Supercharged fluorescent biosensors for analyte detection

Abstract

Disclosed are compositions and methods for detecting nucleic acid, or detecting the presence or absence of a nuclease. Specifically, disclosed herein are nucleic acid-metal nanocluster probes, which comprise a nucleic acid probe with metal nanoclusters associated therewith. These nucleic acid-metal nanocluster probes can be used to detect a variety of cleavage events, and can also be used to detect hybridization to the probe.

Description

[0001] Supercharged Fluorescent Biosensors for Analyte Detection

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of priority to U.S. Provisional Application No. 63 / 611,409, filed December 18, 2023, which is incorporated by reference herein in its entirety.

[0004] GOVERNMENT SUPPORT CLAUSE

[0005] This invention was made with government support under Grant numbers W911NF- 23-2-0089 and W91 INF-22-2-0246 awarded by the Army Research Office, and Grant numbers W912HZ-22-2-0037 and W912HZ-22-Q-0016 awarded by the U. S. Army Engineer Research And Development Center. The government has certain rights in the invention.

[0006] REFERENCE TO SEQUENCE LISTING

[0007] The sequence listing submitted on December 18, 2024, as an .XML file entitled “10046-552W01.xml” created on December 18, 2024, and having a file size of 20,829 bytes is hereby incorporated by reference pursuant to 37 C.F.R. § 1.52(e)(5).

[0008] BACKGROUND

[0009] Rare earth elements (REEs) are critically important to the world economy on account of their unique luminescent, magnetic, and catalytic properties. Comprising the fifteen elements of the lanthanide series, as well as scandium and yttrium, REEs have emerged as non-substitutable elements in a variety of consumer and defense applications, encompassing technologies as diverse as wind turbines, solar cells, high-capacity batteries, image-guided cancer therapies, telecommunications, and night-vision systems.

[0010] Biomining presents a sustainable, potentially scalable path towards bolstering global lanthanide reserves via unconventional sources, though the absence of a high-affinity system with appropriate sensitivity for detecting lanthanides both in vitro and in vivo poses a significant barrier to practical biosensing and microbial engineering. (Nguyen, 2021; Schippers, 2013; Martinez-Bellange, 2022; Cockell, 2020). The lanthanide series elements are naturally luminescent and thus capable of ‘self-sensing.’ However, on account of the Laporte selection rule, the radiative 4f-4f orbital transitions are spin-forbidden, and consequently, free lanthanide cations have a diminutive capacity for energy absorption. (Biinzli, 2016). To circumvent this photophysical constraint, lanthanide cations can be complexed with organic ligands to sensitize the lanthanide’s intrinsic luminescence via the well-studied antenna effect. (Moore, 2009; Yip, 2012; Yin, 2019). However, due to the requirement of synthetic chelators and chemical modulation, these systems are difficult to utilize in living systems, cheaply or at scale. Alternatively, lanthanides can themselves function as donors of energy transfer via overlapping and non-overlapping Forster resonance energy transfer (FRET) to closely positioned fluorescent protein acceptors. (Vuojola, 2009). Still, this system requires extensive tempering to bring the lanthanide chelate within the FRET signaling range, thereby limiting its use in vivo or at scale, but highlighting a promising avenue for biosensor development.

[0011] An attestation to the supreme adaptability of biology, the lanthanide-binding protein, lanmodulin (LanM), from the methylotrophic bacterium Methylobacterium extorquens has a sensitivity for lanthanides in the picomolar range. (Cotruvo, 2018). Natively a periplasmic transporter for lanthanide uptake, LanM has been repurposed by biotechnologists for nascent applications in the sensing, separation, and sequestration of lanthanides (Mattocks, 2019; Mattocks, 2023; Daumann, 2021; Featherston, 2021). For instance, the LaMPl fluorescence-based sensor is a tri-fusion protein composed of a citrine and cyan fluorescent protein separated by the intrinsically disordered LanM domain. (Mattocks, 2019). Upon binding to the lanthanide series cations, LanM folds into a tight configuration, bringing the two adjacent fluorescent proteins within signaling range to initiate a measurable FRET. Alternatively, the naturally fluorescent amino acid tryptophan can be harnessed as a molecular antenna to sensitize the luminescence of bivalent terbium, yielding an apparent Kd of 10-100 pM. (Featherston, 2021). Notably, this strategy employs a direct LRET, instead of FRET; nonetheless, only terbium can be detected, and the energy transfer is inefficient due to tryptophan’s low fluorescence quantum yield. (Vivian, 2001). Although LanM confers an ultra-high sensitivity to the fluorescence-based detection of lanthanides, its superlative nature limits its utility as biosensor at greater than picomolar scales of lanthanide occupancy in more concentrated environmental streams. Considering that acid mine drainage and other waste streams can contain an upwards of 20-100 pM of total REEs, a sensitivity range congruent with such conditions is needed for practical biosensing applications. (Hermassi, 2022; Spears, 2013; Stewart, 2017). Moreover, the functionalization of microbes with the ability to concentrate lanthanides necessitates a platform capable of detecting multiple lanthanides in vivo at concentrations potentially spanning 5-5000 micrograms per kilogram of wet biomass or greater. (Garcia-Balboa, 2022). Recently, the capture of lanthanides on the surface of cells has yielded new insights, including, inadvertently, evidence that lanthanide cations can readily bind to the negatively charged side chains of aspartate and glutamate, found commonly in the extracellular matrix. (Xie, 2022).

[0012] Thus, sustainable and scalable compositions and methods for the tunable detection of analytes, including lanthanides, at environmentally and industrially relevant concentrations are needed.

[0013] SUMMARY

[0014] In various aspects, disclosed herein is a detection system for selectively characterizing the presence and / or amount of an analyte in a sample, the system comprising: a supercharged fluorescent protein configured to directly chelate with an analyte having a spectral overlap with the supercharged fluorescent protein, thereby forming a proteinanalyte complex, wherein the protein- analyte complex is configured to emit a fluorescent signal when irradiated with a light source.

[0015] In some aspects, the system further includes a fluorescence detector configured to detect the fluorescent signal from the protein- analyte complex. In some aspects, the fluorescence detector comprises a fluorescent microscope.

[0016] In some aspects, the light source comprises UV light. In some aspects, the UV light has a wavelength from 340 nm to 360 nm.

[0017] In some aspects, the analyte comprises one or more lanthanides. In some aspects, the one or more lanthanides comprise free lanthanide cations. In some aspects, the analyte comprises terbium (Tb), thulium (Tm), dysprosium (Dy), europium (Eu), samarium (Sm), ytterbium (Yb), or a combination thereof.

[0018] In some aspects, the fluorescent protein comprises a green fluorescent protein (GFP), a yellow fluorescent protein (YFP), a cyan fluorescent protein (CFP), or a combination thereof.

[0019] In some aspects, the fluorescent protein comprises an amino acid sequence having at least 85% sequence identity to any of SEQ ID NOS: 1-3. In some aspects, the protein- analyte complex is configured to be measurably fluorescent when the supercharged fluorescent protein and analyte are separated by a distance of 50 nm or less.

[0020] In some aspects, the supercharged fluorescent protein has a negative net charge of from -1 to -50.

[0021] In some aspects, the supercharged fluorescent protein has a net positive charge.

[0022] In some aspects, the analyte comprises a lanthanide-chelate configured to bind to the supercharged fluorescent protein having a net positive charge.

[0023] In some aspects, the protein- analyte complex is configured to provide multiplex detection of a plurality of analytes.

[0024] In some aspects, the supercharged fluorescent protein is a monomeric protein.

[0025] In some aspects, the supercharged fluorescent protein is a multimeric protein. In some aspects, the multimeric protein includes a protomer comprising a pair of supercharged fluorescent proteins having opposite net charges. In some aspects, the multimeric protein comprises a heterodimer.

[0026] In some aspects, the system further includes: one or more sensors configured to detect the fluorescent signal of the protein- analyte complex; a processor; and software executable by said processor such that said system: determines the presence and / or amount of the analyte in the sample based on the fluorescent signal.

[0027] Also disclosed herein is a detection system for selectively characterizing the presence and / or amount of an analyte in a sample, the system comprising: a genetically modified microorganism engineered to express a supercharged fluorescent protein, wherein supercharged fluorescent protein is configured to directly chelate with an analyte having a spectral overlap with the supercharged fluorescent protein, thereby forming a proteinanalyte complex, and wherein the protein- analyte complex is configured to emit a fluorescent signal when irradiated with a light source.

[0028] In some aspects, the system further includes a fluorescence detector configured to detect the fluorescent signal from the protein- analyte complex. In some aspects, the fluorescence detector comprises a fluorescent microscope.

[0029] In some aspects, the light source comprises UV light. In some aspects, the UV light has a wavelength from 340 nm to 360 nm.

[0030] In some aspects, the analyte comprises one or more lanthanides. In some aspects, the one or more lanthanides comprise free lanthanide cations. In some aspects, the analyte comprises terbium (Tb), thulium (Tm), dysprosium (Dy), europium (Eu), samarium (Sm), ytterbium (Yb), or a combination thereof.

[0031] In some aspects, the fluorescent protein comprises a green fluorescent protein (GFP), a yellow fluorescent protein (YFP), a cyan fluorescent protein (CFP), or a combination thereof.

[0032] In some aspects, the fluorescent protein comprises an amino acid sequence having at least 85% sequence identity to any of SEQ ID NOS: 1-3.

[0033] In some aspects, the fluorescent protein has a negative net charge from -1 to -50.

[0034] In some aspects, the supercharged fluorescent protein has a net positive charge.

[0035] In some aspects, the analyte comprises a lanthanide-chelate configured to bind to the supercharged fluorescent protein having a net positive charge.

[0036] In some aspects, the protein- analyte complex is configured to provide multiplex detection of a plurality of analytes.

[0037] In some aspects, the supercharged fluorescent protein is a monomeric protein.

[0038] In some aspects, the supercharged fluorescent protein is a multimeric protein. In some aspects, the multimeric protein includes a protomer comprising a pair of supercharged fluorescent proteins having opposite net charges. In some aspects, the multimeric protein comprises a heterodimer.

[0039] In some aspects, the system further includes: one or more sensors configured to detect the fluorescent signal of the protein- analyte complex; a processor; and software executable by said processor such that said system: determines the presence and / or amount of the analyte in the sample based on the fluorescent signal.

[0040] In various aspects, disclosed herein is a kit comprising: a supercharged fluorescent protein disposed within an assay chamber, wherein the supercharged fluorescent protein is configured to directly chelate with an analyte having a spectral overlap with the supercharged fluorescent protein to form a protein-analyte complex, wherein the proteinanalyte complex is configured to emit a fluorescent signal when irradiated with a light source.

[0041] In some aspects, the kit further includes a fluorescence detector configured to detect the fluorescent signal from the protein-analyte complex. In some aspects, the fluorescence detector comprises a fluorescent microscope.

[0042] In some aspects, the light source comprises UV light. In some aspects, the UV light has a wavelength from 340 nm to 360 nm. In some aspects, the analyte comprises one or more lanthanides. In some aspects, the one or more lanthanides comprise free lanthanide cations. In some aspects, the analyte comprises terbium (Tb), thulium (Tm), dysprosium (Dy), europium (Eu), samarium (Sm), ytterbium (Yb), or a combination thereof.

[0043] In some aspects, the fluorescent protein comprises a green fluorescent protein (GFP), a yellow fluorescent protein (YFP), a cyan fluorescent protein (CFP), or a combination thereof.

[0044] In some aspects, the fluorescent protein comprises an amino acid sequence having at least 85% sequence identity to any of SEQ ID NOS: 1-3.

[0045] In some aspects, the fluorescent protein has a negative net charge from -1 to -50.

[0046] In some aspects, the supercharged fluorescent protein has a net positive charge. In some aspects, the analyte comprises a lanthanide-chelate configured to bind to the supercharged fluorescent protein having a net positive charge.

[0047] In some aspects, the protein- analyte complex is configured to provide multiplex detection of a plurality of analytes.

[0048] In some aspects, the supercharged fluorescent protein is a monomeric protein.

[0049] In some aspects, the supercharged fluorescent protein is a multimeric protein. In some aspects, the multimeric protein includes a protomer comprising a pair of supercharged fluorescent proteins having opposite net charges. In some aspects, the multimeric protein comprises a heterodimer.

[0050] In some aspects, the supercharged fluorescent protein is immobilized within the assay chamber.

[0051] Also disclosed herein is a method for characterizing the presence and / or amount of an analyte in a sample, the method comprising: providing a sample for qualitative or quantitative determination of an analyte having a spectral overlap with a supercharged fluorescent protein; contacting the supercharged fluorescent protein with the sample, the supercharged fluorescent protein being configured to directly chelate with the analyte to form a protein- analyte complex; irradiating the sample with a light source; and determining, using a fluorescence detector, the presence and / or amount of the analyte in the sample.

[0052] In some aspects, the light source comprises UV light. In some aspects, the UV light have a wavelength from 340 nm to 360 nm.

[0053] In some aspects, the fluorescence detector comprises a fluorescent microscope. In some aspects, the analyte comprises one or more lanthanides. In some aspects, the one or more lanthanides comprises free lanthanide cations. In some aspects, the analyte comprises terbium (Tb), thulium (Tm), dysprosium (Dy), europium (Eu), samarium (Sm), ytterbium (Yb), or a combination thereof.

[0054] In some aspects, the supercharged fluorescent protein comprises a green fluorescent protein (GFP), a yellow fluorescent protein (YFP), a cyan fluorescent protein (CFP), or a combination thereof.

[0055] In some aspects, the fluorescent protein comprises an amino acid sequence having at least 85% sequence identity to any of SEQ ID NOS: 1-18.

[0056] In some aspects, the fluorescent protein comprises an amino acid sequence having at least 85% sequence identity to any of SEQ ID NOS: 1-3.

[0057] In some aspects, the fluorescent protein has a negative net charge.

[0058] In some aspects, the supercharged fluorescent protein has a net positive charge.

[0059] In some aspects, the analyte comprises a lanthanide-chelate configured to bind to the supercharged fluorescent protein having a net positive charge.

[0060] In some aspects, the protein- analyte complex is configured to provide multiplex detection of a plurality of analytes.

[0061] In some aspects, the supercharged fluorescent protein is a monomeric protein.

[0062] In some aspects, the supercharged fluorescent protein is a multimeric protein.

[0063] In some aspects, the multimeric protein includes a protomer comprising a pair of supercharged fluorescent proteins having opposite net charges.

[0064] In some aspects, the multimeric protein comprises a heterodimer.

[0065] In some aspects, the method further includes quantitatively determining an amount of the analyte in the sample based on a fluorescent signal emitted by the protein-analyte complex.

[0066] In some aspects, the amount of the analyte is determined using a normalized ratio between the fluorescent signal emitted by the protein- analyte complex and a baseline fluorescent signal emitted by the supercharged fluorescent protein alone.

[0067] In some aspects, the protein- analyte complex is measurably fluorescent when the analyte and supercharged protein are separated by a distance of 50 nm or less. DESCRIPTION OF DRAWINGS

[0068] FIGURES 1A-1B depict a schema for energy transfer. FIG. 1A shows a schema of the posited lanthanide-based resonance energy transfer between the lanthanide antenna and the fluorescent protein. Antenna excitation gives rise to a detectable signal if the lanthanide and the biosensor are separated by 10-100 A. FIG. IB shows spectral overlaps between terbium, thulium, dysprosium, and YFP, GFP, and CFP. The solid dashes represent select free-ion energy levels of trivalent terbium, thulium, and dysprosium. Error bars denote the approximate excitation ranges of supercharged YFP, GFP and CFP.

[0069] FIGURES 2A-2B depict that charge engineering introduces lanthanide chelation sites. FIG. 2A shows charge engineering schema for generating 18 protein biosensor variants. FIG. 2B shows an illustration of potential lanthanide binding interactions and proximity to the protein chromophore. Solvent-exposed residues are mutated to increase the net charge while preserving the structure and function of the fluorescent protein. The charged surface enables lanthanide binding at various distances to the internal chromophore of the fluorescent protein.

[0070] FIGURES 3A-3B depict supercharged proteins fluoresce in the presence of lanthanides. FIG. 3A shows supemegative sensors fluoresce in the presence of 1 mM of lanthanides. Fluorescent protein concentration of 0.1 mg / mL (-3.7 pM) in 50 mM Tris-HCl at pH of 7. FIG. 3B shows lanthanide sensitivities of biosensors depend on the excitation wavelength.

[0071] FIGURE 4A depicts tryptophan’s proximity to lanthanide binding sites. The proximity of the intrinsically fluorescent Trp57 residue to numerous binding pockets for lanthanides, as well as the internal chromophore of the fluorescent protein, lends to a potential radiative transfer in which Trp57 sensitizes the luminescence of the lanthanides, which in turn excites the chromophore. The labeled aspartate and glutamate residues are positioned approximately 15-30 A away from both Trp57 and the chromophore. FIGURE 4B depicts a hypothetical model mechanism by which the lanthanide, upon excitation at 280 nm, is functionalized into an antenna for energy transfer to the fluorescent protein chromophore. FIGURE 4C depicts the lanthanide binding microenvironment. The posited lanthanide binding sites (i.e., aspartate and glutamate) are surrounded by an electronically dense network of potentially sensitizing aromatics. In conjunction with Trp57, this unique architecture could account for the signal response upon excitation at or near 280 nm. FIGURES 5A-5B depict time-resolved spectroscopy reveals long-lived lanthanide luminescence signaling. FIG. 5A shows that supemegative GFP-10 sensors fluoresce in the presence of ImM of Tb3+. Time-resolved luminescence responses measured at 0 ps, 100 ps, and 300 ps. GFP-10 concentration of 0.1 mg / mL (-3.7 pM) in 50 mM Tris-HCl at pH of 7. FIG. 5B shows that supernegative YFP-31 sensors fluoresce in the presence of 1 mM of Tb3+. Time-resolved luminescence responses measured at 0 ps, 100 ps, and 300 ps. YFP-31 concentration of 0.1 mg / mL (-3.7 pM) in 50 mM Tris-HCl at pH of 7.

[0072] FIGURES 6A-6D depict that time-resolved spectroscopy reveals long-lived detection of Tb3+. FIG. 6A shows GFP-10 fluorescence in the presence of 1 mM of Tb3+upon excitation in the 250-400 nm range. The signal remains detectable with time delay of 100 ps. Fluorescent protein concentration of 0.1 mg / mL (-3.7 pM) in 50 mM Tris-HCl at pH of 7. FIG. 6B shows GFP-10 fluorescence upon excitation in the 250-400 nm range with no lanthanide present. The signal is not detectable with time delay of 100 ps. Fluorescent protein concentration of 0.1 mg / mL (-3.7 pM) in 50 mM Tris-HCl at pH of 7. FIG. 6C shows YFP-31 fluorescence in the presence of 1 mM of Tb3+upon excitation in the 250-400 nm range. The signal remains detectable with time delay of 100 ps. Fluorescent protein concentration of 0.1 mg / mL (-3.7 pM) in 50 mM Tris-HCl at pH of 7. FIG. 6D shows YFP-31 fluorescence upon excitation in the 250-400 nm range with no lanthanide present. The signal is not detectable with time delay of 100 ps. Fluorescent protein concentration of 0.1 mg / mL (-3.7 pM) in 50 mM Tris-HCl at pH of 7.

[0073] FIGURES 7A-7B depict residual Tb3+detection after extended time delay. FIG. 7A shows GFP-10 fluorescence in the presence of 1 mM of Tb3+upon excitation in the 250- 400 nm range. The signal remains detectable with time delays of 100 ps and 300 ps. Fluorescent protein concentration of 0.1 mg / mL (-3.7 pM) in 50 mM Tris-HCl at pH of 7. FIG. 7B shows YFP-31 fluorescence in the presence of 1 mM of Tb3+upon excitation in the 250-400 nm range. The signal remains detectable with time delays of 100 ps and 300 ps. Fluorescent protein concentration of 0.1 mg / mL (-3.7 pM) in 50 mM Tris-HCl at pH of 7.

[0074] FIGURES 8A-8C depict lanthanide sensitivities of supercharged protein biosensors. FIG. 8A shows ratiometric excitation responses of supemegative and superpositive protein sensors in the presence of 1 mM Tb3+, Tm3+, or Dy3+in 50 mM Tris- HCl at pH 7. Fluorescent protein concentration of 0.1 mg / mL (-3.7 pM). FIG. 8B shows GFP-10 biosensor ratiometric responses to Tb3+, Tm3+, Dy3+, Eu3+, Sm3+, and Yb3+in the 1 nM-10 mM range. GFP-10 concentration of 0.1 mg / mL (-3.7 pM) in 50 mM Tris-HCl at pH of 7. FIG. 8C shows YFP-31 biosensor ratiometric responses to Tb3+, Tm3+, Dy3+, EU3+, Sm3+, and Yb3+in the 1 nM-10 mM range. YFP-31 concentration of 0.1 mg / mL (-3.7 pM) in 50 mM Tris-HCl at pH of 7.

[0075] FIGURES 9A-9C depict supercharged protein biosensor responsivities. FIG. 9A shows factor change in excitation ratio (ex. 340 nm) of supercharged GFP variants in the presence of 1 mM Tb3+, Tm3+, and Dy3+. GFP concentration of 0.1 mg / mL (-3.7 pM) in 50 mM Tris-HCl at pH of 7. FIG. 9B shows factor change in excitation ratio (ex. 340 nm) of supercharged YFP variants in the presence of 1 mM Tb3+, Tm3+, and Dy3+. YFP concentration of 0.1 mg / mL (-3.7 pM) in 50 mM Tris-HCl at pH of 7. FIG. 9C shows factor change in excitation ratio (ex. 360 nm) of supercharged CFP variants in the presence of 1 mM Tb3+, Tm3+, and Dy3+. CFP concentration of 0.1 mg / mL (-3.7 pM) in 50 mM Tris- HCl at pH of 7.

[0076] FIGURES 10A-10D depict fluorescence response in the presence of REEs. FIG. 10A shows GFP-10 fluorescence (ex. 465 nm) in the presence of Tb3+in the 1 nM-1 mM range. FIG. 10B shows GFP-10 fluorescence (ex. 340 nm) in the presence of Tb3+in the 1 nM-1 mM range. FIG. 10C shows GFP-10 excitation ratio is correlated with the fluorescence output in response to excitation at 340 nm. FIG. 10D shows GFP-10 sensitivity to Tb3+in an expanded concentration range.

[0077] FIGURE 11 depicts emission spectra of GFP-10 and YFP-31 fluorescence responses to Tb3+(ex. 340 nm and 360 nm, respectively). GFP-10 and YFP-31 concentration of 0.1 mg / mL (-3.7 pM) in 50 mM Tris-HCl at pH of 7.

[0078] FIGURES 12A-12C depict responsivities of biosensors in the presence of interferents. FIG. 12A shows supemegative YFP series emission spectrum (ex. 280 nm) in the presence of equimolar (1 mM) amounts of Tb3+or Al3+. FIG. 12B shows GFP-10 and YFP-31 biosensor responses to 1 mM of Tb3+versus a 10-fold excess of contaminants (10 mM) found in mine drainage from the Virginia Canyon. 50 mM Tris-HCl at pH of 7. FIG. 12C shows Virginia Canyon (VC) and modified Virginia Canyon (mVC) interference of the GFP-10 ratiometric signal in the presence of 1 mM Tb3+. GFP-10 concentration of 0.1 mg / mL (-3.7 pM) in 50 mM Tris-HCl at pH of 7. FIG. 12D shows Virginia Canyon (VC) and modified Virginia Canyon (mVC) interference of the YFP-31 ratiometric signal in the presence of 1 mM Tb3+. YFP-31 concentration of 0.1 mg / mL (-3.7 pM) in 50 mM Tris- HCl at pH of 7. FIGURES 13A-13B depict responsivities of biosensors in the presence of aluminum and iron interferents. FIG. 13A shows that aluminum and iron interferents are not luminescent and therefore do not transmit an LRET signal. FIG. 13B shows Al3+and Fe2+interference of the GFP-10 ratiometric signal in the presence of 1 mM Tb3+. GFP-10 concentration of 0.1 mg / mL (-3.7 pM) in 50 mM Tris-HCl at pH of 7.

[0079] FIGURE 14 depicts positive and negative signal interference in the presence of aluminum. Tb3+:GFP-10 competitive and non-competitive response to aluminum interference. 1) Fluorescence output after UV excitation (340 nm) in the presence of 1 mM Tb3+or 0.1 mM Tb3+. 2) GFP-10 sensor can discriminate between the presence of 1 mM Al3+and a tenfold drop in Tb3+concentration. 3) Positive response to aluminum interference ± 1 SD. 4) Negative response to aluminum interference ± 1 SD. 5) GFP-10 response to tenfold decrease in Tb3+concentration. 6) GFP-10 sensor can discriminate between the presence of 0.1 mM Al3+and 0.1 mM Tb3+.

[0080] FIGURES 15A-15B depict responsivities of biosensors in the presence of Virginia Canyon and modified Virginia Canyon contaminants at pH of 5. FIG. 15A shows Virginia Canyon (VC) and modified Virginia Canyon (mVC) interference of the GFP-10 ratiometric signal in the presence of 1 mM Tb3+. FIG. 15B shows Virginia Canyon (VC) and modified Virginia Canyon (mVC) interference of the YFP-31 ratiometric signal in the presence of 1 mM Tb3+.

[0081] FIGURES 16A-16B depict responsivities of biosensors in the presence of common interferents. FIG. 16A shows Mg2+, Ca2+, Mn2+, Zn2+, and Cu2+interference of the GFP-10 ratiometric signal in the presence of 1 mM Tb3+. GFP-10 concentration of 0.1 mg / mL (-3.7 pM) in 50 mM Tris-HCl at pH of 7. FIG. 16B shows Mg2+, Ca2+, Mn2+, Zn2+, and Cu2+interference of the YFP-31 ratiometric signal in the presence of 1 mM Tb3+. YFP-31 concentration of 0.1 mg / mL (-3.7 pM) in 50 mM Tris-HCl at pH of 7.

[0082] FIGURE 17 depicts YFP-31 biosensor discrimination of lanthanide versus interferent. YFP-31 emission spectrum (ex. 360 nm) in the presence of equimolar (1 mM) amounts of Tb3+versus Mg2+, Ca2+, Mn2+, or Zn2+. YFP-31 concentration of 0.1 mg / mL (-3.7 pM) in 50 mM Tris-HCl at pH of 7.

[0083] FIGURES 18A-18B depict responsivities of biosensors in the presence of the non- luminescent lanthanide lanthanum. FIG. 18A shows La3+interference of the GFP-10 ratiometric signal in the presence of 1 mM Tb3+. La3+and Tb3+mixture prepared prior to the addition of GFP-10 to ensure equal binding opportunity. GFP-10 concentration of 0.1 mg / mL (-3.7 p M) in 50 mM Tris-HCl at pH of 7. FIG. 18B shows La3+interference of the YFP-31 ratiometric signal in the presence of 1 mM Tb3+. La3+and Tb3+mixture prepared prior to the addition of GFP-10 to ensure equal binding opportunity. YFP-31 concentration of 0.1 mg / mL (-3.7 pM) in 50 mM Tris-HCl at pH of 7.

[0084] FIGURE 19 depicts aggregation effect of REEs and interf erents. Response of supercharged GFP variants to the introduction of 1 mM of Tb3+or Al3+after an incubation time of 30 minutes. GFP concentration of 0.1 mg / mL (-3.7 pM) in 50 mM Tris-HCl at pH of 7.

[0085] FIGURES 20A-20D depict NaCl disruption of biosensor-lanthanide signaling.

[0086] FIG. 20A shows NaCl disruption of signaling between 0.1 mg / mL of GFP-10 and 1 mM of Tb3+with GFP-10 and Tb3+combined first and incubated for 30 minutes. FIG. 20B shows NaCl disruption of signaling between 0.1 mg / mL of GFP-10 and 1 mM of Tb3+with NaCl and Tb3+combined first and incubated for 30 minutes. FIG. 20C shows NaCl disruption of signaling between 0.1 mg / mL of YFP-31 and 1 mM of Tb3+with YFP-31 and Tb3+combined first and incubated for 30 minutes. FIG. 20D shows NaCl disruption of signaling between 0.1 mg / mL of YFP-10 and 1 mM of Tb3+with NaCl and Tb3+combined first and incubated for 30 minutes.

[0087] FIGURES 21A-21B depict temperature disruption of biosensor-lanthanide signaling. FIG. 21A shows temperature disruption of signaling between 0.1 mg / mL of GFP-10 and 1 mM of Tb3+. GFP-10 concentration of 0.1 mg / mL (-3.7 pM) in 50 mM Tris-HCl at pH of 7. FIG. 21B shows temperature disruption of signaling between 0.1 mg / mL of YFP-31 and 1 mM of Tb3+. YFP-31 concentration of 0.1 mg / mL (-3.7 pM) in 50 mM Tris-HCl at pH of 7.

[0088] FIGURES 22A-22B depict pH disruption of biosensor-lanthanide signaling. FIG. 22A shows pH disruption of signaling between 0.1 mg / mL of GFP-10 and 1 mM of Tb3+. FIG. 22B shows pH disruption of signaling between 0.1 mg / mL of YFP-31 and 1 mM of Tb3+.

[0089] FIGURES 23A-23B depict the formation of the supercharged protomer. FIG. 23A shows the supercharged GFP fluorescence emission spectrum (ex. 433 nm) confirming the formation of the CFP+32 / GFP-31 protomer. FIG. 23B shows the FRET interaction between CFP+32 / GFP-31 is more clearly seen after subtracting the fluorescence of CFP+32 on its own. FIGURES 24A-24C depict supercharged protomer fluorescence in the presence of lanthanides. FIG. 24A shows a schema of the posited LRET interaction between the lanthanide and the CFP+32 / GFP-31 protomer. FIG. 24B shows lanthanide and aluminum (1 mM) interference of the protomer signal (ex. 433 nm). An increased signal relative to the unperturbed protomer FRET is observed when exciting at 340 nm. Protomer concentration of 3.7 pM in 50 mM Tris-HCl at pH of 7. FIG. 24C shows that the FRET signal is ‘restored’ in varying degrees when exciting at 340 nm versus 433 nm.

[0090] DETAILED DESCRIPTION

[0091] General Definitions

[0092] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs.

[0093] Ranges can be expressed herein as from “about” one particular value, and / or to “about” another particular value. By “about” is meant within 10% of the value, e.g., within 9, 8, 8, 7, 6, 5, 4, 3, 2, or 1% of the value. When such a range is expressed, another aspect includes from the one particular value and / or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another aspect. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed.

[0094] The term “comprising” and variations thereof as used herein is used synonymously with the term “including” and variations thereof and are open, non-limiting terms. Although the terms “comprising” and “including” have been used herein to describe various embodiments, the terms “consisting essentially of’ and “consisting of’ can be used in place of “comprising” and “including” to provide for more specific embodiments and are also disclosed. Throughout the description and claims of this specification the word “comprise” and other forms of the word, such as “comprising” and “comprises,” means including but not limited to, and is not intended to exclude, for example, other additives, components, integers, or steps. As used in the specification and claims, the singular form “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise. For example, the term “an analyte” includes a plurality of analytes, including mixtures thereof.

[0095] As used herein, the terms "may," "optionally," and "may optionally" are used interchangeably and are meant to include cases in which the condition occurs as well as cases in which the condition does not occur. Thus, for example, the statement that a formulation "may include an excipient" is meant to include cases in which the formulation includes an excipient as well as cases in which the formulation does not include an excipient.

[0096] "Inhibit," "inhibiting," and "inhibition" mean to decrease an activity, response, condition, disease, or other biological parameter. This can include but is not limited to the complete ablation of the activity, response, condition, or disease. This may also include, for example, a 10% reduction in the activity, response, condition, or disease as compared to the native or control level. Thus, the reduction can be a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount of reduction in between as compared to native or control levels.

[0097] By “reduce,” or “abrogate,” (used interchangeably) or other forms of the word, such as “reducing” or “reduction,” or “abrogating” or “abrogation” is meant lowering of an event or characteristic. It is understood that this is typically in relation to some standard or expected value, in other words it is relative, but that it is not always necessary for the standard or relative value to be referred to.

[0098] By “increase” or other forms of the word, such as “increasing,” is meant raising or elevating. It is understood that this is typically in relation to some standard or expected value, in other words it is relative, but that it is not always necessary for the standard or relative value to be referred to.

[0099] “Detecting” is used herein to identify the existence, presence, or fact of something. General methods of detecting are known to the skilled artisan and may be supplemented with the protocols and reagents disclosed herein. For example, included herein are methods of detecting an analyte in a sample. Detection can include a physical readout, such as fluorescence output.

[0100] The terms “protein” and “polypeptide” are used herein in a generic sense to include polymers of amino acid residues of any length. The term “peptide” is used herein to refer to polypeptides having less than 250 amino acid residues, typically less than 100 amino acid residues, more typically less than 15 amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residues are an artificial chemical analog of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers. The peptide or protein may be further conjugated to or complexed with other moieties such as dyes, haptens, radioactive isotopes, natural and synthetic polymers (including microspheres), glass, metals and metallic particles, proteins and nucleic acids.

[0101] The term “supercharged protein” as used herein refers to any protein with a modification that results in the increase or decrease of the overall net charge of the protein when compared with the parent protein. Modifications include, but are not limited to, alterations in amino acid sequence or addition of charged moieties (e.g., carboxylic acid groups, phosphate groups, sulfate groups, amino groups). Supercharged proteins may be naturally occurring (i.e., wild-type) or synthetically modified. In some aspects, the supercharged protein is translated from a gene encoding the supercharged protein. Genes encoding supercharged fluorescent proteins can be synthesized using any known method in the art.

[0102] As used herein, the term “fluorescent protein” refers to any protein that can fluoresce when excited with an appropriate electromagnetic radiation (e.g., UV light) In general, a fluorescent protein useful for preparing a composition of the invention or for use in a method of the invention is a protein that derives its fluorescence from autocatalytically forming a chromophore. A fluorescent protein can contain amino acid sequences that are naturally occurring or that have been engineered (i.e., variants or mutants). When used in reference to a fluorescent protein, the term “mutant” or “variant” refers to a protein that is different from a reference protein. For example, a spectral variant of Aequorea GFP can be derived from the naturally occurring GFP by engineering mutations such as amino acid substitutions into the reference GFP protein.

[0103] The term “naturally-occurring” refers to a protein, nucleic acid molecule, cell, or other material that occurs in nature. A naturally occurring material can be in its form as it exists in nature, and can be modified by the hand of man such that, for example, it is in an isolated form.

[0104] The term “mutant” or “variant” is used herein in reference to a fluorescent protein that contains a mutation with respect to a corresponding wild type fluorescent protein. In addition, reference is made herein to a “spectral variant” or “spectral mutant” of a fluorescent protein to indicate a mutant fluorescent protein that has a different fluorescence characteristic with respect to the corresponding wild type fluorescent protein. Two or more amino acid sequences or two or more nucleotide sequences are considered to be “substantially identical” or “substantially similar” if the amino acid sequences or the nucleotide sequences share at least 80% sequence identity with each other, or with a reference sequence over a given comparison window. Thus, substantially similar sequences include those having, for example, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or at least 99% sequence identity.

[0105] Two or more amino acid sequences or two or more nucleotide sequences are considered to be “similar” if the amino acid sequences or the nucleotide sequences share at least 50% sequence identity with each other, or with a reference sequence over a given comparison window. Thus, substantially similar sequences include nucleotide sequences considered to be “substantially identical” or “substantially similar”.

[0106] The term “chelate” is defined as a coordination complex where a single central ion is coordinated (or multiple central ions are coordinated) to at least one ligand with at least one coordination bond (each). Accordingly, the term “chelator” can refer to any molecule which possesses at least one functional group which can coordinate to a metal, either covalently or non-covalently. The chelator may be multidentate or coordinate in a unidentate manner.

[0107] As used herein, the term “light source” refers to any device that emits electromagnetic radiation. In some embodiments, the system, kits, and methods disclosed herein may comprise one or a plurality of light sources. Such light source may be LEDS, incandescent lamps, lasers, or the like or any other device that can excite wavelengths of light.

[0108] The term “fluorescence resonance energy transfer” (FRET) refers to a non-radiative transfer of excitation energy from a donor to an acceptor molecule. After excitation of the donor, energy is transmitted to the acceptor in a non-radiative manner and emitted by the acceptor.

[0109] As used herein, the term “immobilized,” when used with respect to a biomolecule or biological or chemical substance, includes substantially attaching the biomolecule or biological or chemical substance at a molecular level to a surface. For example, a biomolecule or biological or chemical substance may be immobilized to a surface of the substrate material using adsorption techniques including non-covalent interactions (e.g., electrostatic forces, van der Waals, and dehydration of hydrophobic interfaces) and covalent binding techniques where functional groups or linkers facilitate attaching the biomolecules to the surface. Immobilizing biomolecules or biological or chemical substances to a surface of a substrate material may be based upon the properties of the substrate surface, the liquid medium carrying the biomolecule or biological or chemical substance, and the properties of the biomolecules or biological or chemical substances themselves. In some cases, a substrate surface may be functionalized (e.g., chemically or physically modified) to facilitate immobilizing the biomolecules (or biological or chemical substances) to the substrate surface. The substrate surface may be first modified to have functional groups bound to the surface. The functional groups may then bind to biomolecules or biological or chemical substances to immobilize them thereon. A substance can be immobilized to a surface via a gel, for example, as described in US Patent Publ. No. US 2011 / 0059865 Al, which is incorporated herein by reference.

[0110] The term “affinity” as used herein refers to the strength of the binding interaction of two molecules, such as a metal-chelating compound and a metal ion or a positively charged moiety and a negatively charged moiety.

[0111] Certain compounds of the present invention can exist in unsolvated forms as well as solvated forms, including hydrated forms. In general, the solvated forms are equivalent to unsolvated forms and are encompassed within the scope of the present invention. Certain compounds of the present invention may exist in multiple crystalline or amorphous forms. In general, all physical forms are equivalent for the uses contemplated herein and are intended to be within the scope of the present invention.

[0112] Certain compounds of the present invention possess asymmetric carbon atoms (optical centers) or double bonds; the racemates, diastereomers, geometric isomers and individual isomers are encompassed within the scope of the present invention.

[0113] The compounds of the invention may be prepared as a single isomer (e.g., enantiomer, cis-trans, positional, diastereomer) or as a mixture of isomers. In a preferred embodiment, the compounds are prepared as substantially a single isomer. Methods of preparing substantially isomerically pure compounds are known in the art. For example, enantiomerically enriched mixtures and pure enantiomeric compounds can be prepared by using synthetic intermediates that are enantiomerically pure in combination with reactions that either leave the stereochemistry at a chiral center unchanged or result in its complete inversion. Alternatively, the final product or intermediates along the synthetic route can be resolved into a single stereoisomer. Techniques for inverting or leaving unchanged a particular stereocenter, and those for resolving mixtures of stereoisomers are well known in the art and it is well within the ability of one of skill in the art to choose and appropriate method for a particular situation.

[0114] The term “amino” or “amine group” refers to the group — NR'R" (or NRR'R") where R, R' and R" are independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, aryl, substituted aryl, aryl alkyl, substituted aryl alkyl, heteroaryl, and substituted heteroaryl. A substituted amine being an amine group wherein R' or R" is other than hydrogen. In a primary amino group, both R' and R" are hydrogen, whereas in a secondary amino group, either, but not both, R' or R" is hydrogen. In addition, the terms “amine” and “amino” can include protonated and quatemized versions of nitrogen, comprising the group — NRR'R" and its biologically compatible anionic counterions.

[0115] The term “aqueous solution” as used herein refers to a solution that is predominantly water and retains the solution characteristics of water. Where the aqueous solution contains solvents in addition to water, water is typically the predominant solvent.

[0116] The term “buffer” as used herein refers to a system that acts to minimize the change in acidity or basicity of the solution against addition or depletion of chemical substances.

[0117] The term “complex” as used herein refers to the association of two or more molecules, usually by non-covalent bonding.

[0118] The term “detectable response” as used herein refers to a change in or an occurrence of, a signal that is directly or indirectly detectable either by observation or by instrumentation. Typically, the detectable response is an optical response resulting in a change in the wavelength distribution patterns or intensity of absorbance or fluorescence or a change in light scatter, fluorescence lifetime, fluorescence polarization, or a combination of the above parameters. Alternatively, the detectable response is an occurrence of a signal wherein the fluorophore is inherently fluorescent and does not produce a change in signal upon binding to a metal ion. Alternatively, the detectable response is the result of a signal, such as color, fluorescence, radioactivity or another physical property of the detectable label becoming spatially localized in a subset of a sample such as in a gel, on a blot, or an array, in a well of a micoplate, in a microfluidic chamber, or on a microparticle. The term “measurably fluorescent” as used herein refers to a change in or an occurrence of a fluorescent signal that is directly or indirectly detectable either by observation or by instrumentation.

[0119] The term “directly detectable” as used herein refers to the presence of a detectable label or the signal generated from a detectable label that is immediately detectable by observation, instrumentation, or film without requiring chemical modifications or additional substances. For example, a fluorophore produces a directly detectable response.

[0120] The term “fluorophore” as used herein refers to a compound that is inherently fluorescent or demonstrates a change in fluorescence upon binding to a biological compound or metal ion, i.e., Anorogenic. Numerous Auorophores are known to those skilled in the art and include, but are not limited to, coumarin, acridine, furan, indole, quinoline, cyanine, benzofuran, quinazolinone, benzazole, borapolyazaindacene and xanthenes, with the latter including Auoroscein, rhodamine, rhodol, rosamine and derivatives thereof as well as other Auorophores known to those of skill in the art.

[0121] The term “kit” as used herein refers to a packaged set of related components, typically one or more compounds or compositions.

[0122] As used herein, the term “modified” or “engineered” refers to any manipulation of a microorganism, wherein the manipulation includes but not limited to inserting a polynucleotide and / or polypeptide heterologous to the microorganism and mutating a polynucleotide and / or polypeptide native to the microorganism. The term “genetically modified microorganism” refers to a microorganism having at least one genetic alteration not normally found in the wild-type strain of the reference species, for example, involving rational pathway design and assembly of biosynthetic genes, genes associated with operons, and control elements of such polynucleotides, for the production of a desired product (e.g., a supercharged Auorescent protein). As used herein, the term “microorganism” is intended to mean any organism that exists as a microscopic cell that is included within the domains of archaea, bacteria or eukarya. Therefore, the term is intended to encompass prokaryotic or eukaryotic cells or organisms having a microscopic size and includes bacteria, archaea and eubacteria of all species as well as eukaryotic microorganisms such as yeast and fungi.

[0123] The term “sample” as used herein refers to any material that may contain an analyte, as defined above. The term sample includes, but is not limited to, biological samples and environmental samples. Biological samples include any number of biological Auids. Nonlimiting examples of biological Auids include serum, plasma, whole blood, urine, saliva, milk, tears, sweat, joint Auid, cerebrospinal Auid, semen, vaginal Auid, ascetic Auid and amniotic Auid. Environmental samples include environmental material such as surface matter, soil, water, crystals and industrial samples. These examples are not to be construed as limiting the sample types applicable to the present disclosure. By “contacting” is meant placement in direct physical association, for example solid, liquid or gaseous forms. Contacting includes, for example, direct physical association of fully- and partially-solvated molecules.

[0124] Detection Systems

[0125] In various aspects, disclosed herein is a detection system for selectively characterizing the presence and / or amount of an analyte in a sample, the system comprising: a supercharged fluorescent protein configured to directly chelate with an analyte having a spectral overlap with the supercharged fluorescent protein, thereby forming a proteinanalyte complex, wherein the protein- analyte complex is configured to emit a fluorescent signal when irradiated with a light source.

[0126] The supercharged protein of the present systems shares a spectral overlap with an analyte. As used herein, the term “spectral overlap” refers to the overlap between the emission spectrum of a donor fluorophore (e.g., an analyte) and the excitation spectrum of the acceptor fluorophore (e.g., a supercharged fluorescent protein). Such overlaps enable acceptor emission (i.e., fluorescent signal readout) via donor excitation (i.e., UV irradiation).

[0127] In some aspects, the system further includes a fluorescence detector configured to detect the fluorescent signal from the protein- analyte complex. As used herein, the term “fluorescence detector” is inclusive of any device (e.g., fluorescence detecting camera, an array of fluorescence detecting cameras, fluorescence detecting sensor, an array of fluorescence detecting sensors, etc.) that is capable of detecting fluorescence emitted from the protein-analyte complex. In some aspects, the fluorescence detector comprises a fluorescent microscope.

[0128] In some aspects, the light source comprises UV light (i.e., 250-400 nm). In some aspects, the UV light has a wavelength from 340 nm to 360 nm. Generally, the particular wavelength of light is dependent on the specific supercharged fluorescent protein and analyte and can be determined using routine experimentation.

[0129] As used herein, the term “analyte” refers to a chemical or biological species that an experiment or device is intended to detect and / or measure. Analytes can include, for example, surfactants, ions, oils, microorganisms and toxins produced thereby, metal cations, peptides, organophosphates, organic pollutants, chemical agents, pharmaceutically active compounds, proteins and biological ligands. However, as known to a person skilled in the art, additional analytes may be suitable for detection according to the present systems, and therefore these examples are not to be construed as limiting the analyte-types applicable to the present systems.

[0130] In some aspects, the analyte comprises one or more lanthanides. The term “lanthanide” is intended to broadly refers elements or compounds comprising one or more of neodymium, praseodymium, samarium, europium, promethium, gadolinium, terbium, dysprosium, holmium, erbium, thulium, ytterbium and yttrium, especially erbium, praseodymium, thulium, and / or ytterbium. In some aspects, the one or more lanthanides comprise free lanthanide cations. In some aspects, the analyte comprises terbium (Tb), thulium (Tm), dysprosium (Dy), or a combination thereof.

[0131] Unlike other lanthanide detection methods, the presently disclosed system can distinguish the analyte from non-targeted interferents. Interf erent as used herein refers to a chemical or biological species that is an interfering agent in the analysis of the analyte, that is present in the sample and that contributes error to the detection or measurement. Examples of interferents include metal cations such as Al3+and Fe2+.

[0132] Various fluorescent proteins can be used for the detection of analytes according to the present disclosure. For example, some suitable fluorescent proteins can include, but are not limited to, enhanced green fluorescent protein (EGFP), AcGFP, TurboGFP, Emerald, Azami Green, ZsGreen, EBFP, Sapphire, T-Sapphire, ECFP, mCFP, Cerulean, CyPet, AmCyanl, Midori-Ishi Cyan, mTFPl (Teal), enhanced yellow fluorescent protein (EYFP), Topaz, Venus, mCitrine, YPet, PhiYFP, ZsYellowl, mBanana, Kusabira Orange, mOrange, dTomato, dTomato-Tandem, DsRed, DsRed2, DsRed-Express (Tl), DsRed-Monomer, mTangerine, mStrawberry, AsRed2, mRFPl, JRed, mCherry, HcRedl, mRaspberry, HcRedl, HcRed-Tandem, mPlum, and AQ143. In some aspects, the fluorescent protein comprises a green fluorescent protein (GFP), a yellow fluorescent protein (YFP), a cyan fluorescent protein (CFP), or a combination thereof.

[0133] In some aspects, the fluorescent protein comprises an amino acid sequence having at least 85% (e.g., at least 90%, at least 95%, at least 97%, at least 99%) sequence identity to any of SEQ ID NOS: 1-18. In some aspects, the fluorescent protein comprises the amino acid sequence of SEQ ID NO: 1. In some aspects, the fluorescent protein comprises the amino acid sequence of SEQ ID NO: 2. In some aspects, the fluorescent protein comprises the amino acid sequence of SEQ ID NO: 3. In some aspects, the fluorescent protein comprises the amino acid sequence of SEQ ID NO: 4. In some aspects, the fluorescent protein comprises the amino acid sequence of SEQ ID NO: 5. In some aspects, the fluorescent protein comprises the amino acid sequence of SEQ ID NO: 6. In some aspects, the fluorescent protein comprises the amino acid sequence of SEQ ID NO: 7. In some aspects, the fluorescent protein comprises the amino acid sequence of SEQ ID NO: 8. In some aspects, the fluorescent protein comprises the amino acid sequence of SEQ ID NO: 9. In some aspects, the fluorescent protein comprises the amino acid sequence of SEQ ID NO: 10. In some aspects, the fluorescent protein comprises the amino acid sequence of SEQ ID NO: 11. In some aspects, the fluorescent protein comprises the amino acid sequence of SEQ ID NO: 12. In some aspects, the fluorescent protein comprises the amino acid sequence of SEQ ID NO: 13. In some aspects, the fluorescent protein comprises the amino acid sequence of SEQ ID NO: 14. In some aspects, the fluorescent protein comprises the amino acid sequence of SEQ ID NO: 15. In some aspects, the fluorescent protein comprises the amino acid sequence of SEQ ID NO: 16. In some aspects, the fluorescent protein comprises the amino acid sequence of SEQ ID NO: 17. In some aspects, the fluorescent protein comprises the amino acid sequence of SEQ ID NO: 18.

[0134] In some aspects, the fluorescent protein comprises an amino acid sequence having at least 85% sequence identity to any of SEQ ID NOS: 1-3. In some aspects, the fluorescent protein comprises an amino acid sequence having at least 90% sequence identity to any of SEQ ID NOS: 1-3. In some aspects, the fluorescent protein comprises an amino acid sequence having at least 95% sequence identity to any of SEQ ID NOS: 1-3. In some aspects, the fluorescent protein comprises an amino acid sequence having at least 97% sequence identity to any of SEQ ID NOS: 1-3. In some aspects, the fluorescent protein comprises an amino acid sequence having at least 98% sequence identity to any of SEQ ID NOS: 1-3. In some aspects, the fluorescent protein comprises an amino acid sequence having at least 99% sequence identity to any of SEQ ID NOS: 1-3.

[0135] In some aspects, the fluorescent protein comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 1. In some aspects, the fluorescent protein comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 1. In some aspects, the fluorescent protein comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 1. In some aspects, the fluorescent protein comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 1. In some aspects, the fluorescent protein comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 1. In some aspects, the fluorescent protein comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 1.

[0136] In some aspects, the fluorescent protein comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 2. In some aspects, the fluorescent protein comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 2. In some aspects, the fluorescent protein comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 2. In some aspects, the fluorescent protein comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 2. In some aspects, the fluorescent protein comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 2. In some aspects, the fluorescent protein comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 2.

[0137] In some aspects, the fluorescent protein comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 3. In some aspects, the fluorescent protein comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 3. In some aspects, the fluorescent protein comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 3. In some aspects, the fluorescent protein comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 3. In some aspects, the fluorescent protein comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 3. In some aspects, the fluorescent protein comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 3.

[0138] In some aspects, the protein- analyte complex is configured to be measurably fluorescent when the supercharged fluorescent protein and analyte are separated by a distance of 50 nm or less (e.g., 45 nm or less, 40 nm or less, 35 nm or less, 30 nm or less, 25 nm or less, 20 nm or less, 15 nm or less, or 10 nm or less).

[0139] In some aspects, the supercharged fluorescent protein has a negative net charge of from -1 to -50 (e.g., from -4 to -31, from -10 to -31, or from -17 to -31).

[0140] In some aspects, the supercharged fluorescent protein has a net positive charge. In some aspects, the analyte comprises a lanthanide-chelate configured to bind to the supercharged fluorescent protein having a net positive charge. As used herein, the term “bind” refers to any physical attachment or close association, which may be permanent or temporary. The binding can result from hydrogen bonding, hydrophobic forces, van der Waals forces, covalent, or ionic bonding, for example. In some aspects, the protein- analyte complex is configured to provide multiplex detection of a plurality of analytes. The term “multiplex detection” as used herein indicates detection and / or quantification of multiple analytes in a sample or multiple samples. For example, the disclosed systems can be configured to detect and / or quantify multiple different lanthanides.

[0141] In some aspects, the supercharged fluorescent protein is a monomeric protein. As used herein, the term “monomeric protein / polypeptide” refers to a single, non- aggregated protein or polypeptide molecule, including any species thereof, such as phosphorylated species.

[0142] In some aspects, the supercharged fluorescent protein is a multimeric protein. The term “multimeric protein” refers to a protein that may exist as a multimer consisting of two or more subunits. In the multimer, the subunits may be linked by covalent bonds such as disulfide bonds, linked by non-covalent bonds such as hydrogen bonds and hydrophobic interaction, or linked by a combination thereof. The multimer includes one or more intermolecular disulfide bonds. The multimer may be a homo-multimer having a single kind of subunit, or may be a hetero-multimer having two or more kinds of subunits. In the case where the multimeric protein is a hetero-multimer, it is sufficient that at least one subunit selected from the subunits constituting the hetero-multimer is a heterologous protein. That is, all the subunits may be heterologous, or only some of subunits may be heterologous. In some aspects, the multimeric protein includes a protomer comprising a pair of supercharged fluorescent proteins having opposite net charges. Opposite net charges do not necessitate that the magnitudes of the charges be the same. In some aspects, the multimeric protein comprises a heterodimer.

[0143] In some aspects, the system further includes: one or more sensors configured to detect the fluorescent signal of the protein- analyte complex; a processor; and software executable by said processor such that said system: determines the presence and / or amount of the analyte in the sample based on the fluorescent signal.

[0144] The processor of the described system, for processing information associated with the process(es) or method(s) described herein, may be, for example, any computer processor is known in the art capable of performing calculations and directing functions for interpreting and / or performing input, output, calculation, and display of data in accordance with the disclosed methods. The processor may comprise any type of processing unit, such as typical computer processor(s), controller(s), microcontroller(s), microprocessor s), and / or programmable logic controllers (PLCs). The information to be processed by the processor may include, for example, information contained in analog or digital signals and / or translated signals and / or information contained in a data storage. Processing of the information may involve, for example, performing calculations on received signals such as, but not limited to, vector analysis, picture identification, pattern recognition, frequency analysis / Fourier transforms, numerical computations, machine learning, or, as described herein, applying predetermined or recursively fit correlative algorithms to received sensor data. In some embodiments, the system comprises more than one processor, and the reference herein to “processor” includes reference to multiple processors and vice versa.

[0145] In an embodiment, the processor is in communication with the one or more sensors of the detection system. In another embodiment, the processor may also be in communication with data storage(s), and, optionally, display(s). The components of the system, such as the sensors, computing device(s), processor(s), feedback controller(s), data storage(s), and / or display(s), and any other components of the system or computing device, may communicate using any electronic wired or wireless means or protocols for communication known in the art, including but not limited to Ethernet™, Bluetooth™, WiFi™, infrared, near-field communications (NFC), radio-frequency identification (RFID), WiMAX™ (fixed or mobile), cellular communications protocols such as GSM, EDGE, GPRS, CDMA, EMTS, LTE, LTE-A, IMS, and any other cellular communications protocols including, but not limited to, up to and including 5G protocols as established under the 3 GPP, for example, and any other communications protocols suitable for the method(s) and system(s) described herein, including any proprietary protocols. Components of the system may exist on the same network or on separate networks, and the network(s) may include any type of network suitable for the system(s) and method(s) described herein, including but not limited to wired or wireless personal area networks (PANs), local area networks (LANs), mesh or ad hoc networks, wide area networks (WANs), metropolitan area networks (MANs), virtual private networks (VPNs), and any other suitable network type, as well as any suitable network configuration or topology (e.g., token ring, star, bus, mesh, tree, etc.). The presently described system(s) further includes any components necessary to effect the communication and / or network type employed, such as wireless or wired routers and access points.

[0146] The presently described methods and systems may be implemented on a secure network to which access may be limited to authorized users by any known means and which may be protected by known security measures, such as by the use of firewalls. In some embodiments, authentication may be required before granting access to authorized users, such as where autologous cell manufacturing and / or patient data is involved and / or where compliance with government regulations is mandated (such as Title 21 of the U.S. Code of Federal Regulations). Such authentication may be implemented for any one or more of the system components, such as for access to a computing device or machine housing the processor, access to data storage, access to a database of the data storage, access to any of the sensors or sensor readings of the detection system, access to a graphical user interface (GUI) of the system, access to the detection system, etc. Security of the presently described methods and systems may be further provided for by encrypting communications among system components by any means or protocols known to persons skilled in the art, such as Internet Protocol Security (IPSec), Transport Layer Security (TLS), Secure Sockets Layer (SSL), etc., in order to reduce the potential for the tampering with or corruption of the preprogrammed correlative algorithm(s), for example.

[0147] The presently described system may also include a data storage for storing information associated with the described methods. The data storage may include, for example, various types of local or remote memory devices such as a hard disk or hard drive (of any type, including electromechanical magnetic disks and solid-state disks), a memory chip, including, e.g., random-access memory (RAM) and / or read-only memory (ROM), flash memory, optical memory such as CD(s) and DVD(s), floppy disks, and any other form of optical, physical, electronic, and / or magnetic memory devices in or on which information may be stored. The data storage may comprise non-volatile memory. In some embodiments, the data storage may only be accessed via secure data transfer, which may be accomplished using one or more known server platforms and security protocols. The information to be stored in the data storage may comprise, for example, one or more predetermined algorithms for correlating sensor inputs to various outputs, such as present and / or amount of an analyte, and records of such determinations along with associated actions (such as feedings) and / or associated timestamps, unique identifiers, authorized users and associated authentication information for use in authenticating users for authorized access to the data storage or any other system component, and any other pertinent information. In operation, the data storage is in communication with the processor.

[0148] The system may also include a display (which may be co-located with the processor, e.g., where the processor and display are part of a computer or server used for carrying out the method steps described herein) for visually presenting information associated with the described methods. The display may comprise, for example, a computer monitor (e.g., LCD, a CRT monitor, a projection (e.g., heads-up display (HUD) laser), etc. In some embodiments, the visual display may comprise, for example, that of a mobile device such as a tablet computer, cellular phone, smartphone, personal digital assistant (PDA), personal computer (PC), laptop computer, augmented reality display (e.g., Google™ Glass™ or Microsoft™ HoloLens™), etc. The information presented on the display may include any other information collected in the course of carrying out the methods described herein, prompts for information entry associated with one or more steps of the described methods, and / or any predetermined formulae or algorithms, as previously described. The display may also be capable of receiving input (such as, e.g., where the display includes a touch-screen and is capable of receiving touch input and accordingly transmitting information to the processor).

[0149] It will be appreciated that components of the described system, such as a computer or machine housing the processor(s), include components known in the art that is required for their operation, such as a power supply, a network interface (such as a network interface card), network connectivity components (e.g., a modem, Ethernet cards, USB interface cards, FDDI cards, WLAN cards, etc.), a receiver, a transmitter, local memory, e.g., RAM, ROM, flash memory, cache or buffer memory, and / or other types of memory as previously described, a processing unit which may be in communication with input / output (EO) devices, and all required circuitry, including bus(es). The detection system may also include components specific to the sensor type, as would be known to the skilled person in the art. A computing device, such as a computer or machine, housing the processor(s) may also include, for example, memory (e.g., hard disk storage, RAM, ROM, flash memory, cache or buffer memory, and / or other types of memory as previously described), attached input device(s) (e.g., a mouse, keyboard, microphone, etc.), attached output device(s) (e.g., a display monitor), and local memory for the processor(s) (e.g., registers, cached RAM, such as LI cache, L2 cache, etc.). Depending on the system component, other components may also be present (e.g., a device in communication with a cellular network may include an antenna, etc.), and it will be appreciated that such components would be known to the skilled person in the art.

[0150] The described systems may comprise one or more redundant sensors for each of the one or more sensors in the detection system to provide further data for detecting the fluorescent signal or another metric of the system. Where, e.g., the sensor information is consistent among the redundant sensors, it may be stored in the data storage (e.g., in a database thereof), and where the information captured is not consistent among the redundant sensors, the respective sensor readings may be averaged or ignored altogether, or one of the redundant sensor readings, if not generally in line with the trajectory of the other received sensor data, may be discarded as an outlier, as previously described. Redundancy of the data storage and / or a database thereof is expected to help ensure the persistence of stored data and reduce the risk of data loss.

[0151] It will be appreciated that any method step, module, component, or system described herein that is suitable for computer implementation or required to be computer- implemented so as to effect real-time or substantially real-time execution may be implemented using computer readable / executable instructions or operations that may be stored or otherwise held by computer-readable media, as described herein.

[0152] Also disclosed herein is a detection system for selectively characterizing the presence and / or amount of an analyte in a sample, the system comprising: a genetically modified microorganism engineered to express a supercharged fluorescent protein, wherein supercharged fluorescent protein is configured to directly chelate with an analyte having a spectral overlap with the supercharged fluorescent protein, thereby forming a proteinanalyte complex, and wherein the protein- analyte complex is configured to emit a fluorescent signal when irradiated with a light source.

[0153] In some aspects, the system further includes a fluorescence detector configured to detect the fluorescent signal from the protein- analyte complex. The fluorescence detector can include any device (e.g., fluorescence detecting camera, an array of fluorescence detecting cameras, fluorescence detecting sensor, an array of fluorescence detecting sensors, etc.) that is capable of detecting fluorescence emitted from the protein- analyte complex. In some aspects, the fluorescence detector comprises a fluorescent microscope.

[0154] In some aspects, the light source comprises UV light (i.e., 250-400 nm). In some aspects, the UV light has a wavelength from 340 nm to 360 nm. Generally, the particular wavelength of light is dependent on the specific supercharged fluorescent protein and analyte and can be determined using routine experimentation.

[0155] In some aspects, the analyte comprises one or more lanthanides. In some aspects, the one or more lanthanides comprise free lanthanide cations. In some aspects, the analyte comprises terbium (Tb), thulium (Tm), dysprosium (Dy), europium (Eu), samarium (Sm), ytterbium (Yb), or a combination thereof.

[0156] In some aspects, the fluorescent protein comprises a green fluorescent protein (GFP), a yellow fluorescent protein (YFP), a cyan fluorescent protein (CFP), or a combination thereof.

[0157] In some aspects, the fluorescent protein comprises an amino acid sequence having at least 85% (e.g., at least 90%, at least 95%, at least 97%, at least 99%) sequence identity to any of SEQ ID NOS: 1-18. In some aspects, the fluorescent protein comprises the amino acid sequence of SEQ ID NO: 1. In some aspects, the fluorescent protein comprises the amino acid sequence of SEQ ID NO: 2. In some aspects, the fluorescent protein comprises the amino acid sequence of SEQ ID NO: 3. In some aspects, the fluorescent protein comprises the amino acid sequence of SEQ ID NO: 4. In some aspects, the fluorescent protein comprises the amino acid sequence of SEQ ID NO: 5. In some aspects, the fluorescent protein comprises the amino acid sequence of SEQ ID NO: 6. In some aspects, the fluorescent protein comprises the amino acid sequence of SEQ ID NO: 7. In some aspects, the fluorescent protein comprises the amino acid sequence of SEQ ID NO: 8. In some aspects, the fluorescent protein comprises the amino acid sequence of SEQ ID NO: 9. In some aspects, the fluorescent protein comprises the amino acid sequence of SEQ ID NO: 10. In some aspects, the fluorescent protein comprises the amino acid sequence of SEQ ID NO: 11. In some aspects, the fluorescent protein comprises the amino acid sequence of SEQ ID NO: 12. In some aspects, the fluorescent protein comprises the amino acid sequence of SEQ ID NO: 13. In some aspects, the fluorescent protein comprises the amino acid sequence of SEQ ID NO: 14. In some aspects, the fluorescent protein comprises the amino acid sequence of SEQ ID NO: 15. In some aspects, the fluorescent protein comprises the amino acid sequence of SEQ ID NO: 16. In some aspects, the fluorescent protein comprises the amino acid sequence of SEQ ID NO: 17. In some aspects, the fluorescent protein comprises the amino acid sequence of SEQ ID NO: 18.

[0158] In some aspects, the fluorescent protein comprises an amino acid sequence having at least 85% sequence identity to any of SEQ ID NOS: 1-3. In some aspects, the fluorescent protein comprises an amino acid sequence having at least 90% sequence identity to any of SEQ ID NOS: 1-3. In some aspects, the fluorescent protein comprises an amino acid sequence having at least 95% sequence identity to any of SEQ ID NOS: 1-3. In some aspects, the fluorescent protein comprises an amino acid sequence having at least 97% sequence identity to any of SEQ ID NOS: 1-3. In some aspects, the fluorescent protein comprises an amino acid sequence having at least 98% sequence identity to any of SEQ ID NOS: 1-3. In some aspects, the fluorescent protein comprises an amino acid sequence having at least 99% sequence identity to any of SEQ ID NOS: 1-3.

[0159] In some aspects, the fluorescent protein comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 1. In some aspects, the fluorescent protein comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 1. In some aspects, the fluorescent protein comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 1. In some aspects, the fluorescent protein comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 1. In some aspects, the fluorescent protein comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 1. In some aspects, the fluorescent protein comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 1.

[0160] In some aspects, the fluorescent protein comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 2. In some aspects, the fluorescent protein comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 2. In some aspects, the fluorescent protein comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 2. In some aspects, the fluorescent protein comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 2. In some aspects, the fluorescent protein comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 2. In some aspects, the fluorescent protein comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 2.

[0161] In some aspects, the fluorescent protein comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 3. In some aspects, the fluorescent protein comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 3. In some aspects, the fluorescent protein comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 3. In some aspects, the fluorescent protein comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 3. In some aspects, the fluorescent protein comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 3. In some aspects, the fluorescent protein comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 3.

[0162] In some aspects, the fluorescent protein has a negative net charge from -1 to -50 (e.g., from -4 to -31, from -10 to -31, or from -17 to -31). In some aspects, the supercharged fluorescent protein has a net positive charge.

[0163] In some aspects, the analyte comprises a lanthanide-chelate configured to bind to the supercharged fluorescent protein having a net positive charge.

[0164] In some aspects, the protein- analyte complex is configured to provide multiplex detection of a plurality of analytes.

[0165] In some aspects, the supercharged fluorescent protein is a monomeric protein.

[0166] In some aspects, the supercharged fluorescent protein is a multimeric protein. In some aspects, the multimeric protein includes a protomer comprising a pair of supercharged fluorescent proteins having opposite net charges. In some aspects, the multimeric protein comprises a heterodimer.

[0167] In some aspects, the system further includes: one or more sensors configured to detect the fluorescent signal of the protein- analyte complex; a processor; and software executable by said processor such that said system: determines the presence and / or amount of the analyte in the sample based on the fluorescent signal.

[0168] The sample used for detection is generally a representative cell population, fluid or liquid suspension that is known or suspected to contain the analyte. Representative samples include intracellular fluids such as in blood cells, cultured cells, muscle tissue, neurons and the like; extracellular fluids in areas immediately outside of cells; in vesicles; in vascular tissue of plants and animals; in biological fluids such as blood, saliva, and urine; in biological fermentation media; in environmental samples such as water, soil, waste water and sea water; in industrial samples such as pharmaceuticals, foodstuffs and beverages; and in chemical reactors. Detection and quantitation of the target ion in a sample can help characterize the identity of an unknown sample, or facilitate quality control of a sample of known origin.

[0169] Kits

[0170] In various aspects, disclosed herein is a kit comprising: a supercharged fluorescent protein disposed within an assay chamber, wherein the supercharged fluorescent protein is configured to directly chelate with an analyte having a spectral overlap with the supercharged fluorescent protein to form a protein-analyte complex, wherein the proteinanalyte complex is configured to emit a fluorescent signal when irradiated with a light source. In some aspects, the kit further includes a fluorescence detector configured to detect the fluorescent signal from the protein-analyte complex. The fluorescence detector can include any device (e.g., fluorescence detecting camera, an array of fluorescence detecting cameras, fluorescence detecting sensor, an array of fluorescence detecting sensors, etc.) that is capable of detecting fluorescence emitted from the protein- analyte complex. In some aspects, the fluorescence detector comprises a fluorescent microscope.

[0171] In some aspects, the light source comprises UV light (i.e., 250-400 nm). In some aspects, the UV light has a wavelength from 340 nm to 360 nm. Generally, the particular wavelength of light is dependent on the specific supercharged fluorescent protein and analyte and can be determined using routine experimentation.

[0172] In some aspects, the analyte comprises one or more lanthanides. In some aspects, the one or more lanthanides comprise free lanthanide cations. In some aspects, the analyte comprises terbium (Tb), thulium (Tm), dysprosium (Dy), europium (Eu), samarium (Sm), ytterbium (Yb), or a combination thereof.

[0173] In some aspects, the fluorescent protein comprises a green fluorescent protein (GFP), a yellow fluorescent protein (YFP), a cyan fluorescent protein (CFP), or a combination thereof.

[0174] In some aspects, the fluorescent protein comprises an amino acid sequence having at least 85% (e.g., at least 90%, at least 95%, at least 97%, at least 99%) sequence identity to any of SEQ ID NOS: 1-18. In some aspects, the fluorescent protein comprises the amino acid sequence of SEQ ID NO: 1. In some aspects, the fluorescent protein comprises the amino acid sequence of SEQ ID NO: 2. In some aspects, the fluorescent protein comprises the amino acid sequence of SEQ ID NO: 3. In some aspects, the fluorescent protein comprises the amino acid sequence of SEQ ID NO: 4. In some aspects, the fluorescent protein comprises the amino acid sequence of SEQ ID NO: 5. In some aspects, the fluorescent protein comprises the amino acid sequence of SEQ ID NO: 6. In some aspects, the fluorescent protein comprises the amino acid sequence of SEQ ID NO: 7. In some aspects, the fluorescent protein comprises the amino acid sequence of SEQ ID NO: 8. In some aspects, the fluorescent protein comprises the amino acid sequence of SEQ ID NO: 9. In some aspects, the fluorescent protein comprises the amino acid sequence of SEQ ID NO: 10. In some aspects, the fluorescent protein comprises the amino acid sequence of SEQ ID NO: 11. In some aspects, the fluorescent protein comprises the amino acid sequence of SEQ ID NO: 12. In some aspects, the fluorescent protein comprises the amino acid sequence of SEQ ID NO: 13. In some aspects, the fluorescent protein comprises the amino acid sequence of SEQ ID NO: 14. In some aspects, the fluorescent protein comprises the amino acid sequence of SEQ ID NO: 15. In some aspects, the fluorescent protein comprises the amino acid sequence of SEQ ID NO: 16. In some aspects, the fluorescent protein comprises the amino acid sequence of SEQ ID NO: 17. In some aspects, the fluorescent protein comprises the amino acid sequence of SEQ ID NO: 18.

[0175] In some aspects, the fluorescent protein comprises an amino acid sequence having at least 85% sequence identity to any of SEQ ID NOS: 1-3. In some aspects, the fluorescent protein comprises an amino acid sequence having at least 90% sequence identity to any of SEQ ID NOS: 1-3. In some aspects, the fluorescent protein comprises an amino acid sequence having at least 95% sequence identity to any of SEQ ID NOS: 1-3. In some aspects, the fluorescent protein comprises an amino acid sequence having at least 97% sequence identity to any of SEQ ID NOS: 1-3. In some aspects, the fluorescent protein comprises an amino acid sequence having at least 98% sequence identity to any of SEQ ID NOS: 1-3. In some aspects, the fluorescent protein comprises an amino acid sequence having at least 99% sequence identity to any of SEQ ID NOS: 1-3.

[0176] In some aspects, the fluorescent protein comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 1. In some aspects, the fluorescent protein comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 1. In some aspects, the fluorescent protein comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 1. In some aspects, the fluorescent protein comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 1. In some aspects, the fluorescent protein comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 1. In some aspects, the fluorescent protein comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 1.

[0177] In some aspects, the fluorescent protein comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 2. In some aspects, the fluorescent protein comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 2. In some aspects, the fluorescent protein comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 2. In some aspects, the fluorescent protein comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 2. In some aspects, the fluorescent protein comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 2. In some aspects, the fluorescent protein comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 2.

[0178] In some aspects, the fluorescent protein comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 3. In some aspects, the fluorescent protein comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 3. In some aspects, the fluorescent protein comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 3. In some aspects, the fluorescent protein comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 3. In some aspects, the fluorescent protein comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 3. In some aspects, the fluorescent protein comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 3.

[0179] In some aspects, the fluorescent protein has a negative net charge from -1 to -50 (e.g., from -4 to -31, from -10 to -31, or from -17 to -31).

[0180] In some aspects, the supercharged fluorescent protein has a net positive charge. In some aspects, the analyte comprises a lanthanide-chelate configured to bind to the supercharged fluorescent protein having a net positive charge.

[0181] In some aspects, the protein- analyte complex is configured to provide multiplex detection of a plurality of analytes.

[0182] In some aspects, the supercharged fluorescent protein is a monomeric protein.

[0183] In some aspects, the supercharged fluorescent protein is a multimeric protein. In some aspects, the multimeric protein includes a protomer comprising a pair of supercharged fluorescent proteins having opposite net charges. In some aspects, the multimeric protein comprises a heterodimer.

[0184] In some aspects, the supercharged fluorescent protein is immobilized within the assay chamber.

[0185] The kit can include various buffers and other reagents as described herein. Moreover, a kit can comprise the systems described herein and / or instructions for performing any of the methods disclosed herein. Finally, kits can be designed to be especially useful for a portable detection kit (e.g., for environmental monitoring) or for an individual's home use, a laboratory, or a hospital use or use in a doctor's office. Methods

[0186] Also disclosed herein is a method for characterizing the presence and / or amount of an analyte in a sample, the method comprising: providing a sample for qualitative or quantitative determination of an analyte having a spectral overlap with a supercharged fluorescent protein; contacting the supercharged fluorescent protein with the sample, the supercharged fluorescent protein being configured to directly chelate with the analyte to form a protein- analyte complex; irradiating the sample with a light source; and determining, using a fluorescence detector, the presence and / or amount of the analyte in the sample.

[0187] In some aspects, the light source comprises UV light (i.e., 250-400 nm). In some aspects, the UV light has a wavelength from 340 nm to 360 nm. Generally, the particular wavelength of light is dependent on the specific supercharged fluorescent protein and analyte and can be determined using routine experimentation.

[0188] In some aspects, the fluorescence detector comprises a fluorescent microscope.

[0189] In some aspects, the analyte comprises one or more lanthanides. In some aspects, the one or more lanthanides comprises free lanthanide cations. In some aspects, the analyte comprises terbium (Tb), thulium (Tm), dysprosium (Dy), europium (Eu), samarium (Sm), ytterbium (Yb), or a combination thereof.

[0190] In some aspects, the supercharged fluorescent protein comprises a green fluorescent protein (GFP), a yellow fluorescent protein (YFP), a cyan fluorescent protein (CFP), or a combination thereof.

[0191] In some aspects, the fluorescent protein comprises an amino acid sequence having at least 85% (e.g., at least 90%, at least 95%, at least 97%, at least 99%) sequence identity to any of SEQ ID NOS: 1-18. In some aspects, the fluorescent protein comprises the amino acid sequence of SEQ ID NO: 1. In some aspects, the fluorescent protein comprises the amino acid sequence of SEQ ID NO: 2. In some aspects, the fluorescent protein comprises the amino acid sequence of SEQ ID NO: 3. In some aspects, the fluorescent protein comprises the amino acid sequence of SEQ ID NO: 4. In some aspects, the fluorescent protein comprises the amino acid sequence of SEQ ID NO: 5. In some aspects, the fluorescent protein comprises the amino acid sequence of SEQ ID NO: 6. In some aspects, the fluorescent protein comprises the amino acid sequence of SEQ ID NO: 7. In some aspects, the fluorescent protein comprises the amino acid sequence of SEQ ID NO: 8. In some aspects, the fluorescent protein comprises the amino acid sequence of SEQ ID NO: 9. In some aspects, the fluorescent protein comprises the amino acid sequence of SEQ ID NO: 10. In some aspects, the fluorescent protein comprises the amino acid sequence of SEQ ID NO: 11. In some aspects, the fluorescent protein comprises the amino acid sequence of SEQ ID NO: 12. In some aspects, the fluorescent protein comprises the amino acid sequence of SEQ ID NO: 13. In some aspects, the fluorescent protein comprises the amino acid sequence of SEQ ID NO: 14. In some aspects, the fluorescent protein comprises the amino acid sequence of SEQ ID NO: 15. In some aspects, the fluorescent protein comprises the amino acid sequence of SEQ ID NO: 16. In some aspects, the fluorescent protein comprises the amino acid sequence of SEQ ID NO: 17. In some aspects, the fluorescent protein comprises the amino acid sequence of SEQ ID NO: 18.

[0192] In some aspects, the fluorescent protein comprises an amino acid sequence having at least 85% sequence identity to any of SEQ ID NOS: 1-3. In some aspects, the fluorescent protein comprises an amino acid sequence having at least 90% sequence identity to any of SEQ ID NOS: 1-3. In some aspects, the fluorescent protein comprises an amino acid sequence having at least 95% sequence identity to any of SEQ ID NOS: 1-3. In some aspects, the fluorescent protein comprises an amino acid sequence having at least 97% sequence identity to any of SEQ ID NOS: 1-3. In some aspects, the fluorescent protein comprises an amino acid sequence having at least 98% sequence identity to any of SEQ ID NOS: 1-3. In some aspects, the fluorescent protein comprises an amino acid sequence having at least 99% sequence identity to any of SEQ ID NOS: 1-3.

[0193] In some aspects, the fluorescent protein comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 1. In some aspects, the fluorescent protein comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 1. In some aspects, the fluorescent protein comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 1. In some aspects, the fluorescent protein comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 1. In some aspects, the fluorescent protein comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 1. In some aspects, the fluorescent protein comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 1.

[0194] In some aspects, the fluorescent protein comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 2. In some aspects, the fluorescent protein comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 2. In some aspects, the fluorescent protein comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 2. In some aspects, the fluorescent protein comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 2. In some aspects, the fluorescent protein comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 2. In some aspects, the fluorescent protein comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 2.

[0195] In some aspects, the fluorescent protein comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 3. In some aspects, the fluorescent protein comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 3. In some aspects, the fluorescent protein comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 3. In some aspects, the fluorescent protein comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 3. In some aspects, the fluorescent protein comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 3. In some aspects, the fluorescent protein comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 3.

[0196] In some aspects, the fluorescent protein has a negative net charge.

[0197] In some aspects, the supercharged fluorescent protein has a net positive charge.

[0198] In some aspects, the analyte comprises a lanthanide-chelate configured to bind to the supercharged fluorescent protein having a net positive charge.

[0199] In some aspects, the protein- analyte complex is configured to provide multiplex detection of a plurality of analytes.

[0200] In some aspects, the supercharged fluorescent protein is a monomeric protein.

[0201] In some aspects, the supercharged fluorescent protein is a multimeric protein.

[0202] In some aspects, the multimeric protein includes a protomer comprising a pair of supercharged fluorescent proteins having opposite net charges. Opposite net charges do not necessitate that the magnitudes of the charges be the same.

[0203] In some aspects, the multimeric protein comprises a heterodimer.

[0204] In some aspects, the method further includes quantitatively determining an amount of the analyte in the sample based on a fluorescent signal emitted by the protein-analyte complex.

[0205] In some aspects, the amount of the analyte is determined using a normalized ratio between the fluorescent signal emitted by the protein-analyte complex and a baseline fluorescent signal emitted by the supercharged fluorescent protein alone. In some aspects, the protein- analyte complex is measurably fluorescent when the analyte and supercharged protein are separated by a distance of 50 nm or less (e.g., 45 nm or less, 40 nm or less, 35 nm or less, 30 nm or less, 25 nm or less, 20 nm or less, 15 nm or less, or 10 nm or less).

[0206] In some aspects, the method is carried out at a temperature of 0°C or more (e.g., 5°C or more, 10°C or more, 15°C or more, 20°C or more, 25°C or more, 30°C or more, 35°C or more, 40°C or more, 45°C or more, 50°C or more, 55°C or more, 60°C or more, 65°C or more, 70°C or more). In some aspects, the method is carried out at a temperature of 70°C or less (e.g., 65°C or less, 60°C or less, 55°C or less, 50°C or less, 45°C or less, 40°C or less, 35°C or less, 30°C or less, 25°C or less, 20°C or less, 15°C or less, 10°C or less, 5°C or less, 0°C or less).

[0207] It is considered that the method can be carried out at a temperature ranging from any of the minimum values described above to any of the maximum values described above. For example, in some aspects, the method is carried out at a temperature of from 0°C to 70°C (e.g., from 5°C to 65°C, from 10°C to 60°C, from 15°C to 55°C, from 20°C to 50°C, from 25°C to 45°C, from 30°C to 40°C, from 0°C to 35°C, from 5°C to 30°C, from 10°C to 25°C, from 15°C to 20°C, from 35°C to 70°C, from 40°C to 65°C, from 45°C to 60°C4from 50°C to 55°C, from 0°C to 60°C, from 5°C to 55°C, from 10°C to 50°C, from 15°C to 45°C, from 20°C to 40°C, from 25°C to 35°C, from 0°C to 30°C, from 5°C to 25°C, from 10°C to 20°C, from 30°C to 60°C, from 35°C to 55°C, from 40°C to 50°C).

[0208] In some aspects, the sample has a pH of 5 or greater (e.g., 5.2 or greater, 5.4 or greater, 5.6 or greater, 5.8 or greater, 6 or greater, 6.2 or greater, 6.4 or greater, 6.6 or greater, 6.8 or greater, 7 or greater, 7.2 or greater, 7.4 or greater, 7.6 or greater, 7.8 or greater, 8 or greater, 8.2 or greater, 8.4 or greater, 8.6 or greater, 8.8 or greater, 9 or greater, 9.2 or greater, 9.4 or greater, 9.6 or greater, 9.8 or greater, 10 or greater).

[0209] In some aspects, the method further comprises adjusting the pH of the sample, for example, to a pH of 5 or greater.

[0210] EXAMPLES

[0211] Example 1: Supercharged fluorescent proteins detect lanthanides via direct antennae signaling

[0212] Disclosed herein is a simple yet robust strategy for using fluorescent proteins as biosensors for lanthanides. The net charge of proteins can be augmented to a significant degree through extensively mutating the molecular surface. (Der, 2007). This process, termed ‘supercharging’, generally preserves the overall structure and function of the protein while opening doors to customizable intermolecular behaviors that can meet specific engineering needs. Hence, the study modulated the surface of green fluorescent protein (GFP), yellow fluorescent protein (YFP), and cerulean fluorescent protein (CFP) to enable direct chelation between negatively charged residues and free lanthanide cations. Ultraviolet (UV) excitation resulted in a detectable, dose-dependent signal transmission between trivalent terbium, thulium, dysprosium, and the chromophores of YFP and GFP. Notably, supercharged variants of YFP and GFP are responsive to lanthanides in the environmentally relevant 10 pM-5 mM range. The robust detection of lanthanides via a ready signal transmission to fluorescent proteins suggests that engineered protein surfaces may provide a green path forward in REE extraction, as well as broad opportunities in the development of new biosensors and optically active materials.

[0213] Materials and Methods

[0214] Cloning and purification of supercharged fluorescent proteins.

[0215] Synthetic genes encoding supercharged variants of YFP, GFP, and CFP were obtained from Twist Bioscience and Integrated DNA Technologies. E. coli DH10B and BL21 (New England Biolabs) were used for cloning and expression, respectively. Expression plasmids based on the pET system were either constructed through Golden Gate assembly or acquired directly from Twist Bioscience.

[0216] Starter cultures for expression were prepared by picking single colonies of E. coli for overnight growth in Luria-Bertani broth at 37°C and 250 r.p.m. The E. coli were then inoculated into 250 mL of broth and grown at 37°C and 250 r.p.m. until an ODeoo of 0.6 was achieved. The cultures were cooled at 4°C for 45 minutes before 1 mM isopropyl-P-D-1- thiogalactopyranoside was added to induce protein expression. Following 16 hours of incubation at 18°C and 250 r.p.m., the cells were harvested by centrifugation (6,000g, 4°C, 30 minutes). The pellets were then either stored at -20°C for later use or resuspended in 30 mL of phosphate buffer (2 M NaCl, 20 mM imidazole, 5 mM MgSO4, pH 7.5). Once resuspended, the cells were subjected to a sonication routine for cell lysis, and the supernatant containing the soluble proteins was obtained by centrifugation (35,000g, 4°C, 30 minutes). The proteins were subsequently purified using HisPur Ni-NTA Resin (Thermo Fisher Scientific) and custom wash (50 mM phosphate buffer, 2M NaCl, 20 mM imidazole, pH 7.5) and elution buffers (50 mM phosphate buffer, 2M NaCl, 500 mM imidazole, pH 7.5). Desalting and buffer exchange were performed using Amicon Ultra Centrifugal Filter Units (MilliporeSigma), and the purified proteins were concentrated to a volume of 0.4 mL in 50 mM Tris-HCl (pH 7.0). The concentration of the proteins was measured using the Pierce Coomassie Plus Bradford Assay (Thermo Fisher Scientific), and the purity was confirmed through sodium dodecyl sulfate-poly acrylamide gel electrophoresis.

[0217] Assaying fluorescence in the presence of lanthanides and interferents.

[0218] Fluorescence measurements were obtained at room temperature using a Cytation 5 microplate reader. The sample volume was 0.1 mL unless otherwise specified, and the experiments were conducted using 96-well clear bottom polystyrene assay plates (Corning). At least three independent measurements were obtained for each of the conditions tested. Solutions of TbCh, TmCh, DyCL, A1CL, and FeCh were prepared by dissolving the compounds in 50 mM Tris-HCl (pH 7.0). To minimize aggregation, the proteins were carefully agitated by pipetting up and down before diluting the concentrated stock solutions to 0.1 mg / mL in 50 mM Tris-HCl (pH 7.0). The ratio of the protein biosensor’s fluorescence upon excitation at a wavelength outside of its normal excitation range versus at a wavelength within its normal excitation range was obtained as a proxy for the magnitude of the lanthanide-based energy transfer (LRET) from the lanthanide to the biosensor. This quantity, termed the ‘excitation ratio’, normalizes the signal observed when exciting the lanthanide antenna to the signal observed when exciting the fluorescent protein, thereby enabling comparisons of LRET across reaction conditions.

[0219] Results

[0220] Supercharged fluorescent proteins demonstrate energy transfer with lanthanide antennae.

[0221] Contrary to the established paradigm, in which lanthanides are the acceptors of resonance energy transfer, the study unexpectedly detected that lanthanides bound by electrostatic forces to a fluorescent protein surface could be functionalized into antennae for receiving an electromagnetic signal outside the normal excitation range of the internal chromophore (FIG. 1A). Subsequently, the signal could be transmitted via lanthanide-based resonance energy transfer (LRET) to the fluorescent protein for a quantifiable emission in the visible range. Without wishing to be bound by theory, the efficiency of LRET, like FRET, is generally observed to be inversely dependent on distance35-37. The energy transfer is measurable by fluorescence microscopy when the lanthanide and the protein are separated o by a nanometer-scale distance (e.g., 10-100 A).

[0222] To interface with the lanthanide antennae, the study aimed at establishing an expansive biosensor excitation range for a maximal chance of favorable spectral overlap with the emissions of Tb3+, Tm3+, and Dy3+(FIG. IB). To that end, the mutations T65G, V68L, S72A, and T203Y were introduced to red-shift and stabilize the series of supercharged GFP variants with net charges of -4, -10, -17, -31, +16, and +33 at pH 7.0, obtaining a series of YFP variants with identical net charges32,39. Likewise, the mutations Y66W, F146G, N147I, H149D were introduced to blue-shift and stabilize the same series of GFP variants, obtaining a series of CFP variants with net charges of -5, -11, -18, -32, +15, and +32 at pH 7.O32,40-42. This set of eighteen fluorescent protein biosensors then served as the starting point for selecting the optimal candidates for detecting lanthanides (FIGS. 2A- 2B).

[0223] Initially, the fluorescence output of the biosensors was measured in response to excitation in the UV range (250-400 nm). When combined with any of the lanthanides (Tb3+, Tm3+, Dy3+), the negatively charged GFP and YFP variants exhibited a 2-3-fold increase in fluorescence intensity at select excitation wavelengths (FIG. 3A and TABLE 1). Similarly, the lanthanides Yb3+, Eu3+’ and Sm3+elicited a roughly 2-3-fold increase in fluorescence intensity when paired with the GFP- 10 and YFP-31 variants (TABLE 1). In contrast, the addition of Al3+produced virtually unchanged excitation spectra at a concentration of 100 pM (FIG. 3A), a value set to approximate or even exceed the levels of Al3+commonly found in acid mine drainage43. Consistent with the Laporte rule, the lanthanides did not measurably luminesce on their own at any excitation wavelength14. The ratio of the fluorescence intensity upon adding the lanthanides to the fluorescence of the biosensors alone appeared to peak in the 340-400 nm excitation range, with 350 nm and 390 nm being the optimal wavelengths for achieving a maximal factor change in response upon exciting the lanthanide antennae bound to the GFP and YFP variants, respectively (FIG. 3B). However, given that the wavelengths corresponding to these ratiometric maxima encroach on the excitation ranges of GFP and YFP, the study opted to excite the lanthanide- bound system deeper in the UV range, at 340 nm and 360 nm for GFP and YFP, respectively. It was believed that this approach would minimize crosstalk between FRET donor and acceptor channels while still staying within range for a sufficiently robust ratiometric response37. Interestingly, a peak was observed in the fluorescence output of YFP and GFP at an excitation wavelength of 280 nm, with or without the presence of lanthanides, though the addition of lanthanides enhanced the signal response across all variant conditions (FIG. 3A). Hypothetically, the tryptophan residue at position 57, o positioned approximately 12-13 A apart from the chromophore within each of the fluorescent protein variants, could act as an antenna at this excitation wavelength for transferring Forster resonance energy directly to the internal chromophore, or in the presence of lanthanides, to the lanthanide, which in turn transmits the signal to the chromophore for an even greater response (FIGS. 4A-4C)44. A byproduct of the use of excitation wavelengths of 340 nm and 360 nm, the experiment excluded the confounding effect of the tryptophan antenna in further analyses.

[0224] TABLE 1. Fluorescence response of supercharged proteins to lanthanides. Mean apparent Kd (Kd,app) for three technical replicates. Fold response (F / Fo) defined as ratio of fluorescence response in the presence of 1 mM of lanthanide to fluorescence response of supercharged protein alone.

[0225] Importantly, time-resolved fluorescence spectroscopy revealed long-lived luminescence transmission from the lanthanide to the fluorescent protein biosensor across the excitation range of 250-400 nm (FIGS. 5A-5B), consistent with the intrinsically slow decay of the excited state of lanthanide series elements. In the presence of Tb3+, the GFP-10 and YFP-31 biosensors produced a markedly distinct fluorescence response relative to the protein alone. This difference in response was magnified by time delays of 100 ps and 300 s between excitation and response detection. Of note, the biosensors alone produced negligible fluorescence when subjected to time-delayed detection, while the Tb3+:biosensor pairs exhibited a comparably modest attenuation in the signal response (FIGS. 5A-5B, FIGS. 6A-6D, FIGS. 7A-7B). Thus, the long-lived signal responses of the GFP-10 and YFP-31 biosensors in the presence Tb3+support the notion that signal detection is dependent on excitation and transmission via the lanthanide antennae.

[0226] The excitation ratio, defined as the fluorescence of GFP at its emission maximum (510-520 nm) upon exciting at 340 nm versus 465 nm or the fluorescence of YFP (530-540 nm) upon exciting at 360 nm versus 500 nm, is a measure of LRET efficiency that permits comparisons between lanthanide:biosensor and biosensor-only conditions32. The factor change in excitation ratio, defined as the ratio of the excitation ratio calculated for the lanthanide:biosensor condition versus the biosensor-only condition, permits comparisons across biosensor variants. The addition of Tb3+to YFP-31 produced the highest factor change in excitation ratio, 1.97 ± 0.25, whereas YFP variants with net charges of -17, -10, and -4 produced factor changes in the excitation ratio of 1.58 ± 0.25, 1.48 ± 0.15, and 1.30 ± 0.17, respectively (FIG. 8A and FIGS. 9A-9C). As expected, the non-interacting positively charged variants of YFP, +16 and +33, produced factor changes in excitation ratio of 1.13 + 0.04 and 1.05 + 0.03, respectively, indicating decreased binding and detection of lanthanides compared to the wild-type YFP. The addition of Tm3+and Dy3+to the YFP series resulted in a similarly stepwise increase in signal as the magnitude of negative surface charge increased, with YFP-31 being the top-performing sensor for each of the lanthanides. The addition of Tb3+to GFP-10 produced a factor change in excitation ratio of 2.58 + 0.09, the highest signal among the GFP variants, whereas GFP-31, -17, and -4 produced factor changes in excitation ratio of 1.60 + 0.06, 2.32 + 0.25, and 1.82 + 0.08, respectively, and the non-interacting GFP+16 and +33 produced responses of 1.06 + 0.24 and 0.93 + 0.11, respectively (FIG. 8A, FIGS. 9A-9C, FIGS. 10A-10D). Again, the addition of Tm3+and Dy3+to the GFP series manifested a similar trend, with the entire series of negatively supercharged GFP variants exhibiting an enhanced signal response relative to the positively supercharged and wild-type sensors (FIG. 8A and FIGS. 9A-9C). Importantly, the top performing sensors, YFP-31 and GFP-10 (FIG. 11), demonstrated an approximately 2.5-fold or greater increase in their signal response to Tb3+, Tm3+, and Dy3+across a dose-dependent 10 pM-5 mM range, consistent with typical ranges of total REEs found in highly polluted streams (FIGS. 8B-8C). Additional testing with the lanthanides Eu3+, Sm3+, and Yb3+ revealed comparable response ratios and an apparent Kd of 25-30 pM for YFP-31 and 210-500 pM for GFP-10 across all six lanthanide species (FIGS. SB- 8C and TABLE 1). In summary, the findings suggest that negatively supercharging YFP and GFP enables robust lanthanide binding and subsequent antennae signaling via (i) specific and non-specific sensitizing interactions within the electronically manipulable framework of the protein interior and (ii) a near-UV range stimulus resulting in the excitation of lanthanide cations and subsequent energy transfer to the protein chromophore.

[0227] Notably, the CFP series was non-performing, irrespective of the net charge (FIG. 8A and FIGS. 9A-9C). The inability of the CFP variants to detect lanthanides could be attributed to the absence of spectral overlap with the radiative energy levels of Tb3+and Tm3+, as well as the excess overlap with the emission of Dy3+(FIG. IB)45. In the absence of overlap, the energy received by the lanthanide antenna cannot be transmitted to the acceptor fluorophore, even if the oppositely charged parties are positioned within range for LRET signaling18. Likewise, excess overlap prevents biosensor readout, but in this case, the antenna excitation outputs a signal that is not readily distinguishable from the signal arising from the biosensor-specific excitation37. Conversely, the excitation ranges of YFP and GFP appear to favorably overlap with potentially radiative configurations of bivalent terbium, thulium, and dysprosium in their excited states (FIG. IB), thereby enabling the successful transmission of energy from the lanthanide antenna to the fluorescent biosensor. Supercharged fluorescent protein biosensors do not respond to common interferents.

[0228] Lanthanide-containing waste streams and deposits, such as mine drainage and electronic scrap, contain a surplus of potentially interfering, soluble metal compounds, including, most commonly, the chloride salts of aluminum and iron43'46-49. A biomining operation would therefore benefit from a system capable of distinguishing between the capture of lanthanides versus non-target metals.

[0229] To evaluate the response of the biosensor platform to interfering metals, the study measured the fluorescence output of the top performing sensors, GFP- 10 and YFP-31, in the presence of Al3+and Fe2+, first as separate, non-interacting entities (FIG. 12A and FIG. 13A), and second, as species competing for binding and detection (FIG. 13B). Upon UV excitation, the YFP-31 sensor in combination with 1 mM of Tb3+exhibited a nearly twofold increase in fluorescence output compared to YFP-31 in combination with 1 mM of non-target Al3+(FIG. 12A). Notably, the Al3+: YFP-31 condition produced an emission spectrum that was only slightly perturbed compared to the YFP-31 -only condition, indicating that the YFP-31 sensor can readily discriminate between Tb3+and a nonlanthanide, non-luminescent metal species of the same ionic charge (FIG. 12A). Next, a competitive interaction was established between Tb3+and Al3+or Fe2+, holding the signal transmission between 1 mM of Tb3+and the GFP-10 sensor constant as 1 nM-1 mM of Al3+or Fe2+were introduced in separate microplate wells (FIG. 13B and FIG. 14). As the concentration of Al3+was titrated up, the excitation ratio remained constant, with a scale increase in the concentration of six orders of magnitude producing an unperturbed excitation ratio of 0.013 ± 0.001 (FIG. 13B). Likewise, the presence of 10 pM of Fe2+did not impact LRET signaling between GFP-10 and Tb3+, although concentrations greater than 10 M resulted in a modest diminution of the signal response. At 1 mM of Fe2+, the signal weakened even more, indicating that an equimolar concentration of Fe2+in the environment could disrupt the detection of lanthanides (FIG. 13B). It should be noted that under the reaction conditions presented herein, Fe2+ is potentially readily oxidized to Fe3+. Irrespective of this detail, iron removal via pH-controllable precipitation presents a viable pre-processing strategy to minimize signal interference prior to biosensor readout47.

[0230] Importantly, the GFP-10 and YFP-31 biosensors retained functionality for detecting total lanthanides when subjected to mixed ion solutions resembling actual mining outflows (FIGS. 12B-12D and FIGS. 15A-15B). First, to isolate any potential inhibitory effects of the contaminant metals, we measured the dose responses of Mg2+, Ca2+, Mn2+, Zn2+, and Cu2+as single species in a competitive assay with GFP-10:Tb3+and YFP-31 :Tb3+. Apart from moderate perturbations in the fluorescence signal at 1 mM of Zn2+and 100 p M of Cu2+, the contaminants interfered minimally with Tb3+detection, even at a 10-fold excess of contaminant relative to the lanthanide (FIGS. 16A-16B). Interestingly, Cu2+appeared to interfere more significantly with Tb3+detection by the GFP sensor compared to the YFP sensor (FIGS. 16A-16B). Additionally, our analysis yielded no false positive indications; the contaminants did not elicit biosensor fluorescence on their own (i.e., in the absence of Tb3+) as expected (FIG. 17). To examine the GFP-10 and YFP-31 response to a true-to-nature mixed ion solution, we approximated the composition of acid mine drainage found in the Virginia Canyon47. With fidelity to the ratio of contaminants, we subjected GFP-10:Tb3+and YFP-31 :Tb3+to mixtures of Al3+, Fe2+, Mn2+, Zn2+, and Cu2+, termed ‘VC’, as well as Mn2+, Zn2+, and Cu2+, termed ‘mVC’ (TABLE 2). At an adjusted pH of 7, a 5-fold excess of the VC mixture elicited minimal perturbation of Tb3+detection (FIGS. 12C-12D). Of note, an approximately equimolar amount of the mVC mixture produced modest interference with GFP-10 detection of Tb3+(FIG. 12C), and at an adjusted pH of 5, the contaminant mixtures disrupted the detection of 1 mM of Tb3+at approximately 10 pM and 1 mM of contaminants for GFP-10:Tb3+and YFP-31:Tb3+, respectively (FIGS. 15A-15B). In addition to transition metal contaminants, REE-rich process streams often contain a high concentration of the non-luminescent lanthanide lanthanum (La3+)47. Upon measuring the dose response of La3+in a competitive assay with GFP-10:Tb3+and YFP-31:Tb3+, we observed no significant perturbation of Tb3+detection at a 5-fold excess of La3+relative to Tb3+(FIGS. 18A-18B).

[0231] TABLE 2. Compositions of mixtures inspired by REE-rich Virginia Canyon groundwater. Virginia Canyon (VC) and modified Virginia Canyon (mVC) mixtures of transition metal interferents, modeled after the influents arising from Virginia Canyon in Idaho Springs, CO, an AMD source with high loads of REEs. The mixtures were formulated and boosted to 10 mM and 20 mM of total interferents, respectively.

[0232] At concentrations of lanthanides high enough to elicit a strong biosensor response, it was believed that aggregates could form due to the interaction of near equimolar amounts of oppositely charged species. To evaluate the effect of potential aggregation on fluorescence signal detection, we incubated mixtures of supercharged GFP variants and 1 mM of Al3+for 30 minutes prior to fluorescence readout. The Tb3+:GFP pairs exhibited excitation ratios on par with an immediate readout, whereas the A13+:GFP pairs produced slightly increased excitation ratios compared to the GFP-only conditions. Still, the GFP-10 biosensor, as well as the other GFP variants, can discriminate between Tb3+and Al3+following the extended incubation period, highlighting the versatility of the present system (FIG. 19).

[0233] To further validate the functionality of our biosensors in real-world conditions, we assessed other potentially interfering factors, such as ionic strength, temperature, and pH changes (FIGS. 20A-20D, FIGS. 21A-21B, FIGS. 22A-22B). Of note, concentrations of NaCl from 0 to 800 mM were found to minimally perturb GFP-10 and YFP-31 detection of 1 mM of Tb3+(FIGS. 20A-20D). Additionally, the temperature range from 0°C to 70°C was conducive to stability in Tb3+:GFP-10 signal transduction while the temperature range from 0°C to 60°C was conducive to stability in Tb3+: YFP-31 signal transduction (FIGS. 21A-21B). Finally, the pH dependence of Tb3+detection was such that below a pH of 5, Tb3+detection was rendered unlikely and virtually indistinguishable from the absence of Tb3+(FIGS. 22A-22B).

[0234] Higher order structures can also detect lanthanides.

[0235] Symmetry in protein oligomers gives rise to remarkable complexity in biological systems50-52. Given the ubiquity of multimeric protein assemblies and their capacity for diverse structural and transport functions within living organisms, we set out to create a new, higher order protein architecture for detecting lanthanides53-56. Using a method termed the ‘supercharged protein assembly’ (SuPrA), we selected two oppositely charged fluorescent proteins from the set of biosensors, CFP+32 and GFP-31, for assembly into a globular CFP+32 / GFP-31 protomer (FIGS. 23A-23B)32. After combining equimolar volumes of CFP+32 and GFP-31, we observed a unique FRET interaction on par with the signal manifested in the assembly of a 16-unit CFP+32 / GFP-17 protomer with eight-fold symmetry (FIG. 24A)32'57.

[0236] Upon introducing 1 mM of Tb3+, Tm3+, Dy3+, or Al3+, we disrupted the FRET output of GFP-31 in response to excitation at 433 nm, the optimal excitation wavelength of the donor CFP+32. The pattern of disruption in FRET signaling between CFP+32 and GFP-31, in which each of the lanthanides exhibited a stronger effect than aluminum, implies some combined effect of competitive binding, protomer disassembly, and fluorescence modulation. Notably, upon exciting the protomer system in the UV range, following the protocol for transmitting a signal from a lanthanide antenna to its acceptor fluorophore, the fluorescence modulation appeared to reverse course, with each Ln3+:protomer pair manifesting some degree of signal restoration (FIG. 24B). The UV-excited GFP-31 still responded to the lanthanides with a decrease in fluorescence output, although this time, the disruption in FRET between CFP+32 and GFP-31 appeared to be masking a response arising from potentially new signaling between the CFP+32 / GFP-31 protomer and the lanthanide antennae. To quantitate this masked signal, we calculated the percentage of GFP-31 fluorescence relative to the unperturbed signal. Dy3+produced the greatest masked signal, a factor change of 1.01 + 0.09 in the fluorescence output of UV-excited GFP-31 compared to a factor change of 0.77 + 0.04 for GFP-31 excited at 433 nm. Similarly, Tm3+induced a factor change of 0.94 + 0.05 in the fluorescence of UV-excited GFP-31 compared to a factor change of 0.72 + 0.11 for GFP-31 excited at 433 nm. Tb3+appeared to demonstrate a diminutive increase in the UV-excited signal, albeit not significantly (FIG. 24C). Hypothetically, the formation of the supramolecular assembly of fluorescent proteins could have induced a spectral shift towards less overlap with the radiative transitions of Tb3+and, in turn, decreased signal transmission.

[0237] Discussion

[0238] The lanthanide series elements are in high global demand, though a green and scalable path forward in the quantifiable capture of lanthanides from the environment remains elusive. While previous methods of lanthanide sensing and luminescence modulation have advanced sophisticated forms of chelation, sensitization, and semisynthetic modification, such systems are not scalable, difficult to utilize in living systems, or lack an appropriate sensitivity range for detecting lanthanides in highly concentrated streams16,18'23'58. By demonstrating that free lanthanide cations can bind to charge- engineered protein surfaces can function and act directly as excitable antennae in the absence of a specific luminescence sensitizer, organic or otherwise, we instantiate an underutilized model for energy transfer and notably shift the paradigm for lanthanide luminescence signaling. Future computational modeling of the interactions between lanthanides and monomeric and multimeric protein architectures should reveal more precise appositions in which the interactions between lanthanides, surface charge, and electronically dense organic scaffolding give rise to enhanced radiative transfers.

[0239] Unlike existing biosensors for lanthanides, the supercharged protein interface exhibits dual functionality in binding and detecting lanthanides in the millimolar range, thereby implicating its utility in monitoring the development and efficiencies of high load lanthanide processing streams. Even amid the suite of ultra- selective LanM-based sensors, the supercharged fluorescent biosensor offers several unique advantages. Particularly, in environments where the amount of total lanthanides exceeds the upper bound for detection afforded by previously developed LanM derivatives, such as certain acidic discharges and highly concentrated filtration streams, the supercharged series of biosensors retains high capacity for quantitatively reporting on lanthanides25 28. At present, the platform’s operational scope encompasses the environmentally and industrially significant micromolar to millimolar range, though future machine learning guided predictions could enable the broad, structure-based modulation of lanthanide sensitivities to meet specific processing needs. In contrast, the lanthanide biosensor LaMPl, composed of a FRET donor-acceptor pair joined by a collapsible LanM arm, has a considerably higher affinity for lanthanides, potentially predisposing it to oversaturation in more concentrated processing streams20. Likewise, the tryptophan-substituted LanM biosensor boasts an impressive picomolar sensitivity for terbium but meets its upper bound for a dose-dependent fluorescence readout in the nanomolar range, again rendering it prone to saturation23. It should be noted, however, that the upper bound of detection will be condition-dependent; the tryptophansubstituted sensor indeed achieves a maximum sensitivity in 1-10 pM range, approaching that of the YFP-31 sensor, when subjected to conditions like those employed in this study (e.g., -3.7 pM of protein).

[0240] Even in the presence of common interferents that plague other sensor platforms, such as aluminum and iron, the supercharged series of biosensors retains its micromolar to millimolar sensitivity for lanthanides, pointing to its utility in environments mirroring not only this broad range of lanthanide concentrations but also the presence of equimolar amounts of competing metals. Even then, the removal of potentially problematic interferents, such as iron and aluminum, could be readily achieved via a pH-based preprocessing strategy, wherein the competing metal ions are precipitated out of the filtration stream in a minimally obtrusive manner47. More complex conditions, such as the presence of a greater than 10-fold excess of multiple competing metals, will necessitate additional study, and may require more aggressive refinement to be made compatible with the present sensors.

[0241] Importantly, the adaptation of the novel sensing paradigm to organized nanoassemblies suggests the intriguing possibility of utilizing these sensors in engineered materials, or even just protein precipitates that could be readily applied to detectors. Future insights into cross-linking, self-assembly, and modular design could reveal broad opportunities in the development of optically active nanomaterials and macroscale filtration systems alike. In addition, the possibilities for utilizing these simple, genetically encoded biosensors in organisms that could concentrate and detect surface lanthanides are manifest.

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Protein Oligomer Engineering: A New Frontier for Studying Protein Structure, Function, and Toxicity. Angew Chem Int Ed 62, e202216480

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[0247] NPG Asia Mater 10, 52-70 (2018).

[0248] SEQUENCES

[0249] SEO ID NO: 1 - sfGFP

[0250] MSKGEELFTG VVPILVELDG DVNGHKFSVR GEGEGDATNG KLTLKFICTT

[0251] GKLPVPWPTL VTTLTYGVQC FSRYPDHMKR HDFFKSAMPE GYVQERTISF

[0252] KDDGTYKTRA EVKFEGDTLV NRIELKGIDF KEDGNILGHK LEYNFNSHNV

[0253] YITADKQKNG IKANFKIRHN VEDGSVQLAD HYQQNTPIGD GPVLLPDNHY LSTQSVLSKD PNEKRDHMVL LEFVTAAGIT HGMDELYK

[0254] SEP ID NO: 2 - sfYFP

[0255] MSKGEELFTG VVPILVELDG DVNGHKFSVR GEGEGDATNG KLTLKFICTT

[0256] GKLPVPWPTL VTTLTYGVQC FSRYPDHMKR HDFFKSAMPE GYVQERTISF

[0257] KDDGTYKTRA EVKFEGDTLV NRIELKGIDF KEDGNILGHK LEYNFNSHNV

[0258] YITADKQKNG IKANFKIRHN VEDGSVQLAD HYQQNTPIGD GPVLLPDNHY LSYQSVLSKD PNEKRDHMVL LEFVTAAGIT HGMDELYK

[0259] SEQ ID NO: 3 - sfCFP

[0260] MSKGEELFTG VVPILVELDG DVNGHKFSVR GEGEGDATNG KLTLKFICTT

[0261] GKLPVPWPTL VTTLTWGVQC FSRYPDHMKR HDFFKSAMPE GYVQERTISF

[0262] KDDGTYKTRA EVKFEGDTLV NRIELKGIDF KEDGNILGHK LEYNFNSHNV

[0263] YITADKQKNG IKANFKIRHN VEDGSVQLAD HYQQNTPIGD GPVLLPDNHY LSTQSVLSKD PNEKRDHMVL LEFVTAAGIT HGMDELYK

[0264] SEQ ID NO: 4 - sfGFP-10

[0265] MSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDADNGKLDLKFICTTGKLPVP

[0266] WPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGTYKTRAE VKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKEKNGIKANFKIR

[0267] HNVEDGSVQLADHYQQNTPIGDGPDLLPDEHYLSTQSVLSKDPNEKRDHMVLLEF VTAAGITEGHHHHHHHH

[0268] SEQ ID NO: 5 - sfGFP- 17 MSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDADNGKLDLKFICTTGKLPVP

[0269] WPTLVTTLTYGVQCFSRYPDHMKEHDFFKSAMPEGYVQERTISFKDDGTYKTRAE

[0270] VKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHEVYITADDEKNGIKAEFKIR

[0271] HNVEDGSVQLADHYQQNTPIGDGPDLLPDEHYLSTQSVLSKDPNEKRDHMVLLEF

[0272] VTADGITEGHHHHHHHH

[0273] SEQ ID NO: 6 - sfGFP-31

[0274] MSKGEELFEGVVPILVELDGDVNGHKFEVRGEGEGDADNGKLDLKFICTTGELPVP

[0275] WPTLVTTLTYGVQCFSRYPDHMKEHDFFKSAMPEGYVQERTISFDDDGTYETRAE

[0276] VKFEGDTLVNRIELKGIDFKEDGNILGHKLEYDFNSHEVYIEADDEKNGIKAEFKIE

[0277] HNVEDGSVQLADHYQQNTPIGDGPDLLPDEHYLSTQSVLSKDPNEERDHMVLLEF

[0278] VTADGITEGHHHHHHHH

[0279] SEQ ID NO: 7 - sfGFP+16

[0280] MSKGEELFKGVVPILVELKGDVNGHKFKVRGEGEGDAKNGKLRLKFICTTGKLPV

[0281] PWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGTYKTRA

[0282] EVKFEGDTLVNRIELKGIKFKKDGNILGHKLEYNFNSHKVYITADKKKNGIKAKFKI

[0283] RHNVKDGSVQLADHYQQNTPIGDGPKLLPRKHYLSTQSVLSKDPNEKRDHMVLLE

[0284] FVTAAGITKGHHHHHHHH

[0285] SEQ ID NO: 8 - sfGFP+33

[0286] MSKGERLFRGKVPILVELKGDVNGHKFSVRGKGKGDATNGKLTLKFICTTGKLPVP

[0287] WPTLVTTLTYGVQCFARYPKHMKRHDFFKSAMPKGYVQERTISFKKDGTYKTRAE

[0288] VKFEGRTLVNRIKLKGRDFKEKGNILGHKLRYNFNSHKVYITADKRKNGIKAKFKI

[0289] RHNVKDGSVQLADHYQQNTPIGRGPVLLPRNHYLSTRSVLSKDPKEKRDHMVLLE

[0290] FVTAAGIKHGRDERYKLEHHHHHH

[0291] SEQ ID NO: 9 - sfYFP-10

[0292] MSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDADNGKLDLKFICTTGKLPVP

[0293] WPTLVTTLGYGLQCFARYPDHMKRHDFFKSAMPEGYVQERTISFKDDGTYKTRAE

[0294] VKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKEKNGIKANFKIR

[0295] HNVEDGSVQLADHYQQNTPIGDGPDLLPDEHYLSYQSVLSKDPNEKRDHMVLLEF

[0296] VTAAGITEGHHHHHHHH SEQ ID NO: 10 - sfYFP-17

[0297] MSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDADNGKLDLKFICTTGKLPVP

[0298] WPTLVTTLGYGLQCFARYPDHMKEHDFFKSAMPEGYVQERTISFKDDGTYKTRAE

[0299] VKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHEVYITADDEKNGIKAEFKIR

[0300] HNVEDGSVQLADHYQQNTPIGDGPDLLPDEHYLSYQSVLSKDPNEKRDHMVLLEF

[0301] VTADGITEGHHHHHHHH

[0302] SEQ ID NO: 11 - sfYFP-31

[0303] MSKGEELFEGVVPILVELDGDVNGHKFEVRGEGEGDADNGKLDLKFICTTGELPVP

[0304] WPTLVTTLGYGLQCFARYPDHMKEHDFFKSAMPEGYVQERTISFDDDGTYETRAE

[0305] VKFEGDTLVNRIELKGIDFKEDGNILGHKLEYDFNSHEVYIEADDEKNGIKAEFKIE

[0306] HNVEDGSVQLADHYQQNTPIGDGPDLLPDEHYLSYQSVLSKDPNEERDHMVLLEF

[0307] VTADGITEGHHHHHHHH

[0308] SEQ ID NO: 12 - sfYFP+16

[0309] MSKGEELFKGVVPILVELKGDVNGHKFKVRGEGEGDAKNGKLRLKFICTTGKLPV

[0310] PWPTLVTTLGYGLQCFARYPDHMKRHDFFKSAMPEGYVQERTISFKDDGTYKTRA

[0311] EVKFEGDTLVNRIELKGIKFKKDGNILGHKLEYNFNSHKVYITADKKKNGIKAKFKI

[0312] RHNVKDGSVQLADHYQQNTPIGDGPKLLPRKHYLSYQSVLSKDPNEKRDHMVLLE

[0313] FVTAAGITKGHHHHHHHH

[0314] SEQ ID NO: 13 - sfYFP+33

[0315] MSKGERLFRGKVPILVELKGDVNGHKFSVRGKGKGDATNGKLTLKFICTTGKLPVP

[0316] WPTLVTTLGYGLQCFARYPKHMKRHDFFKSAMPKGYVQERTISFKKDGTYKTRAE

[0317] VKFEGRTLVNRIKLKGRDFKEKGNILGHKLRYNFNSHKVYITADKRKNGIKAKFKI

[0318] RHNVKDGSVQLADHYQQNTPIGRGPVLLPRNHYLSYRSVLSKDPKEKRDHMVLLE

[0319] FVTAAGIKHGRDERYKLEHHHHHH

[0320] SEQ ID NO: 14 - sfCFP-11

[0321] MASKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDADNGKLDLKFICTTGKLPV

[0322] PWPTLVTTLTWGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGTYKTRA

[0323] EVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNGISDNVYITADKEKNGIKANFKI RHNVEDGSVQLADHYQQNTPIGDGPDLLPDEHYLSTQSVLSKDPNEKRDHMVLLE

[0324] FVTAAGITEGHHHHHHHH

[0325] SEQ ID NO: 15 - sfCFP-18

[0326] MASKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDADNGKLDLKFICTTGKLPV

[0327] PWPTLVTTLTWGVQCFSRYPDHMKEHDFFKSAMPEGYVQERTISFKDDGTYKTRA

[0328] EVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNGISDEVYITADDEKNGIKAEFKIR

[0329] HNVEDGSVQLADHYQQNTPIGDGPDLLPDEHYLSTQSVLSKDPNEKRDHMVLLEF

[0330] VTADGITEGHHHHHHHH

[0331] SEQ ID NO: 16 - sfCFP-32

[0332] MASKGEELFEGVVPILVELDGDVNGHKFEVRGEGEGDADNGKLDLKFICTTGELPV

[0333] PWPTLVTTLTWGVQCFSRYPDHMKEHDFFKSAMPEGYVQERTISFDDDGTYETRA

[0334] EVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYDGISDEVYIEADDEKNGIKAEFKIE

[0335] HNVEDGSVQLADHYQQNTPIGDGPDLLPDEHYLSTQSVLSKDPNEERDHMVLLEF

[0336] VTADGITEGHHHHHHHH

[0337] SEQ ID NO: 17 - sfCFP+15

[0338] MASKGEELFKGVVPILVELKGDVNGHKFKVRGEGEGDAKNGKLRLKFICTTGKLP

[0339] VPWPTLVTTLTWGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGTYKTR

[0340] AEVKFEGDTLVNRIELKGIKFKKDGNILGHKLEYNGISDKVYITADKKKNGIKAKFK

[0341] IRHNVKDGSVQLADHYQQNTPIGDGPKLLPRKHYLSTQSVLSKDPNEKRDHMVLL

[0342] EFVTAAGITKGHHHHHHHH

[0343] SEQ ID NO: 18 - sfCFP+32

[0344] MASKGERLFRGKVPILVELKGDVNGHKFSVRGKGKGDATNGKLTLKFICTTGKLP

[0345] VPWPTLVTTLTWGVQCFARYPKHMKRHDFFKSAMPKGYVQERTISFKKDGTYKT

[0346] RAEVKFEGRTLVNRIKLKGRDFKEKGNILGHKLRYNGISDKVYITADKRKNGIKAK

[0347] FKIRHNVKDGSVQLADHYQQNTPIGRGPVLLPRNHYLSTRSVLSKDPKEKRDHMV

[0348] LLEFVTAAGIKHGRDERYKLEHHHHHH

Claims

WHAT IS CLAIMED IS:

1. A detection system for selectively characterizing the presence and / or amount of an analyte in a sample, the system comprising: a supercharged fluorescent protein configured to directly chelate with an analyte having a spectral overlap with the supercharged fluorescent protein, thereby forming a protein- analyte complex, wherein the protein-analyte complex is configured to emit a fluorescent signal when irradiated with a light source.

2. The system of claim 1, further comprising a fluorescence detector configured to detect the fluorescent signal from the protein- analyte complex.

3. The system of claim 2, wherein the fluorescence detector comprises a fluorescent microscope.

4. The system of any one of claims 1-3, wherein the light source comprises UV light.

5. The system of claim 4, wherein the UV light has a wavelength from 340 nm to 360 nm.

6. The system of any one of claims 1-5, wherein the analyte comprises one or more lanthanides.

7. The system of claim 6, wherein the one or more lanthanides comprise free lanthanide cations.

8. The system of any one of claims 1-7, wherein the analyte comprises terbium (Tb), thulium (Tm), dysprosium (Dy), europium (Eu), samarium (Sm), ytterbium (Yb), or a combination thereof.

9. The system of any one of claims 1-8, wherein the fluorescent protein comprises a green fluorescent protein (GFP), a yellow fluorescent protein (YFP), a cyan fluorescentprotein (CFP), or a combination thereof.

10. The system of any one of claims 1-9, wherein the fluorescent protein comprises an amino acid sequence having at least 85% sequence identity to any one of SEQ ID NOS: 1- 18.

11. The system of any one of claims 1-10, wherein the protein-analyte complex is configured to be measurably fluorescent when the supercharged fluorescent protein and analyte are separated by a distance of 50 nm or less.

12. The system of any one of claims 1-11, wherein the supercharged fluorescent protein has a negative net charge of from -1 to -50.

13. The system of any one of claims 1-11, wherein the supercharged fluorescent protein has a net positive charge.

14. The system of claim 13, wherein the analyte comprises a lanthanide-chelate configured to bind to the supercharged fluorescent protein having a net positive charge.

15. The system of any one of claims 1-14, wherein the protein-analyte complex is configured to provide multiplex detection of a plurality of analytes.

16. The system of any one of claims 1-15, wherein the supercharged fluorescent protein is a monomeric protein.

17. The system of any one of claims 1-16, where.in the supercharged fluorescent protein is a multimeric protein.

18. The system of claim 17, wherein the multimeric protein includes a protomer comprising a pair of supercharged fluorescent proteins having opposite net charges.

19. The system of any one of claims 17-18, wherein the multimeric protein comprises a heterodimer.

20. The system of any one of claims 1-19, wherein said system further comprises: one or more sensors configured to detect the fluorescent signal of the protein-analyte complex; a processor; and software executable by said processor such that said system: determines the presence and / or amount of the analyte in the sample based on the fluorescent signal.

21. A detection system for selectively characterizing the presence and / or amount of an analyte in a sample, the system comprising: a genetically modified microorganism engineered to express a supercharged fluorescent protein, wherein supercharged fluorescent protein is configured to directly chelate with an analyte having a spectral overlap with the supercharged fluorescent protein, thereby forming a protein- analyte complex, and wherein the protein- analyte complex is configured to emit a fluorescent signal when irradiated with a light source.

22. The system of claim 21, further comprising a fluorescence detector configured to detect the fluorescent signal from the protein- analyte complex.

23. The system of claim 22, wherein the fluorescence detector comprises a fluorescent microscope.

24. The system of any one of claims 21-23, wherein the light source comprises UV light.

25. The system of claim 24, wherein the UV light has a wavelength from 340 nm to 360 nm.

26. The system of any one of claims 21-25, wherein the analyte comprises one or more lanthanides.

27. The system of claim 26, wherein the one or more lanthanides comprise free lanthanide cations.

28. The system of any one of claims 21-27, wherein the analyte comprises terbium (Tb), thulium (Tm), dysprosium (Dy), europium (Eu), samarium (Sm), ytterbium (Yb), or a combination thereof.

29. The system of any one of claims 21-28, wherein the fluorescent protein comprises a green fluorescent protein (GFP), a yellow fluorescent protein (YFP), a cyan fluorescent protein (CFP), or a combination thereof.

30. The system of any one of claims 21-29, wherein the fluorescent protein comprises an amino acid sequence having at least 85% sequence identity to any one of SEQ ID NOS: 1-18.

31. The system of any one of claims 21-30, wherein the fluorescent protein has a negative net charge from -1 to -50.

32. The system of any one of claims 21-30, wherein the supercharged fluorescent protein has a net positive charge.

33. The system of claim 32, wherein the analyte comprises a lanthanide-chelate configured to bind to the supercharged fluorescent protein having a net positive charge.

34. The system of any one of claims 21-33, wherein the protein-analyte complex is configured to provide multiplex detection of a plurality of analytes.

35. The system of any one of claims 21-34, wherein the supercharged fluorescent protein is a monomeric protein.

36. The system of any one of claims 21-35, wherein the supercharged fluorescent protein is a multimeric protein.

37. The system of claim 36, wherein the multimeric protein includes a protomer comprising a pair of supercharged fluorescent proteins having opposite net charges.

38. The system of any one of claims 36-37, wherein the multimeric protein comprises a heterodimer.

39. The system of any one of claims 21-38, wherein said system further comprises: one or more sensors configured to detect the fluorescent signal of the protein-analyte complex; a processor; and software executable by said processor such that said system: determines the presence and / or amount of the analyte in the sample based on the fluorescent signal.

40. A kit comprising: a supercharged fluorescent protein disposed within an assay chamber, wherein the supercharged fluorescent protein is configured to directly chelate with an analyte having a spectral overlap with the supercharged fluorescent protein to form a protein- analyte complex, wherein the protein-analyte complex is configured to emit a fluorescent signal when irradiated with a light source.

41. The kit of claim 40, further comprising a fluorescence detector configured to detect the fluorescent signal from the protein-analyte complex.

42. The kit of claim 41, wherein the fluorescence detector comprises a fluorescent microscope.

43. The kit of any one of claims 40-42, wherein the light source comprises UV light.

44. The kit of claim 43, wherein the UV light has a wavelength from 340 nm to 360 nm.

45. The kit of any one of claims 40-44, wherein the analyte comprises one or more lanthanides.

46. The kit of claim 45, wherein the one or more lanthanides comprise free lanthanide cations.

47. The kit of any one of claims 40-46, wherein the analyte comprises terbium (Tb), thulium (Tm), dysprosium (Dy), europium (Eu), samarium (Sm), ytterbium (Yb), or a combination thereof.

48. The kit of any one of claims 40-47, wherein the fluorescent protein comprises a green fluorescent protein (GFP), a yellow fluorescent protein (YFP), a cyan fluorescent protein (CFP), or a combination thereof.

49. The kit of any one of claims 40-48, wherein the fluorescent protein comprises an amino acid sequence having at least 85% sequence identity to any one of SEQ ID NOS: 1- 18.

50. The kit of any one of claims 40-49, wherein the fluorescent protein has a negative net charge from -1 to -50.

51. The kit of any one of claims 40-49, wherein the supercharged fluorescent protein has a net positive charge.

52. The kit of claim 51, wherein the analyte comprises a lanthanide-chelate configured to bind to the supercharged fluorescent protein having a net positive charge.

53. The kit of any one of claims 40-52, wherein the protein- analyte complex is configured to provide multiplex detection of a plurality of analytes.

54. The kit of any one of claims 40-53, wherein the supercharged fluorescent protein is a monomeric protein.

55. The kit of any one of claims 40-54, wherein the supercharged fluorescent protein is a multimeric protein.

56. The kit of claim 55, wherein the multimeric protein includes a protomer comprising a pair of supercharged fluorescent proteins having opposite net charges.

57. The kit of any one of claims 55-56, wherein the multimeric protein comprises a heterodimer.

58. The kit of any one of claims 40-57, wherein the supercharged fluorescent protein is immobilized within the assay chamber.

59. A method for characterizing the presence and / or amount of an analyte in a sample, the method comprising: providing a sample for qualitative or quantitative determination of an analyte having a spectral overlap with a supercharged fluorescent protein; contacting the supercharged fluorescent protein with the sample, the supercharged fluorescent protein being configured to directly chelate with the analyte to form a proteinanalyte complex; irradiating the sample with a light source; and determining, using a fluorescence detector, the presence and / or amount of the analyte in the sample.

60. The method of claim 59, wherein the light source comprises UV light.

61. The method of claim 60, wherein the UV light have a wavelength from 340 nm to 360 nm.

62. The method of any one of claims 59-61, wherein the fluorescence detector comprises a fluorescent microscope.

63. The method of any one of claims 59-62, wherein the analyte comprises one or more lanthanides.

64. The method of claim 63, wherein the one or more lanthanides comprises free lanthanide cations.

65. The method of any one of claims 59-64, wherein the analyte comprises terbium (Tb), thulium (Tm), dysprosium (Dy), europium (Eu), samarium (Sm), ytterbium (Yb), or a combination thereof.

66. The method of any one of claims 59-65, wherein the supercharged fluorescent protein comprises a green fluorescent protein (GFP), a yellow fluorescent protein (YFP), a cyan fluorescent protein (CFP), or a combination thereof.

67. The method of any one of claims 59-66, wherein the fluorescent protein comprises an amino acid sequence having at least 85% sequence identity to any one of SEQ ID NOS: 1-18.

68. The method of any one of claims 59-67, wherein the fluorescent protein has a negative net charge.

69. The method of any one of claims 59-67, wherein the supercharged fluorescent protein has a net positive charge.

70. The method of claim 69, wherein the analyte comprises a lanthanide-chelate configured to bind to the supercharged fluorescent protein having a net positive charge.

71. The method of any one of claims 59-70, wherein the protein-analyte complex is configured to provide multiplex detection of a plurality of analytes.

72. The method of any one of claims 59-71, wherein the supercharged fluorescent protein is a monomeric protein.

73. The method of any one of claims 59-72, wherein the supercharged fluorescent protein is a multimeric protein.

74. The method of claim 73, wherein the multimeric protein includes a protomer comprising a pair of supercharged fluorescent proteins having opposite net charges.

75. The method of any one of claims 73-74, wherein the multimeric protein comprises a heterodimer.

76. The method of any one of claims 59-75, further comprising quantitatively determining an amount of the analyte in the sample based on a fluorescent signal emitted by the protein-analyte complex.

77. The method of claim 76, wherein the amount of the analyte is determined using a normalized ratio between the fluorescent signal emitted by the protein-analyte complex and a baseline fluorescent signal emitted by the supercharged fluorescent protein alone.

78. The method of any one of claims 59-77, wherein the protein-analyte complex is measurably fluorescent when the analyte and supercharged protein are separated by a distance of 50 nm or less.

79. The method of any one of claims 59-78, wherein the method is carried out at a temperature of from 0°C to 70°C.

80. The method of any one of claims 59-79, wherein the sample has a pH of 5 or greater.

81. The method of claim 80, further comprising adjusting the pH of the sample.