pH-insensitive glucose indicator protein
First Claim
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1. A biosensor for sensing glucose in a solution, comprising:
- at least two different glucose indicator proteins (GIP), each glucose indicator protein comprising;
a glucose binding protein that changes conformation upon exposure to glucose;
at least one pH insensitive fluorescence donor coupled to a first portion of the glucose binding protein; and
at least one pH insensitive fluorescence acceptor coupled to a second portion of the glucose binding protein,wherein;
the changes of conformation of the glucose binding protein upon binding glucose causes an alteration in energy transfer between the at least one fluorescence donor coupled to the first portion of the glucose binding protein and the at least one fluorescence acceptor coupled to the second portion of the glucose binding protein, resulting in a detectable change in the fluorescence; and
the glucose binding protein having a conformation saturation response to glucose concentration which is stable over a pH range of at least about 5.2 to 7.3 and has a non-linear regression with respect to a relationship of glucose concentration to fluorescence resonance energy transfer response of R2≧
0.846 over a concentration range of at least 32;
an illuminator configured to excite fluorescence of the at least two different glucose indicator proteins, while in contact with a physiological solution; and
a detector configured to quantitatively detect excited fluorescence from the at least two different glucose indicator proteins, wherein the indicative of a glucose concentration.
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Abstract
The present invention encompasses a glucose indicator protein, a biosensor comprising one or more glucose indicator proteins, and methods of use thereof.
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Citations
20 Claims
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1. A biosensor for sensing glucose in a solution, comprising:
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at least two different glucose indicator proteins (GIP), each glucose indicator protein comprising; a glucose binding protein that changes conformation upon exposure to glucose; at least one pH insensitive fluorescence donor coupled to a first portion of the glucose binding protein; and at least one pH insensitive fluorescence acceptor coupled to a second portion of the glucose binding protein, wherein; the changes of conformation of the glucose binding protein upon binding glucose causes an alteration in energy transfer between the at least one fluorescence donor coupled to the first portion of the glucose binding protein and the at least one fluorescence acceptor coupled to the second portion of the glucose binding protein, resulting in a detectable change in the fluorescence; and the glucose binding protein having a conformation saturation response to glucose concentration which is stable over a pH range of at least about 5.2 to 7.3 and has a non-linear regression with respect to a relationship of glucose concentration to fluorescence resonance energy transfer response of R2≧
0.846 over a concentration range of at least 32;an illuminator configured to excite fluorescence of the at least two different glucose indicator proteins, while in contact with a physiological solution; and a detector configured to quantitatively detect excited fluorescence from the at least two different glucose indicator proteins, wherein the indicative of a glucose concentration. - View Dependent Claims (2, 3, 4, 5, 6, 7)
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8. A biosensor, comprising:
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at least two different glucose indicator proteins (GIP), each glucose indicator protein comprising; a genetically engineered glucose binding protein which selectively changes conformation upon exposure to glucose, having an amino acid sequence wherein at least one amino acid in a wild-type glucose binding protein which undergoes a pH-sensitive change in conformation corresponding to position 16 or 183 of the mature E. coli glucose/galactose binding protein, causing an unstable response of the glucose binding protein at a pH of 5.2 in response to exposure to glucose, is replaced with at least one amino acid which reduces the pH-sensitive change in conformation in response to exposure to glucose with respect to the wild-type glucose binding protein, selected from the group consisting of valine, cysteine, threonine, and leucine, and alanine, causing a stable response of the glucose binding protein at a pH of 5.2 in response to exposure to glucose; at least one fluorescence donor coupled to a first portion of said glucose binding protein; and at least one fluorescence acceptor coupled to a second portion of said glucose binding protein, wherein the at least one fluorescence donor has a fluorescence emission spectrum which overlaps an absorption spectrum of the at least one fluorescence acceptor, and has an interaction with the at least one fluorescence acceptor which varies dependent on a concentration of glucose over a sensing range dependent on a disassociation constant (Kd) of the genetically engineered glucose binding protein; and a detector, for detecting an intensity of fluorescent emissions from each respective glucose indicator protein while exposed to a sample; wherein the change in conformation of each respective genetically engineered glucose binding protein upon exposure to glucose in the sample produces a detectable and reversible change in the detected intensities of fluorescent emissions from the at least one fluorescence donor and the at least one fluorescence acceptor having a non-linear regression with respect to a relationship of glucose concentration to fluorescence resonance energy transfer response of R2≧
0.846 over a concentration range of at least 32. - View Dependent Claims (9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20)
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Specification