US2022411847A1PendingUtilityA1
Glucose/galactose biosensors and methods of using same
Est. expiryMar 5, 2035(~8.6 yrs left)· nominal 20-yr term from priority
C12Q 1/54C07H 3/02G01N 33/582G01N 33/5438C12N 15/66
67
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Claims
Abstract
Provided herein are glucose and galactose biosensors and methods of making and using the same.
Claims
exact text as granted — not AI-modifiedWe claim:
1 . A biosensor comprising:
a) a polypeptide comprising a ligand binding site and (i) one or more mutations as compared to SEQ ID NO:112 (wild-type E. coli GGBP) that alter the ligand binding affinity of the polypeptide; and b) a reporter conjugated to the polypeptide, wherein when the polypeptide consists of a single mutation, the single mutation is F16C, wherein the ligand-bound biosensor results in a reporter-generated signal that is different from the unbound biosensor, and wherein the ligand is selected from the group consisting of glucose, galactose, and a combination thereof.
2 . The biosensor of claim 1 , wherein the reporter is conjugated to F16C.
3 . The biosensor of claim 1 , wherein the polypeptide further comprises (ii) at least one additional mutation that replaces an amino acid with a cysteine.
4 . The biosensor of claim 3 , wherein the reporter is conjugated to the cysteine.
5 . The biosensor of any one of the preceding claims, wherein the biosensor comprises a single reporter.
6 . The biosensor of any one of the preceding claims, wherein the reporter comprises a fluorophore and wherein the signal is a fluorescent signal.
7 . The biosensor of claim 6 , wherein the fluorophore is selected from the group consisting of acrylodan and badan.
8 . The biosensor of claim 6 , wherein the signal comprises an emission intensity of the fluorophore recorded at one or more wavelengths.
9 . The biosensor of claim 6 , wherein the change in signal comprises a shift in the one or more wavelengths.
10 . The biosensor of claim 6 , wherein the signal comprises a ratio of emission intensities recorded at two or more wavelengths.
11 . The biosensor of claim 6 , wherein the change in signal comprises a shift in two or more wavelengths.
12 . The biosensor of any one of claims 3 - 11 , wherein the at least one additional mutation (ii) is W183C.
13 . The biosensor of claim 12 , wherein the reporter is conjugated to W183C.
14 . The biosensor of any one of claims 3 - 13 , wherein each mutation (i) is a mutation to an amino acid selected from the group consisting of Y10, D14, F16, N91, K92, E149, H152, D154, A155, R158, M182, N211, D236, and N256, and combinations thereof.
15 . The biosensor of any one of claims 3 - 14 , wherein each mutation (i) is selected from the group consisting of Y10A, D14A, D14Q, D14N, D14S, D14T, D14E, D14H, D14L, D14Y, D14F, F16L, F16A, N91A, K92A, E149K, E149Q, E149S, H152A, H152F, H152Q, H152N, D154A, D154N, A155S, A155H, A155L, A155F, A155Y, A155N, A155K, A155M, A155W, A155Q, R158A, R158K, M182W, N211F, N211W, N211K, N211Q, N211S, N211H, N211M, D236A, D236N, N256A, and N256D, and combinations thereof.
16 . The biosensor of any one of claims 3 - 15 , wherein the mutation (i) affects the interaction of the polypeptide with bound glucose, wherein the interaction is with a portion of the glucose selected from the group consisting of 1-hydroxyl, 2-hydroxyl, 3-hydroxyl, 4-hydroxyl, 6-hydroxyl, pyranose ring, and combinations thereof.
17 . The biosensor of any one of claims 3 - 16 , wherein the mutation (i) affects the interaction of the mutant polypeptide with the reporter group.
18 . The biosensor of any one of claims 3 - 17 , wherein the mutation (i) affects the interaction of the mutant polypeptide with a water molecule.
19 . The biosensor of any one of the preceding claims, wherein the polypeptide has an affinity (K D ) for glucose within the concentration range of glucose in vivo for a subject.
20 . The biosensor of any one of the preceding claims, wherein the polypeptide has an affinity (K D ) for galactose within the concentration range of galactose in vivo for a subject.
21 . The biosensor of any one of claims 19 and 20 , wherein the subject is a mammal.
22 . The biosensor of any one of claims 19 and 20 , wherein the subject is a primate or non-primate.
23 . The biosensor of claim 22 , wherein the subject is a non-primate selected from a cow, pig, camel, llama, horse, goat, rabbit, sheep, hamsters, guinea pig, cat, dog, rat, and mouse.
24 . The biosensor of claim 22 , wherein the subject is a primate selected from a monkey, chimpanzee, and human.
25 . The biosensor of claim 24 , wherein the subject is a human.
26 . The biosensor of any one of the preceding claims, wherein the polypeptide has an affinity (K D ) for glucose in the range of about 0.2 mM to about 100 mM.
27 . The biosensor of any one of the preceding claims, wherein the polypeptide has an affinity (K D ) for galactose in the range of about 0.8 mM to about 100 mM.
28 . The biosensor of any one of the preceding claims, wherein the biosensor is capable of detecting glucose in the hypoglycemic, hyperglycemic, and hyperglycemic-hyperosmotic ranges.
29 . The biosensor of any one of the preceding claims, wherein the biosensor is capable of detecting glucose in the range of about 0.1 mmol/L to about 120 mmol/L.
30 . The biosensor of any one of the preceding claims, wherein the biosensor is capable of detecting glucose in the range of about 4 mmol/L to about 33 mmol/L.
31 . The biosensor of any one of the preceding claims, wherein the biosensor is capable of detecting galactose in the range of about 0.2 mM to about 400 mM.
32 . The biosensor of any one of the preceding claims, wherein the mutant polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-54.
33 . A polypeptide comprising the amino acid sequence of SEQ ID NO: 1.
34 . A polypeptide comprising the amino acid sequence of SEQ ID NO: 2.
35 . A polypeptide comprising the amino acid sequence of SEQ ID NO: 3.
36 . A polypeptide comprising the amino acid sequence of SEQ ID NO: 4.
37 . A polypeptide comprising the amino acid sequence of SEQ ID NO: 5.
38 . A polypeptide comprising the amino acid sequence of SEQ ID NO: 6.
39 . A polypeptide comprising the amino acid sequence of SEQ ID NO: 7.
40 . A polypeptide comprising the amino acid sequence of SEQ ID NO: 8.
41 . A polypeptide comprising the amino acid sequence of SEQ ID NO: 9.
42 . A polypeptide comprising the amino acid sequence of SEQ ID NO: 10.
43 . A polypeptide comprising the amino acid sequence of SEQ ID NO: 11.
44 . A polypeptide comprising the amino acid sequence of SEQ ID NO: 12.
45 . A polypeptide comprising the amino acid sequence of SEQ ID NO: 13.
46 . A polypeptide comprising the amino acid sequence of SEQ ID NO: 14.
47 . A polypeptide comprising the amino acid sequence of SEQ ID NO: 15.
48 . A polypeptide comprising the amino acid sequence of SEQ ID NO: 16.
49 . A polypeptide comprising the amino acid sequence of SEQ ID NO: 17.
50 . A polypeptide comprising the amino acid sequence of SEQ ID NO: 18.
51 . A polypeptide comprising the amino acid sequence of SEQ ID NO: 19.
52 . A polypeptide comprising the amino acid sequence of SEQ ID NO: 20.
53 . A polypeptide comprising the amino acid sequence of SEQ ID NO: 21.
54 . A polypeptide comprising the amino acid sequence of SEQ ID NO: 22.
55 . A polypeptide comprising the amino acid sequence of SEQ ID NO: 23.
56 . A polypeptide comprising the amino acid sequence of SEQ ID NO: 24.
57 . A polypeptide comprising the amino acid sequence of SEQ ID NO: 25.
58 . A polypeptide comprising the amino acid sequence of SEQ ID NO: 26.
60 . A polypeptide comprising the amino acid sequence of SEQ ID NO: 27.
61 . A polypeptide comprising the amino acid sequence of SEQ ID NO: 28.
62 . A polypeptide comprising the amino acid sequence of SEQ ID NO: 29.
63 . A polypeptide comprising the amino acid sequence of SEQ ID NO: 30.
64 . A polypeptide comprising the amino acid sequence of SEQ ID NO: 31.
65 . A polypeptide comprising the amino acid sequence of SEQ ID NO: 32.
66 . A polypeptide comprising the amino acid sequence of SEQ ID NO: 33.
67 . A polypeptide comprising the amino acid sequence of SEQ ID NO: 34.
68 . A polypeptide comprising the amino acid sequence of SEQ ID NO: 35.
69 . A polypeptide comprising the amino acid sequence of SEQ ID NO: 36.
70 . A polypeptide comprising the amino acid sequence of SEQ ID NO: 37.
71 . A polypeptide comprising the amino acid sequence of SEQ ID NO: 38.
72 . A polypeptide comprising the amino acid sequence of SEQ ID NO: 39.
73 . A polypeptide comprising the amino acid sequence of SEQ ID NO: 40.
74 . A polypeptide comprising the amino acid sequence of SEQ ID NO: 41.
75 . A polypeptide comprising the amino acid sequence of SEQ ID NO: 42.
76 . A polypeptide comprising the amino acid sequence of SEQ ID NO: 43.
77 . A polypeptide comprising the amino acid sequence of SEQ ID NO: 44.
78 . A polypeptide comprising the amino acid sequence of SEQ ID NO: 45.
79 . A polypeptide comprising the amino acid sequence of SEQ ID NO: 46.
80 . A polypeptide comprising the amino acid sequence of SEQ ID NO: 47.
81 . A polypeptide comprising the amino acid sequence of SEQ ID NO: 48.
82 . A polypeptide comprising the amino acid sequence of SEQ ID NO: 49.
83 . A polypeptide comprising the amino acid sequence of SEQ ID NO: 50.
84 . A polypeptide comprising the amino acid sequence of SEQ ID NO: 51.
85 . A polypeptide comprising the amino acid sequence of SEQ ID NO: 52.
86 . A polypeptide comprising the amino acid sequence of SEQ ID NO: 53.
87 . A polypeptide comprising the amino acid sequence of SEQ ID NO: 54.
88 . A polynucleotide encoding the polypeptide of any one of the preceding claims.
89 . The polynucleotide of claim 88 , wherein the polynucleotide comprises at least one sequence selected from the group consisting of SEQ ID NOs: 56-109.
90 . A vector comprising the polynucleotide of claim 88 or 89 .
91 . A panel comprising a plurality of biosensors according to any one of claims 1 - 32 .
92 . The panel of claim 91 , wherein the panel comprises a composite sensor or an array.
93 . The panel of claim 92 , wherein the array is selected from a multichannel array or multiplexed array.
94 . The panel of any one of claims 91 - 93 , wherein each biosensor comprises the same reporter group.
95 . The panel of any one of claims 91 - 93 , wherein each biosensor comprises a different reporter group.
96 . The panel of claim 92 , wherein the array comprises a plurality of sensor elements, each sensor element comprising a biosensor different from or the same as those of the other sensor elements.
97 . The panel of claim 92 , wherein the composite sensor comprises a plurality of sensor elements, each sensor element comprising a mixture of different biosensors.
98 . The panel of claim 92 , wherein the composite sensor comprises a single sensor element, the single sensor element comprising a mixture of different biosensors.
99 . A method of determining the concentration of glucose, galactose, or a combination thereof, in a sample from a subject, the method comprising applying the sample to the panel of any one of claims 91 - 97 .
100 . The method of claim 99 , wherein the sample is from a subject.
101 . The method according to claim 99 or 100 , wherein the sample comprises a biological fluid.
102 . The method according to claim 101 , wherein the biological fluid is selected from the group consisting of blood, urine, interstitial fluid, saliva, sweat, tears, gastric lavage, fecal matter, emesis, bile, or combinations thereof.
103 . The method according to claim 100 , wherein the sample comprises skin.
104 . A method of detecting the presence of a ligand in a sample, the method comprising:
a) contacting the biosensor of any one of claims 1 - 32 with the sample; b) measuring a signal from the biosensor; and c) comparing the signal to a ligand-free control, wherein a difference in signal indicates the presence of ligand in the sample, and wherein the ligand is selected from the group consisting of glucose, galactose, and a combination thereof.
105 . A method of determining the concentration of a ligand in a sample, the method comprising:
a) contacting the biosensor of any one of claims 1 - 32 with the sample; b) measuring a signal from the biosensor; and c) comparing the signal to a standard hyperbolic ligand binding curve to determine the concentration of ligand in the test sample, wherein the standard hyperbolic ligand binding curve is prepared by measuring the signal transduced by the biosensor when contacted with control samples containing known concentrations of ligand, and wherein the ligand is selected from the group consisting of glucose, galactose, and a combination thereof.
106 . A method of episodically or continuously monitoring the presence of a ligand in a reaction, the method comprising:
a) contacting the biosensor of any one of claims 1 - 32 with the reaction; b) maintaining the reaction under conditions such that the polypeptide is capable of binding ligand present in the reaction; and c) episodically or continuously monitoring the signal from the biosensor in the reaction, wherein the ligand is selected from the group consisting of glucose, galactose, and a combination thereof.
107 . A method of episodically or continuously monitoring the presence of a ligand in a reaction, the method comprising:
a) contacting the biosensor of any one of claims 1 - 32 with the reaction; b) maintaining the reaction under conditions such that the polypeptide is capable of binding ligand present in the reaction; c) episodically or continuously monitoring the signal from the biosensor in the reaction; and d) comparing the signal to a standard hyperbolic ligand binding curve to determine the concentration of ligand in the test sample, wherein the standard hyperbolic ligand binding curve is prepared by measuring the signal transduced by the biosensor when contacted with control samples containing known concentrations of ligand, wherein the ligand is selected from the group consisting of glucose, galactose, and a combination thereof.
108 . The method of any one of claims 104 - 107 , wherein the biosensor is placed in contact with a subject's skin or mucosal surface.
109 . The method of any one of claims 104 - 107 , wherein the biosensor is implanted in a subject's body.
110 . The method of any one of claims 104 - 107 , wherein the biosensor is implanted in a subject's blood vessel, vein, eye, natural or artificial pancreas, alimentary canal, stomach, intestine, esophagus, or skin.
111 . The method of any one of claims 104 - 107 , wherein the biosensor is configured within or on the surface of a contact lens.
112 . The method of any one of claims 104 - 107 , wherein the biosensor is configured to be implanted in the skin.
113 . The method of any one of claims 104 - 107 , wherein the biosensor is implanted in a subject with an optode.
114 . The method of any one of claims 104 - 107 , wherein the biosensor is implanted in a subject with a microbead.
115 . The method of any one of claims 104 - 107 , wherein the biosensor generates the signal transdermally.
116 . The method of claim 106 , wherein the method further comprises:
d) comparing the signal to a ligand-free control, wherein a difference in signal indicates the presence of ligand in the reaction.
117 . The method of claim 106 , wherein the method further comprises:
d) comparing the signal to a standard hyperbolic ligand binding curve to determine the concentration of ligand in the test sample, wherein the standard hyperbolic ligand binding curve is prepared by measuring the signal transduced by the biosensor when contacted with control samples containing known concentrations of ligand.
118 . The method of any one of claims 99 , 104 - 107 , and 116 - 117 , wherein the sample comprises a fermentation sample.
119 . The method of any one of claims 99 , 104 - 107 , and 116 - 117 , wherein the sample comprises food or beverage.
120 . The method of claim 119 , wherein the sample comprises a beverage selected from soft drink, fountain beverage, water, coffee, tea, milk, dairy-based beverage, soy-based beverage, almond-based beverage, vegetable juice, fruit juice, fruit juice flavored drink, energy drink, sport drink, and alcoholic product, and combinations thereof.
121 . The method of claim 120 , wherein the sample comprises water selected from flavored water, mineral water, spring water, sparkling water, and tonic water, and combinations thereof.
122 . The method of claim 120 , wherein the sample comprises an alcoholic product selected from beer, malt beverage, liqueur, whiskey, and wine, and combinations thereof.
123 . The method of claim 119 , wherein the sample comprises food comprising a semi-solid or liquid form.
124 . The method of claim 119 wherein the sample comprises food selected from yogurt, soup, ice cream, broth, purees, shakes, smoothies, batter, condiments, and sauce, and combinations thereof.
125 . The method of any one of claims 99 , 103 - 106 , and 115 - 122 , wherein the sample is from food engineering.Join the waitlist — get patent alerts
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