NBD, NBDE, Acrylodan (6-acryloyl 1-2- dimethylaminonaphthalene), Badan (6-bromo-acetyl-2-dimethylamino-naphthalene), JPW4039, JPW4042, or JPW4045.
  • the fluorophore comprises a derivative of a Prodan-based fluorophore such as Acrylodan or Badan.
  • the excitation and emission properties of the Prodan-based fluorophores Acrylodan and Badan can be altered by manipulating the fluorescent ring system, while preserving the dimethylamino donor group, and the twistable carbonyl acceptor (Klymchenko 2013 Progress in Molecular Biology and Trans lational Science, 35-58).
  • Replacement of the two-ring naphthalene with a three-ring anthracene Li 2006 J. Org. Chem., 71, 9651-9657
  • fluorene Koreanak 2010 J. Phys. Chem.
  • prodan analogues include 2-cyano-6- dihexylaminoanthracene and 2-propionyl-6-dihexylaminoanthracene, as well as fluorophores comprising the following structures:
  • the fluorophore comprises a fluorescent protein.
  • fluorescent proteins that emit blue, cyan, green, yellow, orange, red, far-red, or near infrared radiation when contacted with excitation radiation are known in the art and commercially available as proteins and via the expression of vectors that encode the fluorescent protein.
  • Non-limiting examples of fluorescent proteins include Blue Fluorescent Protein (BFP), TagBFP, mTagBFP2, Azurite, Enhanced Blue Florescent Protein 2 (EBFP2), mKalamal, Sirius, Sapphire, T-Sapphire, Cyan Fluorescent Protein (CFP); Enhanced Cyan Fluorescent Protein (ECFP), Cerulean, SCFP3A, mTurquoise, mTurquoise2, monomeric Midoriishi-Cyan, TagCFP, mTFPl, AmCyanl, Green Fluorescent Protein (GFP), Enhanced Green Fluorescent Protein (EGFP), Emerald, Superfolder GFP, AcGFPl, ZsGreenl, Monomeric Azami Green, TagGFP2, mUKG, mWasabi, Clover, mNeonGreen, Yellow Fluorescent Protein (YFP), Enhanced Yellow Fluorescent Protein (EYFP), Citrine, Venus, Super Yellow Fluorescent Protein 2 (SYFP2), TagYFP, ZsYellow
  • the fluorophore comprises a quantum dot (Medintz et al. 2005) (Sapsford, Berti and Medintz 2006 Angew Chem Int Ed Engl, 45, 4562-89; Resch-Genger et al. 2008 Nat Methods, 5, 763-75).
  • the emission properties of the conjugated protein are enhanced by immobilization on or near metallic nanoparticles (Zeng et al. 2014 Chem Soc Rev, 43, 3426-52; Shen et al. 2015 Nanoscale, 7, 20132-41).
  • the peak emission wavelength and/or the emission intensity of the biosensor change when the ligand binds to the ligand-binding protein.
  • the biosensor exhibits a dichromatic signaling change when the ligand binds to the ligand-binding protein.
  • the peak emission wavelength of the biosensor shifts by at least about 5, 10, 15, 20, 30, 40, 50, or by about 5-50 nm when the biosensor binds to ligand.
  • the emission intensity of the biosensor increases by at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, or 300% when the biosensor binds to ligand.
  • the signal produced by the reporter group persists for at least 1 nanoseconds (ns), 5 ns, 10 ns, 25 ns, 50 ns, 75 ns, 100 ns, 200 ns, 300 ns, 400 ns, 500 ns, 600 ns, 700 ns, 800 ns, 900 ns, 0.001 milliseconds (ms), 0.01 ms, 0.1 ms, 1 ms, 5 ms, 10 ms, 20 ms, 25 ms, 50 ms, 100 ms, or 500 ms when the ligand binds to the ligand-binding protein.
  • biosensors comprising a ligand-binding protein that binds a ligand of interest.
  • ligands include sugars (such as glucose, galactose, lactose, arabinose, ribose, and maltose), lactate, urea, anions (e.g., bicarbonate, phosphate, sulfate, and halide anions such as chloride, fluoride, iodide, astatide, ununseptide, and bromide), cations (e.g., calcium, iron, and hydrogen ions), dipeptides, and amino acids (such as histidine, glutamine, glutamate, aspartate).
  • the ligand-binding protein may comprise a naturally occurring protein or a protein that is modified compared to a naturally occurring protein.
  • the ligand-binding protein may comprise one or more mutations compared to a naturally occurring protein.
  • the naturally occurring protein is a naturally occurring counterpart of the ligand-binding protein (e.g., the ligand-binding protein is a mutant of the naturally occurring counterpart).
  • a "naturally occurring counterpart" of a mutant polypeptide is a polypeptide produced in nature from which the mutant polypeptide has been or may be derived (e.g. , by one or more mutations).
  • the naturally occurring counterpart is an endogenous polypeptide produced by an organism in nature, wherein the endogenous polypeptide typically does not have one or more of the mutations present in the mutant polypeptide.
  • a naturally occurring counterpart may be referred to herein for the purpose of comparison and to illustrate the location and/or presence of one or more mutations, binding activities, and/or structural features.
  • a “mutation” is a difference between the amino acid sequence of a modified polypeptide/protein and a naturally occurring counterpart.
  • a polypeptide having a mutation may be referred to as a "mutant.”
  • Non-limiting examples of mutations include insertions, deletions, and substitutions.
  • the term “mutation” excludes (i) the addition of amino acids to the N-terminus or C-terminus of a polypeptide, and (ii) the omission/deletion/replacement of a polypeptide's signal peptide (e.g., replacement with another signal peptide or with a methionine).
  • amino acids to the N-terminus or C-terminus of a protein via a peptide bond may be referred to herein as a "fusion" of the amino acids to the protein.
  • an exogenous protein fused to amino acids e.g., another protein, a fragment, a tag, or a polypeptide moiety
  • the added amino acids may comprise a non-native polypeptide, e.g., a polypeptide reporter group such as a fluorescent protein, a moiety that facilitates the isolation or modification of a polypeptide, or a moiety that facilitates the attachment of a polypeptide to a substrate or surface.
  • a non-native polypeptide e.g., a polypeptide reporter group such as a fluorescent protein
  • non-native when referring to the added amino acids (e.g., a polypeptide reporter group such as a fluorescent protein, a moiety that facilitates the isolation or modification of a polypeptide, or a moiety that facilitates the attachment of
  • polypeptide of a fusion protein indicates that the polypeptide is not naturally part of the protein to which it is fused in the fusion protein.
  • sequence of a non-native polypeptide (“added amino acids") that is fused to a protein is encoded by an organism other than the organism from which the protein is derived, is not known to be naturally encoded by any organism, or is encoded by a gene other than the wild-type gene that encodes an endogenous version of the protein.
  • signal peptide refers to a short (e.g., 5-30 or 10-100 amino acids long) stretch of amino acids at the N-terminus of a protein that directs the transport of the protein. In various embodiments, the signal peptide is cleaved off during the post- translational modification of a protein by a cell. Signal peptides may also be referred to as “targeting signals,” “leader sequences,” “signal sequences,” “transit peptides,” or
  • the signal peptide may optionally be considered to be, e.g., the first 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 5-15, 5-20, 5-25, 5-100, 10-15, 10-20, 10-25, 10-50, 10-100, 25- 50, 25-75, or 25-100 amino acids from the N-teiminus of the translated protein (compared to a protein that has not had the signal peptide removed, e.g., compared to a naturally occurring protein).
  • the ligand-binding protein comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 1-10, 1-15, 1-20, 5-15, 5-20, 10-25, 10-50, 20-50, 25-75, 25-100 or more mutations compared to a naturally occurring protein while retaining at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5%, or about 100% of the activity of the naturally occurring protein. Mutations include but are not limited to substitutions, insertions, and deletions.
  • Non-limiting examples of ligand-binding proteins may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 1-10, 1-15, 1-20, 5-15, 5-20, 10-25, 10-50, 20-50, 25-75, 25-100, or more substitution mutations compared to a naturally occurring protein while retaining at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5%, or about 100% of the activity of the naturally occurring protein.
  • a ligand-binding protein may include one or more mutations that remove a cysteine, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more substitutions or deletions of a cysteine compared to a naturally occurring protein.
  • the ligand-binding protein is not a mutant.
  • a reporter group is fused to the N-terminus or the C-terminus of the ligand-binding protein.
  • a ligand-binding protein may comprise a stretch of amino acids (e.g., the entire length of the ligand-binding protein or a portion comprising at least about 50, 100, 200, 250, 300, or 350 amino acids) in a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, or 99.5% identical to an amino acid sequence of a naturally occurring protein.
  • amino acids e.g., the entire length of the ligand-binding protein or a portion comprising at least about 50, 100, 200, 250, 300, or 350 amino acids
  • the mutations are conservative, and the present subject matter includes many ligand-binding proteins in which the only mutations are substitution mutations.
  • a ligand-binding protein has no deletions or insertions compared to a naturally occurring protein (e.g. , a naturally occurring counterpart).
  • a ligand-binding protein may have (i) less than about 5, 4, 3, 2, or 1 inserted amino acids, and/or (ii) less than about 5, 4, 3, 2, or 1 deleted amino acids compared to a naturally occurring protein.
  • the prokaryotic ligand-binding protein is a mutant, fragment, or variant of a natural (i.e., wild-type) bacterial protein.
  • the bacterial ligand-binding protein is from a thermophilic, mesophilic, or cryophilic prokaryotic microorganism (e.g., a thermophilic, mesophilic, or cryophilic bacterium).
  • thermophilic if it is capable of surviving, growing, and reproducing at temperatures between 41 and 140 °C (106 and 284 °F), inclusive.
  • a thermophilic organism has an optimal growth temperature between 41 and 140°C, or that is at least about 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, or 140°C.
  • Many thermophiles are archaea. Thermophilic eubacteria are suggested to have been among the earliest bacteria.
  • thermophiles are found in various geothermally heated regions of the Earth, such as hot springs and deep sea hydrothermal vents, as well as decaying plant matter, such as peat bogs and compost. Unlike other types of microorganisms, thermophiles can survive at much hotter temperatures, whereas other bacteria would be damaged and sometimes killed if exposed to the same temperatures.
  • Thermophiles maybe classified into three groups: (1) obligate thermophiles; (2) facultative thermophiles; and (3) hyperthermophiles.
  • Obligate thermophiles also called extreme thermophiles
  • facultative thermophiles also called moderate thermophiles
  • Hyperthermophiles are particularly extreme thermophiles for which the optimal temperatures are above 80°C.
  • thermotolerant biosensor if it is capable of surviving exposure to temperatures above 41°C.
  • a thermotolerant biosensor retains its function and does not become denatured when exposed to a temperature of about 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, or 140°C for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30 or more minutes.
  • thermotolerant compound survives exposure to 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, or 140°C under pressure.
  • a microorganism is "mesophilic" if it is capable of surviving, growing, and reproducing at temperatures between 20 and 40°C (68 and 104°F), inclusive.
  • Psychrophiles or “cryophiles” are microorganisms that are capable of growth and reproduction in cold temperatures.
  • a psychrophile is capable of growth and reproduction at a temperature of 10°C or less, e.g., between -20 °C and +10 °C.
  • the microbial protein is produced by a bacterial
  • a biosensor comprises a modified (e.g., mutated, fused, and/or conjugated) periplasmic binding protein or a cytoplasmic binding protein.
  • aspects of the present subject matter provide a ligand-binding protein with a mutation that alters the interaction of the ligand-binding protein with a ligand.
  • the ligand-binding protein comprises a mutation that alters the interaction of the ligand-binding protein with the ligand compared to a naturally occurring counterpart.
  • the ligand-binding protein comprises a mutation that alters the interaction of an amino acid of the ligand-binding protein with a water molecule compared to a naturally occurring counterpart.
  • the ligand-binding protein does not comprise a signal peptide.
  • the signal peptide e.g., that is present in a naturally occurring counterpart
  • Exemplary implementations relate to a ligand such as sugars (such as glucose, galactose, lactose, arabinose, ribose, and maltose), lactate, urea, anions (e.g., chloride, bicarbonate, phosphate, and sulfate), cations (e.g., calcium and iron), dipeptides, amino acids (such as histidine, glutamine, glutamate, and aspartate).
  • the biosensor may comprise a mutant of, a fragment of, or a fusion protein comprising a microbial ligand- binding protein.
  • the ligand-binding protein is not a mutant or fragment to which a non-native polypeptide has been attached or added.
  • the ratiometric reagentless biosensors produce precise measurements over extended concentration ranges, e.g. from 0.0001 mM to 100 mM, in sample volumes of less than about 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 ⁇ .
  • the ligand-binding protein comprises a mutation that alters (e.g., increases or decreases) the interaction of the mutant with bound ligand compared to a naturally occurring protein (e.g., a microbial ligand-binding protein).
  • the ligand-binding protein comprises a mutation that alters (e.g., increases or decreases) the mutant's affinity and/or specificity for ligand compared to a unmutated ligand-binding protein (e.g., a microbial ligand-binding protein).
  • the ligand-binding protein comprises a mutation that alters the interaction between the protein and bound ligand, a mutation that alters the equilibrium between the open and closed states of the ligand-binding protein, a mutation that alters the interaction between the ligand-binding protein and a reporter group (such as a fluorescent conjugate, e.g., the interaction with a carbonyl group or a naphthalene ring of a prodan-derived fluorophore such as Acrylodan or Badan), and/or a mutation that impacts indirect interactions that alter the geometry of the ligand binding site.
  • the mutation does not reduce, or negligibly impacts, the thermostability of the ligand-binding protein.
  • the mutation alters the thermostability of the ligand-binding protein by less than about 1, 2, 3, 4, 5, or 10°C.
  • the present subject matter provides a glucose-galactose binding protein GGBP that is or is a mutant of: an Escherichia sp. (e.g., E. albertii, E. coli, E.fergusonii, E. hermannii, or E. vulneris) GGBP; a Thermoanaerobacter sp. (e.g., T. acetoethylicus, T. brockii, T.
  • Escherichia sp. e.g., E. albertii, E. coli, E.fergusonii, E. hermannii, or E. vulneris
  • GGBP glucose-galactose binding protein
  • Thermoanaerobacter sp. e.g., T. acetoethylicus, T. brockii, T.
  • thermosaccharolyticum T. uzonensis, or T. wiegelii
  • GGBP a Clostridium sp. (e.g., C.
  • C. methoxybenzovorans mayombei, C. methoxybenzovorans, C. methylpentosum, C. neopropionicum, C. nexile, C. nitrophenolicum, C. novyi, C. oceanicum, C. orbiscindens, C. oroticum, C. oxalicum, C. papyrosolvens, C. paradoxum, C. paraperfringens, C. paraputrificum, C. pascui, C.
  • phytofermentans C. piliforme, C. polysaccharolyticum, C. populeti, C. propionicum, C. proteoclasticum, C. proteolyticum, C. psychrophilum, C. puniceum, C. purinilyticum, C. putrefaciens, C. putrificum, C. quercicolum, C. quinii, C. ramosum, C. rectum, C. roseum, C. saccharobutylicum, C. saccharogumia, C. saccharolyticum, C. saccharoperbutylacetonicum,
  • C. sulfidigenes C. symbiosum, C. tagluense, C. tepidiprofundi, C. termitidis, C. tertium, C. tetani, Clostridium tetanomorphum, C. thermaceticum, C. thermautotrophicum, C.
  • thermoalcaliphilum C. thermobutyricum, C. thermocellum, C. thermocopriae, C.
  • thermohydrosulfuricum C. thermolacticum, C. thermopalmarium, C. thermopapyrolyticum,
  • thermosaccharolyticum C. thermosuccinogenes, C. thermosulfurigenes, C.
  • C. viride, C. xylanolyticum, or C. xylanovorans GGBP; a Salmonella sp. [e.g., S. bongori, S. enterica, S. enterica subspecies enterica, S. enterica subspecies salamae, S. enterica
  • S. enterica subspecies arizonae S. enterica subspecies diarizonae
  • S. enterica subspecies houtenae S. enterica subspecies indica
  • S. enterica subspecies enterica serovar Typhimurium S.
  • GGBP Caldicellulosiruptor sp. (e.g., C. saccharolyticus, C. acetigenus, C. bescii, C. changbaiensis, C. hydrothermalis, Caldicellulosiruptor hydrother, C.
  • Caldicellulosiruptor sp. e.g., C. saccharolyticus, C. acetigenus, C. bescii, C. changbaiensis, C. hydrothermalis, Caldicellulosiruptor hydrother, C.
  • Paenibacillus sp. e.g., P. agarexedens, P. agaridevorans, P. alginolyticus, P. alkaliterrae, P. alvei, P. amylolyticus, P. anaericanus, P. antarcticus, P. assamensis, P. azoreducens, P. azotofixans, P. barcinonensis, P. borealis, P. brasilensis, P. brassicae, P. campinasensis, P. chinjuensis, P. chitinolyticus, P. chondroitinus, P. cineris, P. cookii, P. curdlanolyticus, P. daejeonensis, P. dendritiformis, P. durum, P. ehimensis, P. elgii, P.favisporus, P.
  • glucanolyticus P. glycanilyticus, P. gordonae, P. graminis, P. granivorans, P. hodogayensis,
  • Butyrivibrio sp. e.g., B. proteoclasticus, B. crossotus, B.fibrisolvens, or B. hungatei
  • GGBP Butyrivibrio sp. (e.g., B. proteoclasticus, B. crossotus, B.fibrisolvens, or B. hungatei)
  • GGBP Butyrivibrio sp. (e.g., B. proteoclasticus, B. crossotus, B.fibrisolvens, or B. hungatei) GGBP;
  • a Roseburia sp. e.g., R. intestinalis, R.faecis, R. hominis, or R. inulinivorans
  • Faecalibacterium sp. e.g., F. prausnitzii
  • GGBP GGBP
  • Erysipelothrix sp. e.g., E.
  • rhusiopathiae E. inopinata, orE. tonsillarum
  • GGBP Eubacterium sp.
  • E. acidaminophilum, E. nodatum, E. oxidoreducens, or E.foedans GGBP.
  • the present subject matter provides a urea-binding protein that is or is a mutant of: an
  • Marinomas sp. e.g., M. posidonica
  • a Marinobacter sp. e.g., M. adhaerens, M. algicola, M. alkaliphilus, M. antarcticus, M. arcticus, M.aromaticivorans, M. bryozoorum, M. daepoensis, M. daqiaonensis, M. excellens, M.flavimaris, M. gudaonensis,
  • B. acidiceler e.g., B. acidiceler, B. acidicola, B. acidiproducens, B. acidocaldarius, B. acidoterrestris, B. aeolius, B. aerius, B. aerophilus, B. agar adhaerens, B. agri, B. aidingensis, B. akibai, B. alcalophilus, B. algicola, B. alginolyticus, B. alkalidiazotrophicus, B. alkalinitrilicus, B. alkalisediminis, B. alkalitelluris, B. altitudinis, B. alveayuensis, B. alvei, B.
  • amyloliquefaciens B. a. subsp. amyloliquefaciens, B. a. subsp. plantarum, B. amylolyticus, B. andreesenii, B. aneurinilyticus, B. anthracis, B. aquimaris, B. arenosi, B. arseniciselenatis,
  • B. chagannorensis B. chitinolyticus, B. chondroitinus, B. choshinensis, B. chungangensis, B. cibi, B. circulans, B. clarkii, B. clausii, B. coagulans, B. coagarnsis, B. cohnii, B. composti,
  • decolor ationis B. deserti, B. dipsosauri, B. drentensis, B. edaphicus, B. ehimensis, B.
  • herbersteinensis B. horikoshii, B. horneckiae, B. horti, B. huizhouensis, B. humi, B.
  • B. hwajinpoensis B. idriensis, B. indicus, B. infantis, B. infernus, B. insolitus, B. invictae, B. iranensis, B. isabeliae, B. isronensis, B.jeotgali, B. kaustophilus, B. kobensis, B. kochii, B. kokeshiiformis, B. koreensis, B. korlensis, B. kribbensis, B. krulwichiae, B. laevolacticus, B. larvae, B. laterosporus, B. lautus, B.
  • lehensis B. lentimorbus, B. lentus, B. licheniformis, B. ligniniphilus, B. litoralis, B. locisalis, B. luciferensis, B. luteolus, B. luteus, B. macauensis, B. macerans, B. macquariensis, B. macyae, B. malacitensis, B. mannanilyticus, B. marisflavi, B. marismortui, B. marmarensis, B. massiliensis, B. megaterium, B. mesonae, B. methanolicus,
  • pantothenticus B. parabrevis, B. paraflexus, B. pasteurii, B. patagoniensis, B. peoriae, B. persepolensis, B. persicus, B. pervagus, B. plakortidis, B. pocheonensis, B. polygoni, B. polymyxa, B. popilliae, B. pseudalcalophilus, B. pseudofirmus, B. pseudomycoides, B.
  • pulvifaciens B. pumilus, B. purgationiresistens, B. pycnus, B. qingdaonensis, B. qingshengii, B. reuszeri, B. rhizosphaerae, B. rigui, B. runs, B. safensis, B. salarius, B. salexigens, B. saliphilus, B. schlegelii, B. sediminis, B. selenatarsenatis, B. selenitireducens, B.
  • subtilis subtilis, B. taeanensis, B. tequilensis, B. thermantarcticus, B. thermoaerophilus, B.
  • thermoamylovorans B. thermocatenulatus, B. thermocloacae, B. thermocopriae, B.
  • thermodenitrificans B. thermoglucosidasius, B. thermolactis, B. thermoleovorans, B.
  • thermophilus B. thermoruber, B. thermosphaericus, B. thiaminolyticus, B. thioparans, B. thuringiensis, B. tianshenii, B. trypoxylicola, B. tusciae, B. validus, B. vallismortis, B.
  • B. vedderi B. velezensis, B. vietnamensis, B. vireti, B. vulcani, B. wakoensis, B.
  • weihenstephanensis B. xiamenensis, B. xiaoxiensis, or B. zhanjiangensis
  • urea-binding protein a Desulfotomaculum sp. ⁇ e.g., D. ruminis, D. nigrificans, D. australicum, D.
  • thermobenzoicum D. geothermicum, D. thermocisternum, D. aeronauticum, D. halophilum,
  • D. sp. CYP1 D. sp. CYP9, D. sp. IS3205, D. sp. Srb55, D. sp. Iso-W2, D. sp. 2, D.
  • Clostridium sp. e.g., C. absonum, C. aceticum, C. acetireducens, C. acetobutylicum, C. acidisoli, C. aciditolerans, C. aciduria, C. aerotolerans, C. aestuarii, C. akagii, C. aldenense, C. aldrichii, C. algidicarni,
  • C. cellulofermentans C. cellulolyticum, C. cellulosi, C. cellulovorans, C. chartatabidum, C. chauvoei, C. chromiireducens, C. citroniae, C. clariflavum, C. clostridioforme, C. coccoides,
  • phytofermentans C. piliforme, C. polysaccharolyticum, C. populeti, C. propionicum, C. proteoclasticum, C. proteolyticum, C. psychrophilum, C. puniceum, C. purinilyticum, C. putrefaciens, C. putrificum, C. quercicolum, C. quinii, C. ramosum, C. rectum, C. roseum, C. saccharobutylicum, C. saccharogumia, C. saccharolyticum, C. saccharoperbutylacetonicum,
  • C. sulfidigenes C. symbiosum, C. tagluense, C. tepidiprofundi, C. termitidis, C. tertium, C. tetani, Clostridium tetanomorphum, C. thermaceticum, C. thermautotrophicum, C.
  • thermoalcaliphilum C. thermobutyricum, C. thermocellum, C. thermocopriae, C.
  • thermohydrosulfuricum C. thermolacticum, C. thermopalmarium, C. thermopapyrolyticum,
  • thermosaccharolyticum C. thermosuccinogenes, C. thermosulfurigenes, C.
  • C. viride, C. xylanolyticum, or C. xylanovorans urea-binding protein
  • a Caldicellulosiruptor sp. e.g., C. acetigenus, C. bescii, C. changbaiensis, C. hydrothermalis, C. kristjanssonii, C. kronotskyensis, C. lactoaceticus, C. owensensis, or C. saccharolyticus
  • Thermocrinis sp. e.g., T. ruber, T. albus, or T. minervae
  • Synechoccus sp. e.g., S. ambiguus, S. arcuatus var. calcicolus, S. bigranulatus, S.
  • Thermosynechococcus sp. e.g., T. elongatus or T. vulcanus
  • the present subject matter provides a glucose-binding protein that is or is a mutant of: an Thermus sp. (e.g., T. caldophilus, T. eggertssonii, T. kawarayensis, T. murrieta, T.
  • an Thermus sp. e.g., T. caldophilus, T. eggertssonii, T. kawarayensis, T. murrieta, T.
  • T. parvatiensis nonproteolyticus
  • T. rehai nonproteolyticus
  • T. yunnanensis nonproteolyticus
  • T. amyloliquefaciens T.
  • T. tengchongensis, or T. thermophilus glucose-binding protein
  • a Deinococcus sp. e.g., D. aquivivus, D. puniceus, D. soli, D. xibeiensis, D. aerius, D. aerolatus, D. aerophilus, D. aetherius, D. alpinitundrae, D. altitudinis, D. apachensis, D. aquaticus, D. aquatilis, D. aquiradiocola, D. caeni, D. cellulosilyticus, D. claudionis, D. daejeonensis, D.
  • depolymerans D. deserti, D. erythromyxa, D.ficus, D.frigens, D. geothermalis, D. gobiensis, D. grandis, D. hohokamensis, D. hopiensis, D. indicus, D. maricopensis, D.
  • marmoris D. metalli, D. misasensis, D. murrayi, D. navajonensis, D. papagonensis, D.
  • alkalitelluris B. altitudinis, B. alveayuensis, B. alvei, B. amyloliquefaciens, B. a. subsp. amyloliquefaciens, B. a. subsp. plantarum, B. amylolyticus, B. andreesenii, B.
  • aneurinilyticus B. anthracis, B. aquimaris, B. arenosi, B. arseniciselenatis, B. arsenicus, B. aurantiacus, B. arvi, B. aryabhattai, B. asahii, B. atrophaeus, B. axarquiensis, B. azotofixans,
  • B.fordii B.formosus, B.fortis, B. fumarioli, B. funiculus, B.fusiformis, B. galactophilus, B. galactosidilyticus, B. galliciensis, B. gelatini, B. gibsonii, B. ginsengi, B. ginsengihumi, B. ginsengisoli, B. globisporus, B. g. subsp. globisporus, B. g. subsp. marinus, B.
  • glucanolyticus B. gordonae, B. gottheilii, B. graminis, B. halmapalus, B. haloalkaliphilus, B. halochares, B. halodenitrificans, B. halodurans, B. halophilus, B. halosaccharovorans, B. hemicellulosilyticus, B. hemicentroti, B. herbersteinensis, B. horikoshii, B. horneckiae, B. horti, B. huizhouensis, B. humi, B. hwajinpoensis, B. idriensis, B. indicus, B.
  • infantis B. infernus, B. insolitus, B. invictae, B. iranensis, B. isabeliae, B. isronensis, B.jeotgali, B. kaustophilus, B. kobensis, B. kochii, B. kokeshiiformis, B. koreensis, B. korlensis, B.
  • B. megaterium B. mesonae
  • B. methanolicus B. methylotrophicus
  • B. migulanus B. mojavensis, B. mucilaginosus, B. muralis, B. murimartini, B. mycoides, B. naganoensis, B. nanhaiensis, B. nanhaiisediminis, B. nealsonii, B. neidei, B. neizhouensis, B. niabensis, B. niacini, B. novalis, B. oceanisediminis, B. odysseyi, B. okhensis, B. okuhidensis, B. oleronius, B. oryzaecorticis, B. oshimensis, B.
  • B. pakistanensis B. pallidus, B. pallidus, B. panacisoli, B. panaciterrae, B. pantothenticus, B. parabrevis, B. paraflexus, B. pasteurii, B. patagoniensis, B. peoriae, B. persepolensis, B. persicus, B. pervagus, B.
  • shacheensis B. shackletonii, B. siamensis, B. silvestris, B. simplex, B. siralis, B. smithii, B. soli, B. solimangrovi, B. solisalsi, B. songklensis, B. sonorensis, B. sphaericus, B.
  • thermocatenulatus B. thermocloacae, B. thermocopriae, B. thermodenitrificans, B.
  • thermoglucosidasius B. thermolactis, B. thermoleovorans, B. thermophilus, B. thermoruber,
  • thermosphaericus B. thiaminolyticus, B. thioparans, B. thuringiensis, B. tianshenii, B. trypoxylicola, B. tusciae, B. validus, B. vallismortis, B. vedderi, B. velezensis, B.
  • chlorophenolicus A. citreus, A. cryoconiti, A. cryotolerans, A. crystallopoietes, A. cumminsii,
  • nasiphocae A. nicotinovorans, A. nitroguajacolicus, A. oryzae, A. parietis, A. pascens, A. pigmenti, A. pityocampae, A. psychrochitiniphilus, A. psychrolactophilus, A. ramosus, A. rhombi, A. roseus, A. russicus, A. sanguinis, A. soli, A. stack brandtii, A. subterraneus, A. tecti, A. tumbae, A. viscosus, or A. woluwensis) glucose-binding protein.
  • the present subject matter provides a lactate-binding protein that is or is a mutant of: a Thermus sp. ⁇ e.g., T. caldophilus, T. eggertssonii, T. kawarayensis, T. murrieta, T.
  • T. parvatiensis nonproteolyticus
  • T. rehai nonproteolyticus
  • T. yunnanensis nonproteolyticus
  • T. amyloliquefaciens T.
  • T. shimai T. tengchongensis, or T. thermophilus
  • lactate-binding protein e.g., T. denitrificans, T. halophilus, T.jannaschii, T. nitratireducens, T. nitratis, T.
  • R. sp. BS90 R. sp. CI 15, R. sp. C23, R. sp. CCS2, R. sp. COL2P, R. sp. COLSP, R. sp.
  • R. sp. MBT22 i?. sp. MED001, R. sp. MED006, i?. sp. MED007, R. sp. MED008, R. sp.
  • PRLISYOl R. sp. PRLISY03, R. sp. QSSC9-8, R. sp. RED 15, R. sp. RED1, R. sp. RED59, R. sp. RED68, i?. sp. RED85, R. sp. S03, i?. sp. SC-B2-2, i?. sp. SCB28, i?. sp. SCB31, i.. sp.
  • SCB34 R. sp. SCB48, R. sp. SDBC1, R. sp. SDBC6, R. sp. SFLA13, i?. sp. SIO, R. sp.
  • SYOPl i.. sp. SYOP2, i?. sp. TM1035, i.. sp. TM1038, i?. sp. TM1040, i.. sp. ⁇ 1042.
  • ⁇ . sp. TP9 R. sp. UAzPsJAC-lb, R. sp. UAzPsK-5, R. sp. WED10.10, R. sp. WEDl.l, R. sp.
  • M.aromaticivorans M. bryozoorum, M. daepoensis, M. daqiaonensis, M. excellens, M.
  • hydrocarbonoclasticus M. koreensis, M. lacisalsi, M. lipolyticus, M. litoralis, M. lutaoensis,
  • M. sediminum M. segnicrescens, M. shengliensis, M. squalenivorans, M. similis, M.
  • lactate-binding protein a Anaeromyxobacter sp. ⁇ e.g., A. dehalogenans) lactate-binding protein, aPolymorphum sp. (e.g., P. gilvum ) lactate-binding protein, aPseudomonas sp. (e.g., P. aeruginosa, P. alcaligenes, P. anguilliseptica, P. argentinensis, P. borbori, P.
  • citronellolis P. flavescens, P. mendocina, P. nitroreducens, P. oleovorans, P.
  • panacis P. protegens, P. proteolytica, P. rhodesiae, P. synxantha, P. thivervalensis, P.
  • P. tomato, or P. viridiflava lactate-binding protein
  • aRhodobacter sp. e.g., R. aestuarii, R. azotoformans, R. blasticus, R. capsulatus, R.johrii, R. maris, R. megalophilus, R. ovatus, R. sphaeroides, R. veldkampii, R. vinaykumarii, or R. viridis
  • lactate-binding protein a Flexistipes sp. ⁇ e.g., F. sinusarabici) lactate-binding protein, or a Thermanaerovibrio sp. ⁇ e.g., T. acidaminovorans or T. velox) lactate-binding protein.
  • the present subject matter provides a ligand-binding protein that is or is a mutant of: a Synechocystis sp. ⁇ e.g., S. sp. PCC6803) bicarbonate-binding protein, a
  • Thermosynechococcus sp. ⁇ e.g., T. vulcanus, T. elongatus, or T. elongatus BP-1) bicarbonate- binding protein, a Chroococcidiopsis sp. ⁇ e.g., C. thermalis, C. gigantea, C. cubana, or C. codiicold) bicarbonate-binding protein, a Calothrix sp. (e.g., C. aberrans, C. adscencens, C. aeruginea, C. africana, C. allorgei, C. australiensis, C. baileyi, C. bharadwajae, C. borealis, C. braunii, C. breviarticulata, C. calida, C. castellii, C. capitularis, C. cavernarum
  • gelatinosa C. gracilis, C. intricata, C. litoralis, C. marchica, C. nodulosa, C. obtusa, C. rhizosoleniae, or C. schweickertii
  • bicarbonate-binding protein a Anabaena sp. ⁇ e.g., A. aequalis, A. affinis, A. angstumalis angstumalis, A. angstumalis marchita, A. aphanizomendoides, A. azollae, A. bornetiana, A. catenula, A. cedrorum, A. circinalis, A. confervoides, A. constricta, A. cyanobacterium, A. cycadeae, A. cylindrica, A. echinispora, A.felisii, A.
  • lemmermannii lemmermannii, A. levanderi, A. limnetica, A. macrospora macrospora, A. macrospora robusta, A. monticulosa, A. nostoc, A. oscillarioides, A. planctonica, A. raciborskii, A.
  • scheremetievi A. sphaerica, A. spiroides crassa, A. spiroides spiroides, A. subcylindrica, A. torulosa, A. unispora, A. variabilis, A. verrucosa, A. viguieri, A. wisconsinense, or A.
  • zierlingii) bicarbonate-binding protein or a Chamaesiphon sp. ⁇ e.g., C. africanus, C. amethystinus, C. britannicus, C. carpaticus, C. confervicola, C. cylindricus, C.
  • ocobyrsiodes C. polonicus, C. polymorphus, C. starmachii, C. stratosus, or C. subglobosus bicarbonate-binding protein.
  • the present subject matter provides a ligand-binding protein that is or is a mutant of: a Mannheimia sp. ⁇ e.g., M. caviae, M. glucosida, M. granulomatis, M. haemolytica, M.
  • E. acetylicum E. aestuarii
  • E. alkaliphilum E. antarcticum
  • E. aquaticum E. artemiae
  • E. aurantiacum E. enclense
  • E. indicum E. marinum
  • E. mexicanum E. oxidotolerans
  • E. profundum E. sibiricum
  • E. soli or E. undae
  • bicarbonate and iron binding protein an Exiguobacterium sp. ⁇ e.g., E. acetylicum, E. aestuarii, E. alkaliphilum, E. antarcticum, E. aquaticum, E. artemiae, E. aurantiacum, E. enclense, E. indicum, E. marinum, E. mexicanum, E. oxidotolerans, E. profundum, E. sibiricum, E. soli, or E. undae) bicarbonate and iron
  • Thermosynechococcus sp. ⁇ e.g., T. vulcanus, T. elongatus, or T. elongatus BP-1) bicarbonate and iron binding protein, a Candidatus Nitrospira sp. ⁇ e.g., Candidatus Nitrospira defluvii, Candidatus Nitrospira nitrificans, Candidatus Nitrospira nitrosa, Candidatus Nitrospira inopinata, Candidatus Magnetobacterium casensis, Candidatus Magnetobacterium bavaricum, Candidatus Magnetoovum chiemensis) bicarbonate and iron binding protein, a Thermus sp. ⁇ e.g., T. caldophilus, T. eggertssonii, T. kawarayensis, T. murrieta, T.
  • T. parvatiensis nonproteolyticus
  • T. rehai nonproteolyticus
  • T. yunnanensis nonproteolyticus
  • T. amyloliquefaciens T.
  • a polypeptide may comprise an amino acid sequence which is at least 60%, 65%, 70%, 75%, 76%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to the reference SEQ ID NO or to each of the reference SEQ ID NOs.
  • a polypeptide may comprise an amino acid sequence which is less than 75%, 70%, 65%, 60%, 59%, 58%, 57%, 56%, 55%, 54%, 53%, 52%, 51%, 50%, 49%, 48%, 47%, 46%, 45%, 44%, 43%, 42%, 41%, 40%, 39%, 38%, 37%, 36%, 35%, 34%, 33%, 32%, 31%, 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, or 15% identical to the reference SEQ ID NO or to each of the reference SEQ ID NOs.
  • a polypeptide comprises amino acids in a sequence that is preferably at least about 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, or 30% and less than about 75%, 70%, 65%, 60%, 55%, 50%, 45%, 44%, 43%, 42%, 41%, 40%, 39%, 38%, 37%, 36%, 35%, 34%, 33%, 32%, 31%, or 30% identical to the reference SEQ ID NO or to each of the reference SEQ ID NOs.
  • reference proteins and amino acid sequences disclosed herein include:
  • a glucose-galactose binding protein from Escherichia coli ecGGBP; genome, NC_002695; protein, WP_032329053, SEQ ID NO: 87);
  • thermosaccharolyticum ttGGBP; genome, NC 014410; protein, YP_003852930.1, SEQ ID NO: 88);
  • chyGGBP a glucose-galactose binding protein from Caldicellulosiruptor hydrothermalis
  • NC_013406 protein, YP 003243743.1, SEQ ID NO: 92);
  • xiii a glucose-galactose binding protein from Roseburia intestinalis (rinGGBP B; genome, NC_021012; protein, YP 007778124.1, SEQ ID NO: 99);
  • xv a glucose-galactose binding protein from Eubacterium rectale (ereGGBP; genome, NC 012781 ; protein, YP_002936409.1, SEQ ID NO: 101);
  • NC_015660 protein, YP_004588319.1 ; SEQ ID NO: 106);
  • a urea-binding protein from Caldicellulosiruptor saccharolyticus csUBP; genome, NC_009437, protein, YP_001181243.1 ; SEQ ID NO: 108
  • a urea-binding protein from Thermocrinis albus taUBP; genome,
  • teUBP urea-binding protein from Thermosynechococcus elongatus
  • NC_004113 protein, YP_681910.1 ; SEQ ID NO: 112);
  • xliii a lactate-binding protein from Anaeromyxobacter dehalogens (adLacBP; genome, NC_007760, protein YP_466_099.1; SEQ ID NO: 129);
  • (xlix) a bicarbonate-binding protein from Synechocystis sp. (synBicarbBPl ; genome,
  • NC_017052 protein YP 005410477.1; SEQ ID NO: 135);
  • lix a bicarbonate and iron binding protein from Thermus thermophilus (ttFeBP5; genome, NC_006461, protein, YPJ44894.1 ; SEQ ID NO: 145);
  • lx a bicarbonate and iron binding protein from Meiothermus silvanus (msFeBP6; genome, NC_014212, protein, YP_003686074.1 ; SEQ ID NO: 146);
  • the ligand-binding proteins disclosed herein may optionally be fused ⁇ e.g., at their N- terminal and/or C-terminal ends) to a motif comprising a stretch of amino acids that facilitates the isolation or other manipulation such as conjugation to a moiety or
  • a substrate such as a plastic, a cellulose product such as paper, polymer, metal, noble metal, semi-conductor, or quantum dot ⁇ e.g., a fluorescent quantum dot
  • a stretch of amino acids has the sequence: GGSHHHHHH (SEQ ID NO: 152). This motif is not required for, is not believed to influence or affect ligand-binding activity or signal transduction, and may be omitted from any ligand-binding protein or biosensor disclosed herein.
  • each of SEQ ID NOs: 1-41 (and the non-limiting examples of other proteins used in the experiments disclosed herein) comprises this motif (SEQ ID NO: 152).
  • a ligand-binding protein may be fused to a non-native polypeptide or "added amino acids" that facilitates the attachment thereof to a surface, such as the surface of a device.
  • a polypeptide comprises 1, 2, 3, 4, 5, or more substitutions or deletions of a cysteine compared to the naturally occurring counterpart of the polypeptide (i.e., 1, 2, 3, 4, 5, or more native cysteines have been removed), e.g., 1, 2, 3, 4, 5, or more cysteine to alanine substitutions compared to the naturally occurring counterpart of the polypeptide.
  • all of the cysteines of a polypeptide have been deleted and/or substituted compared to its natural counterpart.
  • one or more cysteines of a polypeptide have been substituted with an alanine, a serine, or a threonine.
  • the amino acid sequence of a protein comprises no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50 mutations compared to its naturally occurring counterpart. In some embodiments, less than 50, 45, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, or 2 of the mutations is a deletion or insertion of 1, 2, 3, 4, or 5 or no more than 1, 2, 3, 4, or 5 amino acids. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more of the mutations is a substitution mutation. In certain embodiments, less than 50, 45, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, or 2 of the mutations is a deletion or insertion of 1, 2, 3, 4, or 5 or no more than 1, 2, 3, 4, or 5 amino acids. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more of the mutations is a substitution mutation. In certain
  • every mutation to a protein compared to its naturally occurring counterpart is a substitution mutation.
  • 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more or all of the mutations to a protein compared to its naturally occurring counterpart is a conservative substitution mutation.
  • a polypeptide does not have any insertion or deletion compared to its natural counterpart, other than (optionally) the removal of the signal peptide and/or the fusion of compounds such as another polypeptide at the N-terminus or C-terminus thereof.
  • biosensors comprise ligand-binding proteins, such as proteins that have altered amino acid sequences compared to their naturally occurring counterparts.
  • ligand-binding proteins such as proteins that have altered amino acid sequences compared to their naturally occurring counterparts.
  • proteins are conjugated to reporter groups.
  • the C a root-mean-square deviation (RMSD) between the backbone of a ligand-binding protein and its naturally occurring counterpart is, e.g., between about 0-3 A, 0-1 A, 0-1.5 A, 0-2 A, 0.1-3 A, 0.5-1 A, 0.5-1.5 A, or 0.5-2 A, or less than about 0.1 A, 0.2 A, 0.3 A, 0.4 A, 0.5 A, 0.6 A, 0.7 A, 0.8 A, 0.9 A, 1.0 A, 1.5 A, 1.6 A, 1.7 A, 1.8 A, 1.9 A, 2.0 A, 2.5 A, or 3 A.
  • RMSD root-mean-square deviation
  • the N-terminal domain i.e., the portion of the protein at the N-terminal side of the binding domain hinge
  • the corresponding domain of its naturally occurring counterpart is, e.g., between about 0-3 A, 0-1 A, 0-1.5 A, 0-2 A, 0.1-3 A, 0.5-1 A, 0.5-1.5 A, or 0.5-2 A, or less than about 0.1 A, 0.2 A, 0.3 A, 0.4 A, 0.5 A, 0.6 A, 0.7 A, 0.8 A, 0.9 A, 1.0 A, 1.5 A, 1.6 A, 1.7 A, 1.8 A, 1.9 A, 2.0 A, 2.5 A, or 3 A.
  • the C a RMSD between the C-terminal domain (i.e., the portion of the protein at the C-terminal side of the binding domain hinge) backbone of the ligand-binding polypeptide and the corresponding domain of its naturally occurring counterpart is, e.g., between about 0-3 A, 0-1 A, 0-1.5 A, 0-2 A, 0.1-3 A, 0.5-1 A, 0.5-1.5 A, or 0.5-2 A, or less than about 0.1 A, 0.2 A, 0.3 A, 0.4 A, 0.5 A, 0.6 A, 0.7 A, 0.8 A, 0.9 A, 1.0 A, 1.5 A, 1.6 A, 1.7 A, 1.8 A, 1.9 A, 2.0 A, 2.5 A, or 3 A.
  • Non-limiting considerations relating to the sequence and structural differences between homologous proteins are discussed in Chothia and Lesk (1986) The EMBO Journal, 5(4):823-826, the entire content of which is incorporated herein by reference.
  • Non-limiting examples of ligand-binding polypeptides that are useful in biosensors provided herein include variants of the naturally occurring proteins disclosed herein that comprise cysteine substitutions and/or N-terminal and/or C-terminal fusions (e.g., to a fiuorophore attachment moiety).
  • a biosensor comprises a modified ligand- binding protein having an amino acid substitution compared to its naturally occurring counterpart, such that the polypeptide has a cysteine at one or more of position 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 101, 102, 103
  • the dissociation constant of the mutant ligand-binding polypeptide differs by at least about 1 ⁇ , 5 ⁇ , 10 ⁇ , 20 ⁇ , 25 ⁇ , 30 ⁇ , 35 ⁇ , 40 ⁇ , 45 ⁇ , 50 ⁇ , 75 ⁇ , 100 ⁇ , 200 ⁇ , 300 ⁇ , 400 ⁇ , 500 ⁇ , 600 ⁇ , 700 ⁇ , 800 ⁇ , 900 ⁇ , 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, or 100 mM (increase or decrease) compared to its naturally occurring counterpart.
  • biosensors and ligand-binding proteins provided herein are robust and useful at a wide range of physical conditions, e.g., pressure, temperature, salinity, osmolality, and pH conditions.
  • biosensors and ligand-binding proteins provided herein may survive substantial periods of time after being dried or exposed to high temperatures.
  • the biosensor maintains at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or more of its signal transduction activity after exposure to a temperature of about 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, or 125, or 40-125°C for about 1, 2, 3, 4, 5, 6, 15, 30, 60, 120, 180, 240, or 360 minutes. In certain embodiments, the biosensor maintains at least about 75%, 80%, 85%, 90%, 95%,
  • the biosensor maintains at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or more of its signal transduction activity after storage at a temperature of between 20-37°C for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, 24, or 1-24 months in dry form.
  • the optimal functional temperature of the biosensor is between 41 and 122°C, between 20 and 40°C, or less than about 10°C (e.g., between -20 and +10 °C).
  • Devices, compositions, and biosensors provided herein may be stored, e.g., with or without protection from exposure to light. In some embodiments, the devices, compositions, and biosensors are stored in the dark, e.g., with protection from light.
  • Non-limiting examples of glucose-binding proteins include variants of ecGGBP, ttGGBP, stGGBP, chyGGBP, cobGGBP, pspGGBP, csaGGBP, bprGGBP, rinGGBP A, rinGGBP B, fprGGBP, cljGGBP, cauGGBP, erhGGBP, ereGGBP, and chyGGBP.
  • a biosensor comprises a modified ecGGBP.
  • the modified ecGGBP may comprise one or more, or any combination of the following substitutions compared to its naturally occurring counterpart: Y10X, D14X, N15X, F16X, P70X, N91X, K92X, SI 12X, SI 15X, E149X, H152X, P153X, D154X, A155X, R158X, M182X, W183X, N21 IX, D212X, D236X, L238X, L255X, N256X, D257X, P294X, and V296X, where X is any amino acid, an amino acid that results in a conservative substitution, or a cysteine, and where each position is counted in ecGGBP without including the signal peptide (SEQ ID NO: 153).
  • the modified ecGGBP comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 of the following substitutions: Y10A, Y10C, D14C, DMA, D14Q, D14N, D14S, D14T, D14E, D14H, D14L, D14Y, D14F, N15C, F16L, F16A, F16C, F16Y, N91C, N91A, K92A, K92C, E93C, S112A, S115A, E149C, E149K, E149Q, E149S, H152C, H152A, H152F, H152Q, H152N, D154C, D154A, D154N, A155C, A155S, A155H, A155L, A155F, A155Y, A155K, A155M, A155W, A155Q, R158C, R158A, R158K, M182C, M182W, W183C, W183A,
  • a biosensor comprises a modified ttGGBP.
  • the modified ttGGBP may comprise one or more, or any combination of the following substitutions compared to its naturally occurring counterpart: Yl IX, D15X, T16X, F17X, G20X, N42X, V67X, R69X, R91X, E92X, Al 1 IX, Q148X, H151X, Q152X, A154X, N181X, W182X, D183X, D211X, T237X, T240X, L257X, N258X, D259X, A260X, and K300X where X is any amino acid, an amino acid that results in a conservative substitution, or a cysteine, and where each position is counted in ttGGBP with the signal peptide replaced with a methionine (SEQ ID NO: 154).
  • the modified ttGGBP comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11
  • a biosensor comprises a modified stGGBP.
  • the modified stGGBP may comprise one or more, or any combination of the following substitutions compared to its naturally occurring counterpart: Yl IX, Y13X, Dl 5X, N16X, F17X, P71X, N92X, K93X, P113X, S116X, E150X, H153X, P154X, D155X, A156X, R159X, M183X, W184X, N21 IX, N212X, D213X, A214X, D237X, L239X, D258X, P295X, and V297X where X is any amino acid, an amino acid that results in a conservative substitution, or a cysteine, and where each position is counted in stGGBP with the signal peptide replaced with a methionine (SEQ ID NO: 155).
  • the modified stGGBP comprises 1, 2 or 3 of the following
  • a biosensor comprises a modified chyGGBP.
  • the modified chyGGBP may comprise one or more, or any combination of the following substitutions compared to its naturally occurring counterpart: F12X, D14X, T15X, F16X, R68X, N89X, R90X, A110X, S113X, E147X, H150X, Q151X, D152X, A153X, R156X, M180X, W181X, N207X, N208X, D209X, D210X, D237X, T239X, D258X, V296X, and Y298X where X is any amino acid, an amino acid that results in a conservative substitution, or a cysteine, and where each position is counted in chyGGBP with the signal peptide replaced with a methionine (SEQ ID NO: 156).
  • the modified chyGGBP comprises 1, 2, or 3 of the following mutations: F12C, F16C,
  • a biosensor comprises a modified cobGGBP.
  • the modified cobGGBP may comprise one or more, or any combination of the following substitutions compared to its naturally occurring counterpart: F12X, D14X, T15X, F16X, C39X, R68X, N89X, R90X, A110X, S113X, E147X, H150X, Q151X, D152X, A153X, R156X, C173X, M180X, W181X, C206X, N207X, N208X, D209X, D210X, D237X, T239X, D258X, P297X, and Q299X where X is any amino acid, an amino acid that results in a conservative substitution, or a cysteine, and where each position is counted in cobGGBP with the signal peptide replaced with a methionine (SEQ ID NO: 157).
  • the modified cobGGBP comprises 1, 2, or 3 of the
  • a biosensor comprises a modified pspGGBP.
  • the modified pspGGBP may comprise one or more, or any combination of the following substitutions compared to its naturally occurring counterpart: F9X, DUX, T12X, F13X, R65X, N86X, R87X, A107X, S110X, E144X, H147X, Q148X, D149X, A150X, R153X, M177X, W178X, N204X, N205X, D206X, D207X, D234X, T236X, 255X, A294X, and K296X where X is any amino acid, an amino acid that results in a conservative substitution, or a cysteine, and where each position is counted in pspGGBP with the signal peptide replaced with a methionine (SEQ ID NO: 158).
  • the modified pspGGBP comprises 1, 2, or 3 of the following mutations: F9C,
  • a biosensor comprises a modified csaGGBP.
  • the modified csaGGBP may comprise one or more, or any combination of the following substitutions compared to its naturally occurring counterpart: Y14X, D16X, F18X, C62X, I72X, C82X, N93X, R94X, C113A, S118X, A121X, E152X, N155X, E156X, D157X, S158X, R161X, N185X, W186X, C211X, D241X, L243X, D262X, D290X, I292X, I297X, F299X, Q301X, and T302X where X is any amino acid, an amino acid that results in a conservative substitution, or a cysteine, and where each position is counted in csaGGBP with the signal peptide replaced with a methionine (SEQ ID NO: 159).
  • SEQ ID NO: 159 methionine
  • the modified csaGGBP comprises 1, 2, 3, 4, 5, 6, 7, or 8 of the following mutations: Y14C, F18C, C62A, C82A, CI 13A, W186C, and C211 A.
  • a biosensor comprises a modified bprGGBP.
  • the modified bprGGBP may comprise one or more, or any combination of the following substitutions compared to its naturally occurring counterpart: C8X, K12X, D14X, N15X, F16X, S72X, N93X, R94X, C112X, C116X, A118X, S121X, A153X, N156X, I157X, D158X, A159X, C179X, N186X, W187X, C211X, N212X, N213X, D214X, A215X, D241X, D243X, K251X, C289X, D290X, and V292X where X is any amino acid, an amino acid that results in a conservative substitution, or a cysteine, and where each position is counted in bprGGBP with the signal peptide replaced with a methionine (SEQ ID NO: 160).
  • the modified bprGGBP comprises 1, 2, 3, 4, 5, 6, 7, 8, or 9 of the following mutations: C8A, K12C, F16C, CI 12A, CI 16A, C179A, W187C, C211A, and C289A.
  • a biosensor comprises a modified rinGGBP A.
  • the modified rinGGBP A may comprise one or more, or any combination of the following substitutions compared to its naturally occurring counterpart: C6X, F10X, D12X, N13X, F14X, S70X, N91X, R92X, C114X, A116X, Q118X, D151X, N154X, V155X, D156X, A157X, R160X, C177X, N184X, W185X, C210X, N211X, N212X, D213X, A214X, D240X, L242X, L250X, C288X, D289X, and V291X where X is any amino acid, an amino acid that results in a conservative substitution, or a cysteine, and where each position is counted in rinGGBP A with the signal peptide replaced with a methionine (SEQ ID NO: 161).
  • the modified rinGGBP A comprises 1, 2, 3, 4, 5, 6, 7, or 8 of the following mutations: C6A, F10C, F14C, CI 14A, C177A, W185C, C210A, and C288A.
  • a biosensor comprises a modified rinGGBP B.
  • the modified rinGGBP B may comprise one or more, or any combination of the following substitutions compared to its naturally occurring counterpart: Q13X, D15X, T16X, F17X, C66X, C70A, R76X, N97X, R98X, A118X, S121X, E155X, H158X, Q159X, D160X, A161X, R164X, N188X, W189X, N215X, N216X, D217X, D218X, D244X, T246X,
  • D265X, P301X, A303X, and C306X where X is any amino acid, an amino acid that results in a conservative substitution, or a cysteine, and where each position is counted in rinGGBP B with the signal peptide replaced with a methionine (SEQ ID NO: 165).
  • X is any amino acid, an amino acid that results in a conservative substitution, or a cysteine, and where each position is counted in rinGGBP B with the signal peptide replaced with a methionine (SEQ ID NO: 165).
  • the modified rinGGBP B comprises 1, 2, 3, 4, 5, or 6 of the following mutations: Q13C, F17C, C66A, C70A, W189C, and C306A.
  • a biosensor comprises a modified fprGGBP.
  • the modified fprGGBP may comprise one or more, or any combination of the following substitutions compared to its naturally occurring counterpart: C8A, F12X, D14X, N15X, F16X, T69X, N90X, R91X, C105X, C106X, A113X, S116X, C143X, D146X, N149X, I150X, D151X, A152X, R155X, N179X, W180X, C205A, N206X, N207X, D208X, A209X, D235X, L237X, N243X, D284X, and V286X where X is any amino acid, an amino acid that results in a conservative substitution, or a cysteine, and where each position is counted in fprGGBP with the signal peptide replaced with a methionine (SEQ ID NO: 162).
  • the modified fprGGBP may comprise one or more,
  • a biosensor comprises a modified cljGGBP.
  • the modified cljGGBP may comprise one or more, or any combination of the following substitutions compared to its naturally occurring counterpart: Fl IX, N13X, T14X, W15X, V67X, C77X, N88X, R89X, A109X, SI 12X, E142X, N145X, Q146X, D147X, A148X, R151X, M175X, W176X, C198X, N201X, N202X, D203X, D204X, D231X,
  • the modified cljGGBP comprises 1, 2, 3, 4, or 5 of the following mutations: Fl 1C, W15C, C77A, W176C, and C198A.
  • a biosensor comprises a modified cauGGBP.
  • the modified cauGGBP may comprise one or more, or any combination of the following substitutions compared to its naturally occurring counterpart: F12X, N14X, T15X, W16X, V68X, C78X, N89X, R90X, Al 10X, SI 13X, E143X, N146X, Q147X, D148X, A149X, R152X, M176X, W177X, C199X, N203X, N204X, D205X, D206X, D233X, T235X, D254X, D293X, and K295X where X is any amino acid, an amino acid that results in a conservative substitution, or a cysteine, and where each position is counted in cauGGBP with the signal peptide replaced with a methionine (SEQ ID NO: 164).
  • SEQ ID NO: 164 methionine
  • the modified cauGGBP comprises 1, 2, 3, 4, or 5 of the following mutations: F12C, W16C, C78A, W177C, and C199A.
  • a biosensor comprises a modified erhGGBP.
  • the modified erhGGBP may comprise one or more, or any combination of the following substitutions compared to its naturally occurring counterpart: F13X, D15X, N16X, F17X, P76X, N97X, R98X, Al 19X, S122X, D153X, N156X, V157X, D158X, A159X, R162X, N187X, W188X, N214X, N215X, D216X, G217X, D243X, I245X, D264X, E312X, and V314X where X is any amino acid, an amino acid that results in a conservative substitution, or a cysteine, and where each position is counted in erhGGBP with the signal peptide replaced with a methionine (SEQ ID NO: 166).
  • the modified erhGGBP comprises 1, 2, or 3 of the following mutations: F13C, F17
  • a biosensor comprises a modified ereGGBP.
  • the modified ereGGBP may comprise one or more, or any combination of the following substitutions compared to its naturally occurring counterpart: Q13X, D15X, T16X, F17X, C29X, C65X, C69X, R75X, N96X, R97, Al 17X, S120X, E154X, H157X, Q158X, D159X, A160X, R163X, C183X, N187X, W188X, N214X, N215X, D216X, A217X, D243X, T245X, D264X, P301X, and E303X where X is any amino acid, an amino acid that results in a conservative substitution, or a cysteine, and where each position is counted in ereGGBP with the signal peptide replaced with a methionine (SEQ ID NO: 167).
  • the modified ereGGBP comprises 1, 2, 3, 4, 5, 6, 7, or 8 of the following mutations: CIOA, Q13C, F17C, C29A, C65A, C69A, C183A, and W188C.
  • biosensors comprise fluorescent proteins, such as fluorescent proteins that have altered amino acid sequences compared to their naturally occurring counterparts.
  • fluorescent proteins are conjugated to reporter groups.
  • the proteins are not conjugated to a reporter group (i.e., a biosensor comprising the fluorescent protein that does not undergo tgmFRET or ngmFRET is provided).
  • a biosensor for ligand comprising a ligand-binding protein, wherein the ligand-binding protein is a fluorescent protein, and wherein binding of the ligand to a ligand-binding domain of the fluorescent protein causes a change in fluorescence by the fluorescent protein.
  • the biosensor further comprises a reporter group, e.g., a fluorophore that acts as a ngmFRET donor fluorophore or a ngmFRET acceptor fluorophore with respect to the fluorescent protein.
  • Green Fluorescent Protein (GFP) and its derivatives such as Yellow Fluorescent Protein (YFP) form their internal fluorophore through an autocatalytic, posttranslational cyclization of a tripeptide from its own amino acid sequence (M. Zimmer, 2002, Chem. Rev. 102, 759-781). This process entails three steps: a nucleophilic attack to create a cyclic peptide, dehydration, and a final oxidation to introduce conjugation (D.P. Barondeau et al., 2003, Proc. Natl. Acad. Sci. USA, 100, 12111-12116). The formation of GFP's or YFP's fluorophore is an autocatalytic process that requires no catalyst external to these proteins.
  • the ligand comprises a halide anion such as a fluoride (F-), chloride (CI-), a bromide (Br-), an iodide (I-), astatide (At-) anion, or an ununseptide (Ts-) anion.
  • the fluorescent protein has an affinity (Kd) for the halide anion that is within the concentration range of the halide anion in a subject.
  • fluorescent proteins that bind halide anions include Yellow Fluorescent Protein (YFP; SEQ ID NO: 149) and mutants thereof.
  • the fluorescent protein comprises a mutation that alters the interaction of the mutant with a bound halide anion compared to YFP.
  • the fluorescent protein comprises 1 halide anion binding site.
  • the fluorescent protein comprises at least 2, 3, 4, or 5 halide anion binding sites.
  • at least one amino acid of the YFP has been substituted with a cysteine.
  • YFP is a non-limiting reference protein respect to fluorescence proteins.
  • a polypeptide of the present disclosure comprises (a) amino acids in a sequence that is preferably (i) at least about 10%, 11%, 12%, 13%, 14%, or 15%, and (ii) less than about 75%, 70%, 65%, 60%, 55%, 50%, 45%, 44%, 43%, 42%, 41%, 40%, 39%, 38%, 37%, 36%, or 35% identical to YFP;
  • the fluorescent polypeptide comprises a ⁇ -barrel.
  • the ⁇ -barrel comprises 9, 10, 11, or 12 ⁇ -strands.
  • the fluorescent protein comprises a cysteine within the first ⁇ -strand ⁇ ), the second ⁇ -strand ( ⁇ 2 ), the third ⁇ -strand ( ⁇ 3 ), the fourth ⁇ -strand ( ⁇ 4 ), the fifth ⁇ -strand (fe), the sixth ⁇ -strand ( ⁇ 6 ), the seventh ⁇ -strand ( ⁇ 7 ), the eighth ⁇ -strand ( ⁇ 8 ), the ninth ⁇ -strand 9), the tenth ⁇ - strand ( ⁇ 10 ), or the eleventh ⁇ -strand ( ⁇ ) of a YFP.
  • the polypeptide comprises (i) 1, 2, or 3 amino acid substitutions between ⁇ and ⁇ 2; (ii) 1, 2, or 3 amino acid substitutions between ⁇ 2 and ⁇ 3; (iii) 1, 2, or 3 amino acid substitutions between the ⁇ 3 and ⁇ 4; (iv) 1, 2, or 3 amino acid substitutions between the ⁇ 4 and ⁇ 5; (v) 1, 2, or 3 amino acid substitutions between ⁇ 5 and ⁇ 6; (vi) 1, 2, or 3 amino acid substitutions between ⁇ 6 and ⁇ 7; (vii) 1, 2, or 3 amino acid substitutions between the ⁇ 7 and ⁇ 8; (viii) 1, 2, or 3, amino acid substitutions between ⁇ 8 and ⁇ 9; (ix) 1, 2, or 3 amino acid substitutions between the ⁇ 9 and ⁇ ; and/or (x) 1, 2, or 3 amino acid substitutions between ⁇ and ⁇ 1.
  • the 1 or more substitutions comprise a substitution with cysteine.
  • the cysteine follows ⁇ in the amino acid sequence
  • Alpha-helical and ⁇ -strand segments assignments are calculated from a three- dimensional protein structure as follows, and as described in C.A.F. Andersen, B. Rost, 2003, Structural Bioinformatics, 341-363, P.E. Bourne, ed., Wiley, the entire content of which is incorporated herein by reference.
  • the backbone trace angle, r is calculated, defined as the dihedral angle between the four successive C a atom positions of residues in the linear protein sequence i, i+l, i+2, i+3. These values are calculated for all residues.
  • Second, the residues that form backbone hydrogen bonds with each other are recorded.
  • a hydrogen bond is scored if the distance between the backbone amide nitrogen and carbonyl oxygen of two different residues in the protein is calculated to be 2.5A or less, and if the calculated angle between the nitrogen, its amide proton, and the carbonyl is greater than 120°.
  • a residue is deemed to be in an a-helix, if 35 ⁇ ⁇ 65 , and it makes a backbone hydrogen bond with its ⁇ ' +4 ⁇ neighbor in the linear amino acid sequence. It is deemed to be in a ⁇ -strand, if the absolute t value falls in the interval 120 ⁇ ⁇ ⁇ 180 and if it makes at least one hydrogen bond with another residue with the same rvalue range.
  • Alpha-helical segments comprise at least four residues; ⁇ -strand residues comprise at least three residues.
  • a biosensor comprises a modified YFP polypeptide having an amino acid substitution compared to its naturally occurring counterpart, such that the polypeptide has a cysteine at position 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 101, 102, 103, 104,
  • a biosensor comprises a modified YFP.
  • the modified YFP may comprise one or more, or any combination of the following substitutions compared to its naturally occurring counterpart: E17X, E32X, T43X, F64X, G65X, L68X, Q69X, A72X, H77X, K79X, R80X, E95X, R109X, R122X, D133X, H148X, N149X, V163X, N164X, D173X, Y182X, Q183X, Y203X, Q204X, L221X, and H231X, where X is any amino acid or is an amino acid that results in a conservative substitution. In some embodiments X is cysteine.
  • the modified YFP comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 of the following substitutions: F64L, G65T, L68V, Q69T, A72S, K79R, R80Q, H148Q, H148G, V163A, H231L, H148Q, or Q183A, wherein each YFP amino acid position is numbered as in SEQ ID NO: 150.
  • the fluorescent protein comprises an R at the 96 position, a Y at the 203 position, a S at the 205 position, and an E at the 222 position compared to YFP, wherein each YFP amino acid position is numbered as in SEQ ID NO: 150.
  • C1BP1 (also referred to as laYFP) comprises L68V, K79R, R80Q, H2131L compared to YFP (as numbered in SEQ ID NO: 150).
  • biosensor for a ligand comprising (a) a polypeptide; (b) a directly responsive fluorophore, wherein binding of a ligand to the directly responsive fluorophore causes a change in signaling by the directly responsive fluorophore (i.e., the fluorophore is chemoresponsive); and (b) an indirectly responsive fluorophore.
  • the directly responsive fluorophore may be a donor fluorophore or an acceptor fluorophore.
  • the directly responsive fluorophore is a donor fluorophore and the indirectly responsive fluorophore is an acceptor fluorophore.
  • the directly responsive fluorophore is an acceptor fluorophore and the indirectly responsive fluorophore is a donor fluorophore.
  • ngmFRET occurs between the donor fluorophore and the acceptor fluorophore when the donor fluorophore is contacted with radiation comprising the excitation wavelength of the donor fluorophore.
  • any polypeptide may be used to link a directly responsive fluorophore (e.g., a chemoresponsive fluorophore) with an indirectly responsive fluorophore.
  • a directly responsive fluorophore e.g., a chemoresponsive fluorophore
  • an indirectly responsive fluorophore e.g., a directly responsive fluorophore
  • an adaptor protein e.g., chemoresponsive fluorophore
  • the polypeptide comprises a stretch of at least 2, 3, 4, 5, 6, 47, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, or 500 amino acids.
  • the polypeptide comprises a stretch of at least 50, 60, 70, 80, 90, or 100 amino acids in a sequence that is at least about 85%, 90%, 95%, or 99% identical to the amino acid sequence of ecTRX (SEQ ID NO: 151).
  • the polypeptide comprises at least 1, 2, or 3 thiol groups; at least 1, 2, or 3 cysteines that each comprise a sulfhydryl group; at least 1, 2, or 3 primary amine groups; or at least 1, 2, or 3 lysines that each comprise a primary amine.
  • the polypeptide comprises a mutant of ecTRX comprising a D3X, K4X, K19X, D27X, K37X, K53X, K58X, K70X, R74X, K83X, K91X, K97X, or K101X mutation, or any combination thereof, wherein X is any amino acid, and wherein each ecTRX amino acid position is numbered as in SEQ ID NO: 151.
  • the polypeptide comprises a mutant of ecTRX comprising a
  • each ecTRX amino acid position is numbered as in SEQ ID NO: 151.
  • the polypeptide comprises a mutant of ecTRX that does not comprise a lysine.
  • the polypeptide further comprises a hexahistidine tag.
  • the polypeptide comprises amino acids in the sequence of any one of SEQ ID NOS:24-41 or 151.
  • the biosensor is a pH biosensor and the ligand comprises a hydrogen ion.
  • the directly responsive fluorophore is pH-sensitive.
  • the fully excited emission intensity of the directly responsive fluorophore is different at a pH less than about 7.0 compared to a pH of 7.5.
  • the directly responsive fluorophore transitions from a monoanion to a dianion at a pH that is less than 7.0 in an aqueous solution.
  • the directly responsive fluorophore comprises a pH-sensitive fluorophore comprising fluorescein or a derivative thereof.
  • aspects of the present subject matter provide a method of assaying for a ligand in a sample.
  • the method may include contacting the sample with a biosensor disclosed herein under conditions such that the ligand-binding protein of the biosensor binds to the ligand if ligand is present in the sample.
  • the method also comprises detecting (i) whether a signal is produced by a reporter group of the biosensor; and/or (ii) the a signal produced by a reporter group of the biosensor.
  • a reporter group of the biosensor is fluorescent, and the method further comprises contacting the reporter group with
  • electromagnetic radiation having a wavelength that comprises a wavelength within the band of excitation wavelengths of the reporter group.
  • the method further comprises (i) comparing a signal produced by a reporter group of the biosensor when the biosensor is contacted with the sample with a signal produced by a control sample containing a known quantity of ligand; and (ii) detecting the presence or absence of ligand in the sample based on this comparison.
  • the method further comprises (i) comparing a signal produced by a reporter group of the biosensor when the biosensor is contacted with the sample with signals produced by a series of control samples containing known quantities of ligand; and (ii) determining the quantity of ligand in the sample based on this comparison.
  • the series of control samples comprises at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 control samples, and wherein each control sample comprises a different quantity of ligand.
  • the method further comprises determining the concentration of a ligand in a sample, wherein determining the concentration of the ligand in the sample comprises comparing the signal to a standard hyperbolic ligand binding curve to determine the concentration of the ligand in the test sample, wherein the standard hyperbolic ligand binding curve is prepared by measuring the signal produced by the reporter group of the biosensor when the biosensor is contacted with control samples containing known concentrations of ligand.
  • the method comprises (i) measuring a ratiometric change (AR) and/or an intensity change ( ⁇ ) of a signal produced by the reporter group.
  • the method includes quantitating the level of ligand present in the sample.
  • aspects of the present subject matter also provide a method of assaying for multiple ligands in a sample, wherein the multiple ligands comprise a first ligand and a second ligand.
  • Such a method may include contacting the sample with (i) a first biosensor a first ligand provided herein and (ii) a second biosensor for the second ligand, under conditions such that the ligand-binding protein of the first biosensor binds to the first ligand, if the first ligand is present in the sample, and detecting (i) a signal produced by a reporter group of the first biosensor, or (ii) whether a signal is produced by a reporter group of the first biosensor.
  • the second biosensor is also a biosensor provided herein, and the second biosensor is contacted with the second ligand under conditions such that the ligand-binding protein of the second biosensor binds to the second ligand it is present in the sample.
  • the method may further comprise detecting (i) a signal produced by a reporter group of the second biosensor, or (ii) whether a signal is produced by a reporter group of the second biosensor.
  • the signal produced by the reporter group of the first biosensor is different than the signal produced by the reporter group of the second biosensor.
  • the reporter group of the first biosensor and the reporter group of the second biosensor are each fluorescent, and the peak emission wavelength of the reporter group of the first biosensor is at least about 10, 25, 50, 75, or 100 run greater or lower than the peak emission wavelength of the reporter group of the second biosensor.
  • biosensors include biosensors with ligand-binding proteins comprising a GGBP (e.g., an E. coli GGBP) or a derivative or mutant thereof; (ii) an E. coli arabinose binding protein (e.g., an E. coli arabinose binding protein) or a derivative or mutant thereof; (iii) a dipeptide binding protein (e.g., an E. coli dipeptide binding protein) or a derivative or mutant thereof; (iv) a histidine binding protein (e.g., an E. coli, histidine binding protein) or a derivative or mutant thereof; (v) a ribose binding protein (e.g., an E.
  • a GGBP e.g., an E. coli GGBP
  • an E. coli arabinose binding protein e.g., an E. coli arabinose binding protein
  • a dipeptide binding protein e.g., an E. coli dipeptide binding protein
  • coli ribose binding protein or a derivative or mutant thereof;
  • a sulfate binding protein e.g., an E. coli sulfate binding protein
  • a maltose binding protein e.g., an E. coli maltose binding protein
  • a glutamine binding protein e.g., an E. coli glutamine binding protein
  • a glutamate/aspartate binding protein e.g., an E. coli glutamate/aspartate binding protein
  • a derivative or mutant thereof a phosphate binding protein (e.g., an E.
  • the second biosensor comprises an E. coli GGBP having a Y10A, Y10C, D14C, DMA, D14Q, D14N, D14S, D14T, D14E, D14H, D14L, D14Y, D14F, N15C, F16L, F16A, F16C, F16Y, N91C, N91A, K92A, K92C, E93C, S112A, S115A, E149C,
  • coli arabinose binding protein having a D257C, F23C, K301C, L253C, or L298C mutation (e.g., comprising 1, 2, 3, 4, or 5 of these mutations) (see, e.g., U.S. Patent Application Publication No. 2004/0118681, the entire contents of which are incorporated herein by reference) (see, e.g., U.S. Patent Application Publication No. 2004/0118681 , the entire contents of which are incorporated herein by reference); (iii) an E.
  • coli dipeptide binding protein having a D450C, K394C, R141C, SI 11C, T44C, or W315C mutation (e.g., comprising 1, 2, 3, 4, 5 or 6 of these mutations) (see, e.g., U.S. Patent Application Publication No. 2004/0118681, the entire contents of which are incorporated herein by reference); (iv) an E. coli, histidine binding protein having a E167C, K229C, V163C, Y230C, F231C, Y88C mutation ⁇ e.g., comprising 1, 2, 3, 4, 5 or 6 of these mutations) ⁇ see, e.g., U.S. Patent Application Publication No.
  • an E. coli ribose binding protein having a T135C, D165C, E192C, A234C, L236C, or L265C mutation ⁇ e.g., comprising 1, 2, 3, 4, 5 or 6 of these mutations) ⁇ see, e.g., U.S. Patent Application Publication No. 2004/0118681, the entire contents of which are incorporated herein by reference);
  • an E. coli ribose binding protein having a T135C, D165C, E192C, A234C, L236C, or L265C mutation ⁇ e.g., comprising 1, 2, 3, 4, 5 or 6 of these mutations) ⁇ see, e.g., U.S. Patent Application Publication No. 2004/0118681, the entire contents of which are incorporated herein by reference);
  • an E. coli ribose binding protein having a T135C, D165C, E192C, A234C, L236C, or L265
  • coli sulfate binding protein having a L65C, N70C, Q294C, R134C, W290C, or Y67C mutation ⁇ e.g., comprising 1, 2, 3, 4, 5 or 6 of these mutations) ⁇ see, e.g., U.S. Patent Application Publication No. 2004/0118681 the entire content of which is incorporated herein by reference); (vii) an E. coli maltose binding protein having a D95C, F92C, E163C, G174C, D29C, or S233C mutation ⁇ e.g., comprising 1, 2, 3, 4, 5 or 6 of these mutations) ⁇ see, e.g., U.S. Patent Application Publication No.
  • coli glutamate/aspartate binding protein having a A207C, A210C, E119C, F126C, F131C, F270C, G211C, K268C, Q123C, or T129C mutation ⁇ e.g., comprising 1, 2, 3, 4, 5 or more of these mutations) ⁇ see, e.g., U.S. Patent Application Publication No. 2004/0118681 the entire content of which is incorporated herein by reference); (x) an E. coli phosphate binding protein having a A225C, N223C, N226C, S164C, or S39C mutation ⁇ e.g., comprising 1, 2, 3, 4, or 5 of these mutations) ⁇ see, e.g., U.S.
  • Patent Application Publication No. 2004/0118681 the entire content of which is incorporated herein by reference); or (xi) a Haemophilus influenza (H. influenzae) iron binding protein having a E203C, K202C, K85C, or V287C mutation ⁇ e.g., comprising 1, 2, 3, or 4 of these mutations) ⁇ see, e.g., U.S. Patent Application Publication No. 2004/0118681 the entire content of which is incorporated herein by reference).
  • the sample is suspected of comprising a ligand, such as a ligand disclosed, described, or otherwise mentioned herein.
  • a sample may comprise a reaction product, a buffer, and/or a solvent.
  • the solvent is an aqueous solvent.
  • the solvent comprises a non-polar solvent, a polar aprotic solvent, and/or a polar protic solvent.
  • a sample may comprise water, liquid ammonia, liquid sulfur dioxide, sulfuryl chloride, sulfuryl chloride fluoride, phosphoryl chloride, dinitrogen tetroxide, antimony trichloride, bromine pentafluoride, hydrogen fluoride, dimethyl sulfoxide, hexane, benzene, toluene, 1,4-dioxane, chlorogoim, diethyl ether, dichloromethane, N-methylpyrrolidone, tetrahydrofuran, ethyl acetate, acetone, dimethylfoimamide, acetonitrile, tormic acid, n- butanol, isopropanol, nitromethane, ethanol, methanol, and/or acetic acid.
  • a sample comprises a Newtonian liquid, a shear thickening liquid, a shear thinning liquid, a thixotropic liquid, a rheopectic liquid, or a Bingham plastic.
  • a sample has a dynamic viscosity of at least about 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, or 2 pascal-seconds (Pa-s) or less than about 2, 1.5, 1.4, 1.3, 1.2, 1.1, 1, 0.9, 0.8, 0.7, 0.6, 0.5 Pa s; and/or a kinematic viscosity of at least about 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, or 2 centistokes (cSt) or less than about 2, 1.5, 1.4, 1.3, 1.2, 1.1, 1, 0.9, 0.8, 0.7, 0.6, 0.5 c
  • the sample comprises a biological sample.
  • the sample may comprise, e.g., a clinical sample (i.e., a sample collected in a clinical or veterinary setting, e.g., by or at the request or supervision or direction of a doctor, nurse, aid worker, or medic) and/or a physiological sample (a sample collected from an organism, e.g., a mammal such as a human).
  • a clinical sample i.e., a sample collected in a clinical or veterinary setting, e.g., by or at the request or supervision or direction of a doctor, nurse, aid worker, or medic
  • a physiological sample a sample collected from an organism, e.g., a mammal such as a human.
  • the biological sample comprises or has been provided or obtained from a skin surface or a mucosal surface.
  • the biological sample comprises a biological fluid.
  • Non-limiting examples of biological fluids include sweat, tear fluid, blood, serum, plasma, interstitial fluid, amniotic fluid, sputum, gastric lavage, skin oil, milk, fecal matter, emesis, bile, saliva, urine, mucous, semen, lymph, spinal fluid, synovial fluid, a cell lysate, venom, hemolymph, and fluid obtained from plants such as the fluid transported in xylem cells or phloem sieve tube elements of a plant (e.g. sap).
  • the present subject matter also provides biosensors, methods, compositions, and devices useful for measuring the level of a ligand within a liquid solution or suspension or composition comprising cultured cells or tissue or a supernatant of such a solution or suspension, e.g., a sample of conditioned media or a sample of growth media in which a population of cells was cultured.
  • the sample is within a culture (e.g., inserted into a bioreactor) or provided from a media, culture, or reaction, e.g., in a bioreactor.
  • the sample may be within or provided from a fermenter such as a culture or culture supernatant from a fermentation reaction (e.g., an ongoing fermentation).
  • Bioreactors include devices or systems that support a biologically active environment.
  • a bioreactor may comprise a vessel in which a chemical process is carried out which involves organisms or biochemically active substances derived from such organisms. Such a process can either be aerobic or anaerobic.
  • Organisms growing in bioreactors may be, e.g.,
  • Suspension bioreactors can use a wider variety of organisms, since special attachment surfaces are not needed, and can operate at much larger scale than immobilized cultures. However, in a continuously operated process the organisms will be removed from the reactor with the effluent. Immobilization is a general term describing a wide variety of cell or particle attachment or entrapment. It can be applied to basically all types of biocatalysis including enzymes, cellular organelles, and cells (e.g., animal cells, plant cells, fungal cells, and bacterial cells).
  • a bioreactor may also refer to a device or system meant to grow cells or tissues in the context of cell culture. The interrogation and/or monitoring of ligand levels in such samples permits the evaluation of the status of growth of the cells or production of secreted products by the cells to inform harvest or feeding or other modification of the culture.
  • aspects of the present subject matter relate to the use of methods and biosensors provided herein to detect contamination.
  • the sample comprises an environmental sample.
  • an environmental sample comprises a solute obtained from a biological composition, such as bone, nail, hair, shell, or cartilage.
  • an environmental sample comprises a solute obtained from an environmental substance and/or an environmental surface.
  • the solute may be dissolved/obtained from the environmental substance and/or an environmental surface using an aqueous or nonaqueous solution.
  • an aqueous may optionally comprise a nonaqueous solvent (e.g., mixed with an aqueous solvent).
  • Non-limiting examples of environmental substances include rock, soil, clay, sand, meteorites, asteroids, dust, plastic, metal, mineral, fossils, sediment, and wood.
  • Non-limiting examples of environmental surfaces include the surface of a vehicle such as a civilian vehicle (e.g., a satellite, a bike, a rocket, an automobile, a truck, a motorcycle, a yacht, a bus, or a plane) or a military vehicle (e.g., a tank, an armored personnel carrier, a transport truck, a jeep, a mobile artillery unit, a mobile antiaircraft unit, a minesweeper, a Mine-Resistant Ambush Protected (MRAP) vehicle, a lightweight tactical all-terrain vehicle, a high mobility multipurpose wheeled vehicle, a mobile multiple rocket launch system, an amphibious landing vehicle, a ship, a hovercraft, a submarine, a transport plane, a fighter jet, a helicopter, a rocket, or an Unmanned A
  • the sample comprises an environmental fluid.
  • environmental fluids include marine water, well water, drinking well water, water at the bottom of well dug for petroleum extraction or exploration, melted ice water, pond water, aquarium water, pool water, lake water, mud, stream water, river water, brook water, waste water, treated waste water, reservoir water, rain water, and ground water.
  • waste water comprises sewage water, septic tank water, agricultural runoff, water from an area in which chemical or oil spill has or is suspected of having occurred (e.g., an oil spill into a marine environment), water from an area where a radiation leak has or is suspected of having occurred (e.g., coolant from a nuclear reactor), water within the plumbing of a building, water within or exiting a research facility, and/or water within or exiting a manufacturing facility such as a factory.
  • chemical or oil spill e.g., an oil spill into a marine environment
  • water from an area where a radiation leak has or is suspected of having occurred e.g., coolant from a nuclear reactor
  • water within the plumbing of a building e.g., water within or exiting a research facility, and/or water within or exiting a manufacturing facility such as a factory.
  • test e.g., a test other than a method or assay provided herein
  • occurrence e.g., that is likely to or that may cause the event such as an emergency, leak, accident, flood, earthquake, storm, fire, malfunction, sunk vessel, or crash
  • report e.g., by a witness, informant, or observer
  • the sample comprises a food or beverage additive and/or a food or beverage composition.
  • the food or beverage composition comprises a fermented composition.
  • the sample comprises a fluid obtained from a food composition.
  • the sample may comprise a solute dissolved from a food composition.
  • a solute is or has been dissolved from a food composition with an aqueous or nonaqueous solution.
  • an aqueous solution may optionally comprise a nonaqueous solvent.
  • a sample comprises a food composition in semisolid or liquid form.
  • a sample is a food engineering process (e.g., obtained from a food design, storage, transport, or production process or from equipment intended to process, transport, or store food).
  • a food composition may comprise, e.g., a plant or a composition isolated from a plant, and/or an animal or a composition isolated from an animal.
  • a sample comprises a beverage composition.
  • Non-limiting examples of beverage compositions include soft drinks, fountain beverages, water, coffee, tea, milk, dairy- based beverages, soy-based beverages (e.g., soy milk), almond-based beverages (e.g., almond milk), vegetable juice, fruit juice, fruit juice-flavored drinks, energy drinks, sports and fitness drinks, alcoholic products, and beverages comprising any combination thereof.
  • beverage compositions comprising water include purified water (e.g., filtered water, distilled water, or water purified by reverse osmosis), flavored water, mineral water, spring water, sparkling water, tonic water, and any combination thereof.
  • the sample comprises alcohol.
  • Non-limiting examples of such samples include samples comprising or obtained/provided from beer, malt beverages, liqueur, wine, spirits, and any combination thereof.
  • a sample comprises a nutritional or supplement composition.
  • the nutritional or supplement composition comprises an omega-3 fatty acid, a vitamin, a mineral, a protein powder, or a meal supplement.
  • a biosensor is implanted in a subject's body.
  • a biosensor may be implanted in a subject's blood vessel, vein, eye, natural or artificial pancreas, alimentary canal, stomach, intestine, esophagus, or skin (e.g., within the skin or under the skin).
  • the biosensor is configured within or on the surface of a contact lens.
  • the biosensor is configured to be implanted in or under the skin.
  • the biosensor is implanted in a subject with an optode and/or a microbead.
  • the biosensor generates a signal transdermally.
  • aspects of the present subject matter provide a method for assaying the level of ligand in a subject.
  • the method may comprise contacting a biological sample from the subject with a biosensor for ligand under conditions such that the biosensor binds to ligand present in the biological sample.
  • the biosensor comprises reporter group that is attached to a ligand binding protein, and binding of ligand to a ligand-binding domain of the ligand binding protein causes a change in signaling by the reporter group.
  • the subject has or is suspected of having a disease or disorder, such as abnormal kidney function, abnormal adrenal gland function, diabetes, hypochloremia, bromism, hypothyroidism, hyperthyroidism, cretinism, depression, fatigue, obesity, a low basal body temperature, a goiter, a fibrocystic breast change, lactic acidosis, septic shock, carbon monoxide poisoning, asthma, a lung disease, respiratory insufficiency, Chronic Obstructive Pulmonary Disease (COPD), regional hypoperfusion, ischemia, severe anemia, cardiac arrest, heart failure, a tissue injury, thrombosis, or a metabolic disorder, diarrhea, shock, ethylene glycol poisoning, methanol poisoning, diabetic ketoacidosis, hypertension, Cushing syndrome, liver failure, cancer, or an infection.
  • a disease or disorder such as abnormal kidney function, abnormal adrenal gland function, diabetes, hypochloremia, bromism, hypothyroidism, hyperthyroidism, cretinism,
  • tissue with respect to a subject's condition (e.g., disease or injury) means that the subject has at least one symptom or test (e.g., a test other than an assay or method provided herein) that is consistent with the condition.
  • a symptom or test e.g., a test other than an assay or method provided herein
  • the biological sample comprises blood, plasma, serum, sweat, tear fluid, or urine.
  • the biological sample is present in or on the surface of the subject.
  • the biosensor is applied onto or inserted into the subject.
  • the biosensor may be tattooed into the subject or is in or on a device that is implanted into the subject.
  • the biosensor may be present in or on a contact lens that is worn by the subject.
  • the present subject matter includes a method for monitoring the level of a ligand, comprising periodically or continuously detecting the level of the ligand, wherein detecting the level of the ligand comprises (a) providing or obtaining a sample; (b) contacting the sample with a biosensor for the ligand under conditions such that the ligand-binding protein of the biosensor binds to the ligand, and (c) detecting a signal produced by the biosensor.
  • aspects of the present subject matter also provide a method for monitoring the level of a ligand in a subject, comprising periodically detecting the level of the ligand in the subject.
  • Detecting the level of the ligand in the subject may comprise (a) providing or obtaining a biological sample from the subject; (b) contacting the biological sample with a biosensor for the ligand provided herein under conditions such that the ligand-binding protein of the biosensor binds to the ligand, if the ligand is present in the biological sample, and (c) detecting (i) a signal produced by a reporter group of the biosensor, or (ii) whether a signal is produced by a reporter group of the biosensor.
  • the level of the ligand may be detected, e.g., at least once every 1, 2, 3, 6, or 12 hours, at least once every 1, 2, 3, or 4 days, at least once every 1, 2, or three weeks, or at least once every 1, 2, 3, 4, 6, or 12 months.
  • the present subject matter also provides a method for monitoring the level of a ligand in a subject.
  • the method comprises (a) administering a biosensor provided herein or a device comprising a biosensor provided herein to the subject, wherein after administration the biosensor is in contact with a bodily fluid or surface that typically comprises the ligand, and (b) detecting (i) a signal produced by a reporter group of the biosensor continuously or repeatedly at intervals less than about 30 minutes (m), 15m, 10m, 5m, lm, 30 seconds (s), 15s, 10s, 5s, Is, 0.1s, 0.001s, 0.0001s, or 0.00001 apart, and/or (ii) whether a signal is produced by a reporter group of the biosensor continuously or repeatedly at intervals less than about 30m, 15m, 10m, 5m, lm, 30s, 15s, 10s, 5s, Is, 0.1s, 0.001s, 0.0001s, or O.OOOOlapart.
  • Non-limiting aspects of continuously monitoring ligand levels are described in Weidemaier et al. (2011) Biosensors and Bioelectronics 26, 4117-4123 and Judge et al.
  • composition comprising a purified thermostable, ligand- binding fluorescently-responsive sensor protein and a solid substrate, e.g., a particle, a bead such as a magnetic bead, or a planar surface such as a chip or slide, wherein the sensor protein is immobilized onto the solid substrate.
  • a solid substrate solid substrate comprises a cyclic olefin copolymer.
  • thermostable ligand sensor protein is one in which the activity (ligand binding) is retained after exposure to relatively high temperatures.
  • the ligand sensor protein comprises a mid-point thermal melt transition greater than 50°C, greater than 60°C, greater than 70°C, greater than 80°C, greater than 90°C, or greater than 100°C.
  • the sensor protein contains a single cysteine residue.
  • the single cysteine residue is located in a site of the ligand-binding protein, where it responds to ligand binding.
  • the protein comprises the amino acid sequence of SEQ ID NO: 16 (ttGGBP.17C0.bZif) and 19 (ttGGBP.182C0.bZif), and in some examples, the single cysteine is conjugated to Badan, Acrylodan, or a derivative thereof.
  • the derivative comprises a replacement of the two-ring naphthalene of Acrylodan or Badan with a three-ring anthracene, a fluorene, or a styrene.
  • a reporter group is covalently bound to the single cysteine.
  • the solid substrate comprises a plurality of sensor proteins, each of which comprises a different dissociation constant (Kd) for ligand, e.g., for detecting and quantifying ligand levels across many ranges of concentrations.
  • Kd dissociation constant
  • the present subject matter also includes a composition comprising purified ligand sensor protein with less than 65% identity and greater than 27% identity (e.g., 44-48% sequence identity) to any ligand-binding protein disclosed herein, wherein the sensor protein comprises a single cysteine residue, such that the sensor protein is immobilized onto the solid substrate.
  • a reporter group is covalently bound to the single cysteine.
  • solid substrate comprises a plurality of sensor proteins, each of which comprises a different dissociation constant (Kd) for ligand for sensing over a wide range or ranges of ligand concentrations.
  • Kd dissociation constant
  • a method of detecting the presence of or the quantity of ligand in a test sample is carried out using the following steps: contacting the test sample with the biosensor or sensor protein/solid support construct to yield a complex of ligand and the ligand-binding protein or biosensor protein; contacting the complex with an excitation light; measuring an emission intensity of the reporter group from at least two wavelengths;
  • test sample may be obtained from a variety of sources.
  • the test sample may be selected from a bodily fluid, a food, a beverage, or a bioreactor culture broth.
  • the testing method may be carried out in vivo, e.g., using an implantable device or dermal patch, or ex vivo.
  • the subject to be tested is a mammal, e.g., a primate (such as a human, a monkey, a chimpanzee, or a gorilla), a fish, a bird, a reptile, an amphibian, or an arthropod.
  • a primate such as a human, a monkey, a chimpanzee, or a gorilla
  • fish such as a human, a monkey, a chimpanzee, or a gorilla
  • a fish such as a human, a monkey, a chimpanzee, or a gorilla
  • a bird such as a bird, a reptile, an amphibian, or an arthropod.
  • an arthropod such as a human, a monkey, a chimpanzee, or a gorilla
  • the subject is a fish, a cow, a pig, a camel, a llama, a horse, a race horse, a work horse, a goat, a rabbit, a sheep, a hamster, a guinea pig, a cat, a wolf, a dog (e.g., a pet dog, a work dog, a police dog, or a military dog), a rat, a mouse, a seal, a whale, a manatee, a lizard, a snake, a chicken, a goose, a swan, a duck, or a penguin.
  • a dog e.g., a pet dog, a work dog, a police dog, or a military dog
  • a rat e.g., a mouse, a seal, a whale, a manatee, a lizard, a snake, a chicken, a goose, a swan, a duck,
  • the ligand comprises a halide anion and the ligand-binding protein comprises a fluorescent protein.
  • Aspects of the present subject matter provide a method for detecting the level of a halide anion in a sample, comprising contacting the sample with a biosensor for a halide anion under conditions such that the biosensor binds to a halide anion present in the sample.
  • the biosensor comprises a halide anion-binding fluorescent protein, and binding of the halide anion to a halide anion-binding domain of the fluorescent protein causes a change in signaling by the fluorescent protein.
  • the sample is an environmental sample. In a non-limiting example, the sample comprises treated wastewater or drinking water.
  • aspects of the present subject matter further provides a method for assaying the level of chloride in a subject, comprising contacting a biological sample from the subject with a biosensor for chloride under conditions such that the biosensor binds to chloride present in the biological sample.
  • the biosensor may comprise, e.g., a chloride-binding fluorescent protein, and binding of chloride to a chloride-binding domain of the fluorescent protein causes a change in signaling by the fluorescent protein.
  • the subject has or is suspected of having hypochloremia.
  • the subject has or is suspected of having abnormal kidney or adrenal gland function.
  • the biological sample comprises blood, plasma, serum, sweat, tear fluid, or urine.
  • the method is performed as part of a battery of clinical testing.
  • Also provided is a method for assaying the level of iodide in a subject comprising contacting a biological sample from the subject with a biosensor for iodide under conditions such that the biosensor binds to iodide present in the biological sample, wherein the biosensor comprises an iodide-binding fluorescent protein. Binding of iodide to an iodide-binding domain of the fluorescent protein causes a change in signaling by the fluorescent protein.
  • the subject has or is suspected of having hypothyroidism
  • the biological sample comprises blood, plasma, serum, sweat, tear fluid, or urine.
  • the method is performed as part of a battery of clinical testing.
  • the present subject matter further includes a method for assaying the level of bromide in a subject, comprising contacting a biological sample from the subject with a biosensor for bromide under conditions such that the biosensor binds to bromide present in the biological sample, wherein the biosensor comprises a bromide-binding fluorescent protein. Upon binding of bromide to a bromide-binding domain of the fluorescent protein, the signal of the fluorescent protein changes.
  • the subject has or is suspected of having bromism.
  • the biological sample comprises blood, plasma, serum, sweat, tear fluid, or urine.
  • the method is performed as part of a battery of clinical testing. Exemplary Devices and Compositions Comprising Biosensors
  • Such devices may be, e.g., wearable, implantable, portable, or fixed.
  • the device is a nanoparticle or a microparticle comprising the biosensor.
  • devices include devices comprising a test strip, patch, plate, bead, or chip comprising a biosensor provided herein.
  • a device may comprise a desiccated biosensor.
  • the present subject matter also provides a contact lens or a skin patch comprising a biosensor provided herein.
  • the biosensor is throughout the contact lens or skin patch or within a particular region or zone of a contact lens or skin patch (e.g., in one or more shapes (e.g., a square, circle, or star), dots, lines, or zones, located at the periphery or a portion of the periphery of a contact lens or patch).
  • the skin patch comprises an adhesive that facilitates attachment of the patch to the surface of skin.
  • Devices provided herein may include a variety of structural compositions. For example, many polymers (including copolymers), and plastics may be used.
  • compositions useful in certain devices include glass, polystyrene, polypropylene, cyclic olefin copolymers, ethylene-norbornene copolymers, polyethylene, dextran, nylon, amylase, paper, a natural cellulose, a modified cellulose, a polyacrylamide, gabbros, gold, and magnetite (as well as combinations thereof).
  • the device comprises a hydrogel, a cryogel, or a soluble gel.
  • the biosensor may be incorporated into or onto the hydrogel, cryogel, or soluble gel.
  • the device comprises a matrix comprising nanopores, micropores, and/or macropores.
  • the surface of a device comprises a polymer.
  • the surface comprises the surface of a particle or a bead having a diameter of about 0.001-1, 0.001-0.1, 0.01-0.1, 0.001-0.01, 0.1-1, 0.1-0.5, or 0.01-0.5 centimeters (cm).
  • the particle comprises a nanoparticle or a microparticle.
  • Non-limiting examples of polymers include cyclic olefin copolymers, ethylene- norbornene copolymers, polylactic acid, polyglycolic acid, agarose, alginate, poly(lactide-co- glycolide), gelatin, collagen, agarose, natural and synthetic polysaccharides, polyamino acids, poly(lysine), polyesters, polyhydroxybutyrates, polyanhydrides, polyphosphazines, polyvinyl alcohol, polyalkylene oxide, polyethylene oxide, polyallylamines, polyacrylates, modified styrene polymers, poly(4-aminomethylstyrene), pluronic polyols, polyoxamers, polyuronic acid, polyvinylpyrrolidone, hydroxyethyl (meth)acrylate, polyolefins, polyurethane, polystyrene, ethylene/methacrylic acid copolymers, ethylene/methyl methacrylate copolymers, polyester
  • Non-limiting examples of temporary tattoo compositions for application to a subject's skin are discussed in U.S. Patent Application Publication No. 20090325221, published December 31, 2009, and U.S. Patent No. 6,428,797, the entire contents of each of which are incorporated herein by reference.
  • Biosensor disclosed herein may be incorporated into any temporary tattoo or other composition for application to the skin.
  • a temporary tattoo decal for application to a subject's skin and configured to detect the presence of a ligand may comprise, e.g., a base paper or plastic; a water-soluble slip layer applied to the base paper or plastic; a temporary tattoo applied to the water-soluble release layer on the base paper, wherein the temporary tattoo comprises a biosensor disclosed herein; an adhesive layer overlying the temporary tattoo; and a protective sheet overlying the adhesive layer.
  • the device comprises a plastic polymer comprising cyclic olefin copolymer (COC), such as e.g. TOP AS® COC.
  • COC cyclic olefin copolymer
  • TOP AS® COC cyclic olefin copolymer
  • Cyclic olefin copolymers are produced by chain copolymerization of cyclic monomers such as 8,9,10-trinorborn-2-ene (norbornene) or 1, 2,3,4 ,4a,5,8,8a-octahydro- l,4:5,8-dimethanonaphthalene (tetracyclododecene) with ethene (such as TOP AS Advanced Polymer's TOP AS, Mitsui Chemical's APEL), or by ring-opening metathesis polymerization of various cyclic monomers followed by hydrogenation (Japan Synthetic Rubber's ARTON, Zeon Chemical's Zeonex and Zeonor).
  • cyclic monomers such as 8,9,10-trinorborn-2-ene (norbornene) or 1, 2,3,4 ,4a,5,8,8a-octahydro- l,4:5,8-dimethanonaphthalene (tetracyclodode
  • the device is attached to a surface of a device or is not attached to a surface of the device (e.g., the biosensor is present loosely within the device as a component of a solution or powder).
  • a biosensor may be attached to a device via a variety or means, e.g., via attachment motif.
  • the attachment motif is attached to the N-terminus or the C- terminus of the biosensor.
  • the biosensor is linked to an attachment motif via a covalent bond.
  • the biosensor is linked to the attachment motif via a linker.
  • a non-limiting example of a linker is a polyglycine comprising 2, 3, 4, 5, or more glycines and optionally further comprising a serine.
  • the attachment motif comprises a polypeptide.
  • Non-limiting examples of polypeptides useful in attachment moieties include hexahistidine peptides, hexalysine peptides, zinc-finger domains (ZF-QNKs), and disulfide-containing truncated zinc fingers (pZifs).
  • An example of a hexalysine peptide comprises amino acids in the sequence of SEQ ID NO: 45
  • an example of a ZF-QNK comprises amino acids in the sequence of SEQ ID NO: 43
  • an example of a ⁇ comprises amino acids in the sequence of SEQ ID NO: 42.
  • the attachment motif comprises a polypeptide that binds to plastic or cellulose.
  • hexahistidine, hexalysine, ⁇ and QNK-ZF fusions enable FRSs to be immobilized onto chemically functionalized surfaces.
  • Non-limiting aspects of chemically functionalized surfaces are discussed in Biju, V. (2014) Chem Soc Rev, 43, 744-64 and McDonagh (2008) Chem Rev, 108, 400-422, the entire contents of which are incorporated herein by reference. Directed evolution methods have been used to develop peptides that bind directly to non-functionalized surfaces (Care, Bergquist and Sunna 2015 Trends Biotechnol, 33, 259-68; Baneyx 2007 Curr. Opin.
  • Inorganic material include noble metals (Hnilova 2012 Soft Matter, 8, 4327-4334), semi-conductors (Care et al. 2015 Trends Biotechnol, 33, 259-68), and fluorescent quantum dots(Medintz et al. 2005 Nat Mater, 4, 435-46; Lee et al. 2002 Science, 296, 892-5).
  • the attachment motif is attached to a device surface and/or within a matrix of the device.
  • a biosensor is attached to an attachment motif via a covalent bond and the attachment motif is attached to a device via a covalent bond.
  • covalent bonds include disulfide bonds, ester bonds, thioester bonds, amide bonds, and bonds that have been formed by click reactions.
  • Non- limiting examples of a click reaction include a reaction between an azide and an alkyne; an azide and an alkyne in the presence of Cu(I); an azide and a strained cyclooctyne; an azide and a dibenzylcyclooctyne, a difluorooctyne, or a biarylazacyclooctynone; a diaryl-strained- cyclooctyne and a 1,3-nitrone; an azide, a tetrazine, or a tetrazole and a strained alkene; an azide, a tetrazine, or a tretrazole and a oxanorbornadiene, a cyclooctene, or a trans- cycloalkene; a tetrazole and an alkene; or a tetrazole with an amino or styryl group
  • a surface of a device may be modified to contain a moiety (e.g. a reactive group) what facilitates the attachment of a biosensor and/or binds to the biosensor.
  • a moiety e.g. a reactive group
  • the biosensor is attached to a surface via a biotin-avidin interaction.
  • the device comprises a first region for receiving a sample and second a region that comprises the biosensor, wherein the first region is separated from the second region by a filter.
  • the filter is impermeable to compounds greater than about 1, 2, 3, 4, 5, 10, 50, 200, or 250 kiloDalton (kDa) in size.
  • the sample may comprise, e.g., a tube, such as a tube that is configured for centrifugation. When sample is placed into the first region and the device is centrifuged, then a portion of the sample comprising a ligand flows through the filter into the second region where the biosensor is contacted.
  • Non-limiting examples of devices provided herein include endoscopy probes and colonoscopy probes.
  • the device comprises an optode.
  • the optode comprises an optical fiber and a single biosensor or composite biosensor.
  • the single biosensor or composite biosensor is immobilized on the surface or at an end of the optical fiber.
  • the optode is configured for implantation into a subject. Alternatively or in addition, the optode is configured for insertion into a sample.
  • the devices provided herein may optionally comprise a biosensor panel, a composite sensor, a sensor array, and/or a composition comprising a plurality of biosensors.
  • a device comprises multiple ligand biosensors that detect a range of different ligand concentrations in a single sample and/or assay run (i.e., each biosensor has a different affinity for ligand).
  • Devices may provide spatial localization of multiple biosensors to provide the necessary addressability of different elements in a multi-sensor array comprising sensors that differ in their engineered affinities for coverage of a wide range of ligand concentrations, or sensors that each detects distinct analytes.
  • a biosensor panel comprising a plurality of biosensors, wherein the plurality of biosensors comprises at least one biosensor disclosed herein. In some embodiments, the plurality comprises at least about 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 biosensors.
  • the present subject matter also provides a composite sensor.
  • the composite sensor may comprise a sensor element, wherein the sensor element comprises 2 or more biosensors, wherein at least 1 of the 2 or more biosensors is a biosensor disclosed herein.
  • the biosensors are not spatially separated in the sensor element, e.g., the biosensors are mixed within a solution or on a surface of the sensor element.
  • the composite sensor comprises a plurality of sensor elements, wherein each sensor element of the plurality of sensor elements comprises 2 or more biosensors, wherein at least 1 of the 2 or more biosensors is a biosensor provided herein.
  • the plurality of sensor elements comprises at least about 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 sensor elements.
  • a sensor array comprising a plurality of biosensors of the present subject matter.
  • the sensor array may include, e.g., multichannel array or a multiplexed array.
  • the biosensors of the plurality of biosensors are spatially separated from each other.
  • the biosensors are arranged linearly or in a grid on a surface of the array.
  • composition comprising a plurality of biosensors including at least one biosensor disclosed herein.
  • a non-human mammal comprising a biosensor or device disclosed herein.
  • the present subject matter provides polynucleotides encoding any one of the polypeptides disclosed herein.
  • the polypeptides are also provided.
  • the polynucleotides are codon-optimized for expression in a desired host cell, such as bacterial cells (e.g., E. coli), yeast, insect cells, plant cells, algal cells, or mammalian cells.
  • the polypeptides provided herein include polypeptides comprising the amino acid sequence of any one of SEQ ID NOS: 1-41, 87-151, or 153-167.
  • the polynucleotides provided herein include polynucleotides encoding a polypeptide comprising the amino acid sequence of any one of SEQ ID NOS: 1-41, 87-151, or 153-167.
  • polypeptides and biosensors provided herein may be in a variety of forms, e.g., purified in solution, dried (e.g. lyophilized) such as in the form of a powder, and in the form of a crystal (e.g., a crystal suitable for x-ray crystallography).
  • a crystal e.g., a crystal suitable for x-ray crystallography.
  • expression vectors comprising a polynucleotide of the present subject matter and/or encoding a polypeptide disclosed herein.
  • Non-limiting examples of expression vectors include viral vectors and plasmid vectors.
  • an expression vector comprises nucleotides in the sequence set forth as any one of SEQ ID NOS: 46-86.
  • a polynucleotide encoding a ligand-binding protein and/or biosensor is operably linked to a promoter. The promoter may be expressed, e.g., in a prokaryotic and/or a eukaryotic cell.
  • the subject matter further includes an isolated cell comprising an expression vector provided herein.
  • the isolated cell may be, e.g., a bacterial cell, a yeast cell, an algal cell, a plant cell, an insect cell, or a mammalian cell.
  • a non-human multicellular organism such as a plant or an animal (e.g., an insect, a mammal, a worm, a fish, a bird, or a reptile) comprising an expression vector disclosed herein.
  • aspects of the present subject matter provide method of identifying a candidate ligand-binding protein for use in a biosensor, comprising: (a) selecting a first protein having a known amino acid sequence (seed sequence), wherein the first protein is a ligand binding protein; (b) identifying a second protein having an amino acid sequence (hit sequence) with at least 15% sequence identity to the seed sequence; (c) aligning the seed amino acid sequence and the hit sequence, and comparing the hit sequence with the seed sequence at positions of the seed sequence that correspond to at least 5 primary complementary surface (PCS) amino acids, wherein each of the at least 5 PCS amino acids has a hydrogen bond interaction or a van der Waals interaction with ligand when ligand is bound to the first protein; and (d) identifying the second protein to be a candidate ligand-binding protein if the hit sequence comprises at least 5 amino acids that are consistent with the PCS.
  • seed sequence seed sequence
  • hit sequence an amino acid sequence with at least 15% sequence identity to the seed sequence
  • PCS primary complementary surface
  • the present subject matter also includes a method for constructing a candidate biosensor, comprising: (a) providing a candidate ligand-binding protein; (b) generating a structure of the second protein; (c) identifying at least one putative allosteric, endosteric, or peristeric site of the second protein based on the structure; (d) mutating the second protein to substitute an amino acid at the at least one putative allosteric, endosteric, or peristeric site of the second protein with a cysteine; and (e) conjugating a fluorescent compound to the cysteine.
  • the structure comprises a homology model of the second protein generated using a structure of the first protein.
  • the structure comprises a structure experimentally determined by nuclear magnetic resonance spectroscopy or X-ray crystallography.
  • aspects of the present subject matter further provide a method for constructing a biosensor comprising a desired dissociation constant (Kd) for ligand, comprising: (a) providing an initial biosensor that does not comprise the desired Kd for ligand, wherein the initial biosensor is a biosensor provided herein; (b) mutating the initial biosensor to (i) alter a direct interaction in the PCS between the initial biosensor and bound ligand; (ii) manipulate the equilibrium between open and closed states of the initial biosensor; (iii) alter an interaction between the ligand-binding protein and the reporter group of the initial biosensor; or (iv) alter an indirect interaction that alters the geometry of the binding site of the biosensor, to produce a modified biosensor; and (c) selecting the modified biosensor if the modified biosensor comprises the desired Kd for ligand.
  • Kd dissociation constant
  • the reporter comprises Acrylodan, Badan, or a derivative thereof, and mutating the initial biosensor in (b) comprises altering an interaction between the ligand-binding protein and a carbonyl group of the Acrylodan, Badan, or derivative thereof.
  • the reporter group comprises Acrylodan, Badan, or a derivative thereof, and mutating the initial biosensor in (b) comprises altering an interaction between the ligand-binding protein and a naphthalene ring of the Acrylodan, Badan, or derivative thereof.
  • mutating the initial biosensor comprises introducing a substitution mutation into the initial biosensor.
  • the method further comprises immobilizing the affinity-tuned biosensor on a substrate.
  • the second protein comprises (i) amino acids in the sequence of any one of SEQ ID NOS: 1-41, 87-151, or 153-167; (ii) a stretch of amino acids in a sequence that is least about 95, 96, 97, 98, or 99% identical to the sequence of any one of SEQ ID NOS: 1-41, 87-151, or 153-167; (iii) a stretch of at least about 50, 100, 150, 200, 250, 300, 350, or 400 amino acids in a sequence that is at least about 95, 96, 97, 98, or 99% identical to a sequence within any one of SEQ ID NOS: 1-41, 87-151, or 153-167; or (iv) a stretch of at least about 50, 100, 150, 200, 250, 300, 350, or 400 amino acids in a sequence that is identical to a sequence within any one of SEQ ID NOS: 1-41, 87-151, or 153-167.
  • attaching the reporter group to the putative allosteric, endosteric, or peristeric site of the first protein comprises substituting a cysteine at the site with a cysteine.
  • the reporter group is conjugated to the cysteine.
  • attaching a reporter group to the corresponding amino acid of the second protein produces a functional biosensor.
  • aspects also provide method for constructing a biosensor, comprising (a) providing a ligand-binding protein; (b) identifying at least one putative allosteric, endosteric, or peristeric site of the ligand-binding based a structure of the ligand-binding protein; (c) mutating the ligand-binding protein to substitute an amino acid at the at least one putative allosteric, endosteric, or peristeric site of the second protein with a cysteine; (d) conjugating a donor fluorophore or an acceptor fluorophore to the cysteine to produce single labeled biosensor; (e) detecting whether there is a spectral shift or change in emission intensity of the single labeled biosensor upon ligand binding when the donor fluorophore or the acceptor fluorophore is fully excited; and (f) if a spectral shift or change in emission intensity is detected in (g), attaching a donor fluorophore to the second
  • the ligand-binding protein has been identified by (i) selecting a first protein having a known amino acid sequence (seed sequence), wherein the first protein is a ligand-binding protein; (ii) identifying a second protein having an amino acid sequence (hit sequence) with at least 15% sequence identity to the seed sequence; (iii) aligning the seed amino acid sequence and the hit sequence, and comparing the hit sequence with the seed sequence at positions of the seed sequence that correspond to at least 5 primary
  • the spectral shift may comprise, e.g., a monochromatic fluorescence intensity change or a dichromatic spectral shift.
  • Also provided is a method of converting a biosensor that shows a monochromatic response upon ligand binding into a biosensor with a dichromatic response upon ligand binding comprising (a) selecting a biosensor that exhibits a monochromatic response upon ligand binding, wherein the biosensor comprises a ligand-binding protein and a first reporter group; and (b) attaching a second reporter group to the biosensor, wherein the second reporter group has (i) an excitation spectrum that overlaps with the emission spectrum of the first reporter group; or (ii) an emission spectrum that overlaps with the excitation spectrum of the first reporter group.
  • the average distance between the first reporter group and the second reporter group changes by less than about 5, 4, 3, 2, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.05, or 0.01 angstroms (A) compared to when ligand is not bound to the ligand-binding protein.
  • aspects include a method of converting a biosensor that shows a monochromatic response upon ligand binding into a biosensor with a dichromatic response upon ligand binding, the method comprising (a) selecting a biosensor that exhibits a monochromatic response upon ligand binding, wherein the biosensor comprises a ligand-binding fluorescent protein; and (b) attaching an acceptor fluorophore or a donor fluorophore to the biosensor, wherein (i) the acceptor fluorophore has an excitation spectrum that overlaps with the emission spectrum of the fluorescent protein; or (ii) the donor fluorophore has an emission spectrum that overlaps with the excitation spectrum of the fluorescent protein.
  • Also provided is a method of increasing a dichromatic response of a biosensor to ligand binding comprising (a) selecting a biosensor that exhibits a dichromatic response upon ligand binding, wherein the biosensor comprises a ligand-binding protein and a first reporter group; and (b) attaching a second reporter group to the biosensor, wherein the second reporter group has (i) an excitation spectrum that overlaps with the emission spectrum of the first reporter group; or (ii) an emission spectrum that overlaps with the excitation spectrum of the first reporter group.
  • the selected first protein (e.g., the amino acid sequence thereof) may be novel or known. However, in many instances, the function of the first protein will not be known.
  • identifying a protein not previously known to have ligand binding activity may comprise a structurally assisted functional evaluation (SAFE) homolog search method comprising the following steps:
  • PCS primary complementary surface
  • the ligand-binding properties of each of the aligned sequence homologs is determined by measuring their compliance with the PCS sequence filter.
  • a "Hamming distance", H is assigned for each homolog, which specifies the degree of sequence identity of all the residues at the aligned PCS positions.
  • identifying a protein not previously known to have ligand binding activity may comprises the following steps:
  • step (2) using the list from step (1), deriving a search profile containing common sequence and/or structural motifs shared by the members of the list [e.g. by using computer programs such as MEME (Multiple Em for Motif Elicitation available at meme.sdsc.edu/meme/cgi-bin/meme.cgi) or BLAST];
  • MEME Multiple Em for Motif Elicitation available at meme.sdsc.edu/meme/cgi-bin/meme.cgi
  • BLAST BLAST
  • step (3) searching sequence/structural databases, using a derived search profile based on the common sequence or structural motif from step (2) as query (e.g., using computer programs such as BLAST, or MAST (Motif Alignment Search Tool available at meme.sdsc.edu/meme/cgi-bin/mast.cgi), and identifying a candidate sequence, wherein a sequence homology and/or structural similarity to a reference ligand binding protein is a predetermined percentage threshold;
  • the MEME suite of sequence analysis tools includes
  • the ClustalW alignment program may be, e.g., ClustalW alignment program version 2.1.
  • FIGS. 1A and B are cartoons showing fluorescent probes.
  • FIG. 1 A is a cartoon relating to indirect fluorescent responses. Fluorescent biosensors can be constructed by site- specifically attaching a fluorophore to a protein that undergoes a conformational change upon binding ligand (triangle) in a location between the two lobes of the protein (periplasmic binding protein or engineered derivative thereof), such that the shape and intensities of the fluorescent conjugate emission spectra changes.
  • FIG. IB is a cartoon relating to direct fluorescent responses. Fluorescent chemosensors based on fluorophores that interact directly with an analyte.
  • FIGS. 2A-C are graphs illustrating ratiometry. If the fluorescence emission spectrum changes shape in response to binding of an analyte, such as glucose, then the ratio of emission intensities at two appropriately chosen wavelengths reports on analyte
  • FIG. 2A In the absence of ligand, the emitted fluorescence color is predominantly blue, whereas the ligand complex fluoresces green. Arrows indicate the direction of change upon ligand addition.
  • FIG. 2B The ligand dependence of the absolute blue and green intensities.
  • FIG. 2C The ratio of the blue and green intensities reports enables ligand binding to be determined.
  • FIGS. 3A-D are graphs and diagrams showing three dominant factors that affect overall ligand-mediated change in fluorescence emission intensity between donor and acceptors in which one partner responds to ligand binding.
  • FIG. 3A Simplified Jablonski diagram illustrating radiative and non-radiative pathways in the donor and acceptor.
  • the donor excited state (£>*) is formed through illumination by the excitation source (wavy arrow) whereas the acceptor excited state (A*) is formed by resonance energy transfer (dashed arrow).
  • the fluorescence intensity is determined by the ratio of radiative decay (gray arrows) of the excited states (gray lines) to the ground state (black line) relative to all non- radiative processes (black arrows), and the resonance energy transfer rate, k t , from donor to acceptor.
  • FIG. 3B Inter-dipole geometry. Top, energy transfer efficiency
  • 3C Spectral overlap (gray area) between the donor fluorescence emission ( D I, blue) and acceptor fluorescence excitation ( A, black) spectra. This overlap increases with bathochromic or hypsochromic shifts of the donor emission (red arrow) and acceptor excitation (dotted blue arrow) spectra, respectively. Shifts in the opposite directions decreases spectral overlap.
  • FIGS. 4A-C are illustrations of the construction of ngmFRET pairs by combining a fluorophore that responds directly to ligand binding with a non-interacting, indirectly responsive partner.
  • FIG. 4A A glucose-binding protein in which a directly responsive partner is positioned in the vicinity of the glucose-binding site, and the indirectly responsive partner is fused as the protein C-terminus.
  • FIG. 4B The internally positioned fluorophore of Yellow Fluorescent Protein responds directly chloride binding by itself and regardless of the presence of any other fluorophore/partner. Its monochromatic response can be converted to a dichromatic one by positioning a second, indirectly responsive fluorophore on the surface of the protein.
  • FIG. 4C An adaptor protein such as E. coli thioredoxin can be used to position an directly responsive chemosensor next to an indirectly responsive partner, thereby converting a monochromatic into a dichromatic signal.
  • FIGS. 5A-E are cartoons of fusion constructs that enable site-specific labeling of cysteines at two independently addressable sites with distinct, thiol-reactive fluorophores.
  • FIG. 5A In the first labeling step, an unprotected single thiol (circle) reacts with a fluorophore, while thiols at a second site remain protected within a disulfide bridge. In the second labeling step, the disulfide is deprotected by reduction, and fluorophores are coupled to the second site.
  • FIG. 5B C-terminal fusion of a ⁇ domain (slanted lines) to ttGGBP (solid gray).
  • FIG. 5C N-terminal fusion of ⁇ .
  • FIG. 5D C-terminal fusion ecTRX (horizontal lines).
  • FIG. 5E N-terminal fusion of ecTRX.
  • FIG. 6 is a structural depiction of ionization states affecting fluorescence of the YFP fluorophore (by itself and regardless of the presence of any other fluorophore/partner).
  • FIG. 7 shows the sequence of laYFP and the locations of cysteine mutations for the construction of semisynthetic chloride sensors.
  • Gray the tripeptide that forms the fluorophore in the matured protein.
  • Underlined mutations that tune the YFP wavelength relative to GFP.
  • laYFP retains the wild-type GFP residues H148 and V68 which affect chloride location and affinity (Wang 2015). Standard numbering is used in which the start methionine is 0.
  • FIGS. 8 A and B are structures showing the locations of the cysteine mutations on the surface of laYFP.
  • FIG. 8A Side-view showing that the cysteine mutations on the surface of the barrel ⁇ strands form an annulus that approximately encircles the fluorophore in the hydrophobic core.
  • FIG. 8B Top view showing the positions of all the annulus cysteine mutations around the barrel (end mutations omitted for clarity).
  • FIGS. 9A and B are graphs showing the chloride-dependent responses of the emission intensity spectra of representative Acrylodan and Pacific Blue YFP conjugates.
  • Left column normalized corrected emission spectra (see notes to Table 2): purple line, no chloride; red line, high chloride concentration; thin black lines, intermediate concentrations.
  • YFP emission intensity peak is centered at 530 run in both conjugates. Arrows indicate direction of change with increased chloride concentrations.
  • Middle column fit of ratiometric signal (i? 12 ) to a Langmuir binding isotherm (yields app .3 ⁇ 4; see notes to Table 2).
  • FIG. 10 is an illustration of a structure showing positions for introducing cysteine mutations in ttGGBP to which fluorophores can be covalently coupled for reagentless biosensor construction.
  • Positions 17, 91, 151, and 182 are endosteric; positions 11, 16, 42, 67, 92, 111, 148, 152, 181, and 183 are peristeric; and positions 257, 259, and 300 are allosteric
  • FIGS. 11 A-P are illustrations of fluorophore structures.
  • Naphthalene family (arrows indicate known or potential internal twists): FIG. 11 A shows Acrylodan; FIG. 1 IB shows Badan; FIG. 11C shows IAEDANS.
  • Xanthene family FIG. 1 ID shows Fluorescein (5-IAF and 6-IAF); FIG. 1 IE shows Oregon Green; FIG. 1 IF shows Alexa 432; FIG. 11G shows Alexa 532; FIG. 11H shows Alexa 546; FIG. I ll shows Texas Red.
  • Coumarin family FIG. 11 J shows Pacific Blue;
  • FIG. 1 IK shows CPM. benzoxadiazole family: FIG. 11L shows IANBD.
  • FIGS. 12A and B is a pair of graphs showing donor quenching effects.
  • FIG. 12A The normalized fluorescence intensity ( ⁇ ( ⁇ ); purple: apo-protein; red, high glucose; thin black line: intermediate concentrations) of the singly labeled F170 Pacific Blue conjugate increases (blue arrow) in response to glucose binding without significant shifts in the wavelength of the intensity maximum.
  • FIG. 12B In the doubly labeled fusion protein, the fluorescence emission intensities (color scheme as in FIG.
  • FIG. 13A is a cartoon and FIG. 13B is a structural illustration relating to ratiometric sensing using Forster resonance energy transfer between pairs of glucose-responsive and non- responsive fluorophores attached site-specifically in a fusion protein that enables orthogonal site-specific cysteine labeling.
  • FIG. 13 A Fusion of a single cysteine ttGGBP mutant (gray line; two alternative cysteine positions indicated, i.e., F17C and W182C) and a disulfide- containing pZif domain (disulfide indicated), separated by a linker (thin line), enables sequential labeling with two different fluorophores (line sizes indicate relative size of the fusion domains).
  • FIG. 13 A Fusion of a single cysteine ttGGBP mutant (gray line; two alternative cysteine positions indicated, i.e., F17C and W182C) and a disulfide- containing pZif domain (disulfide indicated), separated by a link
  • FIGS. 14A-D are graphs showing acceptor dipole switching and quenching effects in fluorescein conjugates.
  • FIG. 14A W18205-IAF (directly responsive acceptor) pZif Pacific Blue (indirectly responsive donor). Normalized emission intensities are colored according to glucose concentration range: purple line, apo-protein; dotted gray line, emission at saturated glucose level for higher affinity binding site (phase I response, see main text); red line, emission intensity at highest glucose concentration measured; solid black, intermediate glucose concentrations for phase I; dotted black lines, elevated glucose concentrations in the phase II response.
  • Directions of signal change bottom arrow, phase I; top arrow, phase II.
  • Inset Langmuir binding isotherm of ratiometric signal i?
  • FIG. 14B Contour plot of FIG. 14 A, indicating phase I and phase II responses (see main text).
  • FIG. 14C W18200regon Green (directly responsive acceptor) pZif Pacific Blue (indirectly responsive donor) coloring as in FIG. 14 A.
  • FIG. 14D Contour plot of FIG. 14C.
  • FIGS. 15A and B are graphs showing glucose-dependent emission spectra of F17C Badan and W182C -Acrylodan conjugates of ttGGBP. Corrected spectra (apo-protein, dark red; saturated glucose, purple; intermediate glucose concentrations, black). Insets, fit of the ratiometric signal (equation 1 and 2; 20 nm integration bandwidth): gray circles, experimentally observed ratios; black line, calculated fit (baselines; apo-protein, constant; saturated glucose complex, linear).
  • FIG. 15A F17C Badan (hypsochromic; ⁇ ⁇ 5 470 nm; ⁇ 2 , 542 nm; 0.18 mM);
  • FIG. 15B F182C- Acrylodan (bathochromic; ⁇ ⁇ 5 475 nm; ⁇ 2 , 545 nm; a 3 ⁇ 4, 2.2 mM).
  • FIGS. 16A-D are graphs showing glucose dependence of electronic transitions in the fluorescence emission intensity spectra of F17C Badan and W182C -Acrylodan ttGGBP conjugates.
  • FIGS. 16A and B F17C-Badan;
  • FIGS. 16C and D F182C- Acrylodan.
  • frequency transformations of the spectra were decomposed into principal components (equation 31).
  • the spectral emission intensities can be accounted for to a first approximation by two excited state electronic transitions: a low-energy, S ⁇ (Acrylodan: 521 ⁇ 9 nm; Badan: 530 ⁇ 14 nm) and a high-energy, 3 ⁇ 4 (Acrylodan: 477 ⁇ 14 nm; Badan: 477 ⁇ 16 nm) transition. Glucose binding shifts the population of these excited states.
  • Inserts show the population fractions (equation 34) of the Si and i3 ⁇ 4 transitions extracted from the spectra at each titration point (black circles) fit to Langmuir binding isotherms (solid lines) with m K& values constrained to be the same for both populations.
  • the wavelengths of Si or _3 ⁇ 4 transitions are the same in apo-protein and the saturated glucose complex.
  • the residuals indicate that a more extensive treatment is required in which the Si and _3 ⁇ 4 are split into multiple transitions to fully fit the spectra. Wavelength shifts occur if there is a significant redistribution of the two excited state populations in the apo-protein and the saturated ligand complexes.
  • the Si state dominates in the glucose complex (FIG. 16D); in the hypsochromic conjugate 17C- Badan (FIG. 16B) the apo-protein comprises a mixture of the two states, whereas the glucose complex contains almost exclusively the _3 ⁇ 4 state.
  • FIGS. 17A-D are graphs showing glucose dependence of the absorption spectra of ttGGBP Acrylodan and Badan conjugates that undergo wavelength shifts in their fluorescence emission intensities in response to ligand binding
  • FIGS. 17A and B F17C -Badan;
  • FIGS. 17C and D F182C -Acrylodan.
  • frequency transformations of the spectra were decomposed into principal components (equation 31). Inserts show glucose dependence of the fractional contribution of each component (equation 32).
  • FIGS. 18A-F are a set of graphs showing donor dipole switching effects in Acrylodan and Badan conjugates.
  • Four doubly labeled conjugates were constructed, in which Acrylodan or Badan directly responsive donors were combined with Alexa532 or 5-IAF indirectly responsive acceptors.
  • FIG. 18A F17C-Badan pZif-Alexa532.
  • ⁇ ( ⁇ ) normalized emission intensity: purple line, apo-protein; red line, saturating glucose concentration; thin black lines, intermediate glucose concentrations. Blue arrow, direction of change with increased glucose concentration.
  • Langmuir binding isotherm of ratio, i?
  • FIG. 18C W182C-Acrylodan pZif-Alexa532.
  • Normalized emission intensities are colored according to glucose concentration range: purple line, apo-protein; dotted red line, emission at saturated glucose level for higher affinity binding site (phase I response, see main text); red line, emission intensity at highest glucose concentration measured; solid black, intermediate glucose concentrations for phase I; dotted black lines, elevated glucose concentrations in the phase II response.
  • Directions of signal change blue arrow, phase I; red arrow, phase II.
  • Langmuir binding isotherm of ratiometric signal i? 12 at 480 nm and 550 nm, m K & 1.7 mM, constant and linear baselines for apo-protein and ligand complex respectively.
  • FIG. 18D W182C-Acrylodan pZif-5-IAF.
  • FIG. 18F W182C-Acrylodan pZif-Alexa532 contour plot of the glucose dependence of emission intensities, indicating phase I and phase II responses (see main text).
  • FIG. 18F W182C-Acrylodan pZif-Alexa532 contour plot of the glucose dependence of emission intensities, indicating phase I and phase II responses (see main text).
  • FIGS. 19A and B are graphs providing a comparison of singly-labeled
  • FIGS. 20A-D are graphs showing donor dipole switching effects in
  • FIG. 20A Singular value decomposition analysis of the change in the emission intensity of singly labeled W182C-IAEDANS in response to glucose, showing the wavenumber dependence of the invariant (black line) and variant (red line) spectral components. Inset shows change in contribution of the two spectral components with respect to glucose concentration.
  • FIG. 20B The glucose response is accounted for largely by two electronic transitions (green, 542 nm; blue, 485 nm) which were fits as Gaussians to the experimental emission intensities (purple line, in the absence of glucose; red line, saturating glucose). Black lines show residuals between observed and calculated spectra.
  • FIG. 20C W182C-IAEDANS pZif-5-IAF.
  • Normalized emission intensities, ⁇ ( ⁇ ) are colored according to glucose concentration range: purple line, apo-protein; dotted red line, emission at saturated glucose level for higher affinity binding site (phase I response, see main text); red line, emission intensity at highest glucose concentration measured; solid black, intermediate glucose concentrations for phase I; dotted black lines, elevated glucose concentrations in the phase II response.
  • Directions of signal change blue arrow, phase I; red arrow, phase II.
  • FIG. 20D Contour plot of the glucose dependence of W182C-IAEDANS pZif-5-IAF emission intensities, indicating phase I and phase II responses (see main text).
  • FIG. 21 is a structural illustration of ionization equilibria of the fluorescein carboxylate and phenolic hydroxyl (Martin 1975 ).
  • FIG. 22 is an illustration of the structure of E. coli thioredoxin (Katti 1990) showing positions of mutations constructed in the adaptor proteins. Disulfide (C32,C35) is indicated. The D2A, D26A, and K57M background mutations constructed in all adaptor proteins are indicated (D2A removes adventitious N-teiminal Cu(II)-binding site; D26A and K57M remove charges buried in the hydrophobic core). Large gray spheres: surface lysines mutated to arginine in Adaptor2.0a (K4Q and K18Q in Adaptor2.0b). Structure from PDB accession code 2trx.
  • FIG. 23 shows amino acid sequences of the engineered adaptor proteins based on E. coli thioredoxin. Numbering according the X-ray structure of the mature protein, lacking the initial methionine. Wild-type sequence is shown in full, mutations are given below (blank indicates wild-type residue).
  • FIGS. 24A-D are graphs showing the pH dependence of the emission spectra of
  • FIG. 24B Fluorescein attached to the disulfide, and
  • FIGS. 25A-D are graphs showing the pH dependence of the absorption spectra of Adoptorl .0 conjugates.
  • Left column corrected absorbance, ⁇ ( ⁇ ), spectra (see notes to Table 9) of doubly labeled conjugates (purple, pH 4.0; red, pH 9.5; thin black lines, intermediate values at 0.5 pH unit intervals); middle column: ratiometric response, i?
  • FIGS. 26A and B are graphs showing the pH dependence of the fluorescence emission and absorption spectra of Adaptor2.0a conjugate labeled with Fluorescein at the amino terminus, and Acrylodan at the disulfide.
  • Left column corrected spectra (purple, pH 4.0; red, pH 9.5; thin black lines, intermediate values at 0.5 pH unit intervals).
  • Middle column corrected spectra (purple, pH 4.0; red, pH 9.5; thin black lines, intermediate values at 0.5 pH unit intervals).
  • FIGS. 27A-F are graphs showing temperature dependence and pH dependence of Adaptorl .0 and Adaptor 2.0a fluorescent conjugates measured on a Roche LightCycler.
  • Left column Adaptorl .O R73C Pacific Blue, disulfide Fluorescein, fluorescence ratio recorded at 488 nm and 510 nm; right column: Adaptor 2.0 Fluorescein, disulfide Pacific Blue, fluorescent ratio recorded at 488 nm and 580 nm.
  • FIGS. 27 A, B pH- and temperature- dependent landscape of the fluorescence ratio (Z axis): dashed-dotted line, approximate midpoint concentrations (pK a ) of the response to pH; dashed line, approximate mid-point temperatures (I'm) of thermal stability (equation 36).
  • FIGS. 27E, F
  • FIG. 28 is a diagram relating to directly responsive partners and indirectly responsive partners in ngmFRET pathways.
  • FIG. 29 shows the sequence of an exemplary chloride-binding protein (YFP) 1
  • FIG. 30 shows the sequence of an exemplary C1BP2 expression construct (SEQ ID NO: 47).
  • FIG. 31 shows the sequence of an exemplary C1BP3 expression construct (SEQ ID NO: 48).
  • FIG. 32 shows the sequence of an exemplary C1BP4 expression construct (SEQ ID NO: 49).
  • FIG. 33 shows the sequence of an exemplary C1BP5 expression construct (SEQ ID NO: 50).
  • FIG. 34 shows the sequence of an exemplary C1BP6 expression construct (SEQ ID NO: 1
  • FIG. 35 shows the sequence of an exemplary C1BP7 expression construct (SEQ ID NO: 52).
  • FIG. 36 shows the sequence of an exemplary C1BP8 expression construct (SEQ ID NO: 53).
  • FIG. 37 shows the sequence of an exemplary C1BP9 expression construct (SEQ ID NO: 54).
  • FIG. 38 shows the sequence of an exemplary C1BP10 expression construct (SEQ ID NO: 55).
  • FIG. 39 shows the sequence of an exemplary C1BP11 expression construct (SEQ ID NO: 56).
  • FIG. 40 shows the sequence of an exemplary C1BP12 expression construct (SEQ ID NO: 1
  • FIG. 41 shows the sequence of an exemplary C1BP13 expression construct (SEQ ID NO: 58).
  • FIG. 42 shows the sequence of an exemplary C1BP14 expression construct (SEQ ID NO: 59).
  • FIG. 43 shows the sequence of an exemplary ttGGBP.l IC.O.bZif expression construct (SEQ ID NO: 60).
  • FIG. 44 shows the sequence of an exemplary ttGGBP.l 7C.0.bZif expression construct (SEQ ID NO: 61).
  • FIG. 45 shows the sequence of an exemplary ttGGBP. I l l C.O.bZif expression construct (SEQ ID NO: 62).
  • FIG. 46 shows the sequence of an exemplary ttGGBP.151C.0.bZif expression construct (SEQ ID NO: 63).
  • FIG. 47 shows the sequence of an exemplary ttGGBP.182C.0.bZif expression construct (SEQ ID NO: 64).
  • FIG. 48 shows the sequence of an exemplary ttGGBP.17C.3.Trx expression construct (SEQ ID NO: 65).
  • FIG. 49 shows the sequence of an exemplary Trx.ttGGBP.17C3 expression construct (SEQ ID NO: 66).
  • FIG. 50 shows the sequence of an exemplary ttGGBP.182C.2.Trx expression construct (SEQ ID NO: 67).
  • FIG. 51 shows the sequence of an exemplary Trx.ttGGBP.182C.2 expression construct (SEQ ID NO: 68).
  • FIG. 52 shows the sequence of an exemplary AdaptorO expression construct (SEQ ID NO: 69).
  • FIG. 53 shows the sequence of an exemplary Adaptorl .0 expression construct (SEQ ID NO: 70).
  • FIG. 54 shows the sequence of an exemplary Adaptor2.0a expression construct (SEQ ID NO: 71).
  • FIG. 56 shows the sequence of an exemplary Adaptor3.0 expression construct (SEQ
  • FIG. 57 shows the sequence of an exemplary Adaptor4.0 expression construct (SEQ ID NO: 74).
  • FIG. 58 shows the sequence of an exemplary Adaptor5.0 expression construct (SEQ ID NO: 75).
  • FIG. 59 shows the sequence of an exemplary Adaptor6.0 expression construct (SEQ ID NO: 76).
  • FIG. 60 shows the sequence of an exemplary Adaptor7.0 expression construct (SEQ ID NO: 77).
  • FIG. 61 shows the sequence of an exemplary Adaptor8.0 expression construct (SEQ
  • FIG. 62 shows the sequence of an exemplary Adaptor9.0 expression construct (SEQ ID NO: 79).
  • FIG. 63 shows the sequence of an exemplary Adaptor 10.0 expression construct (SEQ ID NO: 80).
  • FIG. 64 shows the sequence of an exemplary Adaptor 11.0 expression construct (SEQ ID NO: 81).
  • FIG. 65 shows the sequence of an exemplary Adaptor 12.0 expression construct (SEQ ID NO: 82).
  • FIG. 66 shows the sequence of an exemplary Adaptorl3.0 expression construct (SEQ
  • FIG. 67 shows the sequence of an exemplary Adaptorl4.0 expression construct (SEQ ID NO: 84).
  • FIG. 68 shows the sequence of an exemplary Adaptor 15.0 expression construct (SEQ ID NO: 85).
  • FIG. 69 shows the sequence of an exemplary Adaptor 16.0 expression construct (SEQ ID NO: 86). DETAILED DESCRIPTION
  • Biosensors are analytical tools that can be used to measure the presence of a single molecular species in a complex mixture by combining the extraordinary molecular recognition properties of biological macromolecules with signal transduction mechanisms that couple ligand binding to readily detectable physical changes (Hall, Biosensors, Prentice-Hall, Englewood Cliffs, N.J.; Scheller et al., Curr. Op. Biotech. 12:35-40, 2001).
  • a biosensor is reagentless and, in contrast to enzyme-based assays or competitive
  • biosensors does not change composition as a consequence of making the measurement (Hellinga & Marvin, Trends Biotech. 16:183-189, 1998).
  • Most biosensors combine a naturally occurring macromolecule such as an enzyme or an antibody, with the identification of a suitable physical signal particular to the molecule in question, and the construction of a detector specific to that system (Meadows, Adv. Drug Deliv. Rev. 21 :177-189, 1996).
  • Escherichia coli periplasmic binding proteins are members of a protein superfamily (bacterial periplasmic binding proteins, bPBPs) (Tarn & Saier, Microbiol. Rev. 57:320-346, 1993). These proteins comprise two domains linked by a hinge region (Quiocho & Ledvina, Molec. Microbiol. 20: 17-25, 1996). The ligand-binding site is located at the interface between the two domains. The proteins typically adopt two conformations: a ligand-free open form, and a ligand-bound closed form, which interconvert via a hinge-bending mechanism upon ligand binding.
  • the present disclosure provides a biosensor for ligand, comprising a ligand-binding protein that is attached to one or more reporter group (e.g., 1, 2, 3, or more reporter groups).
  • a reporter group e.g. 1, 2, 3, or more reporter groups.
  • the binding of a ligand to the ligand-binding domain of the ligand-binding protein causes a change in signaling by the biosensor.
  • the biosensor may produce a signal when a ligand is bound to the ligand binding domain that is not produced (and/or that is different from a signal that is produced) when the ligand is absent from the ligand binding domain.
  • biosensors provided herein produce a dichromatic, ratiometric signal, i.e., the signal is defined as the quotient of the intensities at two independent wavelengths.
  • the advantage of such a signal is that it provides an internally consistent reference.
  • the self- calibrating nature of a ratiometric measurement removes the necessity for carrying out onboard calibration tests prior to each measurement.
  • the biosensors are reagentless in that their monitoring mechanism requires neither an enzyme nor additional substrates for a signal to develop, nor measurement of substrate consumption or product generation rates to determine ligand concentrations.
  • Reagentless, fluorescently responsive biosensors present a number of advantages over enzyme-based biosensors, including elimination of chemical transformations, elimination of substrate requirements, and self-calibration, which together lead to rapid response times, continuous monitoring capabilities, simple sample-handling, and lower cost due to simplified manufacturing and distribution processes.
  • Fluorescent chemosensors based on small-molecule fluorophores that interact directly with an analyte Zhang, Yin and Yoon 2014, Lavis and Raines 2008, Lavis and Raines 2014
  • fluorescent biosensors based on engineered proteins that couple analyte-binding events to changes in the emission properties of fluorophores being fluorescent by themselves and regardless of the presence of any other fluorophore/partner)
  • Okumoto 2012 or semi-synthetically (Wang 2009) incorporated fluorophores have wide-ranging applications in cell biology and analytical chemistry(Borisov and Wolfbeis 2008, Liu 2015, Matzeu 2015, Heo and Takeuchi 2013).
  • ratiometric measurements can be used to monitor analyte concentrations (FIGS. 2A-C). Ratiometry is essential for devices that rely on quantifying changes in fluorescence emission intensities, because it provides an internally consistent reference (Demchenko 2010, Demchenko 2014). The self-calibrating nature of a ratiometric measurement removes the necessity for carrying out on-board calibration tests prior to each measurement (Choleau et al. 2002), obviating the need for multiple components and fluidic circuitry.
  • reagentless, ratiometric fluorescent sensors have many uses in process engineering, environmental or clinical chemistry, including single-use point-of-care applications (Kozma et al. 2013, Ahmed et al. 2014, Mohammed 2011, Ispas 2012, Rogers and Boutelle 2013, Robinson and Dittrich 2013, Arora et al. 2010, Gubala et al. 2012), wearable devices (Badugu, Lakowicz and Geddes 2005), optodes for continuous monitoring (Weidemaier et al. 2011, Judge et al. 2011), or implanted "tattoos" that are interrogated transdermally (Bandodkar et al. 2015).
  • the present subject matter provides methods for converting monochromatic responses into dichromatic responses that enable ratiometric sensing.
  • these methods are based on establishing non-geometrically modulated Forster Resonance Energy Transfer (ngmFRET) between the monochromatic fluorophore (directly responsive partner), and a second fluorophore that neither interacts directly with the ligand, nor is sensitive to ligand-mediated changes in its environment (indirectly responsive partner).
  • ngmFRET non-geometrically modulated Forster Resonance Energy Transfer
  • this arrangement does not rely on analyte-mediated geometrical changes (inter-fluorophore distance or angle) between the donor and acceptor, but instead exploits effects by analyte binding, which alter the photophysics of only the directly responsive partner such as changes in its spectral properties and non-radiative decay rates (FIG. 3).
  • the exemplary and non-limiting studies described herein demonstrate how these ngmFRET effects were used to convert monochromatic into dichromatic responses and thereby improve the ratiometric properties of dichromatic responses, using three classes of examples that illustrate the application of this technique both to biosensors and chemosensors (FIG. 4): 1.
  • the analyte recognition element is a protein that undergoes an analyte-mediated conformational change that is alters the properties of an environmentally responsive directly responsive partner (FIG. 4A): a glucose-binding protein in which a conjugated directly responsive fluorophore respond via a glucose-induced protein conformational change that alters its emission properties.
  • This fluorophore is paired with a second, indirectly responsive partner attached to a fusion domain (such as a fluorophore attachment motif attached to, e.g., the first or the last amino acid of the ligand-binding protein).
  • a fusion domain such as a fluorophore attachment motif attached to, e.g., the first or the last amino acid of the ligand-binding protein.
  • the analyte recognition element is a rigid protein with an analyte-binding site located adjacent to an fluorophore (having fluorescence by itself and regardless of the presence of any other fluorophore/partner; FIG. 4B): the monochromatic response of the fluorophore to chloride ion binding in a yellow fluorescent protein is converted to a dichromatic response using a indirectly responsive extrinsic fluorophore site-specifically attached to the protein surface.
  • the analyte recognition element is a synthetic chemoresponsive fluorophore (FIG.
  • an adaptor protein is engineered to establish ngmFRET between two, site- specifically attached extrinsic fluorophores. The monochromatic response of the directly responsive partner to proton binding is converted into a dichromatic signal.
  • the first example represents a large class of protein-based fluorescent biosensors which undergo ligand-mediated conformational changes that alter the local environment of an attached fluorophore. Such conformational changes are found in many proteins; coupling these to fluorescent responses therefore provides a rich source for engineering fluorescent biosensors.
  • the glucose-binding protein used in this example is a member of the bacterial periplasmic-binding protein (PBP) superfamily which combines a large diversity of ligand specifities with a common structural mechanism (Berntsson et al. 2010) that is well suited to the construction of fluorescent sensors (de Lorimier et al. 2002, Grunewald 2014).
  • PBP bacterial periplasmic-binding protein
  • engineered fluorescent responses are more commonly monochromatic than dichromatic.
  • ligand-binding proteins include proteins that bind sugars (such galactose-binding proteins, lactose-binding proteins, arabinose-binding proteins, ribose-binding proteins, and maltose-binding proteins), urea- binding proteins, bicarbonate-binding proteins, phosphate-binding proteins, sulfate-binding proteins, calcium-binding proteins, dipeptide-binding proteins, amino acid-binding proteins (such as histidine-binding proteins, glutamine-binding proteins, glutamate-binding proteins, and aspartate-binding proteins), and iron-binding proteins.
  • sugars such galactose-binding proteins, lactose-binding proteins, arabinose-binding proteins, ribose-binding proteins, and maltose-binding proteins
  • urea- binding proteins such as galactose-binding proteins, lactose-binding proteins, arabinose-binding proteins, ribose-binding
  • the second example represents a smaller set of proteins that contain fluorophores formed by a self-catalyzed cyclization of a peptide within their sequences such as Green Fluorescent Protein, its engineered variants and homologs(Tsien 1998, Zimmer 2002). Some of these fluorophores function as direct natural chemosensors by interacting with ligands such as protons and halides (Miesenbock, De Angelis and Rothman 1998, Grimley et al. 2013), but typically evince only monochromatic responses.
  • the example demonstrates how ratiometric sensing mechanisms can be engineered into these proteins.
  • FIG. 4C illustrates how protein engineering was used to improve the properties of synthetic directly responsive chemosensors, by incorporating them as extrinsic fluorophores into a protein and combining with a second fluorophore to introduce ngmFRET.
  • the protein therefore functions as an "adaptor"
  • a chemoresponsive (directly responsive) fluorophore may be linked to another fluorophore (an indirectly responsive fluorophore) using virtually any polypeptide sequence.
  • a pH-sensitive chemoresponsive fluorophore is exemplified in the examples, any other chemoresponsive fluorophore may be used.
  • the principles demonstrated for converting the monochromatic response of fluorescein to protons into a dichromatic signal by incorporation into a dually labeled adaptor protein can be extended to other chemoresponsive fluorophores for the detection of a wide variety of analytes.
  • a well-known thiol- or amine-reactive functional group (imidoesters, NHS esters, carbodiimides, maleides, aziridines, arcyloyls; see, e.g., G.T. Hermanson, 2013, Bionjugate Techniques, Academic Press, incorporated herein by reference) may be incorporated into the
  • chemoresponsive fluorophore such that the chemoresponsive fluorophore can be coupled to the adaptor protein
  • Chemoresponsive sensors can bind specifically to small molecules such as ions, monosaccharides, amino acids, and short peptides, using a variety of molecular recognition units. Such units are then linked to a fluorescent group, the properties of which are altered upon binding the ligand (A. P. Demchenko, 2015, Introduction to Fluorescence Sensing, Springer).
  • a variety of schemes can be used to couple proton-binding groups such as amines to fluorescence responses (op. cit.). Fluoresccent chemosensors have been developed for toxic metals such as lead, cadmium, and mercury (K.P. Carter et al., 2014, Chem. Rev., 114, 4564-4601).
  • Chemoresponsive fluorophores have been developed for glucose (X.S. Sun, T.D. James, 2015, Chem. Rev., 115, 8001-8037), and other organic analytes including amines, urea, and guanidinium (T.W. Bell and N.M. Hext, 2004, Chem. Soc. Rev., 33, 589-598).
  • the fluorescent glucose sensor described in the first example has utility in glucose monitoring is essential for the management of diabetes mellitus, a disease that affects at least 366 million people world-wide(Yoo and Lee 2010, Cash and Clark 2010) and is increasing every year.
  • the majority of current glucose-monitoring technologies rely on enzymes for which glucose is one of the substrates(Wang 2008, Bergel, Souppe and Comtat 1989).
  • Glucose concentration measurements therefore are subject to variations in second substrate concentrations consumed in the enzyme reaction, such as oxygen in the case of glucose oxidase(Tang et al. 2001). Additional complications arise in systems where reaction rates are measured for enzymes immobilized on electrodes. In such arrangements, accuracy is compromised by factors that alter the rate at which glucose arrives at the electrode surface interfere with accuracy, such as hematocrit levels(Karon et al. 2008, Tang et al. 2000), or surface "fouling" by deposition of proteins and cells in the foreign body
  • Ratiometric fluorescent glucose sensors obviate these problems, and accordingly have been incorporated successfully in optodes for continuous glucose monitoring in animals and humans.
  • the total signal, S, of a fluorescent sensor (either single-wavelength emission intensities, , or ratios of intensities at two wavelengths, i? 12 ) is the sum of the fluorescence due to the ligand-free (apo) and ligand-bound states:
  • Fluorescence quantum yields are the fractions of photons emitted by the excited state relative to the total absorbed, and correspond to the ratio of the radiative decay rate relative to the sum of the rates of all possible decay pathways (FIGS. 3A-D).
  • Qo bs Qa P o(i-y)+ Q sa ,y 6
  • Q 0 bs, Qa P o and Q sat are the quantum yield of the total system, the apo-protein, and the ligand-bound complex, respectively.
  • the Q avo and Q sat quantum yields each are combinations of their
  • the intensity of the light emitted by a donor or its acceptor is determined by the rate of photon emission from their respective excited states (FIG. 3A).
  • the excited state of a donor is formed by the incident light from the excitation source, and there are three pathways by which this state decays: radiative and non-radiative decay and resonance transfer (by itself and regardless of the presence of any other fluorophore/parter).
  • the rate of formation of the acceptor excited state is determined by the resonance transfer rate from the donor, and there are only two processes that determine its decay rate: the radiative and non- radiative pathways (by itself and regardless of the presence of any other fluorophore/parter).
  • the rate of resonance energy transfer, k t , along a non-radiative pathway between donor and acceptor is a fraction of the donor radiative emission pathway rate (by itself and regardless of the presence of any other fluorophore/parter), D k r (the emission rate in the absence of an acceptor) multiplied by the energy transfer coupling factor, ⁇ , (Lakowicz 2006, Valeur 2012):
  • the energy transfer coupling factor is dependent on the spectral overlap, J, of the donor emission, D em , and acceptor excitation spectrum, A ex, and the variation of the geometry, G, between the donor and acceptor excited state transition dipoles with distance, r, and orientation factor, ⁇ .
  • the intensity of the emitted donor light, ID is

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