D-glucose optical probe, and preparation method therefor and use thereof

Abstract

The present invention relates to a D-glucose optical probe, and a preparation method therefor and the use thereof. Specifically, provided is a D-glucose optical probe, the optical probe containing a D-glucose sensitive polypeptide and an optically active polypeptide, wherein the optically active polypeptide is located within the sequence of the D-glucose sensitive polypeptide.

Description

A D-glucose optical probe, its preparation method and application Technical Field

[0001] This invention relates to the field of optical probe technology, and in particular to a D-glucose optical probe, its preparation method, and its application. Background Technology

[0002] D-glucose (D-glc) is the most important and fundamental energy source known to all living organisms on Earth, from bacteria to humans, and is also the primary energy source for the human brain and nervous system. D-glucose holds a vital position in biology, serving as an essential nutrient for organisms. It is not only a rich source of potential energy but also a precursor to a wide range of metabolic intermediates in biosynthetic pathways. Plants can also produce glucose through photosynthesis. D-glucose is the primary energy source for most tissues, particularly the nervous system, red blood cells, renal medulla, and skeletal muscle. Failure to regulate this nutrient can lead to both hypoglycemia and hyperglycemia, and may even result in death.

[0003] Currently, the most commonly used methods for detecting D-glucose include enzyme-catalyzed probes, electrochemical methods, nanomaterial probes, various wearable glucose detection devices developed based on the above principles, and gene-encoded glucose probes. Among these, while enzyme-catalyzed probes have high selectivity for glucose and are feasible for large-scale production, they suffer from high manufacturing costs, low reproducibility, and poor stability. Electrochemical methods struggle to balance cost and glucose selectivity. Currently developed gene-encoded fluorescent probes have limited specificity, and their affinity for substrates cannot enable in situ real-time monitoring in vivo. Therefore, there is an urgent need to develop genetically encoded fluorescent probes capable of in situ monitoring of the dynamic changes in D-glucose within living cells. Summary of the Invention

[0004] The purpose of this invention is to provide probes and methods for real-time, high-throughput, and quantitative detection of D-glucose inside and outside cells.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0006] The first aspect of this invention provides a variant of a D-glucose-binding protein, wherein:

[0007] (a) Having the sequence shown in SEQ ID NO: 1 and having mutations at one, two, three or more sites selected from the following: W8, W9, A42, H66, K312, V347, H348, said mutations including amino acid modifications, substitutions or deletions.

[0008] (b) is a sequence that has at least 70% sequence identity with the sequence of (a) and has the mutation described in (a) and retains the ability to bind to D-glucose.

[0009] In one or more embodiments, the mutation includes a mutation at a site selected from any of the following groups:

[0010] (1) V347, H348, (2) V347, H348 and K312, (3) V347, H348 and H66, (4) V347, H348 and W9, (5) V347, H348 and E13, (6) V347, H348 and W8, (7) V347, H348 and A42.

[0011] In one or more embodiments, W8 mutates to R, H, E, A, V, L, F, I, M, C, N, G, K, D, or T; in one or more embodiments, W9 mutates to R, H, E, A, V, L, F, I, M, C, P, D, N, G, K, Y, S, or T; in one or more embodiments, A42 mutates to R, E, W, F, M, C, P, N, G, K, Y, or T; in one or more embodiments, H66 mutates to R, E, A, F, M, C, P, Q, or T; in one or more embodiments, K312 mutates to R, A, Q, G, H, or S; in one or more embodiments, V347 mutates to W; in one or more embodiments, H348 mutates to T, G, or S.

[0012] In one or more embodiments, the mutation comprises mutations selected from any of the following groups: (1) V347W and H348T, (2) V347W and H348G, (3) V347W and H348S, (4) V347W, H348T and H66R, (5) V347W, H348T and H66E, (6) V347W, H348T and H66A, (7) V347W, H348T and H66F, (8) V347W, H348T and H66M, (9) V347W, H348T and H66C, (10) V347W, H348T and H66P, (11) V347W, H348T and H66Q, (12) V347W, H348T and H66Q. T and H66T, (13)V347W, H348T and W9R, (14)V347W, H348T and W9H, (15)V347W, H348T and W9E, (16)V347W, H348T and W9A, (17)V347W, H348T and W9V, (18)V347W, H348T and W9 L, (19)V347W, H348T and W9F, (20)V347W, H348T and W9I, (21)V347W, H348T and W9M, (22)V347W, H348T and W9C, (23)V347W, H348T and W9P, (24)V347W, H348T and W9N, (25) (26) V347W, H348T and W9G, (27) V347W, H348T and W9K, (28) V347W, H348T and W9Y, (29) V347W, H348T and W9S, (30) V347W, H348T and W9T, (31) V347 W, H348T and W8R, (32) V347W, H348T and W8H, (33) V347W, H348T and W8E, (34) V347W, H348T and W8A, (35) V347W, H348T and W8V, (36) V347W, H348T and W8L, (37) V347W, H34 8T and W8F, (38)V347W, H348T and W8I, (39)V347W, H348T and W8M, (40)V347W, H348T and W8C, (41)V347W, H348T and W8N, (42)V347W, H348T and W8G, (43)V347W, H348T and W8 K, (44)V347W, H348T and W8T, (45)V347W, H348T and W8D, (46)V347W, H348T and A42R, (47)V347W, H348T and A42E, (48)V347W, H348T and A42W, (49)V347W, H348T and A42F,(50) V347W, H348T and A42M, (51) V347W, H348T and A42C, (52) V347W, H348T and A42P, (53) V347W, H348T and A42N, (54) V347W, H348T and A42G, (55) V347W, H348T and A42K, (56) V347W, H348T and A42Y, (57) (58) V347W, H348T and A42T, (59) V347W, H348T and K312R, (60) V347W, H348T and K312A, (61) V347W, H348T and K312Q, (62) V347W, H348T and K312G, (63) V347W, H348T and K312H, (64) V347W, H348T and K312S.

[0013] Another aspect of the present invention provides a D-glucose optical probe comprising a D-glucose-sensitive polypeptide and an optically active polypeptide, wherein the optically active polypeptide is located within the sequence of the D-glucose-sensitive polypeptide. The D-glucose-sensitive polypeptide is divided into a first part and a second part by the optically active polypeptide.

[0014] In one or more embodiments, the D-glucose optical probe includes a D-glucose-sensitive polypeptide B and an optically active polypeptide A, wherein the optically active polypeptide A is located within the sequence of the D-glucose-sensitive polypeptide B, and the D-glucose-sensitive polypeptide B is divided into a first part B1 and a second part B2 to form a probe structure of type B1-A-B2.

[0015] In one or more embodiments, the optically active polypeptide is located between residues 117-123, 266-288, and / or 340-353 of the D-glucose-sensitive polypeptide, numbered to correspond to the full length of the D-glucose-sensitive polypeptide. Preferably, the optically active polypeptide is located at any one or more of the following sites of the D-glucose-sensitive polypeptide: 266 / 267, 266 / 268, 266 / 269, 266 / 270, 266 / 271, 266 / 272, 266 / 273, 266 / 274, 266 / 275, 266 / 276, 266 / 277, 266 / 278, 266 / 279, 266 / 280, 266 / 281, 266 / 282, 266 / 283, 266 / 284, 266 / 285, 266 / 286, 266 / 287, 266 / 288, 267 / 267, 267 / 268. 267 / 269, 267 / 270, 267 / 271, 267 / 272, 267 / 273, 267 / 274, 267 / 275, 267 / 276, 267 / 277, 267 / 278, 267 / 279, 267 / 280, 267 / 281, 267 / 282, 267 / 283, 267 / 284, 267 / 285, 267 / 286, 267 / 287, 267 / 288, 268 / 267, 268 / 268, 268 / 269, 268 / 270, 268 / 271, 268 / 272, 268 / 273, 268 / 274, 268 / 2 75, 268 / 276, 268 / 277, 268 / 278, 268 / 279, 268 / 280, 268 / 281, 268 / 282, 268 / 283, 268 / 284, 268 / 285, 268 / 286, 268 / 287, 268 / 288, 269 / 267, 269 / 268, 269 / 269, 269 / 270, 269 / 271, 269 / 272, 269 / 273, 269 / 274, 269 / 275, 269 / 276, 269 / 277, 269 / 278, 269 / 279, 269 / 280, 269 / 281, 26 9 / 282, 269 / 283, 269 / 284, 269 / 285, 269 / 286, 269 / 287, 269 / 288, 270 / 267, 270 / 268, 270 / 269, 270 / 270, 270 / 271, 270 / 272, 270 / 273, 270 / 274, 270 / 275, 270 / 276, 270 / 277, 270 / 278, 270 / 279, 270 / 280, 270 / 281, 270 / 282, 270 / 283, 270 / 284, 270 / 285, 270 / 286, 270 / 287, 270 / 288271 / 267、271 / 268、271 / 269、271 / 270、271 / 271、271 / 272、271 / 273、271 / 274、271 / 275、271 / 276、271 / 277、271 / 278、271 / 279、271 / 280、271 / 281、271 / 282、271 / 283、271 / 284、271 / 285、271 / 286、271 / 287、271 / 288、272 / 267、272 / 268、272 / 269、272 / 270、272 / 271、272 / 272、272 / 273、272 / 274、272 / 275、272 / 276、272 / 277、272 / 278、272 / 279、272 / 280、272 / 281、272 / 282、272 / 283、272 / 284、272 / 285、272 / 286、272 / 287、272 / 288、273 / 267、273 / 268、273 / 269、273 / 270、273 / 271、273 / 272、273 / 273、273 / 274、273 / 275、273 / 276、273 / 277、273 / 278、273 / 279、273 / 280、273 / 281、273 / 282、273 / 283、273 / 284、273 / 285、273 / 286、273 / 287、273 / 288、274 / 267、274 / 268、274 / 269、274 / 270、274 / 271、274 / 272、274 / 273、274 / 274、274 / 275、274 / 276、274 / 277、274 / 278、274 / 279、274 / 280、274 / 281、274 / 282、274 / 283、274 / 284、274 / 285、274 / 286、274 / 287、274 / 288、275 / 267、275 / 268、275 / 269、275 / 270、275 / 271、275 / 272、275 / 273、275 / 274、275 / 275、275 / 276、275 / 277、275 / 278、275 / 279、275 / 280、275 / 281、275 / 282、275 / 283、275 / 284、275 / 285、275 / 286、275 / 287、275 / 288、276 / 267、276 / 268、276 / 269、276 / 270、276 / 271、276 / 272、276 / 273、276 / 274、276 / 275、276 / 276、276 / 277、276 / 278、276 / 279、276 / 280、276 / 281、276 / 282、276 / 283、276 / 284、276 / 285、276 / 286、276 / 287、276 / 288、277 / 267、277 / 268、277 / 269、277 / 270、277 / 271、277 / 272、277 / 273、277 / 274、277 / 275、277 / 276、277 / 277、277 / 278、277 / 279、277 / 280、277 / 281、277 / 282、277 / 283、277 / 284、277 / 285、277 / 286、277 / 287、277 / 288、278 / 267、278 / 268、278 / 269、278 / 270、278 / 271、278 / 272、278 / 273、278 / 274、278 / 275、278 / 276、278 / 277、278 / 278、278 / 279、278 / 280、278 / 281、278 / 282、278 / 283、278 / 284、278 / 285、278 / 286、278 / 287、278 / 288、279 / 267、279 / 268、279 / 269、279 / 270、279 / 271、279 / 272、279 / 273、279 / 274、279 / 275、279 / 276、279 / 277、279 / 278、279 / 279、279 / 280、279 / 281、279 / 282、279 / 283、279 / 284、279 / 285、279 / 286、279 / 287、279 / 288、280 / 267、280 / 268、280 / 269、280 / 270、280 / 271、280 / 272、280 / 273、280 / 274、280 / 275、280 / 276、280 / 277、280 / 278、280 / 279、280 / 280、280 / 281、280 / 282、280 / 283、280 / 284、280 / 285、280 / 286、280 / 287、280 / 288、281 / 267、281 / 268、281 / 269、281 / 270、281 / 271、281 / 272、281 / 273、281 / 274、281 / 275、281 / 276、281 / 277、281 / 278、281 / 279、281 / 280、281 / 281、281 / 282、281 / 283、281 / 284、281 / 285、281 / 286、281 / 287、281 / 288、282 / 267、282 / 268、282 / 269、282 / 270、282 / 271、282 / 272、282 / 273、282 / 274、282 / 275、282 / 276、282 / 277、282 / 278、282 / 279、282 / 280、282 / 281、282 / 282、282 / 283、282 / 284、282 / 285、282 / 286、282 / 287、282 / 288、283 / 267、283 / 268、283 / 269、283 / 270、283 / 271、283 / 272、283 / 273、283 / 274、283 / 275、283 / 276、283 / 277、283 / 278、283 / 279、283 / 280、283 / 281、283 / 282、283 / 283、283 / 284、283 / 285、283 / 286、283 / 287、283 / 288、284 / 267、284 / 268、284 / 269、284 / 270、284 / 271、284 / 272、284 / 273、284 / 274、284 / 275、284 / 276、284 / 277、284 / 278、284 / 279、284 / 280、284 / 281、284 / 282、284 / 283、284 / 284、284 / 285、284 / 286、284 / 287、284 / 288、285 / 267、285 / 268、285 / 269、285 / 270、285 / 271、285 / 272、285 / 273、285 / 274、285 / 275、285 / 276、285 / 277、285 / 278、285 / 279、285 / 280、285 / 281、285 / 282、285 / 283、285 / 284、285 / 285、285 / 286、285 / 287、285 / 288、286 / 267、286 / 268、286 / 269、286 / 270、286 / 271、286 / 272、286 / 273、286 / 274、286 / 275、286 / 276、286 / 277、286 / 278、286 / 279、286 / 280、286 / 281、286 / 282、286 / 283、286 / 284、286 / 285、286 / 286、286 / 287、286 / 288、287 / 267、287 / 268、287 / 269、287 / 270、287 / 271、287 / 272、287 / 273、287 / 274、287 / 275、287 / 276、287 / 277、287 / 278、287 / 279、287 / 280、287 / 281、287 / 282、287 / 283、287 / 284、287 / 285、287 / 286、287 / 287、287 / 288，117 / 118、117 / 119、117 / 120、117 / 121、117 / 122、117 / 123、118 / 118、118 / 119、118 / 120、118 / 121、118 / 122、118 / 123、119 / 118、119 / 119、119 / 120、119 / 121、119 / 122、119 / 123、120 / 118、120 / 119、120 / 120、120 / 121、120 / 122、120 / 123、121 / 118、121 / 119、121 / 120、121 / 121、121 / 122、121 / 123、122 / 118、122 / 119、122 / 120、122 / 121、122 / 122、122 / 123，340 / 341、340 / 342、340 / 343、340 / 344、340 / 345、340 / 346、340 / 347、340 / 348、340 / 349、340 / 350、340 / 351、340 / 352、340 / 353、341 / 341、341 / 342、341 / 343、341 / 344、341 / 345、341 / 346、341 / 347、341 / 348、341 / 349、341 / 350、341 / 351、341 / 352、341 / 353、342 / 341、342 / 342、342 / 343、342 / 344、342 / 345、342 / 346、342 / 347、342 / 348、342 / 349、342 / 350、342 / 351、342 / 352、342 / 353、343 / 341、343 / 342、343 / 343、343 / 344、343 / 345、343 / 346、343 / 347、343 / 348、343 / 349、343 / 350、343 / 351、343 / 352、343 / 353、344 / 341、344 / 342、344 / 343、344 / 344、344 / 345、344 / 346、344 / 347、344 / 348、344 / 349、344 / 350、344 / 351、344 / 352、344 / 353、345 / 341、345 / 342、345 / 343、345 / 344、345 / 345、345 / 346、345 / 347、345 / 348、345 / 349、345 / 350、345 / 351、345 / 352、345 / 353、346 / 341、346 / 342、346 / 343、346 / 344、346 / 345、346 / 346、346 / 347、346 / 348、346 / 349、346 / 350、346 / 351、346 / 352、346 / 353, 347 / 341, 347 / 342, 347 / 343, 347 / 344, 347 / 345, 347 / 346, 347 / 347, 347 / 348, 347 / 349, 347 / 350, 347 / 351, 347 / 352, 347 / 353, 348 / 341, 348 / 342, 348 / 343, 348 / 344, 348 / 345, 348 / 34 6, 348 / 347, 348 / 348, 348 / 349, 348 / 350, 348 / 351, 348 / 352, 348 / 353, 349 / 341, 349 / 342, 349 / 343, 349 / 344, 349 / 345, 349 / 346, 349 / 347, 349 / 348, 349 / 349, 349 / 350, 349 / 351, 349 / 352, 349 / 353, 350 / 341, 350 / 342, 350 / 343, 350 / 344, 350 / 345, 350 / 346, 350 / 347, 350 / 348, 350 / 349, 350 / 350, 350 / 351, 350 / 352, 350 / 353, 351 / 341, 351 / 342, 351 / 343, 351 / 344, 351 / 345, 351 / 346, 35 1 / 347, 351 / 348, 351 / 349, 351 / 350, 351 / 351, 351 / 352, 351 / 353, 352 / 341, 352 / 342, 352 / 343, 352 / 344, 352 / 345, 352 / 346, 352 / 347, 352 / 348, 352 / 349, 352 / 350, 352 / 351, 352 / 352, and 352 / 353.

[0016] In one or more embodiments, the D-glucose-sensitive polypeptide is a D-glucose-binding protein or a functional variant thereof.

[0017] In one or more embodiments, the D-glucose-sensitive polypeptide has:

[0018] (1) The sequence shown in SEQ ID NO: 1, or a sequence that has at least 70% sequence identity with them and retains D-glucose binding activity,

[0019] (2) The sequence of the D-glucose-binding protein variant described in any embodiment of the first aspect of this document, or

[0020] (3) has at least 70% sequence identity with the sequence described in (2) and has the mutation described in (2) and retains the sequence sensitive to D-glucose.

[0021] In one or more embodiments, the optically active polypeptide is a fluorescent protein or a functional variant thereof, wherein the functional variant of the fluorescent protein has a mutation within 3 amino acids at the linker to the optically active polypeptide.

[0022] In one embodiment, the fluorescent protein is selected from yellow fluorescent protein, green fluorescent protein, blue fluorescent protein, and apple red fluorescent protein. In one embodiment, the fluorescent protein has the sequence shown in any of SEQ ID NO: 2-9. Preferably, the fluorescent protein has the sequence shown in any of SEQ ID NO: 2, 6, 7, and 9.

[0023] In one or more embodiments, the functional variant of the fluorescent protein has a mutation at amino acid positions 1-3, preferably position 1. Preferably, the functional variant of the fluorescent protein includes a mutation in the amino acid position 1 of the fluorescent protein to A or G.

[0024] In one or more embodiments, a functional variant of the fluorescent protein has the sequence shown in SEQ ID NO: 2 and a mutation at the Y1 site. Preferably, the mutation is Y1A or Y1G.

[0025] In one or more embodiments, the fluorescent protein has the sequence shown in SEQ ID NO: 2 or a variant Y1A or Y1G with any of the following mutations at its first amino acid position, and the optically active polypeptide is located at position 348 / 352 of the D-glucose-sensitive polypeptide.

[0026] In one embodiment, the optical probe further comprises one or more linkers flanking the optically active polypeptide. The linkers of this invention can be any amino acid sequence of any length. In one embodiment, the optically active polypeptide flanking the linker comprises a linker of no more than 5 amino acids, for example, linkers of 0, 1, 2, 3, or 4 amino acids. In one embodiment, the linker flanking the optically active polypeptide comprises amino acid Y. In one embodiment, linker Y is located at the N-terminus and / or C-terminus of the optically active polypeptide. In one embodiment, the optical probe is as follows: a first portion B1 of a D-glucose-sensitive polypeptide, Y, an optically active polypeptide A, and a second portion B2 of a D-glucose-sensitive polypeptide. In one embodiment, the optical probe of this invention does not comprise a linker.

[0027] In one embodiment, the optical probe of the present invention further includes a positioning sequence for positioning the probe to a specific organelle, such as a cell.

[0028] In one or more embodiments, the sequence of the D-glucose-sensitive polypeptide is as shown in SEQ ID NO: 1, and the optically active polypeptide is as shown in any one of SEQ ID NO: 2, 6, 7, 9, wherein the optically active polypeptide is located at any one or more sites of the D-glucose-sensitive polypeptide selected from the following: 266 / 267, 266 / 268, 266 / 269, 266 / 270, 266 / 271, 266 / 272, 266 / 273, 266 / 274, 266 / 275, 266 / 276, 266 / 277, 266 / 278, 266 / 279, 266 / 280, 266 / 281, 266 / 282, 266 / 283, 266 / 284, 266 / 285, 266 / 286, 266 / 287, 266 / 288, 267 / 267. 267 / 268, 267 / 269, 267 / 270, 267 / 271, 267 / 272, 267 / 273, 267 / 274, 267 / 275, 267 / 276, 267 / 277, 267 / 278, 267 / 279, 267 / 280, 267 / 281, 267 / 282, 267 / 283, 267 / 284, 267 / 285, 267 / 286, 267 / 287, 267 / 288, 268 / 267, 268 / 268, 268 / 269, 268 / 270, 268 / 271, 268 / 272, 268 / 273, 268 / 274, 26 8 / 275, 268 / 276, 268 / 277, 268 / 278, 268 / 279, 268 / 280, 268 / 281, 268 / 282, 268 / 283, 268 / 284, 268 / 285, 268 / 286, 268 / 287, 268 / 288, 269 / 267, 269 / 268, 269 / 269, 269 / 270, 269 / 271, 269 / 272, 269 / 273, 269 / 274, 269 / 275, 269 / 276, 269 / 277, 269 / 278, 269 / 279, 269 / 280, 269 / 281, 269 / 2 82, 269 / 283, 269 / 284, 269 / 285, 269 / 286, 269 / 287, 269 / 288, 270 / 267, 270 / 268, 270 / 269, 270 / 270, 270 / 271, 270 / 272, 270 / 273, 270 / 274, 270 / 275, 270 / 276, 270 / 277, 270 / 278, 270 / 279, 270 / 280, 270 / 281, 270 / 282, 270 / 283, 270 / 284, 270 / 285, 270 / 286, 270 / 287, 270 / 288, 271 / 267271 / 268、271 / 269、271 / 270、271 / 271、271 / 272、271 / 273、271 / 274、271 / 275、271 / 276、271 / 277、271 / 278、271 / 279、271 / 280、271 / 281、271 / 282、271 / 283、271 / 284、271 / 285、271 / 286、271 / 287、271 / 288、272 / 267、272 / 268、272 / 269、272 / 270、272 / 271、272 / 272、272 / 273、272 / 274、272 / 275、272 / 276、272 / 277、272 / 278、272 / 279、272 / 280、272 / 281、272 / 282、272 / 283、272 / 284、272 / 285、272 / 286、272 / 287、272 / 288、273 / 267、273 / 268、273 / 269、273 / 270、273 / 271、273 / 272、273 / 273、273 / 274、273 / 275、273 / 276、273 / 277、273 / 278、273 / 279、273 / 280、273 / 281、273 / 282、273 / 283、273 / 284、273 / 285、273 / 286、273 / 287、273 / 288、274 / 267、274 / 268、274 / 269、274 / 270、274 / 271、274 / 272、274 / 273、274 / 274、274 / 275、274 / 276、274 / 277、274 / 278、274 / 279、274 / 280、274 / 281、274 / 282、274 / 283、274 / 284、274 / 285、274 / 286、274 / 287、274 / 288、275 / 267、275 / 268、275 / 269、275 / 270、275 / 271、275 / 272、275 / 273、275 / 274、275 / 275、275 / 276、275 / 277、275 / 278、275 / 279、275 / 280、275 / 281、275 / 282、275 / 283、275 / 284、275 / 285、275 / 286、275 / 287、275 / 288、276 / 267、276 / 268、276 / 269、276 / 270、276 / 271、276 / 272、276 / 273、276 / 274、276 / 275、276 / 276、276 / 277、276 / 278、276 / 279、276 / 280、276 / 281、276 / 282、276 / 283、276 / 284、276 / 285、276 / 286、276 / 287、276 / 288、277 / 267、277 / 268、277 / 269、277 / 270、277 / 271、277 / 272、277 / 273、277 / 274、277 / 275、277 / 276、277 / 277、277 / 278、277 / 279、277 / 280、277 / 281、277 / 282、277 / 283、277 / 284、277 / 285、277 / 286、277 / 287、277 / 288、278 / 267、278 / 268、278 / 269、278 / 270、278 / 271、278 / 272、278 / 273、278 / 274、278 / 275、278 / 276、278 / 277、278 / 278、278 / 279、278 / 280、278 / 281、278 / 282、278 / 283、278 / 284、278 / 285、278 / 286、278 / 287、278 / 288、279 / 267、279 / 268、279 / 269、279 / 270、279 / 271、279 / 272、279 / 273、279 / 274、279 / 275、279 / 276、279 / 277、279 / 278、279 / 279、279 / 280、279 / 281、279 / 282、279 / 283、279 / 284、279 / 285、279 / 286、279 / 287、279 / 288、280 / 267、280 / 268、280 / 269、280 / 270、280 / 271、280 / 272、280 / 273、280 / 274、280 / 275、280 / 276、280 / 277、280 / 278、280 / 279、280 / 280、280 / 281、280 / 282、280 / 283、280 / 284、280 / 285、280 / 286、280 / 287、280 / 288、281 / 267、281 / 268、281 / 269、281 / 270、281 / 271、281 / 272、281 / 273、281 / 274、281 / 275、281 / 276、281 / 277、281 / 278、281 / 279、281 / 280、281 / 281、281 / 282、281 / 283、281 / 284、281 / 285、281 / 286、281 / 287、281 / 288、282 / 267、282 / 268、282 / 269、282 / 270、282 / 271、282 / 272、282 / 273、282 / 274、282 / 275、282 / 276、282 / 277、282 / 278、282 / 279、282 / 280、282 / 281、282 / 282、282 / 283、282 / 284、282 / 285、282 / 286、282 / 287、282 / 288、283 / 267、283 / 268、283 / 269、283 / 270、283 / 271、283 / 272、283 / 273、283 / 274、283 / 275、283 / 276、283 / 277、283 / 278、283 / 279、283 / 280、283 / 281、283 / 282、283 / 283、283 / 284、283 / 285、283 / 286、283 / 287、283 / 288、284 / 267、284 / 268、284 / 269、284 / 270、284 / 271、284 / 272、284 / 273、284 / 274、284 / 275、284 / 276、284 / 277、284 / 278、284 / 279、284 / 280、284 / 281、284 / 282、284 / 283、284 / 284、284 / 285、284 / 286、284 / 287、284 / 288、285 / 267、285 / 268、285 / 269、285 / 270、285 / 271、285 / 272、285 / 273、285 / 274、285 / 275、285 / 276、285 / 277、285 / 278、285 / 279、285 / 280、285 / 281、285 / 282、285 / 283、285 / 284、285 / 285、285 / 286、285 / 287、285 / 288、286 / 267、286 / 268、286 / 269、286 / 270、286 / 271、286 / 272、286 / 273、286 / 274、286 / 275、286 / 276、286 / 277、286 / 278、286 / 279、286 / 280、286 / 281、286 / 282、286 / 283、286 / 284、286 / 285、286 / 286、286 / 287、286 / 288、287 / 267、287 / 268、287 / 269、287 / 270、287 / 271、287 / 272、287 / 273、287 / 274、287 / 275、287 / 276、287 / 277、287 / 278、287 / 279、287 / 280、287 / 281、287 / 282、287 / 283、287 / 284、287 / 285、287 / 286、287 / 287、287 / 288，117 / 118、117 / 119、117 / 120、117 / 121、117 / 122、117 / 123、118 / 118、118 / 119、118 / 120、118 / 121、118 / 122、118 / 123、119 / 118、119 / 119、119 / 120、119 / 121、119 / 122、119 / 123、120 / 118、120 / 119、120 / 120、120 / 121、120 / 122、120 / 123、121 / 118、121 / 119、121 / 120、121 / 121、121 / 122、121 / 123、122 / 118、122 / 119、122 / 120、122 / 121、122 / 122、122 / 123，340 / 341、340 / 342、340 / 343、340 / 344、340 / 345、340 / 346、340 / 347、340 / 348、340 / 349、340 / 350、340 / 351、340 / 352、340 / 353、341 / 341、341 / 342、341 / 343、341 / 344、341 / 345、341 / 346、341 / 347、341 / 348、341 / 349、341 / 350、341 / 351、341 / 352、341 / 353、342 / 341、342 / 342、342 / 343、342 / 344、342 / 345、342 / 346、342 / 347、342 / 348、342 / 349、342 / 350、342 / 351、342 / 352、342 / 353、343 / 341、343 / 342、343 / 343、343 / 344、343 / 345、343 / 346、343 / 347、343 / 348、343 / 349、343 / 350、343 / 351、343 / 352、343 / 353、344 / 341、344 / 342、344 / 343、344 / 344、344 / 345、344 / 346、344 / 347、344 / 348、344 / 349、344 / 350、344 / 351、344 / 352、344 / 353、345 / 341、345 / 342、345 / 343、345 / 344、345 / 345、345 / 346、345 / 347、345 / 348、345 / 349、345 / 350、345 / 351、345 / 352、345 / 353、346 / 341、346 / 342、346 / 343、346 / 344、346 / 345、346 / 346、346 / 347、346 / 348、346 / 349、346 / 350、346 / 351、346 / 352、346 / 353、347 / 341, 347 / 342, 347 / 343, 347 / 344, 347 / 345, 347 / 346, 347 / 347, 347 / 348, 347 / 349, 347 / 350, 347 / 351, 347 / 352, 347 / 353, 348 / 341, 348 / 342, 348 / 343, 348 / 344, 348 / 345, 348 / 346, 348 / 347, 348 / 348, 348 / 349, 348 / 350, 348 / 351, 348 / 352, 348 / 353, 349 / 341, 349 / 342, 349 / 343, 349 / 344, 349 / 345, 349 / 346, 349 / 347, 349 / 348, 349 / 349, 349 / 350, 349 / 351, 349 / 352, 349 / 353 350 / 341, 350 / 342, 350 / 343, 350 / 344, 350 / 345, 350 / 346, 350 / 347, 350 / 348, 350 / 349, 350 / 350, 350 / 351, 350 / 352, 350 / 353, 351 / 341, 351 / 342, 351 / 343, 351 / 344, 351 / 345, 351 / 346, 351 / 347, 351 / 348, 351 / 349, 351 / 350, 351 / 351, 351 / 352, 351 / 353, 352 / 341, 352 / 342, 352 / 343, 352 / 344, 352 / 345, 352 / 346, 352 / 347, 352 / 348, 352 / 349, 352 / 350, 352 / 351, 352 / 352, and 352 / 353.

[0029] In one or more embodiments, the D-glucose-sensitive polypeptide is as shown in SEQ ID NO: 1, and the optically active polypeptide is as shown in any one of SEQ ID NO: 2, 6, 7, 9 or a variant thereof having a mutation selected from one or more of the following at the amino acid position corresponding to the first amino acid of SEQ ID NO: 2: Y1A or Y1G, and the optically active polypeptide is located between residues 117-123, 266-288 and / or 340-353 of the D-glucose-sensitive polypeptide, the numbers corresponding to the full length of the D-glucose-sensitive polypeptide. In one or more embodiments, the optically active polypeptide is located at any one or more sites of the D-glucose-sensitive polypeptide selected from the following: 266 / 267, 266 / 268, 266 / 269, 266 / 270, 266 / 271, 266 / 272, 266 / 273, 266 / 274, 266 / 275, 266 / 276, 266 / 277, 266 / 278, 266 / 279, 266 / 280, 266 / 281, 266 / 282, 266 / 283, 266 / 284, 266 / 285, 266 / 28 6, 266 / 287, 266 / 288, 267 / 267, 267 / 268, 267 / 269, 267 / 270, 267 / 271, 267 / 272, 267 / 273, 267 / 274, 267 / 275, 267 / 276, 267 / 277, 267 / 278, 267 / 279, 267 / 280, 267 / 281, 267 / 282, 267 / 283, 267 / 284, 267 / 285, 267 / 286, 267 / 287, 267 / 288, 268 / 267, 2 68 / 268, 268 / 269, 268 / 270, 268 / 271, 268 / 272, 268 / 273, 268 / 274, 268 / 275, 268 / 276, 268 / 277, 268 / 278, 268 / 279, 268 / 280, 268 / 281, 268 / 282, 268 / 283, 268 / 284, 268 / 285, 268 / 286, 268 / 287, 268 / 288, 269 / 267, 269 / 268, 269 / 269, 269 / 270, 269 / 271, 269 / 272, 269 / 273, 269 / 274, 269 / 275, 269 / 276, 269 / 277, 269 / 278, 269 / 279, 269 / 280, 269 / 281, 269 / 282, 269 / 283, 269 / 284, 269 / 285, 269 / 286, 269 / 287, 269 / 288, 270 / 267, 270 / 268, 270 / 269, 270 / 270, 270 / 271, 270 / 272, 270 / 273, 270 / 274,270 / 275、270 / 276、270 / 277、270 / 278、270 / 279、270 / 280、270 / 281、270 / 282、270 / 283、270 / 284、270 / 285、270 / 286、270 / 287、270 / 288、271 / 267、271 / 268、271 / 269、271 / 270、271 / 271、271 / 272、271 / 273、271 / 274、271 / 275、271 / 276、271 / 277、271 / 278、271 / 279、271 / 280、271 / 281、271 / 282、271 / 283、271 / 284、271 / 285、271 / 286、271 / 287、271 / 288、272 / 267、272 / 268、272 / 269、272 / 270、272 / 271、272 / 272、272 / 273、272 / 274、272 / 275、272 / 276、272 / 277、272 / 278、272 / 279、272 / 280、272 / 281、272 / 282、272 / 283、272 / 284、272 / 285、272 / 286、272 / 287、272 / 288、273 / 267、273 / 268、273 / 269、273 / 270、273 / 271、273 / 272、273 / 273、273 / 274、273 / 275、273 / 276、273 / 277、273 / 278、273 / 279、273 / 280、273 / 281、273 / 282、273 / 283、273 / 284、273 / 285、273 / 286、273 / 287、273 / 288、274 / 267、274 / 268、274 / 269、274 / 270、274 / 271、274 / 272、274 / 273、274 / 274、274 / 275、274 / 276、274 / 277、274 / 278、274 / 279、274 / 280、274 / 281、274 / 282、274 / 283、274 / 284、274 / 285、274 / 286、274 / 287、274 / 288、275 / 267、275 / 268、275 / 269、275 / 270、275 / 271、275 / 272、275 / 273、275 / 274、275 / 275、275 / 276、275 / 277、275 / 278、275 / 279、275 / 280、275 / 281、275 / 282、275 / 283、275 / 284、275 / 285、275 / 286、275 / 287、275 / 288、276 / 267、276 / 268、276 / 269、276 / 270、276 / 271、276 / 272、276 / 273、276 / 274、276 / 275、276 / 276、276 / 277、276 / 278、276 / 279、276 / 280、276 / 281、276 / 282、276 / 283、276 / 284、276 / 285、276 / 286、276 / 287、276 / 288、277 / 267、277 / 268、277 / 269、277 / 270、277 / 271、277 / 272、277 / 273、277 / 274、277 / 275、277 / 276、277 / 277、277 / 278、277 / 279、277 / 280、277 / 281、277 / 282、277 / 283、277 / 284、277 / 285、277 / 286、277 / 287、277 / 288、278 / 267、278 / 268、278 / 269、278 / 270、278 / 271、278 / 272、278 / 273、278 / 274、278 / 275、278 / 276、278 / 277、278 / 278、278 / 279、278 / 280、278 / 281、278 / 282、278 / 283、278 / 284、278 / 285、278 / 286、278 / 287、278 / 288、279 / 267、279 / 268、279 / 269、279 / 270、279 / 271、279 / 272、279 / 273、279 / 274、279 / 275、279 / 276、279 / 277、279 / 278、279 / 279、279 / 280、279 / 281、279 / 282、279 / 283、279 / 284、279 / 285、279 / 286、279 / 287、279 / 288、280 / 267、280 / 268、280 / 269、280 / 270、280 / 271、280 / 272、280 / 273、280 / 274、280 / 275、280 / 276、280 / 277、280 / 278、280 / 279、280 / 280、280 / 281、280 / 282、280 / 283、280 / 284、280 / 285、280 / 286、280 / 287、280 / 288、281 / 267、281 / 268、281 / 269、281 / 270、281 / 271、281 / 272、281 / 273、281 / 274、281 / 275、281 / 276、281 / 277、281 / 278、281 / 279、281 / 280、281 / 281、281 / 282、281 / 283、281 / 284、281 / 285、281 / 286、281 / 287、281 / 288、282 / 267、282 / 268、282 / 269、282 / 270、282 / 271、282 / 272、282 / 273、282 / 274、282 / 275、282 / 276、282 / 277、282 / 278、282 / 279、282 / 280、282 / 281、282 / 282、282 / 283、282 / 284、282 / 285、282 / 286、282 / 287、282 / 288、283 / 267、283 / 268、283 / 269、283 / 270、283 / 271、283 / 272、283 / 273、283 / 274、283 / 275、283 / 276、283 / 277、283 / 278、283 / 279、283 / 280、283 / 281、283 / 282、283 / 283、283 / 284、283 / 285、283 / 286、283 / 287、283 / 288、284 / 267、284 / 268、284 / 269、284 / 270、284 / 271、284 / 272、284 / 273、284 / 274、284 / 275、284 / 276、284 / 277、284 / 278、284 / 279、284 / 280、284 / 281、284 / 282、284 / 283、284 / 284、284 / 285、284 / 286、284 / 287、284 / 288、285 / 267、285 / 268、285 / 269、285 / 270、285 / 271、285 / 272、285 / 273、285 / 274、285 / 275、285 / 276、285 / 277、285 / 278、285 / 279、285 / 280、285 / 281、285 / 282、285 / 283、285 / 284、285 / 285、285 / 286、285 / 287、285 / 288、286 / 267、286 / 268、286 / 269、286 / 270、286 / 271、286 / 272、286 / 273、286 / 274、286 / 275、286 / 276、286 / 277、286 / 278、286 / 279、286 / 280、286 / 281、286 / 282、286 / 283、286 / 284、286 / 285、286 / 286、286 / 287、286 / 288、287 / 267、287 / 268、287 / 269、287 / 270、287 / 271、287 / 272、287 / 273、287 / 274、287 / 275、287 / 276、287 / 277、287 / 278、287 / 279、287 / 280、287 / 281、287 / 282、287 / 283、287 / 284、287 / 285、287 / 286、287 / 287、287 / 288，117 / 118、117 / 119、117 / 120、117 / 121、117 / 122、117 / 123、118 / 118、118 / 119、118 / 120、118 / 121、118 / 122、118 / 123、119 / 118、119 / 119、119 / 120、119 / 121、119 / 122、119 / 123、120 / 118、120 / 119、120 / 120、120 / 121、120 / 122、120 / 123、121 / 118、121 / 119、121 / 120、121 / 121、121 / 122、121 / 123、122 / 118、122 / 119、122 / 120、122 / 121、122 / 122、122 / 123，340 / 341、340 / 342、340 / 343、340 / 344、340 / 345、340 / 346、340 / 347、340 / 348、340 / 349、340 / 350、340 / 351、340 / 352、340 / 353、341 / 341、341 / 342、341 / 343、341 / 344、341 / 345、341 / 346、341 / 347、341 / 348、341 / 349、341 / 350、341 / 351、341 / 352、341 / 353、342 / 341、342 / 342、342 / 343、342 / 344、342 / 345、342 / 346、342 / 347、342 / 348、342 / 349、342 / 350、342 / 351、342 / 352、342 / 353、343 / 341、343 / 342、343 / 343、343 / 344、343 / 345、343 / 346、343 / 347、343 / 348、343 / 349、343 / 350、343 / 351、343 / 352、343 / 353、344 / 341、344 / 342、344 / 343、344 / 344、344 / 345、344 / 346、344 / 347、344 / 348、344 / 349、344 / 350、344 / 351、344 / 352、344 / 353、345 / 341、345 / 342、345 / 343、345 / 344、345 / 345、345 / 346、345 / 347、345 / 348、345 / 349、345 / 350、345 / 351、345 / 352, 345 / 353, 346 / 341, 346 / 342, 346 / 343, 346 / 344, 346 / 345, 346 / 346, 346 / 347, 346 / 348, 346 / 349, 346 / 350, 346 / 351, 346 / 352, 346 / 353, 347 / 341, 347 / 342, 347 / 343, 347 / 344, 347 / 345, 347 / 346, 347 / 347, 347 / 348, 34 7 / 349, 347 / 350, 347 / 351, 347 / 352, 347 / 353, 348 / 341, 348 / 342, 348 / 343, 348 / 344, 348 / 345, 348 / 346, 348 / 347, 348 / 348, 348 / 349, 348 / 350, 348 / 351, 348 / 352, 348 / 353, 349 / 341, 349 / 342, 349 / 343, 349 / 344, 349 / 345, 349 / 346, 349 / 347, 349 / 348, 349 / 349, 349 / 350, 349 / 351, 349 / 352, 349 / 353, 350 / 341, 350 / 342, 350 / 343, 350 / 344, 350 / 345, 350 / 346, 350 / 347, 350 / 348, 350 / 349, 350 / 350, 350 / 351, 350 / 352, 350 / 353, 351 / 341, 351 / 342, 351 / 34 3. 351 / 344, 351 / 345, 351 / 346, 351 / 347, 351 / 348, 351 / 349, 351 / 350, 351 / 351, 351 / 352, 351 / 353, 352 / 341, 352 / 342, 352 / 343, 352 / 344, 352 / 345, 352 / 346, 352 / 347, 352 / 348, 352 / 349, 352 / 350, 352 / 351, 352 / 352, and 352 / 353.

[0030] In one or more embodiments, the optical probe contains a D-glucose-sensitive polypeptide as shown in SEQ ID NO: 1, and has one or more of the following mutations: W8, W9, A42, H66, K312, V347, H348. The optically active polypeptide is shown in SEQ ID NO: 2, 6, 7, 9, or a variant thereof corresponding to SEQ ID NO: 1. The amino acid at position 1 of NO:2 has a variant selected from one or more of the following mutations: Y1A or Y1G. The optically active polypeptide is located at positions 266 / 267, 266 / 268, 266 / 269, 266 / 270, 266 / 271, 266 / 272, 266 / 273, 266 / 274, 266 / 275, 266 / 276, 266 / 277, 266 / 278, 266 / 279, 266 / 280, 266 / 281, 266 / 282, 266 / 283, 266 / 284, 266 / 285, 266 / 2... 86, 266 / 287, 266 / 288, 267 / 267, 267 / 268, 267 / 269, 267 / 270, 267 / 271, 267 / 272, 267 / 273, 267 / 274, 267 / 275, 267 / 276, 267 / 277, 267 / 278, 267 / 279, 267 / 280, 267 / 281, 267 / 282, 267 / 283, 267 / 284, 267 / 285, 267 / 286, 267 / 287, 267 / 288, 268 / 267, 268 / 268, 268 / 26 9, 268 / 270, 268 / 271, 268 / 272, 268 / 273, 268 / 274, 268 / 275, 268 / 276, 268 / 277, 268 / 278, 268 / 279, 268 / 280, 268 / 281, 268 / 282, 268 / 283, 268 / 284, 268 / 285, 268 / 286, 268 / 287, 268 / 288, 269 / 267, 269 / 268, 269 / 269, 269 / 270, 269 / 271, 269 / 272, 269 / 273, 269 / 274 269 / 275, 269 / 276, 269 / 277, 269 / 278, 269 / 279, 269 / 280, 269 / 281, 269 / 282, 269 / 283, 269 / 284, 269 / 285, 269 / 286, 269 / 287, 269 / 288, 270 / 267, 270 / 268, 270 / 269, 270 / 270, 270 / 271, 270 / 272, 270 / 273, 270 / 274, 270 / 275, 270 / 276, 270 / 277, 270 / 278, 270 / 279270 / 280、270 / 281、270 / 282、270 / 283、270 / 284、270 / 285、270 / 286、270 / 287、270 / 288、271 / 267、271 / 268、271 / 269、271 / 270、271 / 271、271 / 272、271 / 273、271 / 274、271 / 275、271 / 276、271 / 277、271 / 278、271 / 279、271 / 280、271 / 281、271 / 282、271 / 283、271 / 284、271 / 285、271 / 286、271 / 287、271 / 288、272 / 267、272 / 268、272 / 269、272 / 270、272 / 271、272 / 272、272 / 273、272 / 274、272 / 275、272 / 276、272 / 277、272 / 278、272 / 279、272 / 280、272 / 281、272 / 282、272 / 283、272 / 284、272 / 285、272 / 286、272 / 287、272 / 288、273 / 267、273 / 268、273 / 269、273 / 270、273 / 271、273 / 272、273 / 273、273 / 274、273 / 275、273 / 276、273 / 277、273 / 278、273 / 279、273 / 280、273 / 281、273 / 282、273 / 283、273 / 284、273 / 285、273 / 286、273 / 287、273 / 288、274 / 267、274 / 268、274 / 269、274 / 270、274 / 271、274 / 272、274 / 273、274 / 274、274 / 275、274 / 276、274 / 277、274 / 278、274 / 279、274 / 280、274 / 281、274 / 282、274 / 283、274 / 284、274 / 285、274 / 286、274 / 287、274 / 288、275 / 267、275 / 268、275 / 269、275 / 270、275 / 271、275 / 272、275 / 273、275 / 274、275 / 275、275 / 276、275 / 277、275 / 278、275 / 279、275 / 280、275 / 281、275 / 282、275 / 283、275 / 284、275 / 285、275 / 286、275 / 287、275 / 288、276 / 267、276 / 268、276 / 269、276 / 270、276 / 271、276 / 272、276 / 273、276 / 274、276 / 275、276 / 276、276 / 277、276 / 278、276 / 279、276 / 280、276 / 281、276 / 282、276 / 283、276 / 284、276 / 285、276 / 286、276 / 287、276 / 288、277 / 267、277 / 268、277 / 269、277 / 270、277 / 271、277 / 272、277 / 273、277 / 274、277 / 275、277 / 276、277 / 277、277 / 278、277 / 279、277 / 280、277 / 281、277 / 282、277 / 283、277 / 284、277 / 285、277 / 286、277 / 287、277 / 288、278 / 267、278 / 268、278 / 269、278 / 270、278 / 271、278 / 272、278 / 273、278 / 274、278 / 275、278 / 276、278 / 277、278 / 278、278 / 279、278 / 280、278 / 281、278 / 282、278 / 283、278 / 284、278 / 285、278 / 286、278 / 287、278 / 288、279 / 267、279 / 268、279 / 269、279 / 270、279 / 271、279 / 272、279 / 273、279 / 274、279 / 275、279 / 276、279 / 277、279 / 278、279 / 279、279 / 280、279 / 281、279 / 282、279 / 283、279 / 284、279 / 285、279 / 286、279 / 287、279 / 288、280 / 267、280 / 268、280 / 269、280 / 270、280 / 271、280 / 272、280 / 273、280 / 274、280 / 275、280 / 276、280 / 277、280 / 278、280 / 279、280 / 280、280 / 281、280 / 282、280 / 283、280 / 284、280 / 285、280 / 286、280 / 287、280 / 288、281 / 267、281 / 268、281 / 269、281 / 270、281 / 271、281 / 272、281 / 273、281 / 274、281 / 275、281 / 276、281 / 277、281 / 278、281 / 279、281 / 280、281 / 281、281 / 282、281 / 283、281 / 284、281 / 285、281 / 286、281 / 287、281 / 288、282 / 267、282 / 268、282 / 269、282 / 270、282 / 271、282 / 272、282 / 273、282 / 274、282 / 275、282 / 276、282 / 277、282 / 278、282 / 279、282 / 280、282 / 281、282 / 282、282 / 283、282 / 284、282 / 285、282 / 286、282 / 287、282 / 288、283 / 267、283 / 268、283 / 269、283 / 270、283 / 271、283 / 272、283 / 273、283 / 274、283 / 275、283 / 276、283 / 277、283 / 278、283 / 279、283 / 280、283 / 281、283 / 282、283 / 283、283 / 284、283 / 285、283 / 286、283 / 287、283 / 288、284 / 267、284 / 268、284 / 269、284 / 270、284 / 271、284 / 272、284 / 273、284 / 274、284 / 275、284 / 276、284 / 277、284 / 278、284 / 279、284 / 280、284 / 281、284 / 282、284 / 283、284 / 284、284 / 285、284 / 286、284 / 287、284 / 288、285 / 267、285 / 268、285 / 269、285 / 270、285 / 271、285 / 272、285 / 273、285 / 274、285 / 275、285 / 276、285 / 277、285 / 278、285 / 279、285 / 280、285 / 281、285 / 282、285 / 283、285 / 284、285 / 285、285 / 286、285 / 287、285 / 288、286 / 267、286 / 268、286 / 269、286 / 270、286 / 271、286 / 272、286 / 273、286 / 274、286 / 275、286 / 276、286 / 277、286 / 278、286 / 279、286 / 280、286 / 281、286 / 282、286 / 283、286 / 284、286 / 285、286 / 286、286 / 287、286 / 288、287 / 267、287 / 268、287 / 269、287 / 270、287 / 271、287 / 272、287 / 273、287 / 274、287 / 275、287 / 276、287 / 277、287 / 278、287 / 279、287 / 280、287 / 281、287 / 282、287 / 283、287 / 284、287 / 285、287 / 286、287 / 287、287 / 288，117 / 118、117 / 119、117 / 120、117 / 121、117 / 122、117 / 123、118 / 118、118 / 119、118 / 120、118 / 121、118 / 122、118 / 123、119 / 118、119 / 119、119 / 120、119 / 121、119 / 122、119 / 123、120 / 118、120 / 119、120 / 120、120 / 121、120 / 122、120 / 123、121 / 118、121 / 119、121 / 120、121 / 121、121 / 122、121 / 123、122 / 118、122 / 119、122 / 120、122 / 121、122 / 122、122 / 123，340 / 341、340 / 342、340 / 343、340 / 344、340 / 345、340 / 346、340 / 347、340 / 348、340 / 349、340 / 350、340 / 351、340 / 352、340 / 353、341 / 341、341 / 342、341 / 343、341 / 344、341 / 345、341 / 346、341 / 347、341 / 348、341 / 349、341 / 350、341 / 351、341 / 352、341 / 353、342 / 341、342 / 342、342 / 343、342 / 344、342 / 345、342 / 346、342 / 347、342 / 348、342 / 349、342 / 350、342 / 351、342 / 352、342 / 353、343 / 341、343 / 342、343 / 343、343 / 344、343 / 345、343 / 346、343 / 347、343 / 348、343 / 349、343 / 350、343 / 351、343 / 352、343 / 353、344 / 341、344 / 342、344 / 343、344 / 344、344 / 345、344 / 346、344 / 347、344 / 348、344 / 349、344 / 350、344 / 351、344 / 352、344 / 353、345 / 341、345 / 342、345 / 343、345 / 344、345 / 345、345 / 346、345 / 347、345 / 348、345 / 349、345 / 350、345 / 351、345 / 352、345 / 353、346 / 341、346 / 342、346 / 343、346 / 344, 346 / 345, 346 / 346, 346 / 347, 346 / 348, 346 / 349, 346 / 350, 346 / 351, 346 / 352, 346 / 353, 347 / 341, 347 / 342, 347 / 343, 347 / 344, 347 / 345, 347 / 346, 347 / 347, 347 / 348, 347 / 349, 347 / 350, 347 / 351, 347 / 352, 347 / 353, 348 / 341, 348 / 342, 348 / 343, 348 / 344, 348 / 345, 348 / 346, 348 / 347, 348 / 348, 348 / 349, 348 / 350, 348 / 351, 348 / 352, 348 / 353, 349 / 341, 349 / 342, 349 / 343, 349 / 344, 349 / 345, 349 / 346, 349 / 347, 349 / 348, 3 49 / 349, 349 / 350, 349 / 351, 349 / 352, 349 / 353, 350 / 341, 350 / 342, 350 / 343, 350 / 344, 350 / 345, 350 / 346, 350 / 347, 350 / 348, 350 / 349, 350 / 350, 350 / 351, 350 / 352, 350 / 353, 351 / 341, 351 / 342, 351 / 343, 351 / 344, 3 Loci 51 / 345, 351 / 346, 351 / 347, 351 / 348, 351 / 349, 351 / 350, 351 / 351, 351 / 352, 351 / 353, 352 / 341, 352 / 342, 352 / 343, 352 / 344, 352 / 345, 352 / 346, 352 / 347, 352 / 348, 352 / 349, 352 / 350, 352 / 351, 352 / 352, and 352 / 353. Preferably, the mutations in the D-glucose-sensitive polypeptide include mutations selected from any of the following groups: (1) V347W and H348T, (2) V347W and H348G, (3) V347W and H348S, (4) V347W, H348T and H66R, (5) V347W, H348T and H66E, (6) V347W, H348T and H66A, (7) V347W, H348T and H66F, (8) V347W, H348T and H66M, (9) V347W, H348T and H66C, (10) V347W, H348T and H66P, (11) V347W, H348T and H66Q, (12) V347W, H348T and H66T, (13) V347W, H348T and W9R, (14) V347W,H348T and W9H, (15) V347W, H348T and W9E, (16) V347W, H348T and W9A, (17) V347W, H348T and W9V, (18) V347W, H348T and W9L, (19) V347W, H348T and W9F, (20) V347W, H348T and W9I, (21) V347W, H348T and W9M, (22) V347W, H348T and W9C, (23) V347W, H348T and W9P, (24) V347W, H348T and W9N, (25) V347W, H348T and W9G, (26) V347W, H348T And W9K, (27)V347W, H348T and W9Y, (28)V347W, H348T and W9S, (29)V347W, H348T and W9T, (30)V347W, H348T and W9D, (31)V347W, H348T and W8R, (32)V347W, H348T and W 8H, (33)V347W, H348T and W8E, (34)V347W, H348T and W8A, (35)V347W, H348T and W8V, (36)V347W, H348T and W8L, (37)V347W, H348T and W8F, (38)V347W, H348T and W8I, (39) V347W, H348T and W8M, (40) V347W, H348T and W8C, (41) V347W, H348T and W8N, (42) V347W, H348T and W8G, (43) V347W, H348T and W8K, (44) V347W, H348T and W8T, (45) V347W, H348T and W8D, (46) V347W, H348T and A42R, (47) V347W, H348T and A42E, (48) V347W, H348T and A42W, (49) V347W, H348T and A42F, (50) V347W, H348T and A42M, (51) V347W, H348T and A42C, (52) V347W, H348T and A42P, (53) V347W, H348T and A42N, (54) V347W, H348T and A42G, (55) V347W, H348T and A42K, (56) V347W, H348T and A42Y, (57) V347W, H348T and A42T, (58) V347W, H348T and K312R, (59) V347W, H348T and K312A, (60) V347W, H348T and K312Q, (61) V347W, H348T and K312G, (62) V347W,H348T and K312H, (63)V347W, H348T and K312S.

[0031] In one or more embodiments, the optical probe contains a D-glucose-sensitive polypeptide as shown in SEQ ID NO: 1, and an optically active polypeptide as shown in SEQ ID NO: 2, 6, 7, and 9. The optically active polypeptide is located at position 348 / 352 of the D-glucose-sensitive polypeptide, and the optical probe has the following mutations: (1) V347W and H348T of the D-glucose-sensitive polypeptide and 1A of the optically active polypeptide; (2) V347W and H348G of the D-glucose-sensitive polypeptide and 1G of the optically active polypeptide; (3) V347W and H348S of the D-glucose-sensitive polypeptide and 1A of the optically active polypeptide; (4) V347W and H348S of the D-glucose-sensitive polypeptide and 1G of the optically active polypeptide; (5) V347W and H348T of the D-glucose-sensitive polypeptide and the optically active polypeptide. (6) D-glucose-sensitive peptides V347W, H348T, H66R, and optically active peptide 1A, (7) D-glucose-sensitive peptides V347W, H348T, H66E, and optically active peptide 1A, (8) D-glucose-sensitive peptides V347W, H348T, H66A, and optically active peptide 1A, (9) D-glucose-sensitive peptides V347W, H348T, H66F, and optically active peptide 1A, (10) D-glucose-sensitive peptides V347W, H348T, H66M, and optically active peptide 1A, (11) D-glucose-sensitive peptides V347W, H348T (12) D-glucose-sensitive peptides V347W, H348T, H66P, and optically active peptide 1A; (13) D-glucose-sensitive peptides V347W, H348T, H66Q, and optically active peptide 1A; (14) D-glucose-sensitive peptides V347W, H348T, H66T, and optically active peptide 1A; (15) D-glucose-sensitive peptides V347W, H348T, W9R, and optically active peptide 1A; (16) D-glucose-sensitive peptides V347W, H348T, W9H, and optically active peptide 1A; (17) D-glucose-sensitive peptides V347W, H348T, W9H, and optically active peptide 1A; (18) D-glucose-sensitive peptides V347W, H348T, W9E, and optically active peptide 1A; (19) D-glucose-sensitive peptides V347W, H348T, W9V, and optically active peptide 1A; (20) D-glucose-sensitive peptides V347W, H348T, W9L, and optically active peptide 1A; (21) D-glucose-sensitive peptides V347W, H348T, W9F, and optically active peptide 1A; (22) D-glucose-sensitive peptides V347W, H348T, W9I, and optically active peptide 1A.(23) D-glucose-sensitive peptides V347W, H348T, W9M, and optically active peptide 1A; (24) D-glucose-sensitive peptides V347W, H348T, W9C, and optically active peptide 1A; (25) D-glucose-sensitive peptides V347W, H348T, W9P, and optically active peptide 1A; (26) D-glucose-sensitive peptides V347W, H348T, W9N, and optically active peptide 1A; (27) D-glucose-sensitive peptides V347W, H348T, W9G, and optically active peptide 1A; (28) D-glucose-sensitive peptides V347W, H348T, W9K, and optically active peptide 1A. (29) D-glucose-sensitive peptides V347W, H348T, W9Y, and optically active peptide 1A; (30) D-glucose-sensitive peptides V347W, H348T, W9S, and optically active peptide 1A; (31) D-glucose-sensitive peptides V347W, H348T, W9T, and optically active peptide 1A; (32) D-glucose-sensitive peptides V347W, H348T, W9D, and optically active peptide 1A; (33) D-glucose-sensitive peptides V347W, H348T, W8R, and optically active peptide 1A; (34) D-glucose-sensitive peptides V347W, H348T, W8H, and optically active peptide 1A. (35) D-glucose-sensitive peptides V347W, H348T, W8E, and optically active peptide 1A; (36) D-glucose-sensitive peptides V347W, H348T, W8A, and optically active peptide 1A; (37) D-glucose-sensitive peptides V347W, H348T, W8V, and optically active peptide 1A; (38) D-glucose-sensitive peptides V347W, H348T, W8L, and optically active peptide 1A; (39) D-glucose-sensitive peptides V347W, H348T, W8F, and optically active peptide 1A; (40) D-glucose-sensitive peptides V347W, H348T, W8I, and optically active peptide 1A. (41) D-glucose-sensitive peptides V347W, H348T, W8M, and optically active peptide 1A; (42) D-glucose-sensitive peptides V347W, H348T, W8C, and optically active peptide 1A; (43) D-glucose-sensitive peptides V347W, H348T, W8N, and optically active peptide 1A; (44) D-glucose-sensitive peptides V347W, H348T, W8G, and optically active peptide 1A; (45) D-glucose-sensitive peptides V347W, H348T, W8K, and optically active peptide 1A; (46) D-glucose-sensitive peptides V347W, H348T, W8T, and optically active peptide 1A.(47) D-glucose-sensitive peptides V347W, H348T, W8D, and optically active peptide 1A; (48) D-glucose-sensitive peptides V347W, H348T, A42R, and optically active peptide 1A; (49) D-glucose-sensitive peptides V347W, H348T, A42E, and optically active peptide 1A; (50) D-glucose-sensitive peptides V347W, H348T, A42W, and optically active peptide 1A; (51) D-glucose-sensitive peptides V347W, H348T, A42F, and optically active peptide 1A. (52) D-glucose-sensitive peptides V347W, H348T, A42M, and optically active peptides 1A, (53) D-glucose-sensitive peptides V347W, H348T, A42C, and optically active peptides 1A, (54) D-glucose-sensitive peptides V347W, H348T, A42P, and optically active peptides 1A, (55) D-glucose-sensitive peptides V347W, H348T, A42N, and optically active peptides 1A, (56) D-glucose-sensitive peptides V347W, H348T, A42N, and optically active peptides 1A, (57) D-glucose-sensitive peptides V347W, H348T, A42M, and optically active peptides 1A, (58) D-glucose-sensitive peptides V347W, H348T, A42M ... T, A42G, and optically active peptide 1A, (57) D-glucose-sensitive peptides V347W, H348T, A42K, and optically active peptide 1A, (58) D-glucose-sensitive peptides V347W, H348T, A42Y, and optically active peptide 1A, (59) D-glucose-sensitive peptides V347W, H348T, A42T, and optically active peptide 1A, (60) D-glucose-sensitive peptides V347W, H348T, K312R, and optically active peptide 1A, (61) D-glucose-sensitive peptides V347W, H348T, K312A, and optically active peptide 1A; (62) D-glucose-sensitive peptides V347W, H348T, K312Q, and optically active peptide 1A; (63) D-glucose-sensitive peptides V347W, H348T, K312G, and optically active peptide 1A; (64) D-glucose-sensitive peptides V347W, H348T, K312H, and optically active peptide 1A; (65) D-glucose-sensitive peptides V347W, H348T, K312S, and optically active peptide 1A.

[0032] In one or more embodiments, the optical probe is as shown in SEQ ID NO: 11.

[0033] Another aspect of the present invention provides a fusion polypeptide comprising the optical probe described herein and other polypeptides. In some embodiments, the other polypeptides are located at the N-terminus and / or C-terminus of the optical probe. In some embodiments, the other polypeptides include a localization sequence (e.g., a polypeptide that localizes the optical probe to a different organelle or sub-organelle), a tag for easy purification, or a tag for use in an immunoreaction (e.g., immunoblotting). A linker may be present between the optical probe and the other polypeptides in the fusion polypeptide described herein.

[0034] Another aspect of the present invention provides a nucleic acid molecule comprising: (a) a coding sequence of a polypeptide or probe as described in any embodiment herein, or (b) a complementary sequence to (a), or (c) a fragment of (a) or (b). The fragment is a primer.

[0035] The present invention also relates to variants of the aforementioned nucleic acid molecules, including fragments, analogs, derivatives, soluble fragments and variants of the present invention encoding the optical probes or fusion proteins of the present invention, or their complementary sequences.

[0036] In another aspect, the present invention also provides nucleic acid constructs comprising the nucleic acid molecules described herein. The nucleic acid sequence encodes the optical probe or fusion polypeptide described herein.

[0037] In one or more embodiments, the nucleic acid construct is a cloning vector, an expression vector, or a recombinant vector.

[0038] In one or more embodiments, the nucleic acid molecule is operatively linked to an expression control sequence.

[0039] In some implementations, the expression vector is selected from prokaryotic expression vectors, eukaryotic expression vectors, and viral vectors.

[0040] In another aspect, the present invention provides a host cell that: (1) expresses the optical probe or fusion polypeptide described in any embodiment of the present invention; (2) contains the nucleic acid molecule described in any embodiment of the present invention; or (3) contains the nucleic acid construct described in any embodiment of the present invention. The host cell is preferably *Escherichia coli*.

[0041] Another aspect of the present invention provides a D-glucose detection kit, comprising the optical probes described herein or fusion peptides or polynucleotides or optical probes prepared as described herein.

[0042] In one or more embodiments, the kit further comprises one or more reagents selected from the following: buffer, culture medium, D-glucose standard.

[0043] Another aspect of the present invention provides a method for preparing the optical probe described herein, comprising: providing a host cell expressing the optical probe or fusion polypeptide described herein, culturing the host cell under conditions of cell expression, and isolating the optical probe or fusion polypeptide.

[0044] In one or more embodiments, the method includes the following steps: 1) incorporating a nucleic acid molecule encoding the D-glucose optical probe described herein into an expression vector; 2) transferring the expression vector into a host cell; 3) culturing the host cell under conditions suitable for expression of the expression vector; and 4) isolating the D-glucose optical probe.

[0045] Another aspect of the present invention provides a method for detecting D-glucose in a sample, comprising: contacting the sample with the optical probe or fusion peptide or host cell described herein, and detecting changes in the optically active peptide. The detection can be performed in vivo, in vitro, subcellular, or in situ. The sample may be, for example, blood.

[0046] This article also provides a method for quantifying D-glucose in a sample, comprising: contacting the optical probe or fusion peptide or host cell described herein with the sample, detecting optical changes in the optically active peptide, and quantifying D-glucose in the sample based on the optical changes in the optically active peptide.

[0047] Another aspect of the present invention provides a method for screening compounds (e.g., drugs), comprising: contacting the optical probe or fusion peptide or host cell described herein with a candidate compound in a system containing D-glucose; detecting optical changes in the optically active peptide; and screening the compound based on the optical changes in the optically active peptide. The method can screen compounds in high throughput.

[0048] In one or more embodiments, the host cells described herein are contacted with the candidate compound in a system containing D-glucose, and optical changes in the optically active peptide indicate whether the candidate compound can regulate the uptake of D-glucose by the cells.

[0049] Another aspect of the present invention provides a method for intracellular and / or extracellular localization of the D-glucose, comprising: contacting a system containing D-glucose with the optical probe or the host cell, and detecting optical changes in the optically active peptide.

[0050] In one or more embodiments, the system is a solution system, a cellular system, or a subcellular system.

[0051] Another aspect of the present invention provides the use of the D-glucose optical probes, fusion peptides, or host cells described herein in the intracellular and / or extracellular detection of D-glucose in samples, screening compounds, or D-glucose. In one or more embodiments, the localization is real-time localization.

[0052] Another aspect of the present invention provides the use of the D-glucose optical probes or fusion peptides or polynucleotides or nucleic acid constructs or host cells described herein in the preparation of kits for detecting D-glucose in samples, screening compounds or intracellular and / or extracellular localization of D-glucose.

[0053] The beneficial effects of this invention are as follows: The D-glucose optical probe provided by this invention is easy to mature, exhibits large fluorescence dynamics, and has good specificity. Furthermore, it can be expressed in cells through gene manipulation, enabling real-time, high-throughput, and quantitative detection of D-glucose both inside and outside cells, eliminating time-consuming sample processing steps. Experimental results show that the D-glucose optical probe provided by this application achieves a response to D-glucose that is more than 10 times that of the control. It can also perform localization, qualitative, and quantitative detection of D-glucose in subcellular structures such as the cytoplasm, nucleus, cell membrane, and mitochondria, and allows for high-throughput compound screening and quantitative detection of D-glucose in blood. Attached Figure Description

[0054] Figure 1 shows an SDS-PAGE image of an exemplary D-glucose optical probe;

[0055] Figure 2 shows the fluorescence spectral properties of an exemplary D-glucose optical probe;

[0056] Figure 3 shows the titration curves of different concentrations of D-glucose using an exemplary D-glucose optical probe;

[0057] Figure 4 is a bar chart showing the specificity of an exemplary D-glucose optical probe for another substrate and sugar metabolism intermediates and similar substances;

[0058] Figure 5 shows a photograph of the subcellular organelle localization of an exemplary D-glucose optical probe in mammalian cells;

[0059] Figure 6 is a schematic diagram of the dynamic monitoring of D-glucose concentration in the cytoplasm of mammalian cells using an exemplary D-glucose optical probe.

[0060] Figure 7 is a dot plot of an exemplary D-glucose optical probe used for high-throughput compound screening at the live cell level;

[0061] Figure 8 is a bar chart showing the quantification of D-glucose in mouse and human blood using an exemplary D-glucose optical probe. Detailed Implementation

[0062] When a value or range is given, the term “about” as used herein means that the value or range is within 20%, 10%, and 5% of the given value or range.

[0063] The terms “comprising,” “including,” and their equivalents as used herein include the meanings of “containing” and “composed of,” for example, a composition “comprising” X may consist of only X or may contain other substances, such as X+Y.

[0064] As used herein, the term "D-glucose-sensitive peptide" refers to a peptide that responds to D-glucose, and the response includes any response to chemical, biological, electrical, or physiological parameters of the peptide in relation to the interaction with the sensitive peptide. Responses include small changes, such as changes in the orientation of amino acids or peptide fragments of the peptide, and changes in the primary, secondary, or tertiary structure of the peptide, including, for example, changes in protonation, electrochemical potential, and / or conformation. "Conformation" is the three-dimensional arrangement of the primary, secondary, and tertiary structures of a molecule containing side groups; a conformational change occurs when the three-dimensional structure of the molecule changes. Examples of conformational changes include a change from an α-helix to a β-sheet or vice versa. It is understood that a detectable change need not be a conformational change, as long as the fluorescence of the fluorescent protein moiety is altered. The D-glucose-sensitive peptides described herein may also include their functional variants. Functional variants of D-glucose-sensitive peptides include, but are not limited to, variants that can interact with D-glucose to undergo the same or similar changes as the parental D-glucose-sensitive peptide.

[0065] The D-glucose-sensitive polypeptides described in this invention include, but are not limited to, the glucose-galactose-binding protein TtGBP derived from the thermophilic bacterium *T. thermophilus*, or variants thereof with more than 90% homology. The D-glucose-binding protein can sense changes in D-glucose concentration, and its spatial conformation also changes during dynamic changes in D-glucose concentration.

[0066] As used herein, the term "optical probe" refers to a D-glucose-sensitive peptide fused to an optically active peptide. The inventors discovered that conformational changes resulting from the specific binding of D-glucose-sensitive peptides, such as D-glucose-binding proteins, to physiological concentrations of D-glucose induce conformational changes in optically active peptides (e.g., fluorescent proteins), thereby altering the optical properties of the optically active peptides. By plotting standard curves using fluorescence data of fluorescent proteins measured at different D-glucose concentrations, the presence and / or level of D-glucose can be detected and analyzed. When describing the optical probes of this invention (e.g., when describing insertion or mutation sites), all amino acid residue numbers are referenced to SEQ ID NO: 1.

[0067] In the optical probes of the present invention, an optically active polypeptide (e.g., a fluorescent protein) is operatively inserted into a D-glucose-sensitive polypeptide. A protein-based "optically active polypeptide" is a polypeptide capable of emitting fluorescence. Fluorescence is an optical property of the optically active polypeptide and can be used as a means of detecting the responsiveness of the optical probes of the present invention. As used herein, the term "fluorescence property" refers to the molar extinction coefficient at an appropriate excitation wavelength, fluorescence quantum efficiency, shape of the excitation or emission spectrum, maximum excitation wavelength and maximum emission wavelength, amplitude of excitation at two different wavelengths, ratio of emission amplitudes at two different wavelengths, excited-state lifetime, or fluorescence anisotropy. A measurable difference in any of these properties between active and inactive states is sufficient for the utility of the fluorescent protein substrate of the present invention in activity assays. The measurable difference can be determined by determining the amount of any quantitative fluorescence property, for example, the amount of fluorescence at a specific wavelength or the integral of fluorescence over the emission spectrum. Preferably, the protein substrate is selected to have fluorescence properties that are easily distinguishable between inactive and activated conformational states. The optically active polypeptides described herein may also include their functional variants. Functional variants of optically active peptides include, but are not limited to, variants that can undergo the same or similar fluorescence property changes as the parent optically active peptide.

[0068] As used herein, the term "fluorescent protein" refers to a protein that emits fluorescence under excitation light. Fluorescent proteins are fundamental detection methods in the field of bioscience. Examples include the commonly used green fluorescent protein GFP and its cyclically rearranged derivatives such as blue fluorescent protein (cpBFP), green fluorescent protein (cpGFP), and yellow fluorescent protein (cpYFP); and the commonly used red fluorescent protein RFP, and its cyclically rearranged derivatives such as cpmApple, cpmOrange, and cpmKate. For example, cpYFP is shown in SEQ ID NO: 2, cpGFP in SEQ ID NO: 6, cpBFP in SEQ ID NO: 7, and cpmApple in SEQ ID NO: 9.

[0069] The fluorescent protein in the optical probe also includes functional variants with mutations, including but not limited to fluorescent proteins with a mutation at the first amino acid corresponding to SEQ ID NO: 2. The mutation at position 1 is preferably A or G. In some embodiments, the functional variant of the fluorescent protein has the sequence shown in any of SEQ ID NO: 2, 6, 7, 9 and has a mutation selected from any of the following groups at the first amino acid corresponding to SEQ ID NO: 2: Y1A or Y1G. 1S indicates that the first amino acid is mutated to S, and so on.

[0070] In the optical probe of the present invention, the optically active polypeptides are located in the NC direction along the residues 266 / 267, 266 / 268, 266 / 269, 266 / 270, 266 / 271, 266 / 272, 266 / 273, 266 / 274, 266 / 275, 266 / 276, 266 / 277, 266 / 278, 266 / 279, 266 / 280, 266 / 281, 266 / 282, 266 / 283, 266 / 284, 266 / 285, 266 / 286, 266 / 287, 266 / 288, 267 / 267, 267 / 268, 267 / 269, and 267 / 270 of the D-glucose-sensitive polypeptide. 267 / 271, 267 / 272, 267 / 273, 267 / 274, 267 / 275, 267 / 276, 267 / 277, 267 / 278, 267 / 279, 267 / 280, 267 / 281, 267 / 282, 267 / 283, 267 / 284, 267 / 285, 267 / 286, 267 / 287, 267 / 288, 268 / 267, 268 / 268, 268 / 269, 268 / 270, 268 / 271, 268 / 272, 268 / 273, 268 / 274, 268 / 275, 268 / 276, 268 / 277, 268 / 278, 268 / 279 268 / 280, 268 / 281, 268 / 282, 268 / 283, 268 / 284, 268 / 285, 268 / 286, 268 / 287, 268 / 288, 269 / 267, 269 / 268, 269 / 269, 269 / 270, 269 / 271, 269 / 272, 269 / 273, 269 / 274, 269 / 275, 269 / 276, 269 / 277, 269 / 278, 269 / 279, 269 / 280, 269 / 281, 269 / 282, 269 / 283, 269 / 284, 269 / 285, 269 / 286, 269 / 287, 269 / 288 270 / 267, 270 / 268, 270 / 269, 270 / 270, 270 / 271, 270 / 272, 270 / 273, 270 / 274, 270 / 275, 270 / 276, 270 / 277, 270 / 278, 270 / 279, 270 / 280, 270 / 281, 270 / 282, 270 / 283, 270 / 284, 270 / 285, 270 / 286, 270 / 287, 270 / 288, 271 / 267, 271 / 268, 271 / 269, 271 / 270, 271 / 271, 271 / 272, 271 / 273, 271 / 274, 271 / 275,271 / 276、271 / 277、271 / 278、271 / 279、271 / 280、271 / 281、271 / 282、271 / 283、271 / 284、271 / 285、271 / 286、271 / 287、271 / 288、272 / 267、272 / 268、272 / 269、272 / 270、272 / 271、272 / 272、272 / 273、272 / 274、272 / 275、272 / 276、272 / 277、272 / 278、272 / 279、272 / 280、272 / 281、272 / 282、272 / 283、272 / 284、272 / 285、272 / 286、272 / 287、272 / 288、273 / 267、273 / 268、273 / 269、273 / 270、273 / 271、273 / 272、273 / 273、273 / 274、273 / 275、273 / 276、273 / 277、273 / 278、273 / 279、273 / 280、273 / 281、273 / 282、273 / 283、273 / 284、273 / 285、273 / 286、273 / 287、273 / 288、274 / 267、274 / 268、274 / 269、274 / 270、274 / 271、274 / 272、274 / 273、274 / 274、274 / 275、274 / 276、274 / 277、274 / 278、274 / 279、274 / 280、274 / 281、274 / 282、274 / 283、274 / 284、274 / 285、274 / 286、274 / 287、274 / 288、275 / 267、275 / 268、275 / 269、275 / 270、275 / 271、275 / 272、275 / 273、275 / 274、275 / 275、275 / 276、275 / 277、275 / 278、275 / 279、275 / 280、275 / 281、275 / 282、275 / 283、275 / 284、275 / 285、275 / 286、275 / 287、275 / 288、276 / 267、276 / 268、276 / 269、276 / 270、276 / 271、276 / 272、276 / 273、276 / 274、276 / 275、276 / 276、276 / 277、276 / 278、276 / 279、276 / 280、276 / 281、276 / 282、276 / 283、276 / 284、276 / 285、276 / 286、276 / 287、276 / 288、277 / 267、277 / 268、277 / 269、277 / 270、277 / 271、277 / 272、277 / 273、277 / 274、277 / 275、277 / 276、277 / 277、277 / 278、277 / 279、277 / 280、277 / 281、277 / 282、277 / 283、277 / 284、277 / 285、277 / 286、277 / 287、277 / 288、278 / 267、278 / 268、278 / 269、278 / 270、278 / 271、278 / 272、278 / 273、278 / 274、278 / 275、278 / 276、278 / 277、278 / 278、278 / 279、278 / 280、278 / 281、278 / 282、278 / 283、278 / 284、278 / 285、278 / 286、278 / 287、278 / 288、279 / 267、279 / 268、279 / 269、279 / 270、279 / 271、279 / 272、279 / 273、279 / 274、279 / 275、279 / 276、279 / 277、279 / 278、279 / 279、279 / 280、279 / 281、279 / 282、279 / 283、279 / 284、279 / 285、279 / 286、279 / 287、279 / 288、280 / 267、280 / 268、280 / 269、280 / 270、280 / 271、280 / 272、280 / 273、280 / 274、280 / 275、280 / 276、280 / 277、280 / 278、280 / 279、280 / 280、280 / 281、280 / 282、280 / 283、280 / 284、280 / 285、280 / 286、280 / 287、280 / 288、281 / 267、281 / 268、281 / 269、281 / 270、281 / 271、281 / 272、281 / 273、281 / 274、281 / 275、281 / 276、281 / 277、281 / 278、281 / 279、281 / 280、281 / 281、281 / 282、281 / 283、281 / 284、281 / 285、281 / 286、281 / 287、281 / 288、282 / 267、282 / 268、282 / 269、282 / 270、282 / 271、282 / 272、282 / 273、282 / 274、282 / 275、282 / 276、282 / 277、282 / 278、282 / 279、282 / 280、282 / 281、282 / 282、282 / 283、282 / 284、282 / 285、282 / 286、282 / 287、282 / 288、283 / 267、283 / 268、283 / 269、283 / 270、283 / 271、283 / 272、283 / 273、283 / 274、283 / 275、283 / 276、283 / 277、283 / 278、283 / 279、283 / 280、283 / 281、283 / 282、283 / 283、283 / 284、283 / 285、283 / 286、283 / 287、283 / 288、284 / 267、284 / 268、284 / 269、284 / 270、284 / 271、284 / 272、284 / 273、284 / 274、284 / 275、284 / 276、284 / 277、284 / 278、284 / 279、284 / 280、284 / 281、284 / 282、284 / 283、284 / 284、284 / 285、284 / 286、284 / 287、284 / 288、285 / 267、285 / 268、285 / 269、285 / 270、285 / 271、285 / 272、285 / 273、285 / 274、285 / 275、285 / 276、285 / 277、285 / 278、285 / 279、285 / 280、285 / 281、285 / 282、285 / 283、285 / 284、285 / 285、285 / 286、285 / 287、285 / 288、286 / 267、286 / 268、286 / 269、286 / 270、286 / 271、286 / 272、286 / 273、286 / 274、286 / 275、286 / 276、286 / 277、286 / 278、286 / 279、286 / 280、286 / 281、286 / 282、286 / 283、286 / 284、286 / 285、286 / 286、286 / 287、286 / 288、287 / 267、287 / 268、287 / 269、287 / 270、287 / 271、287 / 272、287 / 273、287 / 274、287 / 275、287 / 276、287 / 277、287 / 278、287 / 279、287 / 280、287 / 281、287 / 282、287 / 283、287 / 284、287 / 285、287 / 286、287 / 287、287 / 288，117 / 118、117 / 119、117 / 120、117 / 121、117 / 122、117 / 123、118 / 118、118 / 119、118 / 120、118 / 121、118 / 122、118 / 123、119 / 118、119 / 119、119 / 120、119 / 121、119 / 122、119 / 123、120 / 118、120 / 119、120 / 120、120 / 121、120 / 122、120 / 123、121 / 118、121 / 119、121 / 120、121 / 121、121 / 122、121 / 123、122 / 118、122 / 119、122 / 120、122 / 121、122 / 122、122 / 123，340 / 341、340 / 342、340 / 343、340 / 344、340 / 345、340 / 346、340 / 347、340 / 348、340 / 349、340 / 350、340 / 351、340 / 352、340 / 353、341 / 341、341 / 342、341 / 343、341 / 344、341 / 345、341 / 346、341 / 347、341 / 348、341 / 349、341 / 350、341 / 351、341 / 352、341 / 353、342 / 341、342 / 342、342 / 343、342 / 344、342 / 345、342 / 346、342 / 347、342 / 348、342 / 349、342 / 350、342 / 351、342 / 352、342 / 353、343 / 341、343 / 342、343 / 343、343 / 344、343 / 345、343 / 346、343 / 347、343 / 348、343 / 349、343 / 350、343 / 351、343 / 352、343 / 353、344 / 341、344 / 342、344 / 343、344 / 344、344 / 345、344 / 346、344 / 347、344 / 348、344 / 349、344 / 350、344 / 351、344 / 352、344 / 353、345 / 341、345 / 342、345 / 343、345 / 344、345 / 345、345 / 346、345 / 347、345 / 348、345 / 349、345 / 350、345 / 351、345 / 352、345 / 353、346 / 341、346 / 342、346 / 343、346 / 344、346 / 345、346 / 346、346 / 347、346 / 348、346 / 349、346 / 350、346 / 351、346 / 352、346 / 353、347 / 341、347 / 342、347 / 343、347 / 344、347 / 345、347 / 346、347 / 347、347 / 348、347 / 349、347 / 350、347 / 351、347 / 352、347 / 353、348 / 341、348 / 342、348 / 343、348 / 344、348 / 345、348 / 346、348 / 347、348 / 348、348 / 349、348 / 350、348 / 351、348 / 352、348 / 353、349 / 341、349 / 342、349 / 343、349 / 344、349 / 345、349 / 346、349 / 347、349 / 348、349 / 349、349 / 350、349 / 351、349 / 352、349 / 353、350 / 341、350 / 342、350 / 343、350 / 344、350 / 345、350 / 346、350 / 347、350 / 348、350 / 349、350 / 350、350 / 351、350 / 352、350 / 353、351 / 341、351 / 342、351 / 343、351 / 344、351 / 345、351 / 346、351 / 347、351 / 348、351 / 349、351 / 350、351 / 351、351 / 352、351 / 353、352 / 341、352 / 342、352 / 343、352 / 344、352 / 345、352 / 346、352 / 347、352 / 348、352 / 349、352 / 350、352 / 351、Residues in or substituted for 352 / 352 and 352 / 353 are numbered corresponding to the full length of the D-glucose-sensitive polypeptide. In this paper, at sites represented in the form of "X / Y", the optically active polypeptide has portions of the D-glucose-sensitive polypeptide at both ends, wherein the N-terminus of the optically active polypeptide is the N-terminal starting amino acid (e.g., any amino acid from position 1 to 266) to the Xth amino acid of the D-glucose-sensitive polypeptide sequence, and the C-terminus of the optically active polypeptide is the Yth amino acid to its C-terminal ending amino acid (e.g., any amino acid from position Y to 394) of the D-glucose-sensitive polypeptide sequence. In this context, if the two numbers in the "X / Y" format are consecutive integers, it indicates that the optically active polypeptide is located between the amino acids represented by those numbers. For example, insertion site 266 / 267 indicates that the optically active polypeptide is located between amino acids 266 and 267 of the D-glucose-sensitive polypeptide. If the two numbers in the "X / Y" format are not consecutive integers and X is less than Y, it indicates that the optically active polypeptide replaces the amino acids between those numbers. For example, insertion site 266 / 269 indicates that the optically active polypeptide replaces amino acids 267-268 of the D-glucose-sensitive polypeptide. If X is greater than Y in the "X / Y" format, it indicates that the D-glucose-sensitive polypeptide portion located at the N-terminus of the optically active polypeptide terminates at the Xth amino acid in the D-glucose-sensitive polypeptide sequence, while the D-glucose-sensitive polypeptide portion located at the C-terminus of the optically active polypeptide terminates at the Xth amino acid in the D-glucose-sensitive polypeptide sequence. Starting at amino acid position Y; for example, insertion site 346 / 344 indicates that the N-terminus of the optically active polypeptide is fused with the N-terminal starting amino acid (e.g., any amino acid from position 1 to 394) to amino acid 346 of the D-glucose-sensitive polypeptide sequence, and the C-terminus of the optically active polypeptide is fused with the amino acid from position 344 to the C-terminal ending amino acid (e.g., amino acid 394) of the D-glucose-sensitive polypeptide sequence, with an exemplary structure of: (amino acids 1 to 346 of the D-glucose-sensitive polypeptide sequence) - (optically active polypeptide) - (amino acids 344 to 394 of the D-glucose-sensitive polypeptide sequence); if the two numbers in the site represented in the form of "X / Y" are not consecutive integers and X equals Y, it indicates that the optically active polypeptide is inserted between the amino acids indicated by that number, for example, insertion site 267 / 267 indicates that the optically active polypeptide is inserted between amino acids 267 and 268 of the inositol-sensitive polypeptide. In an exemplary embodiment, the optically active polypeptide represented by SEQ ID NO: 2, 6, 7, or 9 is located at any one or more of the following sites of the D-glucose-sensitive polypeptide represented by SEQ ID NO: 1: 266 / 267, 266 / 268, 266 / 269, 266 / 270, 266 / 271, 266 / 272, 266 / 273, 266 / 274, 266 / 275, 266 / 276, 266 / 277, 266 / 278.266 / 279、266 / 280、266 / 281、266 / 282、266 / 283、266 / 284、266 / 285、266 / 286、266 / 287、266 / 288、267 / 267、267 / 268、267 / 269、267 / 270、267 / 271、267 / 272、267 / 273、267 / 274、267 / 275、267 / 276、267 / 277、267 / 278、267 / 279、267 / 280、267 / 281、267 / 282、267 / 283、267 / 284、267 / 285、267 / 286、267 / 287、267 / 288、268 / 267、268 / 268、268 / 269、268 / 270、268 / 271、268 / 272、268 / 273、268 / 274、268 / 275、268 / 276、268 / 277、268 / 278、268 / 279、268 / 280、268 / 281、268 / 282、268 / 283、268 / 284、268 / 285、268 / 286、268 / 287、268 / 288、269 / 267、269 / 268、269 / 269、269 / 270、269 / 271、269 / 272、269 / 273、269 / 274、269 / 275、269 / 276、269 / 277、269 / 278、269 / 279、269 / 280、269 / 281、269 / 282、269 / 283、269 / 284、269 / 285、269 / 286、269 / 287、269 / 288、270 / 267、270 / 268、270 / 269、270 / 270、270 / 271、270 / 272、270 / 273、270 / 274、270 / 275、270 / 276、270 / 277、270 / 278、270 / 279、270 / 280、270 / 281、270 / 282、270 / 283、270 / 284、270 / 285、270 / 286、270 / 287、270 / 288、271 / 267、271 / 268、271 / 269、271 / 270、271 / 271、271 / 272、271 / 273、271 / 274、271 / 275、271 / 276、271 / 277、271 / 278、271 / 279、271 / 280、271 / 281、271 / 282、271 / 283、271 / 284、271 / 285、271 / 286、271 / 287、271 / 288、272 / 267、272 / 268、272 / 269、272 / 270、272 / 271、272 / 272、272 / 273、272 / 274、272 / 275、272 / 276、272 / 277、272 / 278、272 / 279、272 / 280、272 / 281、272 / 282、272 / 283、272 / 284、272 / 285、272 / 286、272 / 287、272 / 288、273 / 267、273 / 268、273 / 269、273 / 270、273 / 271、273 / 272、273 / 273、273 / 274、273 / 275、273 / 276、273 / 277、273 / 278、273 / 279、273 / 280、273 / 281、273 / 282、273 / 283、273 / 284、273 / 285、273 / 286、273 / 287、273 / 288、274 / 267、274 / 268、274 / 269、274 / 270、274 / 271、274 / 272、274 / 273、274 / 274、274 / 275、274 / 276、274 / 277、274 / 278、274 / 279、274 / 280、274 / 281、274 / 282、274 / 283、274 / 284、274 / 285、274 / 286、274 / 287、274 / 288、275 / 267、275 / 268、275 / 269、275 / 270、275 / 271、275 / 272、275 / 273、275 / 274、275 / 275、275 / 276、275 / 277、275 / 278、275 / 279、275 / 280、275 / 281、275 / 282、275 / 283、275 / 284、275 / 285、275 / 286、275 / 287、275 / 288、276 / 267、276 / 268、276 / 269、276 / 270、276 / 271、276 / 272、276 / 273、276 / 274、276 / 275、276 / 276、276 / 277、276 / 278、276 / 279、276 / 280、276 / 281、276 / 282、276 / 283、276 / 284、276 / 285、276 / 286、276 / 287、276 / 288、277 / 267、277 / 268、277 / 269、277 / 270、277 / 271、277 / 272、277 / 273、277 / 274、277 / 275、277 / 276、277 / 277、277 / 278、277 / 279、277 / 280、277 / 281、277 / 282、277 / 283、277 / 284、277 / 285、277 / 286、277 / 287、277 / 288、278 / 267、278 / 268、278 / 269、278 / 270、278 / 271、278 / 272、278 / 273、278 / 274、278 / 275、278 / 276、278 / 277、278 / 278、278 / 279、278 / 280、278 / 281、278 / 282、278 / 283、278 / 284、278 / 285、278 / 286、278 / 287、278 / 288、279 / 267、279 / 268、279 / 269、279 / 270、279 / 271、279 / 272、279 / 273、279 / 274、279 / 275、279 / 276、279 / 277、279 / 278、279 / 279、279 / 280、279 / 281、279 / 282、279 / 283、279 / 284、279 / 285、279 / 286、279 / 287、279 / 288、280 / 267、280 / 268、280 / 269、280 / 270、280 / 271、280 / 272、280 / 273、280 / 274、280 / 275、280 / 276、280 / 277、280 / 278、280 / 279、280 / 280、280 / 281、280 / 282、280 / 283、280 / 284、280 / 285、280 / 286、280 / 287、280 / 288、281 / 267、281 / 268、281 / 269、281 / 270、281 / 271、281 / 272、281 / 273、281 / 274、281 / 275、281 / 276、281 / 277、281 / 278、281 / 279、281 / 280、281 / 281、281 / 282、281 / 283、281 / 284、281 / 285、281 / 286、281 / 287、281 / 288、282 / 267、282 / 268、282 / 269、282 / 270、282 / 271、282 / 272、282 / 273、282 / 274、282 / 275、282 / 276、282 / 277、282 / 278、282 / 279、282 / 280、282 / 281、282 / 282、282 / 283、282 / 284、282 / 285、282 / 286、282 / 287、282 / 288、283 / 267、283 / 268、283 / 269、283 / 270、283 / 271、283 / 272、283 / 273、283 / 274、283 / 275、283 / 276、283 / 277、283 / 278、283 / 279、283 / 280、283 / 281、283 / 282、283 / 283、283 / 284、283 / 285、283 / 286、283 / 287、283 / 288、284 / 267、284 / 268、284 / 269、284 / 270、284 / 271、284 / 272、284 / 273、284 / 274、284 / 275、284 / 276、284 / 277、284 / 278、284 / 279、284 / 280、284 / 281、284 / 282、284 / 283、284 / 284、284 / 285、284 / 286、284 / 287、284 / 288、285 / 267、285 / 268、285 / 269、285 / 270、285 / 271、285 / 272、285 / 273、285 / 274、285 / 275、285 / 276、285 / 277、285 / 278、285 / 279、285 / 280、285 / 281、285 / 282、285 / 283、285 / 284、285 / 285、285 / 286、285 / 287、285 / 288、286 / 267、286 / 268、286 / 269、286 / 270、286 / 271、286 / 272、286 / 273、286 / 274、286 / 275、286 / 276、286 / 277、286 / 278、286 / 279、286 / 280、286 / 281、286 / 282、286 / 283、286 / 284、286 / 285、286 / 286、286 / 287、286 / 288、287 / 267、287 / 268、287 / 269、287 / 270、287 / 271、287 / 272、287 / 273、287 / 274、287 / 275、287 / 276、287 / 277、287 / 278、287 / 279、287 / 280、287 / 281、287 / 282、287 / 283、287 / 284、287 / 285、287 / 286、287 / 287、287 / 288，117 / 118、117 / 119、117 / 120、117 / 121、117 / 122、117 / 123、118 / 118、118 / 119、118 / 120、118 / 121、118 / 122、118 / 123、119 / 118、119 / 119、119 / 120、119 / 121、119 / 122、119 / 123、120 / 118、120 / 119、120 / 120、120 / 121、120 / 122、120 / 123、121 / 118、121 / 119、121 / 120、121 / 121、121 / 122、121 / 123、122 / 118、122 / 119、122 / 120、122 / 121、122 / 122、122 / 123，340 / 341、340 / 342、340 / 343、340 / 344、340 / 345、340 / 346、340 / 347、340 / 348、340 / 349、340 / 350、340 / 351、340 / 352、340 / 353、341 / 341、341 / 342、341 / 343、341 / 344、341 / 345、341 / 346、341 / 347、341 / 348、341 / 349、341 / 350、341 / 351、341 / 352、341 / 353、342 / 341、342 / 342、342 / 343、342 / 344、342 / 345、342 / 346、342 / 347、342 / 348、342 / 349、342 / 350、342 / 351、342 / 352、342 / 353、343 / 341、343 / 342、343 / 343、343 / 344、343 / 345、343 / 346、343 / 347、343 / 348、343 / 349、343 / 350、343 / 351、343 / 352、343 / 353、344 / 341、344 / 342、344 / 343、344 / 344、344 / 345、344 / 346、344 / 347、344 / 348、344 / 349、344 / 350、344 / 351、344 / 352、344 / 353、345 / 341、345 / 342、345 / 343、345 / 344、345 / 345、345 / 346、345 / 347、345 / 348、345 / 349、345 / 350、345 / 351、345 / 352、345 / 353、346 / 341、346 / 342、346 / 343、346 / 344、346 / 345、346 / 346、346 / 347、346 / 348、346 / 349、346 / 350、346 / 351、346 / 352、346 / 353、347 / 341、347 / 342、347 / 343、347 / 344、347 / 345、347 / 346、347 / 347、347 / 348、347 / 349、347 / 350、347 / 351、347 / 352、347 / 353、348 / 341、348 / 342、348 / 343、348 / 344、348 / 345、348 / 346、348 / 347、348 / 348、348 / 349、348 / 350、348 / 351、348 / 352、348 / 353、349 / 341, 349 / 342, 349 / 343, 349 / 344, 349 / 345, 349 / 346, 349 / 347, 349 / 348, 349 / 349, 349 / 350, 349 / 351, 349 / 352, 349 / 353, 350 / 341, 350 / 342, 350 / 343, 350 / 344, 350 / 345, 350 / 346, 350 / 347, 350 / 348, 350 / 349, 350 / 350, 350 / 351, 350 / 352, 350 / 353 351 / 341, 351 / 342, 351 / 343, 351 / 344, 351 / 345, 351 / 346, 351 / 347, 351 / 348, 351 / 349, 351 / 350, 351 / 351, 351 / 352, 351 / 353, 352 / 341, 352 / 342, 352 / 343, 352 / 344, 352 / 345, 352 / 346, 352 / 347, 352 / 348, 352 / 349, 352 / 350, 352 / 351, 352 / 352, and 352 / 353.

[0071] In one or more embodiments, the optical probe comprises, from the N-terminus to the C-terminus, residues 1-X of SEQ ID NO: 1, an optically active polypeptide or a variant thereof shown in any one of SEQ ID NO: 2, 6, 7, 9, and residues Y-394 of SEQ ID NO: 1, wherein X and Y are selected from any one of the following groups:

[0072] (1) X is 266, Y is 267, (2) X is 266, Y is 268, (3) X is 266, Y is 269, (4) X is 266, Y is 270, (5) X is 266, Y is 271, (6) X is 266, Y is 272, (7) X is 266, Y is 273, (8) X is 266, Y is 274, (9) X X is 266, Y is 275, (10) X is 266, Y is 276, (11) X is 266, Y is 277, (12) X is 266, Y is 278, (13) X is 266, Y is 279, (14) X is 266, Y is 280, (15) X is 266, Y is 281, (16) X is 266, Y is 282, (1 7) X is 266, Y is 283, (18) X is 266, Y is 284, (19) X is 266, Y is 285, (20) X is 266, Y is 286, (21) X is 266, Y is 287, (22) X is 266, Y is 288, (23) X is 267, Y is 267, (24) X is 267, Y is 268 (25) X is 267, Y is 269; (26) X is 267, Y is 270; (27) X is 267, Y is 271; (28) X is 267, Y is 272; (29) X is 267, Y is 273; (30) X is 267, Y is 274; (31) X is 267, Y is 275; (32) X is 267, Y is... 276, (33) X is 267, Y is 277, (34) X is 267, Y is 278, (35) X is 267, Y is 279, (36) X is 267, Y is 280, (37) X is 267, Y is 281, (38) X is 267, Y is 282, (39) X is 267, Y is 283, (40) X is 267 Y is 284, (41) X is 267, Y is 285, (42) X is 267, Y is 286, (43) X is 267, Y is 287, (44) X is 267, Y is 288, (45) X is 268, Y is 267, (46) X is 268, Y is 268, (47) X is 268, Y is 269, (48) X is 268, Y is 270, (49) X is 268, Y is 271, (50) X is 268, Y is 272, (51) X is 268, Y is 273, (52) X is 268, Y is 274, (53) X is 268, Y is 275, (54) X is 268, Y is 276, (55) X is 268, Y is 277, (56) (57) X is 268, Y is 278, (58) X is 268, Y is 279, (59) X is 268, Y is 280, (60) X is 268, Y is 281, (61) X is 268, Y is 282, (62) X is 268, Y is 283, (63) X is 268, Y is 284, (64) X is 268, Y is 285.(64) X is 268, Y is 286. (65) X is 268, Y is 287. (66) X is 268, Y is 288. (67) X is 269, Y is 267. (68) X is 269, Y is 268. (69) X is 269, Y is 269. (70) X is 269, Y is 270. (71) X is 269, Y is 270. 71, (72) X is 269, Y is 272, (73) X is 269, Y is 273, (74) X is 269, Y is 274, (75) X is 269, Y is 275, (76) X is 269, Y is 276, (77) X is 269, Y is 277, (78) X is 269, Y is 278, (79) X is 269, Y is 279, (80) X is 269, Y is 280, (81) X is 269, Y is 281, (82) X is 269, Y is 282, (83) X is 269, Y is 283, (84) X is 269, Y is 284, (85) X is 269, Y is 285, (86) X is 269, Y is 286, (87) X is 2 69, Y is 287, (88) X is 269, Y is 288, (89) X is 270, Y is 267, (90) X is 270, Y is 268, (91) X is 270, Y is 269, (92) X is 270, Y is 270, (93) X is 270, Y is 271, (94) X is 270, Y is 272, (95) X is 270, Y is 273, (96) X is 270, Y is 274, (97) X is 270, Y is 275, (98) X is 270, Y is 276, (99) X is 270, Y is 277, (100) X is 270, Y is 278, (101) X is 270, Y is 279, (102) X is 270, Y is 28 0, (103) X is 270, Y is 281, (104) X is 270, Y is 282, (105) X is 270, Y is 283, (106) X is 270, Y is 284, (107) X is 270, Y is 285, (108) X is 270, Y is 286, (109) X is 270, Y is 287, (110) (111) X is 270, Y is 288, (112) X is 271, Y is 267, (113) X is 271, Y is 268, (114) X is 271, Y is 269, (115) X is 271, Y is 270, (116) X is 271, Y is 272, (117) X is 271 Y is 273, (118) X is 271, Y is 274, (119) X is 271, Y is 275, (120) X is 271, Y is 276, (121) X is 271, Y is 277, (122) X is 271, Y is 278, (123) X is 271, Y is 279, (124) X is 271, Y is 280(125) X is 271, Y is 281, (126) X is 271, Y is 282, (127) X is 271, Y is 283, (128) X is 271, Y is 284, (129) X is 271, Y is 285, (130) X is 271, Y is 286, (131) X is 271, Y is 287, (132) X is 271, Y is 288, (133) X is 272, Y is 267, (134) X is 272, Y is 268, (135) X is 272, Y is 269, (136) X is 272, Y is 270, (137) X is 272, Y is 271, (138) X is 272, Y is 272, (139) X is 272 Y is 273, (140) X is 272, Y is 274, (141) X is 272, Y is 275, (142) X is 272, Y is 276, (143) X is 272, Y is 277, (144) X is 272, Y is 278, (145) X is 272, Y is 279, (146) X is 272, Y is 280 (147) X is 272, Y is 281; (148) X is 272, Y is 282; (149) X is 272, Y is 283; (150) X is 272, Y is 284; (151) X is 272, Y is 285; (152) X is 272, Y is 286; (153) X is 272, Y is 287; (154) X is 272, Y is 288, (155) X is 273, Y is 267, (156) X is 273, Y is 268, (157) X is 273, Y is 269, (158) X is 273, Y is 270, (159) X is 273, Y is 271, (160) X is 273, Y is 272, (161) X is 273 Y is 273, (162) X is 273, Y is 274, (163) X is 273, Y is 275, (164) X is 273, Y is 276, (165) X is 273, Y is 277, (166) X is 273, Y is 278, (167) X is 273, Y is 279, (168) X is 273, Y is 280 (169) X is 273, Y is 281; (170) X is 273, Y is 282; (171) X is 273, Y is 283; (172) X is 273, Y is 284; (173) X is 273, Y is 285; (174) X is 273, Y is 286; (175) X is 273, Y is 287; (176) X is 273, Y is 288, (177) X is 274, Y is 267, (178) X is 274, Y is 268, (179) X is 274, Y is 269, (180) X is 274, Y is 270, (181) X is 274, Y is 271, (182) X is 274, Y is 272, (183) X is 274Y is 273, (184) X is 274, Y is 274, (185) X is 274, Y is 275, (186) X is 274, Y is 276, (187) X is 274, Y is 277, (188) X is 274, Y is 278, (189) X is 274, Y is 279, (190) X is 274, Y is 280 (191) X is 274, Y is 281, (192) X is 274, Y is 282, (193) X is 274, Y is 283, (194) X is 274, Y is 284, (195) X is 274, Y is 285, (196) X is 274, Y is 286, (197) X is 274, Y is 287, (198) (199) X is 274, Y is 288, (200) X is 275, Y is 267, (201) X is 275, Y is 268, (202) X is 275, Y is 269, (203) X is 275, Y is 270, (204) X is 275, Y is 272, (205) X is 27 5, Y is 273, (206) X is 275, Y is 274, (207) X is 275, Y is 275, (208) X is 275, Y is 276, (209) X is 275, Y is 277, (210) X is 275, Y is 278, (211) X is 275, Y is 279, (212) X is 275, Y is 2 80, (213) X is 275, Y is 281, (214) X is 275, Y is 282, (215) X is 275, Y is 283, (216) X is 275, Y is 284, (217) X is 275, Y is 285, (218) X is 275, Y is 286, (219) X is 275, Y is 287, (2 20) X is 275, Y is 288, (221) X is 276, Y is 267, (222) X is 276, Y is 268, (223) X is 276, Y is 269, (224) X is 276, Y is 270, (225) X is 276, Y is 271, (226) X is 276, Y is 272, (227) X is 276, Y is 273, (228) X is 276, Y is 274, (229) X is 276, Y is 275, (230) X is 276, Y is 276, (231) X is 276, Y is 277, (232) X is 276, Y is 278, (233) X is 276, Y is 279, (234) X is 276, Y is 275 The answer is 280. (235) X is 276, Y is 281. (236) X is 276, Y is 282. (237) X is 276, Y is 283. (238) X is 276, Y is 284. (239) X is 276, Y is 285. (240) X is 276, Y is 286. (241) X is 276, Y is 287.(242) X is 276, Y is 288, (243) X is 277, Y is 267, (244) X is 277, Y is 268, (245) X is 277, Y is 269, (246) X is 277, Y is 270, (247) X is 277, Y is 271, (248) X is 277, Y is 272, (249) X is 277, Y is 273, (250) X is 277, Y is 274, (251) X is 277, Y is 275, (252) X is 277, Y is 276, (253) X is 277, Y is 277, (254) X is 277, Y is 278, (255) X is 277, Y is 279, (256) X is 277 Y is 280, (257)X is 277, Y is 281, (258)X is 277, Y is 282, (259)X is 277, Y is 283, (260)X is 277, Y is 284, (261)X is 277, Y is 285, (262)X is 277, Y is 286, (263)X is 277, Y is 287 (264) X is 277, Y is 288; (265) X is 278, Y is 267; (266) X is 278, Y is 268; (267) X is 278, Y is 269; (268) X is 278, Y is 270; (269) X is 278, Y is 271; (270) X is 278, Y is 272; (271) X is 278, Y is 273, (272) X is 278, Y is 274, (273) X is 278, Y is 275, (274) X is 278, Y is 276, (275) X is 278, Y is 277, (276) X is 278, Y is 278, (277) X is 278, Y is 279, (278) X is 278 Y is 280, (279)X is 278, Y is 281, (280)X is 278, Y is 282, (281)X is 278, Y is 283, (282)X is 278, Y is 284, (283)X is 278, Y is 285, (284)X is 278, Y is 286, (285)X is 278, Y is 287 (286) X is 278, Y is 288; (287) X is 279, Y is 267; (288) X is 279, Y is 268; (289) X is 279, Y is 269; (290) X is 279, Y is 270; (291) X is 279, Y is 271; (292) X is 279, Y is 272; (293) X is 279, Y is 273, (294) X is 279, Y is 274, (295) X is 279, Y is 275, (296) X is 279, Y is 276, (297) X is 279, Y is 277, (298) X is 279, Y is 278, (299) X is 279, Y is 279, (300) X is 279Y is 280, (301) X is 279, Y is 281, (302) X is 279, Y is 282, (303) X is 279, Y is 283, (304) X is 279, Y is 284, (305) X is 279, Y is 285, (306) X is 279, Y is 286, (307) X is 279, Y is 287 (308) X is 279, Y is 288; (309) X is 280, Y is 267; (310) X is 280, Y is 268; (311) X is 280, Y is 269; (312) X is 280, Y is 270; (313) X is 280, Y is 271; (314) X is 280, Y is 272; (315) (316) X is 280, Y is 273, (317) X is 280, Y is 274, (318) X is 280, Y is 275, (319) X is 280, Y is 276, (320) X is 280, Y is 277, (321) X is 280, Y is 278, (322) X is 280, Y is 279, (323) X is 280, Y is 274, (314) X is 280, Y is 275, (315) X is 280, Y is 276, (316) X is 280, Y is 277, (327) X is 280, Y is 278, (328) X is 280, Y is 279, (329) X is 0, Y is 280, (323) X is 280, Y is 281, (324) X is 280, Y is 282, (325) X is 280, Y is 283, (326) X is 280, Y is 284, (327) X is 280, Y is 285, (328) X is 280, Y is 286, (329) X is 280, Y is 2 87, (330) X is 280, Y is 288, (331) X is 281, Y is 267, (332) X is 281, Y is 268, (333) X is 281, Y is 269, (334) X is 281, Y is 270, (335) X is 281, Y is 271, (336) X is 281, Y is 272, (3 37) X is 281, Y is 273, (338) X is 281, Y is 274, (339) X is 281, Y is 275, (340) X is 281, Y is 276, (341) X is 281, Y is 277, (342) X is 281, Y is 278, (343) X is 281, Y is 279, (344) X is 281, Y is 280, (345) X is 281, Y is 281, (346) X is 281, Y is 282, (347) X is 281, Y is 283, (348) X is 281, Y is 284, (349) X is 281, Y is 285, (350) X is 281, Y is 286, (351) X is 281, Y The answer is 287. (352) X is 281, Y is 288. (353) X is 282, Y is 267. (354) X is 282, Y is 268. (355) X is 282, Y is 269. (356) X is 282, Y is 270. (357) X is 282, Y is 271. (358) X is 282, Y is 272.(359) X is 282, Y is 273, (360) X is 282, Y is 274, (361) X is 282, Y is 275, (362) X is 282, Y is 276, (363) X is 282, Y is 277, (364) X is 282, Y is 278, (365) X is 282, Y is 279, (366) X is 282, Y is 280, (367) X is 282, Y is 281, (368) X is 282, Y is 282, (369) X is 282, Y is 283, (370) X is 282, Y is 284, (371) X is 282, Y is 285, (372) X is 282, Y is 286, (373) X is 282 Y is 287, (374)X is 282, Y is 288, (375)X is 283, Y is 267, (376)X is 283, Y is 268, (377)X is 283, Y is 269, (378)X is 283, Y is 270, (379)X is 283, Y is 271, (380)X is 283, Y is 272 (381) X is 283, Y is 273; (382) X is 283, Y is 274; (383) X is 283, Y is 275; (384) X is 283, Y is 276; (385) X is 283, Y is 277; (386) X is 283, Y is 278; (387) X is 283, Y is 279; (388) X is 283, Y is 280, (389) X is 283, Y is 281, (390) X is 283, Y is 282, (391) X is 283, Y is 283, (392) X is 283, Y is 284, (393) X is 283, Y is 285, (394) X is 283, Y is 286, (395) X is 283 Y is 287, (396)X is 283, Y is 288, (397)X is 284, Y is 267, (398)X is 284, Y is 268, (399)X is 284, Y is 269, (400)X is 284, Y is 270, (401)X is 284, Y is 271, (402)X is 284, Y is 272 (403) X is 284, Y is 273; (404) X is 284, Y is 274; (405) X is 284, Y is 275; (406) X is 284, Y is 276; (407) X is 284, Y is 277; (408) X is 284, Y is 278; (409) X is 284, Y is 279; (410) X is 284, Y is 280, (411) X is 284, Y is 281, (412) X is 284, Y is 282, (413) X is 284, Y is 283, (414) X is 284, Y is 284, (415) X is 284, Y is 285, (416) X is 284, Y is 286, (417) X is 284Y is 287, (418) X is 284, Y is 288, (419) X is 285, Y is 267, (420) X is 285, Y is 268, (421) X is 285, Y is 269, (422) X is 285, Y is 270, (423) X is 285, Y is 271, (424) X is 285, Y is 272 (425) X is 285, Y is 273, (426) X is 285, Y is 274, (427) X is 285, Y is 275, (428) X is 285, Y is 276, (429) X is 285, Y is 277, (430) X is 285, Y is 278, (431) X is 285, Y is 279, (432) (433) X is 285, Y is 280, (434) X is 285, Y is 281, (435) X is 285, Y is 282, (436) X is 285, Y is 283, (437) X is 285, Y is 285, (438) X is 285, Y is 286, (439) X is 28 5, Y is 287, (440) X is 285, Y is 288, (441) X is 286, Y is 267, (442) X is 286, Y is 268, (443) X is 286, Y is 269, (444) X is 286, Y is 270, (445) X is 286, Y is 271, (446) X is 286, Y is 2 72, (447) X is 286, Y is 273, (448) X is 286, Y is 274, (449) X is 286, Y is 275, (450) X is 286, Y is 276, (451) X is 286, Y is 277, (452) X is 286, Y is 278, (453) X is 286, Y is 279, (4 54) X is 286, Y is 280, (455) X is 286, Y is 281, (456) X is 286, Y is 282, (457) X is 286, Y is 283, (458) X is 286, Y is 284, (459) X is 286, Y is 285, (460) X is 286, Y is 286, (461) X is 286, Y is 287, (462) X is 286, Y is 288, (463) X is 287, Y is 267, (464) X is 287, Y is 268, (465) X is 287, Y is 269, (466) X is 287, Y is 270, (467) X is 287, Y is 271, (468) X is 287, Y The answer is 272. (469) X is 287, Y is 273. (470) X is 287, Y is 274. (471) X is 287, Y is 275. (472) X is 287, Y is 276. (473) X is 287, Y is 277. (474) X is 287, Y is 278. (475) X is 287, Y is 279.(476) X is 287, Y is 280; (477) X is 287, Y is 281; (478) X is 287, Y is 282; (479) X is 287, Y is 283; (480) X is 287, Y is 284; (481) X is 287, Y is 285; (482) X is 287, Y is 286; (483) X is 287, Y is 287, (484) X is 287, Y is 288, (485) X is 117, Y is 118, (486) X is 117, Y is 119, (487) X is 117, Y is 120, (488) X is 117, Y is 121, (489) X is 117, Y is 122, (490) X is 117 Y is 123, (491) X is 118, Y is 118, (492) X is 118, Y is 119, (493) X is 118, Y is 120, (494) X is 118, Y is 121, (495) X is 118, Y is 122, (496) X is 118, Y is 123, (497) X is 119, Y is 118 (498) X is 119, Y is 119; (499) X is 119, Y is 120; (500) X is 119, Y is 121; (501) X is 119, Y is 122; (502) X is 119, Y is 123; (503) X is 120, Y is 118; (504) X is 120, Y is 119; (505) X is 120, Y is 120, (506) X is 120, Y is 121, (507) X is 120, Y is 122, (508) X is 120, Y is 123, (509) X is 121, Y is 118, (510) X is 121, Y is 119, (511) X is 121, Y is 120, (512) X is 121 Y is 121, (513) X is 121, Y is 122, (514) X is 121, Y is 123, (515) X is 122, Y is 118, (516) X is 122, Y is 119, (517) X is 122, Y is 120, (518) X is 122, Y is 121, (519) X is 122, Y is 122 (520) X is 122, Y is 123, (521) X is 340, Y is 341, (522) X is 340, Y is 342, (523) X is 340, Y is 343, (524) X is 340, Y is 344, (525) X is 340, Y is 345, (526) X is 340, Y is 346, (527) X is 340, Y is 347, (528) X is 340, Y is 348, (529) X is 340, Y is 349, (530) X is 340, Y is 350, (531) X is 340, Y is 351, (532) X is 340, Y is 352, (533) X is 340, Y is 353, (534) X is 341Y is 341, (535) X is 341, Y is 342, (536) X is 341, Y is 343, (537) X is 341, Y is 344, (538) X is 341, Y is 345, (539) X is 341, Y is 346, (540) X is 341, Y is 347, (541) X is 341, Y is 348 (542) X is 341, Y is 349, (543) X is 341, Y is 350, (544) X is 341, Y is 351, (545) X is 341, Y is 352, (546) X is 341, Y is 353, (547) X is 342, Y is 341, (548) X is 342, Y is 342, (549) (550) X is 342, Y is 343, (551) X is 342, Y is 345, (552) X is 342, Y is 346, (553) X is 342, Y is 347, (554) X is 342, Y is 348, (555) X is 342, Y is 349, (556) X is 34 2, Y is 350, (557) X is 342, Y is 351, (558) X is 342, Y is 352, (559) X is 342, Y is 353, (560) X is 343, Y is 341, (561) X is 343, Y is 342, (562) X is 343, Y is 343, (563) X is 343, Y is 3 44, (564)X is 343, Y is 345, (565)X is 343, Y is 346, (566)X is 343, Y is 347, (567)X is 343, Y is 348, (568)X is 343, Y is 349, (569)X is 343, Y is 350, (570)X is 343, Y is 351, (5 71) X is 343, Y is 352, (572) X is 343, Y is 353, (573) X is 344, Y is 341, (574) X is 344, Y is 342, (575) X is 344, Y is 343, (576) X is 344, Y is 344, (577) X is 344, Y is 345, (578) X is 344, Y is 346, (579) X is 344, Y is 347, (580) X is 344, Y is 348, (581) X is 344, Y is 349, (582) X is 344, Y is 350, (583) X is 344, Y is 351, (584) X is 344, Y is 352, (585) X is 344, Y is 346, (585) X is 344, Y is 346, (586) X is 344, Y is 347, (586) X is 344, Y is 348, (587) X is 344, Y is 349, (586) X is 344, Y is 350, (587) X is 344, Y is The answer is 353. (586) X is 345, Y is 341. (587) X is 345, Y is 342. (588) X is 345, Y is 343. (589) X is 345, Y is 344. (590) X is 345, Y is 345. (591) X is 345, Y is 346. (592) X is 345, Y is 347.(593) X is 345, Y is 348, (594) X is 345, Y is 349, (595) X is 345, Y is 350, (596) X is 345, Y is 351, (597) X is 345, Y is 352, (598) X is 345, Y is 353, (599) X is 346, Y is 341, (600) X is 346, Y is 342, (601) X is 346, Y is 343, (602) X is 346, Y is 344, (603) X is 346, Y is 345, (604) X is 346, Y is 346, (605) X is 346, Y is 347, (606) X is 346, Y is 348, (607) X is 346 Y is 349, (608) X is 346, Y is 350, (609) X is 346, Y is 351, (610) X is 346, Y is 352, (611) X is 346, Y is 353, (612) X is 347, Y is 341, (613) X is 347, Y is 342, (614) X is 347, Y is 343 (615) X is 347, Y is 344; (616) X is 347, Y is 345; (617) X is 347, Y is 346; (618) X is 347, Y is 347; (619) X is 347, Y is 348; (620) X is 347, Y is 349; (621) X is 347, Y is 350; (622) X is 347, Y is 351, (623) X is 347, Y is 352, (624) X is 347, Y is 353, (625) X is 348, Y is 341, (626) X is 348, Y is 342, (627) X is 348, Y is 343, (628) X is 348, Y is 344, (629) X is 348 Y is 345, (630) X is 348, Y is 346, (631) X is 348, Y is 347, (632) X is 348, Y is 348, (633) X is 348, Y is 349, (634) X is 348, Y is 350, (635) X is 348, Y is 351, (636) X is 348, Y is 352 (637) X is 348, Y is 353; (638) X is 349, Y is 341; (639) X is 349, Y is 342; (640) X is 349, Y is 343; (641) X is 349, Y is 344; (642) X is 349, Y is 345; (643) X is 349, Y is 346; (644) X is 349, Y is 347, (645) X is 349, Y is 348, (646) X is 349, Y is 349, (647) X is 349, Y is 350, (648) X is 349, Y is 351, (649) X is 349, Y is 352, (650) X is 349, Y is 353, (651) X is 350Y is 341, (652) X is 350, Y is 342, (653) X is 350, Y is 343, (654) X is 350, Y is 344, (655) X is 350, Y is 345, (656) X is 350, Y is 346, (657) X is 350, Y is 347, (658) X is 350, Y is 348, (659) X is 350, Y is 349, (660) X is 350, Y is 350, (661) (662) X is 350, Y is 351, (663) X is 350, Y is 352, (664) X is 350, Y is 353, (665) X is 351, Y is 341, (666) X is 351, Y is 342, (667) X is 351, Y is 344, (668) X is 351, Y is 345, (669) X is 351, Y is 346, (670) X is 351, Y is 3 47, (671) X is 351, Y is 348, (672) X is 351, Y is 349, (673) X is 351, Y is 350, (674) X is 351, Y is 351, (675) X is 351, Y is 352, (676) X is 351, Y is 353, (677) X is 352, Y is 341, (678) X is 352, Y is 342, (679) X is 352, Y is 343, (680) X is 352, Y is 344, (681) X is 352, Y is 345, (682) X is 352, Y is 346, (683) X is 352, Y is 347, (684) X is 352, Y is 348, (685) X is 352, Y is 349, (686) X is 352, Y is 350, (687) X is 352, Y is 351, (688) X is 352, Y is 352, (689) X is 352, Y is 353.

[0073] In this article, "response fold" refers to the standardized fluorescence ratio. The greater the deviation of the probe's response fold from 1 (whether it increases or decreases), the greater the change in the probe's response ability to the substrate relative to the control, or the greater its responsiveness. For example, in this application, the response fold is calculated by detecting the change in the ratio of fluorescence intensity at 528nm emission from 420nm excitation to fluorescence intensity at 528nm emission from 485nm excitation (Normalized Ratio 420 / 485), as detailed below:

[0074] Fluorescence signal values ​​were corrected by subtracting the detection signal values ​​from cells that did not express the probe protein. pH-sensitive interference was eliminated by dividing the probe detection signals from parallel experimental groups by the control detection signals to obtain corrected data.

[0075] F = F sample -F BLK

[0076] F represents fluorescence intensity. sample F represents the total fluorescence intensity of the sample expressing the fluorescent probe. BLK F represents the background fluorescence intensity of samples without expressed fluorescent probes. cpYFP This indicates the fluorescence intensity of the sample used as a pH control. F 485 F represents the fluorescence intensity of a fluorescent protein sample excited at 485 nm and emitted at 528 nm. 420 This indicates the fluorescence intensity of a fluorescent protein sample excited at 420 nm and emitted at 528 nm. The ratiosensor represents the ratio of the probe's fluorescence intensity. cpYFP This represents the ratio of the fluorescence intensity of the corresponding probe to the pH control fluorescent protein. (NormalizedRatio) 420 / 485 It represents the multiple of the probe's change or the multiple of the response. NormalizedRatio 485 / 420 The greater the deviation from 1 (whether it increases or decreases), the greater the change factor or response factor of the probe.

[0077] When referring to a polypeptide or protein, the term "variant" or "mutant" as used in this invention includes variants that have the same function as the polypeptide or protein but have a different sequence. Variants of polypeptides or proteins may include: homologous sequences, conserved variants, allelic variants, natural mutants, and induced mutants. These variants include, but are not limited to: deletions, insertions, and / or substitutions of one or more (typically 1-30, preferably 1-20, more preferably 1-10, most preferably 1-5) amino acids in the sequence of the polypeptide or protein, and sequences obtained by adding one or more (typically up to 20, preferably up to 10, more preferably up to 5) amino acids to its carboxyl terminus and / or amino terminus. These variants may also comprise polypeptides or proteins with at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% sequence identity with the polypeptide or protein. The goal is to avoid being limited by theory, where changes to amino acid residues do not alter the overall conformation and function of the polypeptide or protein—that is, functionally conserved mutations. For example, in this field, substitution with amino acids of similar or identical properties typically does not change the function of the polypeptide or protein. In this field, amino acids with similar properties often refer to families of amino acids with similar side chains, which are well-defined. These families include amino acids with basic side chains (e.g., lysine, D-glucose, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, D-glucose, phenylalanine, methionine, D-glucose), amino acids with β-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, D-glucose, histidine). For example, adding one or more amino acids to the amino terminus and / or carboxyl terminus generally does not alter the function of a polypeptide or protein. Conserved amino acid substitutions for many common, known non-genetically encoded amino acids are known in the art. Conserved substitutions for other non-coding amino acids can be determined based on a comparison of their physical properties with those of their genetically encoded amino acids.

[0078] "Connector" or "linking region" refers to an amino acid or nucleotide sequence that links two parts in a polypeptide, protein, or nucleic acid of the present invention. Exemplarily, in the present invention, the number of amino acids at the amino terminus of the linking region between the D-glucose-sensitive polypeptide and the optically active polypeptide is selected to be 0-3, and the number of amino acids at the carboxyl terminus is selected to be 0-2; when the recombinant optical probe is used as a basic unit to link with a functional protein, it can be fused to the amino acid or carboxyl terminus of the recombinant optical probe. The connector sequence can be a short peptide chain composed of one or more flexible amino acids, such as Y.

[0079] The inventors have discovered that D-glucose-binding protein variants with mutations at the following sites exhibit different binding activities than D-glucose: W8, W9, E13, A42, H66, D278, K312, V347, and H348 of SEQ ID NO: 1. The amino acid mutations include modifications, substitutions, or deletions of amino acids.

[0080] This invention provides D-glucose-binding protein variants having these mutations and optical probes comprising such D-glucose-binding protein variants as D-glucose-sensitive peptides. Therefore, in one or more embodiments, the D-glucose-sensitive peptide in the optical probe is a D-glucose-binding protein variant as described in any embodiment herein, and the fluorescent protein in the optical probe is as shown in SEQ ID NO: 2-9 or a functional variant thereof. Preferably, the fluorescent protein is as shown in SEQ ID NO: 2, 6, 7, 9 or a functional variant thereof.

[0081] In some specific embodiments, the D-glucose-sensitive polypeptide in the optical probe is shown in SEQ ID NO: 1, and the optically active polypeptide is shown in SEQ ID NO: 1. As shown in NO:2, the optically active peptides are located at positions 266 / 267, 266 / 268, 266 / 269, 266 / 270, 266 / 271, 266 / 272, 266 / 273, 266 / 274, 266 / 275, 266 / 276, 266 / 277, 266 / 278, 266 / 279, 266 / 280, 266 / 281, 266 / 282, 266 / 283, 266 / 284, 266 / 285, 266 / 286, 266 / 287, 266 / 288, 267 / 267, 267 / 268, 267 / 269, 267 / 270, and 267 of the D-glucose-sensitive peptide. / 271, 267 / 272, 267 / 273, 267 / 274, 267 / 275, 267 / 276, 267 / 277, 267 / 278, 267 / 279, 267 / 280, 267 / 281, 267 / 282, 267 / 283, 267 / 284, 267 / 285, 267 / 286, 267 / 287, 267 / 288, 268 / 267, 268 / 268, 268 / 269, 268 / 270, 268 / 271, 268 / 272, 268 / 273, 268 / 274, 268 / 275, 268 / 276, 268 / 277, 268 / 278 268 / 279, 268 / 280, 268 / 281, 268 / 282, 268 / 283, 268 / 284, 268 / 285, 268 / 286, 268 / 287, 268 / 288, 269 / 267, 269 / 268, 269 / 269, 269 / 270, 269 / 271, 269 / 272, 269 / 273, 269 / 274, 269 / 275, 269 / 276, 269 / 277, 269 / 278, 269 / 279, 269 / 280, 269 / 281, 269 / 282, 269 / 283, 269 / 284, 269 / 285, 269 / 286, 269 / 287, 269 / 288, 270 / 267, 270 / 268, 270 / 269, 270 / 270, 270 / 271, 270 / 272, 270 / 273, 270 / 274, 270 / 275, 270 / 276, 270 / 277, 270 / 278, 270 / 279, 270 / 280, 270 / 281, 270 / 282, 270 / 283, 270 / 284, 270 / 285, 270 / 286, 270 / 287, 270 / 288, 271 / 267, 271 / 268, 271 / 269, 271 / 270, 271 / 271271 / 272、271 / 273、271 / 274、271 / 275、271 / 276、271 / 277、271 / 278、271 / 279、271 / 280、271 / 281、271 / 282、271 / 283、271 / 284、271 / 285、271 / 286、271 / 287、271 / 288、272 / 267、272 / 268、272 / 269、272 / 270、272 / 271、272 / 272、272 / 273、272 / 274、272 / 275、272 / 276、272 / 277、272 / 278、272 / 279、272 / 280、272 / 281、272 / 282、272 / 283、272 / 284、272 / 285、272 / 286、272 / 287、272 / 288、273 / 267、273 / 268、273 / 269、273 / 270、273 / 271、273 / 272、273 / 273、273 / 274、273 / 275、273 / 276、273 / 277、273 / 278、273 / 279、273 / 280、273 / 281、273 / 282、273 / 283、273 / 284、273 / 285、273 / 286、273 / 287、273 / 288、274 / 267、274 / 268、274 / 269、274 / 270、274 / 271、274 / 272、274 / 273、274 / 274、274 / 275、274 / 276、274 / 277、274 / 278、274 / 279、274 / 280、274 / 281、274 / 282、274 / 283、274 / 284、274 / 285、274 / 286、274 / 287、274 / 288、275 / 267、275 / 268、275 / 269、275 / 270、275 / 271、275 / 272、275 / 273、275 / 274、275 / 275、275 / 276、275 / 277、275 / 278、275 / 279、275 / 280、275 / 281、275 / 282、275 / 283、275 / 284、275 / 285、275 / 286、275 / 287、275 / 288、276 / 267、276 / 268、276 / 269、276 / 270、276 / 271、276 / 272、276 / 273、276 / 274、276 / 275、276 / 276、276 / 277、276 / 278、276 / 279、276 / 280、276 / 281、276 / 282、276 / 283、276 / 284、276 / 285、276 / 286、276 / 287、276 / 288、277 / 267、277 / 268、277 / 269、277 / 270、277 / 271、277 / 272、277 / 273、277 / 274、277 / 275、277 / 276、277 / 277、277 / 278、277 / 279、277 / 280、277 / 281、277 / 282、277 / 283、277 / 284、277 / 285、277 / 286、277 / 287、277 / 288、278 / 267、278 / 268、278 / 269、278 / 270、278 / 271、278 / 272、278 / 273、278 / 274、278 / 275、278 / 276、278 / 277、278 / 278、278 / 279、278 / 280、278 / 281、278 / 282、278 / 283、278 / 284、278 / 285、278 / 286、278 / 287、278 / 288、279 / 267、279 / 268、279 / 269、279 / 270、279 / 271、279 / 272、279 / 273、279 / 274、279 / 275、279 / 276、279 / 277、279 / 278、279 / 279、279 / 280、279 / 281、279 / 282、279 / 283、279 / 284、279 / 285、279 / 286、279 / 287、279 / 288、280 / 267、280 / 268、280 / 269、280 / 270、280 / 271、280 / 272、280 / 273、280 / 274、280 / 275、280 / 276、280 / 277、280 / 278、280 / 279、280 / 280、280 / 281、280 / 282、280 / 283、280 / 284、280 / 285、280 / 286、280 / 287、280 / 288、281 / 267、281 / 268、281 / 269、281 / 270、281 / 271、281 / 272、281 / 273、281 / 274、281 / 275、281 / 276、281 / 277、281 / 278、281 / 279、281 / 280、281 / 281、281 / 282、281 / 283、281 / 284、281 / 285、281 / 286、281 / 287、281 / 288、282 / 267、282 / 268、282 / 269、282 / 270、282 / 271、282 / 272、282 / 273、282 / 274、282 / 275、282 / 276、282 / 277、282 / 278、282 / 279、282 / 280、282 / 281、282 / 282、282 / 283、282 / 284、282 / 285、282 / 286、282 / 287、282 / 288、283 / 267、283 / 268、283 / 269、283 / 270、283 / 271、283 / 272、283 / 273、283 / 274、283 / 275、283 / 276、283 / 277、283 / 278、283 / 279、283 / 280、283 / 281、283 / 282、283 / 283、283 / 284、283 / 285、283 / 286、283 / 287、283 / 288、284 / 267、284 / 268、284 / 269、284 / 270、284 / 271、284 / 272、284 / 273、284 / 274、284 / 275、284 / 276、284 / 277、284 / 278、284 / 279、284 / 280、284 / 281、284 / 282、284 / 283、284 / 284、284 / 285、284 / 286、284 / 287、284 / 288、285 / 267、285 / 268、285 / 269、285 / 270、285 / 271、285 / 272、285 / 273、285 / 274、285 / 275、285 / 276、285 / 277、285 / 278、285 / 279、285 / 280、285 / 281、285 / 282、285 / 283、285 / 284、285 / 285、285 / 286、285 / 287、285 / 288、286 / 267、286 / 268、286 / 269、286 / 270、286 / 271、286 / 272、286 / 273、286 / 274、286 / 275、286 / 276、286 / 277、286 / 278、286 / 279、286 / 280、286 / 281、286 / 282、286 / 283、286 / 284、286 / 285、286 / 286、286 / 287、286 / 288、287 / 267、287 / 268、287 / 269、287 / 270、287 / 271、287 / 272、287 / 273、287 / 274、287 / 275、287 / 276、287 / 277、287 / 278、287 / 279、287 / 280、287 / 281、287 / 282、287 / 283、287 / 284、287 / 285、287 / 286、287 / 287、287 / 288，117 / 118、117 / 119、117 / 120、117 / 121、117 / 122、117 / 123、118 / 118、118 / 119、118 / 120、118 / 121、118 / 122、118 / 123、119 / 118、119 / 119、119 / 120、119 / 121、119 / 122、119 / 123、120 / 118、120 / 119、120 / 120、120 / 121、120 / 122、120 / 123、121 / 118、121 / 119、121 / 120、121 / 121、121 / 122、121 / 123、122 / 118、122 / 119、122 / 120、122 / 121、122 / 122、122 / 123，340 / 341、340 / 342、340 / 343、340 / 344、340 / 345、340 / 346、340 / 347、340 / 348、340 / 349、340 / 350、340 / 351、340 / 352、340 / 353、341 / 341、341 / 342、341 / 343、341 / 344、341 / 345、341 / 346、341 / 347、341 / 348、341 / 349、341 / 350、341 / 351、341 / 352、341 / 353、342 / 341、342 / 342、342 / 343、342 / 344、342 / 345、342 / 346、342 / 347、342 / 348、342 / 349、342 / 350、342 / 351、342 / 352、342 / 353、343 / 341、343 / 342、343 / 343、343 / 344、343 / 345、343 / 346、343 / 347、343 / 348、343 / 349、343 / 350、343 / 351、343 / 352、343 / 353、344 / 341、344 / 342、344 / 343、344 / 344、344 / 345、344 / 346、344 / 347、344 / 348、344 / 349、344 / 350、344 / 351、344 / 352、344 / 353、345 / 341、345 / 342、345 / 343、345 / 344、345 / 345、345 / 346、345 / 347、345 / 348、345 / 349、345 / 350、345 / 351、345 / 352、345 / 353、346 / 341、346 / 342、346 / 343、346 / 344、346 / 345、346 / 346、346 / 347、346 / 348、346 / 349、346 / 350、346 / 351、346 / 352、346 / 353、347 / 341、347 / 342、347 / 343、347 / 344、347 / 345, 347 / 346, 347 / 347, 347 / 348, 347 / 349, 347 / 350, 347 / 351, 347 / 352, 347 / 353, 348 / 341, 348 / 342, 348 / 343, 348 / 344, 348 / 345, 348 / 346, 348 / 347, 348 / 348, 348 / 349, 348 / 350 348 / 351, 348 / 352, 348 / 353, 349 / 341, 349 / 342, 349 / 343, 349 / 344, 349 / 345, 349 / 346, 349 / 347, 349 / 348, 349 / 349, 349 / 350, 349 / 351, 349 / 352, 349 / 353, 350 / 341, 350 / 342, 350 / 343, 3 50 / 344, 350 / 345, 350 / 346, 350 / 347, 350 / 348, 350 / 349, 350 / 350, 350 / 351, 350 / 352, 350 / 353, 351 / 341, 351 / 342, 351 / 343, 351 / 344, 351 / 345, 351 / 346, 351 / 347, 351 / 348, 351 / 349, 35 The mutations of the optical probe at sites 1 / 350, 351 / 351, 351 / 352, 351 / 353, 352 / 341, 352 / 342, 352 / 343, 352 / 344, 352 / 345, 352 / 346, 352 / 347, 352 / 348, 352 / 349, 352 / 350, 352 / 351, 352 / 352, and 352 / 353 are shown in any row of Table 5.

[0082] In one or more embodiments, the optical probe contains a D-glucose-sensitive polypeptide as shown in SEQ ID NO: 1, and an optically active polypeptide as shown in SEQ ID NO: 2, 6, 7, and 9. The optically active polypeptide is located at position 348 / 352 of the D-glucose-sensitive polypeptide, and the optical probe has the following mutations: (1) V347W and H348T of the D-glucose-sensitive polypeptide and 1A of the optically active polypeptide; (2) V347W and H348G of the D-glucose-sensitive polypeptide and 1G of the optically active polypeptide; (3) V347W and H348S of the D-glucose-sensitive polypeptide and 1A of the optically active polypeptide; (4) V347W and H348S of the D-glucose-sensitive polypeptide and 1G of the optically active polypeptide; (5) V347W and H348T of the D-glucose-sensitive polypeptide and the optically active polypeptide. (6) D-glucose-sensitive peptides V347W, H348T, H66R, and optically active peptide 1A, (7) D-glucose-sensitive peptides V347W, H348T, H66E, and optically active peptide 1A, (8) D-glucose-sensitive peptides V347W, H348T, H66A, and optically active peptide 1A, (9) D-glucose-sensitive peptides V347W, H348T, H66F, and optically active peptide 1A, (10) D-glucose-sensitive peptides V347W, H348T, H66M, and optically active peptide 1A, (11) D-glucose-sensitive peptides V347W, H348T (12) D-glucose-sensitive peptides V347W, H348T, H66P, and optically active peptide 1A; (13) D-glucose-sensitive peptides V347W, H348T, H66Q, and optically active peptide 1A; (14) D-glucose-sensitive peptides V347W, H348T, H66T, and optically active peptide 1A; (15) D-glucose-sensitive peptides V347W, H348T, W9R, and optically active peptide 1A; (16) D-glucose-sensitive peptides V347W, H348T, W9H, and optically active peptide 1A; (17) D-glucose-sensitive peptides V347W, H348T, W9H, and optically active peptide 1A; (18) D-glucose-sensitive peptides V347W, H348T, W9E, and optically active peptide 1A; (19) D-glucose-sensitive peptides V347W, H348T, W9V, and optically active peptide 1A; (20) D-glucose-sensitive peptides V347W, H348T, W9L, and optically active peptide 1A; (21) D-glucose-sensitive peptides V347W, H348T, W9F, and optically active peptide 1A; (22) D-glucose-sensitive peptides V347W, H348T, W9I, and optically active peptide 1A.(23) D-glucose-sensitive peptides V347W, H348T, W9M, and optically active peptide 1A; (24) D-glucose-sensitive peptides V347W, H348T, W9C, and optically active peptide 1A; (25) D-glucose-sensitive peptides V347W, H348T, W9P, and optically active peptide 1A; (26) D-glucose-sensitive peptides V347W, H348T, W9N, and optically active peptide 1A; (27) D-glucose-sensitive peptides V347W, H348T, W9G, and optically active peptide 1A; (28) D-glucose-sensitive peptides V347W, H348T, W9K, and optically active peptide 1A. (29) D-glucose-sensitive peptides V347W, H348T, W9Y, and optically active peptide 1A; (30) D-glucose-sensitive peptides V347W, H348T, W9S, and optically active peptide 1A; (31) D-glucose-sensitive peptides V347W, H348T, W9T, and optically active peptide 1A; (32) D-glucose-sensitive peptides V347W, H348T, W9D, and optically active peptide 1A; (33) D-glucose-sensitive peptides V347W, H348T, W8R, and optically active peptide 1A; (34) D-glucose-sensitive peptides V347W, H348T, W8H, and optically active peptide 1A. (35) D-glucose-sensitive peptides V347W, H348T, W8E, and optically active peptide 1A; (36) D-glucose-sensitive peptides V347W, H348T, W8A, and optically active peptide 1A; (37) D-glucose-sensitive peptides V347W, H348T, W8V, and optically active peptide 1A; (38) D-glucose-sensitive peptides V347W, H348T, W8L, and optically active peptide 1A; (39) D-glucose-sensitive peptides V347W, H348T, W8F, and optically active peptide 1A; (40) D-glucose-sensitive peptides V347W, H348T, W8I, and optically active peptide 1A. (41) D-glucose-sensitive peptides V347W, H348T, W8M, and optically active peptide 1A; (42) D-glucose-sensitive peptides V347W, H348T, W8C, and optically active peptide 1A; (43) D-glucose-sensitive peptides V347W, H348T, W8N, and optically active peptide 1A; (44) D-glucose-sensitive peptides V347W, H348T, W8G, and optically active peptide 1A; (45) D-glucose-sensitive peptides V347W, H348T, W8K, and optically active peptide 1A; (46) D-glucose-sensitive peptides V347W, H348T, W8T, and optically active peptide 1A.(47) D-glucose-sensitive peptides V347W, H348T, W8D, and optically active peptide 1A; (48) D-glucose-sensitive peptides V347W, H348T, A42R, and optically active peptide 1A; (49) D-glucose-sensitive peptides V347W, H348T, A42E, and optically active peptide 1A; (50) D-glucose-sensitive peptides V347W, H348T, A42W, and optically active peptide 1A; (51) D-glucose-sensitive peptides V347W, H348T, A42F, and optically active peptide 1A. (52) D-glucose-sensitive peptides V347W, H348T, A42M, and optically active peptides 1A, (53) D-glucose-sensitive peptides V347W, H348T, A42C, and optically active peptides 1A, (54) D-glucose-sensitive peptides V347W, H348T, A42P, and optically active peptides 1A, (55) D-glucose-sensitive peptides V347W, H348T, A42N, and optically active peptides 1A, (56) D-glucose-sensitive peptides V347W, H348T, A42N, and optically active peptides 1A, (57) D-glucose-sensitive peptides V347W, H348T, A42M, and optically active peptides 1A, (58) D-glucose-sensitive peptides V347W, H348T, A42M ... T, A42G, and optically active peptide 1A, (57) D-glucose-sensitive peptides V347W, H348T, A42K, and optically active peptide 1A, (58) D-glucose-sensitive peptides V347W, H348T, A42Y, and optically active peptide 1A, (59) D-glucose-sensitive peptides V347W, H348T, A42T, and optically active peptide 1A, (60) D-glucose-sensitive peptides V347W, H348T, K312R, and optically active peptide 1A, (61) D-glucose-sensitive peptides V347W, H348T, K312A, and optically active peptide 1A; (62) D-glucose-sensitive peptides V347W, H348T, K312Q, and optically active peptide 1A; (63) D-glucose-sensitive peptides V347W, H348T, K312G, and optically active peptide 1A; (64) D-glucose-sensitive peptides V347W, H348T, K312H, and optically active peptide 1A; (65) D-glucose-sensitive peptides V347W, H348T, K312S, and optically active peptide 1A.

[0083] In two or more polypeptide or nucleic acid sequences, the term "identity" or "percentage of identity" refers to the similarity of two or more sequences or subsequences, or the similarity of a certain percentage of amino acid residues or nucleotides in a specified region, when compared and matched for maximum correspondence using methods known in the art, such as sequence comparison algorithms, through manual alignment and visual inspection (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% similarity, within a comparison window or specified region). For example, preferred algorithms suitable for determining the percentage of sequence identity and the percentage of sequence similarity are the BLAST and BLAST 2.0 algorithms, see Altschul et al. (1977) Nucleic Acids Res. 25: 3389 and Altschul et al. (1990) J. Mol. Biol. 215: 403, respectively.

[0084] As is known to those skilled in the art, gene cloning often requires the design of suitable restriction enzyme sites, which inevitably introduces one or more irrelevant residues at the end of the expressed polypeptide or protein, without affecting the activity of the target polypeptide or protein. Similarly, to construct fusion proteins, promote the expression of recombinant proteins, obtain recombinant proteins that are automatically secreted outside host cells, or facilitate the purification of recombinant proteins, it is often necessary to add certain amino acids to the N-terminus, C-terminus, or other suitable regions within the recombinant protein. These include, but are not limited to, suitable adaptor peptides, signal peptides, leader peptides, terminal extensions, glutathione S-transferase (GST), maltose E-binding proteins, protein A, tags such as 6His or Flag, or proteolytic enzyme sites such as factor Xa, thrombin, or enterokinase.

[0085] As used herein, the terms “functional fragment,” “derivative,” and “analyte” refer to a protein that substantially retains the same biological function or activity as the original polypeptide or protein (e.g., D-glucose-binding protein or fluorescent protein). Functional variants, derivatives, or analogs of the polypeptides or proteins of the present invention (e.g., D-glucose-binding protein or fluorescent protein) may be (i) proteins with one or more conserved or non-conserved amino acid residues (preferably conserved amino acid residues) substituted, such substituted amino acid residues may or may not be encoded by the genetic code; or (ii) proteins having substituent groups in one or more amino acid residues; or (iii) proteins formed by the fusion of a mature protein with another compound (e.g., a compound that extends the protein's half-life, such as polyethylene glycol); or (iv) proteins formed by the fusion of an additional amino acid sequence into the protein sequence (e.g., a secreted sequence or a sequence used to purify the protein or the original protein sequence, or a fusion protein formed with an antigen IgG fragment). Based on the teachings herein, these functional variants, derivatives, and analogs are within the scope well known to those skilled in the art. The analogues also include those having residues different from naturally occurring L-amino acids (such as D-amino acids), and those having non-naturally occurring or synthetic amino acids (such as β- or γ-amino acids). It should be understood that the D-glucose-sensitive polypeptides of the present invention are not limited to the representative proteins, variants, derivatives, and analogues listed above. Modifications (generally without altering the primary structure) include chemically derived forms of proteins, such as acetylation or carboxylation, either in vivo or in vitro. Modifications also include glycosylation, such as those resulting from glycosylation modifications during protein synthesis and processing or further processing steps. This modification can be accomplished by exposing the protein to glycosylation enzymes (such as mammalian glycosylation or deglycosylation enzymes). Modifications also include sequences having phosphorylated amino acid residues (such as phosphotyrosine, phosphotyserine, phosphotythreonine). Proteins modified to improve their resistance to proteolytic hydrolysis or optimize their solubility are also included.

[0086] The fusion polypeptide of this invention comprises the optical probe described herein and other polypeptides. In some embodiments, the optical probe described herein further comprises other polypeptides fused thereto. These other polypeptides do not affect the properties of the optical probe. The other polypeptides may be located at the N-terminus and / or C-terminus of the optical probe. In some embodiments, the other polypeptides include polypeptides for targeting the optical probe to different organelles or subcellular organelles, tags for purification, or tags for immunoblotting. A linker may be present between the optical probe and other polypeptides in the fusion polypeptide described herein.

[0087] The subcellular organelles described herein include the cytoplasm, mitochondria, nucleus, endoplasmic reticulum, cell membrane, Golgi apparatus, lysosomes, and peroxisomes. In some embodiments, the tags used for purification or for immunoblotting include 6-histidine (6*His), glutathione S-transferase (GST), and Flag.

[0088] This invention comprises nucleic acid molecules encoding the D-glucose-sensitive polypeptide or optical probe described herein. The terms "nucleic acid," "nucleotide," "polynucleotide," or "nucleic acid sequence" as used herein can be in DNA or RNA form. DNA form includes cDNA, genomic DNA, or artificially synthesized DNA. DNA can be single-stranded or double-stranded. DNA can be a coding strand or a non-coding strand. When referring to nucleic acids, the term "variant" as used herein can be a naturally occurring allelic variant or a non-naturally occurring variant. These nucleotide variants include degenerate variants, substitution variants, deletion variants, and insertion variants. As is known in the art, an allelic variant is a substitution of a nucleic acid, which may be a substitution, deletion, or insertion of one or more nucleotides, but does not substantially alter the function of the protein it encodes. The nucleic acid of this invention may comprise a nucleotide sequence with at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% sequence identity with the nucleic acid sequence described herein. This invention also relates to nucleic acid fragments that hybridize with the above-described sequences. As used herein, a "nucleic acid fragment" contains at least 15 nucleotides in length, preferably at least 30 nucleotides, more preferably at least 50 nucleotides, and most preferably at least 100 nucleotides or more. The nucleic acid fragments can be used in nucleic acid amplification techniques (such as PCR).

[0089] The full-length sequence or fragment of the optical probe or fusion protein of this invention can typically be obtained by PCR amplification, artificial synthesis, or recombinant methods. The steps and reagents used in conventional PCR, synthesis, and recombinant methods are known in the art. Furthermore, mutations can be introduced into the protein sequence of this invention through methods such as mutagenic PCR or chemical synthesis.

[0090] This invention also relates to nucleic acid constructs containing the polynucleotides described herein, and one or more regulatory sequences operatively linked to these sequences. The polynucleotides described herein can be manipulated in various ways to ensure the expression of the polypeptide or protein. The nucleic acid constructs can be manipulated prior to insertion into a vector, depending on the expression vector or requirements. Techniques for altering polynucleotide sequences using recombinant DNA methods are known in the art.

[0091] In some embodiments, the nucleic acid construct is a vector. The vector can be a cloning vector, an expression vector, or a homologous recombination vector. The polynucleotides of the present invention can be cloned into many types of vectors, such as plasmids, phage particles, phage derivatives, animal viruses, and entrapments.

[0092] Typical expression vectors contain expression control sequences that can be used to regulate the expression of a desired nucleic acid sequence, operatively linked to the nucleic acid sequence described herein or its complement. As used herein, the term "expression control sequence" refers to an element that can be operatively linked to the target gene to regulate the transcription, translation, and expression of the target gene. This can be an origin of replication, promoter, marker gene, or translation control element, including enhancers, operons, terminators, ribosome binding sites, etc. The choice of expression control sequence depends on the host cell used. In recombinant expression vectors, "operative linking" refers to the linking of the target nucleotide sequence to the regulatory sequence in a manner that allows the nucleotide sequence to be expressed. Those skilled in the art are familiar with methods for constructing expression vectors containing the coding sequence of the fusion protein of this invention and suitable transcription / translation control signals. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombination techniques, etc. The DNA sequence can be efficiently linked to an appropriate promoter in the expression vector to guide mRNA synthesis. Representative examples of these promoters include: the lac or trp promoter of *E. coli*; the PL promoter of *λ* phage; eukaryotic promoters including the CMV immediate early promoter, the HSV thymidine kinase promoter, early and late SV40 promoters, the LTR of retroviruses, and other known promoters that control gene expression in prokaryotic or eukaryotic cells or their viruses. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator. In one embodiment, the expression vector may be a commercially available pCDF vector, with no other special requirements. Exemplarily, the nucleotide sequence encoding the optical probe and the expression vector are double-digested with BamHI and EcoRI, respectively, and then the digestion products are ligated to obtain the recombinant expression vector. This invention does not specifically limit the specific steps and parameters of digestion and ligation; conventional steps and parameters in the art can be used.

[0093] After obtaining the recombinant expression vector, the vector is transformed into a host cell to produce a protein or peptide including a fusion protein. This transfer process can be performed using conventional techniques well known to those skilled in the art, such as transformation or transfection. The host cell described in this invention refers to a cell capable of receiving and accommodating recombinant DNA molecules, serving as the site for recombinant gene amplification. Ideally, the recipient cell should meet the conditions of easy acquisition and proliferation. The "host cell" of this invention can include prokaryotic and eukaryotic cells, specifically including bacterial cells, yeast cells, insect cells, and mammalian cells. The host cell is preferably a variety of cells conducive to gene product expression or fermentation production, such cells being well known and commonly used in the art. Specifically, it can be bacterial cells of *Escherichia coli*, *Streptomyces*, *Salmonella typhimurium*, fungal cells such as yeast, plant cells, insect cells of *Drosophila S2* or *Sf9*, animal cells such as CHO, COS, HEK293, HeLa cells, or Bowes melanoma cells, etc. The exemplary host cell used in the embodiments of this invention is *Escherichia coli* strain BL21-DE3. Those skilled in the art will understand how to select appropriate vectors, promoters, enhancers, and host cells.

[0094] The method for transferring DNA to host cells described in this invention is a conventional method in the art, including calcium phosphate or calcium chloride co-precipitation, DEAE-mannan-mediated transfection, lipid transfection, native competent cells, chemically mediated transfer, or electroporation. When the host is a prokaryote such as *Escherichia coli*, the preferred method is the CaCl2 or MgCl2 method, and the steps used are well known in the art. When the host cell is a eukaryotic cell, the following DNA transfection methods can be used: calcium phosphate co-precipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

[0095] This invention involves transforming an expression vector into host cells, followed by amplification and expression culture of the host cells to isolate the D-glucose optical probe. The host cell amplification and expression culture can be performed using conventional methods. Depending on the type of host cells used, the culture medium can be any conventional medium. Culture is carried out under conditions suitable for host cell growth.

[0096] In this invention, the optical probe is expressed intracellularly, on the cell membrane, or secreted extracellularly. If desired, the recombinant protein can be separated or purified using various separation methods based on its physical, chemical, and other properties. This invention does not specifically limit the method for separating the D-glucose fluorescent protein; conventional methods for separating fusion proteins in the art can be used. These methods are well known to those skilled in the art and include, but are not limited to, conventional refolding, salting out, centrifugation, permeation, sonication, ultracentrifugation, molecular sieve chromatography, adsorption chromatography, ion exchange chromatography, high-performance liquid chromatography (HPLC), and various other liquid chromatography techniques and combinations thereof. In one embodiment, the optical probe is separated using His-tagged affinity chromatography.

[0097] This invention also provides the application of the D-glucose optical probe in real-time localization, quantitative detection, and high-throughput compound screening of D-glucose. In one aspect, the D-glucose optical probe is preferably linked to a signal peptide at different sites within the cell and transferred into the cell. Real-time localization of D-glucose is achieved by detecting the intensity of fluorescence signals within the cell; quantitative detection of D-glucose is then performed by combining a standard D-glucose titration curve with changes in fluorescence signals. Changes in fluorescence signals are displayed, for example, by a standardized fluorescence signal ratio. In embodiments involving cpYFP, ​​this ratio is the ratio of the sample's 485 nm fluorescence signal to its 420 nm fluorescence signal to the corresponding ratio of the control. The D-glucose standard titration curve of this invention is plotted based on the fluorescence signals obtained by the D-glucose optical probe at different concentrations of D-glucose. The D-glucose optical probe of this invention is directly transferred into the cell, eliminating the need for time-consuming sample processing during real-time localization and quantitative detection of D-glucose, thus improving accuracy. The D-glucose optical probe of this invention, when used for high-throughput compound screening, involves adding different compounds to cell culture medium and measuring changes in D-glucose content to screen for compounds that affect these changes. The application of the D-glucose optical probe described in this invention for real-time localization, quantitative detection, and high-throughput compound screening is not for diagnostic or therapeutic purposes and does not involve the diagnosis or treatment of diseases.

[0098] This invention also provides a detection kit comprising the optical probes, nucleic acid molecules, nucleic acid constructs, and / or cells described herein. The kit also contains other reagents required for the detection of D-glucose. These other reagents are well known in the art, such as buffers, cell culture media, and D-glucose standards. Exemplary buffers include, for example, 100 mM HEPES and 100 mM NaCl, pH 7.4.

[0099] In this document, concentrations, contents, percentages, and other values ​​are expressed in range form. It should also be understood that this range form is used for convenience and brevity only, and should be flexibly interpreted to include the values ​​explicitly mentioned at the upper and lower limits of the range, as well as all individual values ​​or subranges included within that range.

[0100] Example

[0101] The D-glucose optical probe provided by the present invention will be described in detail below with reference to the embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0102] I. Experimental Materials and Reagents

[0103] The embodiments mainly employ conventional genetic engineering molecular biology cloning methods, cell culture, and imaging methods, which are well known to those skilled in the art. Examples include: Jane Rothcams et al.'s "Molecular Biology Laboratory Reference Manual," J. Sambrook and DW Russell, translated by Huang Peitang et al. (Molecular Cloning Laboratory Manual, 3rd edition, August 2002, Science Press, Beijing); Fereschney et al.'s "Animal Cell Culture: A Basic Technical Guide" (5th edition), translated by Zhang Jingbo and Xu Cunshuan et al.; and J.S. Bonnie Fesnon, M. Dassault et al.'s "A Concise Guide to Cell Biology Laboratory Techniques," translated by Zhang Jingbo et al.

[0104] The pCDF-cpYFP and pCDF-ttGBP plasmids used in the examples were constructed by the Protein Laboratory of East China University of Science and Technology, and the pCDF plasmid vectors were purchased from Invitrogen. All primers used for PCR were synthesized, purified, and identified correctly by mass spectrometry by Shanghai Jierui Biotechnology Co., Ltd. and BGI Genomics. The expression plasmids constructed in the examples were all sequenced, and the sequencing was performed by BGI Genomics and J. Lee Sequencing. The Taq DNA polymerase used in each example was purchased from Dongsheng Biotechnology, the pfu DNA polymerase was purchased from Tiangen Biotech (Beijing) Co., Ltd., and the primeSTAR DNA polymerase was purchased from TaKaRa. The corresponding polymerase buffer and dNTPs were included with the purchase of the three polymerases. Restriction endonucleases such as BamHI, BglII, HindIII, NdeI, XhoI, EcoRI, and SpeI, T4 ligase, and T4 phosphorylase (T4PNK) were purchased from Fermentas, and the corresponding buffers were included with the purchase. The Lip2000 transfection kit was purchased from Invitrogen. D-glucose and other compounds were purchased from Sigma-Aldrich. Unless otherwise stated, inorganic salts and other chemical reagents were purchased from Sigma-Aldrich. HEPES salt, ampicillin (Amp), and puromycin were purchased from Amersco. The 96-well detection blackboard and the 384-well fluorescence detection blackboard were purchased from Grenier.

[0105] The DNA purification kits used in these examples were purchased from BBI (Canada), and the general plasmid extraction kits were purchased from Tiangen Biotech (Beijing) Co., Ltd. The cloned strain Mach1 was purchased from Invitrogen. Nickel affinity chromatography columns and desalting column packing materials were both from GE Healthcare.

[0106] The main instruments used in the examples include: Biotek Synergy2 multi-functional microplate reader (Bio-Tek, USA), X-15R high-speed refrigerated centrifuge (Beckman, USA), Microfuge 22R benchtop high-speed refrigerated centrifuge (Beckman, USA), PCR amplifier (Biometra, Germany), ultrasonic disruptor (Ningbo Xinzhi Co., Ltd.), nucleic acid electrophoresis apparatus (Sheneng Bocai Co., Ltd.), fluorescence spectrophotometer (Varian, USA), CO2 constant temperature cell incubator (SANYO), and inverted fluorescence microscope (Nikon, Japan).

[0107] II. Molecular Biology Methods and Cellular Experimental Methods

[0108] II.1 Polymerase Chain Reaction (PCR):

[0109] 1. PCR amplification of the target fragment:

[0110] This method is mainly used for gene fragment amplification and colony PCR identification of positive clones. The PCR amplification reaction system is as follows: template sequence 0.5-1 μL, forward primer (25 μM) 0.5 μL, reverse primer (25 μM) 0.5 μL, 10×pfu buffer 5 μL, pfu DNA polymerase 0.5 μL, dNTP (10 mM) 1 μL, sterile ultrapure water (ddH2O) 41.5-42 μL, total volume 50 μL. The PCR amplification program is as follows: denaturation at 95℃ for 2-10 minutes, 30 cycles (94-96℃ for 30-45 seconds, 50-65℃ for 30-45 seconds, 72℃ for a certain time (600 bp / min)), extension at 72℃ for 10 minutes.

[0111] 2. Long fragment (>2500bp) amplification PCR:

[0112] The long-fragment amplification used in this invention is mainly a reverse PCR amplification vector, a technique used in the following examples to obtain site-directed mutagenesis. Reverse PCR primers are designed at the mutation site, with one primer containing the mutated nucleotide sequence at its 5' end. The amplified product then contains the corresponding mutation site. The long-fragment amplification PCR reaction system is as follows: template sequence (10 pg-1 ng) 1 μL, forward primer (25 μM) 0.5 μL, reverse primer (25 μM) 0.5 μL, 5×PrimerSTAR buffer 10 μL, PrimerSTAR DNA polymerase 0.5 μL, dNTP (2.5 mM) 4 μL, sterile ultrapure water (ddH2O) 33.5 μL, total volume 50 μL. The PCR amplification program is as follows: denaturation at 95℃ for 5 minutes, 30 cycles (98℃ for 10 seconds, 50-68℃ for 5-15 seconds, 72℃ for a certain time (1000bp / min)), extension at 72℃ for 10 minutes; or denaturation at 95℃ for 5 minutes, 30 cycles (98℃ for 10 seconds, 68℃ for a certain time (1000bp / min)), extension at 72℃ for 10 minutes.

[0113] II.2 Endonuclease digestion reaction:

[0114] The double enzyme digestion system for the plasmid vector is as follows: 20 μL plasmid vector (approximately 1.5 μg), 5 μL 10× buffer, 11-2 μL restriction endonuclease, 21-2 μL restriction endonuclease, and bring the total volume to 50 μL with sterile ultrapure water. Reaction conditions: 37℃, 1-7 hours.

[0115] II.3 Phosphorylation of DNA Fragments at the 5' End

[0116] Plasmids or genomes extracted from microorganisms contain phosphate groups at their ends, while PCR products do not. Therefore, a phosphate addition reaction is required at the 5' end of the PCR product. Only DNA molecules with phosphate groups at their ends can undergo ligation. The phosphorylation reaction system is as follows: 5-8 μL of PCR product DNA sequence, 1 μL of 10×T4 ligase buffer, 1 μL of T4 polynucleotide kinase (T4 PNK), and 0-3 μL of sterile ultrapure water, for a total volume of 10 μL. The reaction conditions are 37°C for 30 minutes to 2 hours, followed by inactivation at 72°C for 20 minutes.

[0117] II.4 Ligation reaction between target fragment and vector

[0118] The methods for connecting different fragments and carriers vary, and this invention uses three connection methods.

[0119] 1. Blunt-end junctions of blunt-ended short fragments and linearized vectors

[0120] The principle of this method is that the blunt-end product obtained by PCR is phosphorylated at the 5' end of a DNA fragment using T4 PNK, and then ligated with a linearized vector using PEG4000 and T4 DNA ligase to obtain a recombinant plasmid. The homologous recombination ligation system is as follows: 4 μL of T4 PNK-treated DNA fragment, 4 μL of linearized vector fragment, 1 μL of PEG4000, 1 μL of 10×T4 ligase buffer, and 1 μL of T4 DNA ligase, for a total of 10 μL. The reaction conditions are 22℃ for 30 minutes.

[0121] 2. Ligation of DNA fragments with sticky ends and vector fragments with sticky ends.

[0122] DNA fragments digested with restriction endonucleases typically produce prominent sticky ends, which can then be ligated to vector fragments containing sequence-complementary sticky ends to form recombinant plasmids. The ligation reaction system is as follows: 1-7 μL of the digested PCR product DNA fragment, 0.5-7 μL of the digested plasmid, 1 μL of 10×T4 ligase buffer, 1 μL of T4 DNA ligase, and sterile ultrapure water to a total volume of 10 μL. The reaction conditions are 16°C for 4-8 hours.

[0123] 3. Ligation reaction involving the self-circularization of DNA fragment products phosphorylated at the 5' end following site-directed mutagenesis using reverse PCR.

[0124] The 5' phosphorylated DNA fragment was ligated to the 3' and 5' ends of the linearized vector via a self-circularization ligation reaction to obtain a recombinant plasmid. The self-circularization ligation reaction system was as follows: 10 μL phosphorylation reaction mixture, 0.5 μL T4 ligase (5 U / μL), total volume 10.5 μL. Reaction conditions: 16℃, 4–16 hours.

[0125] II.5 Preparation and Transformation of Competent Cells

[0126] Preparation of competent cells:

[0127] 1. Pick a single colony (e.g., Mach1) and inoculate it into 5 mL of LB medium. Incubate overnight at 37°C in a shaker.

[0128] 2. Transfer 0.5-1 mL of the overnight culture to 50 mL of LB medium and incubate at 37°C and 220 rpm for 3 to 5 hours, until OD (dose-free survival) is achieved. 600 It reached 0.5.

[0129] 3. Pre-cool the cells in an ice bath for 2 hours.

[0130] Centrifuge at 4.4℃ and 4000rpm for 10 minutes.

[0131] 5. Discard the supernatant, resuspend the cells in 5 mL of pre-cooled buffer, and add resuspending buffer to a final volume of 50 mL after homogenization.

[0132] 6. Ice bath for 45 minutes.

[0133] Centrifuge at 7.4℃ and 4000rpm for 10 minutes, then resuspend the bacteria in 5mL of ice-cold storage buffer.

[0134] 8. Place 100 μL of bacterial culture in each EP tube and store at -80°C or in liquid nitrogen.

[0135] Resuspension buffer: CaCl2 (100mM), MgCl2 (70mM), NaAc (40mM)

[0136] Storage buffer: 0.5 mL DMSO, 1.9 mL 80% glycerol, 1 mL 10×CaCl2 (1 M), 1 mL 10×MgCl2 (700 mM), 1 mL 10×NaAc (400 mM), 4.6 mL ddH2O

[0137] Transformation of competent cells:

[0138] 1. Take 100 μL of competent cells and thaw them on an ice bath.

[0139] 2. Add an appropriate volume of ligation product, gently mix by pipetting, and incubate on ice for 30 minutes. The volume of ligation product added is usually less than 1 / 10 of the competent cell volume.

[0140] 3. Place the bacterial solution in a 42°C water bath for 90 seconds to heat shock, then quickly transfer it to an ice bath and place it for 5 minutes.

[0141] 4. Add 500 μL LB and incubate at 200 rpm for 1 hour on a constant temperature shaker at 37℃.

[0142] 5. Centrifuge the bacterial culture at 4000 rpm for 3 minutes, and keep 200 μL of supernatant. Spread the bacterial cells evenly on the surface of an agar plate containing appropriate antibiotics. Incubate the plate upside down in a 37°C incubator overnight.

[0143] II.6 Protein Expression, Purification, and Fluorescence Detection

[0144] 1. Transform the expression vector (e.g., a pCDF-based D-glucose optical probe expression vector) into BL21(DE3) cells, incubate upside down overnight, pick clones from the plate into 250ml Erlenmeyer flasks, place them on a 37℃ shaker, and incubate at 220rpm until OD = 0.4-0.8. Add 1 / 1000 (v / v) of IPTG (1M) and induce expression at 18℃ for 24-36 hours.

[0145] 2. After induction of expression, centrifuge at 4000 rpm for 30 minutes to collect the bacteria. Resuspend the bacterial pellet in 50 mM phosphate buffer and sonicate until the bacterial cells are clear. Centrifuge at 9600 rpm at 4°C for 20 minutes.

[0146] 3. The supernatant from centrifugation was purified by a self-assembled nickel affinity chromatography column to obtain the protein. The protein after nickel affinity chromatography was then purified by a self-assembled desalting column to obtain the protein dissolved in 100mM HEPES buffer (pH 7.4).

[0147] 4. After SDS-PAGE identification of the purified protein, the probe was diluted with assay buffer (100 mM HEPES, 100 mM NaCl, pH 7.4) to a final concentration of 0.2-5 μM. D-glucose was prepared into a stock solution with a final concentration of 50 mM using assay buffer (100 mM HEPES, 100 mM NaCl, pH 7.4).

[0148] 5. Take 100 μl of 1 μM protein solution, incubate at 37℃ for 10 minutes, add D-glucose for titration, and measure the fluorescence intensity of the protein at 528 nm emission after excitation by 420 nm light and at 528 nm emission after excitation by 485 nm light. The fluorescence excitation and emission measurements of the samples were performed using a multifunctional fluorescent microplate reader.

[0149] 6. Take 100 μl of 1 μM protein solution, incubate at 37℃ for 10 minutes, add D-glucose, and measure the absorption and fluorescence spectra of the protein. The absorption and fluorescence spectra of the samples were measured using a spectrophotometer and a fluorescence spectrophotometer.

[0150] II.7 Transfection and Fluorescence Detection of Mammalian Cells

[0151] 1. The pCDNA3.1+-based D-glucose optical probe plasmid was transfected into HEK293 cells using Lipofectamine 2000 (Invitrogen) and cultured in a 37°C, 5% CO2 cell culture incubator. Fluorescence detection was performed 24–36 h after the exogenous gene was fully expressed.

[0152] 2. After the expression was induced, the adherent HEK293 cells were washed three times with PBS and placed in HBSS solution for fluorescence microscopy and microplate reader detection.

[0153] Example 1: D-glucose binding protein particles

[0154] The TtGBP gene (SEQ ID NO: 1) from *Thermophilus T* was amplified by PCR. The PCR product was recovered after gel electrophoresis and digested with HindIII and XhoI enzymes. The pCDF vector was also double-digested with the corresponding enzymes. Ligation was performed using T4 DNA ligase, and the product was used to transform DH5α cells. The transformed DH5α cells were plated on LB agar plates (streptomycin 100 μg / mL) and incubated overnight at 37°C. Plasmids were extracted from the grown DH5α transformants and identified by PCR. Positive plasmids, after being correctly sequenced, were used for subsequent plasmid construction.

[0155] Example 2: Expression and detection of cpYFP optical probes at different insertion sites

[0156] In this embodiment, based on pCDF-TtGBP, the following sites were selected for inserting cpYFP to obtain the corresponding pCDF-TtGBP-cpYFP plasmids: 266 / 267, 266 / 268, 266 / 269, 266 / 270, 266 / 271, 266 / 272, 266 / 273, 266 / 274, 266 / 275, 266 / 276, 266 / 277, 266 / 278, 266 / 279, 266 / 280, 266 / 281, 266 / 282, 266 / 283, 266 / 284, 266 / 285, 266 / 286, 266 / 287, 266 / 288, 267 / 267, 267 / 268. 8, 267 / 269, 267 / 270, 267 / 271, 267 / 272, 267 / 273, 267 / 274, 267 / 275, 267 / 276, 267 / 277, 267 / 278, 267 / 279, 267 / 280, 267 / 281, 267 / 282, 267 / 283, 26 7 / 284, 267 / 285, 267 / 286, 267 / 287, 267 / 288, 268 / 267, 268 / 268, 268 / 269, 268 / 270, 268 / 271, 268 / 272, 268 / 273, 268 / 274, 268 / 275, 268 / 276, 268 / 277 268 / 278, 268 / 279, 268 / 280, 268 / 281, 268 / 282, 268 / 283, 268 / 284, 268 / 285, 268 / 286, 268 / 287, 268 / 288, 269 / 267, 269 / 268, 269 / 269, 269 / 270, 269 / 271, 269 / 272, 269 / 273, 269 / 274, 269 / 275, 269 / 276, 269 / 277, 269 / 278, 269 / 279, 269 / 280, 269 / 281, 269 / 282, 269 / 283, 269 / 284, 269 / 285, 269 / 286 269 / 287, 269 / 288, 270 / 267, 270 / 268, 270 / 269, 270 / 270, 270 / 271, 270 / 272, 270 / 273, 270 / 274, 270 / 275, 270 / 276, 270 / 277, 270 / 278, 270 / 279, 270 / 280, 270 / 281, 270 / 282, 270 / 283, 270 / 284, 270 / 285, 270 / 286, 270 / 287, 270 / 288, 271 / 267, 271 / 268, 271 / 269, 271 / 270, 271 / 271, 271 / 272, 271 / 273,271 / 274、271 / 275、271 / 276、271 / 277、271 / 278、271 / 279、271 / 280、271 / 281、271 / 282、271 / 283、271 / 284、271 / 285、271 / 286、271 / 287、271 / 288、272 / 267、272 / 268、272 / 269、272 / 270、272 / 271、272 / 272、272 / 273、272 / 274、272 / 275、272 / 276、272 / 277、272 / 278、272 / 279、272 / 280、272 / 281、272 / 282、272 / 283、272 / 284、272 / 285、272 / 286、272 / 287、272 / 288、273 / 267、273 / 268、273 / 269、273 / 270、273 / 271、273 / 272、273 / 273、273 / 274、273 / 275、273 / 276、273 / 277、273 / 278、273 / 279、273 / 280、273 / 281、273 / 282、273 / 283、273 / 284、273 / 285、273 / 286、273 / 287、273 / 288、274 / 267、274 / 268、274 / 269、274 / 270、274 / 271、274 / 272、274 / 273、274 / 274、274 / 275、274 / 276、274 / 277、274 / 278、274 / 279、274 / 280、274 / 281、274 / 282、274 / 283、274 / 284、274 / 285、274 / 286、274 / 287、274 / 288、275 / 267、275 / 268、275 / 269、275 / 270、275 / 271、275 / 272、275 / 273、275 / 274、275 / 275、275 / 276、275 / 277、275 / 278、275 / 279、275 / 280、275 / 281、275 / 282、275 / 283、275 / 284、275 / 285、275 / 286、275 / 287、275 / 288、276 / 267、276 / 268、276 / 269、276 / 270、276 / 271、276 / 272、276 / 273、276 / 274、276 / 275、276 / 276、276 / 277、276 / 278、276 / 279、276 / 280、276 / 281、276 / 282、276 / 283、276 / 284、276 / 285、276 / 286、276 / 287、276 / 288、277 / 267、277 / 268、277 / 269、277 / 270、277 / 271、277 / 272、277 / 273、277 / 274、277 / 275、277 / 276、277 / 277、277 / 278、277 / 279、277 / 280、277 / 281、277 / 282、277 / 283、277 / 284、277 / 285、277 / 286、277 / 287、277 / 288、278 / 267、278 / 268、278 / 269、278 / 270、278 / 271、278 / 272、278 / 273、278 / 274、278 / 275、278 / 276、278 / 277、278 / 278、278 / 279、278 / 280、278 / 281、278 / 282、278 / 283、278 / 284、278 / 285、278 / 286、278 / 287、278 / 288、279 / 267、279 / 268、279 / 269、279 / 270、279 / 271、279 / 272、279 / 273、279 / 274、279 / 275、279 / 276、279 / 277、279 / 278、279 / 279、279 / 280、279 / 281、279 / 282、279 / 283、279 / 284、279 / 285、279 / 286、279 / 287、279 / 288、280 / 267、280 / 268、280 / 269、280 / 270、280 / 271、280 / 272、280 / 273、280 / 274、280 / 275、280 / 276、280 / 277、280 / 278、280 / 279、280 / 280、280 / 281、280 / 282、280 / 283、280 / 284、280 / 285、280 / 286、280 / 287、280 / 288、281 / 267、281 / 268、281 / 269、281 / 270、281 / 271、281 / 272、281 / 273、281 / 274、281 / 275、281 / 276、281 / 277、281 / 278、281 / 279、281 / 280、281 / 281、281 / 282、281 / 283、281 / 284、281 / 285、281 / 286、281 / 287、281 / 288、282 / 267、282 / 268、282 / 269、282 / 270、282 / 271、282 / 272、282 / 273、282 / 274、282 / 275、282 / 276、282 / 277、282 / 278、282 / 279、282 / 280、282 / 281、282 / 282、282 / 283、282 / 284、282 / 285、282 / 286、282 / 287、282 / 288、283 / 267、283 / 268、283 / 269、283 / 270、283 / 271、283 / 272、283 / 273、283 / 274、283 / 275、283 / 276、283 / 277、283 / 278、283 / 279、283 / 280、283 / 281、283 / 282、283 / 283、283 / 284、283 / 285、283 / 286、283 / 287、283 / 288、284 / 267、284 / 268、284 / 269、284 / 270、284 / 271、284 / 272、284 / 273、284 / 274、284 / 275、284 / 276、284 / 277、284 / 278、284 / 279、284 / 280、284 / 281、284 / 282、284 / 283、284 / 284、284 / 285、284 / 286、284 / 287、284 / 288、285 / 267、285 / 268、285 / 269、285 / 270、285 / 271、285 / 272、285 / 273、285 / 274、285 / 275、285 / 276、285 / 277、285 / 278、285 / 279、285 / 280、285 / 281、285 / 282、285 / 283、285 / 284、285 / 285、285 / 286、285 / 287、285 / 288、286 / 267、286 / 268、286 / 269、286 / 270、286 / 271、286 / 272、286 / 273、286 / 274、286 / 275、286 / 276、286 / 277、286 / 278、286 / 279、286 / 280、286 / 281、286 / 282、286 / 283、286 / 284、286 / 285、286 / 286、286 / 287、286 / 288、287 / 267、287 / 268、287 / 269、287 / 270、287 / 271、287 / 272、287 / 273、287 / 274、287 / 275、287 / 276、287 / 277、287 / 278、287 / 279、287 / 280、287 / 281、287 / 282、287 / 283、287 / 284、287 / 285、287 / 286、287 / 287、287 / 288，117 / 118、117 / 119、117 / 120、117 / 121、117 / 122、117 / 123、118 / 118、118 / 119、118 / 120、118 / 121、118 / 122、118 / 123、119 / 118、119 / 119、119 / 120、119 / 121、119 / 122、119 / 123、120 / 118、120 / 119、120 / 120、120 / 121、120 / 122、120 / 123、121 / 118、121 / 119、121 / 120、121 / 121、121 / 122、121 / 123、122 / 118、122 / 119、122 / 120、122 / 121、122 / 122、122 / 123，340 / 341、340 / 342、340 / 343、340 / 344、340 / 345、340 / 346、340 / 347、340 / 348、340 / 349、340 / 350、340 / 351、340 / 352、340 / 353、341 / 341、341 / 342、341 / 343、341 / 344、341 / 345、341 / 346、341 / 347、341 / 348、341 / 349、341 / 350、341 / 351、341 / 352、341 / 353、342 / 341、342 / 342、342 / 343、342 / 344、342 / 345、342 / 346、342 / 347、342 / 348、342 / 349、342 / 350、342 / 351、342 / 352、342 / 353、343 / 341、343 / 342、343 / 343、343 / 344、343 / 345、343 / 346、343 / 347、343 / 348、343 / 349、343 / 350、343 / 351、343 / 352、343 / 353、344 / 341、344 / 342、344 / 343、344 / 344、344 / 345、344 / 346、344 / 347、344 / 348、344 / 349、344 / 350、344 / 351、344 / 352、344 / 353、345 / 341、345 / 342、345 / 343、345 / 344、345 / 345、345 / 346、345 / 347、345 / 348、345 / 349、345 / 350、345 / 351、345 / 352、345 / 353、346 / 341、346 / 342、346 / 343、346 / 344、346 / 345、346 / 346、346 / 347、346 / 348、346 / 349、346 / 350、346 / 351、346 / 352、346 / 353、347 / 341、347 / 342、347 / 343、347 / 344、347 / 345、347 / 346、347 / 347, 347 / 348, 347 / 349, 347 / 350, 347 / 351, 347 / 352, 347 / 353, 348 / 341, 348 / 342, 348 / 343, 348 / 344, 348 / 345, 348 / 346, 348 / 347, 348 / 348, 348 / 349, 348 / 350, 348 / 351 348 / 352, 348 / 353, 349 / 341, 349 / 342, 349 / 343, 349 / 344, 349 / 345, 349 / 346, 349 / 347, 349 / 348, 349 / 349, 349 / 350, 349 / 351, 349 / 352, 349 / 353, 350 / 341, 350 / 342, 350 / 343, 350 / 344, 350 / 345, 350 / 346, 350 / 347, 350 / 348, 350 / 349, 350 / 350, 350 / 351, 350 / 352, 350 / 353, 351 / 341, 351 / 342, 351 / 343, 351 / 344, 351 / 345, 351 / 346, 351 / 347, 351 / 348 351 / 349, 351 / 350, 351 / 351, 351 / 352, 351 / 353, 352 / 341, 352 / 342, 352 / 343, 352 / 344, 352 / 345, 352 / 346, 352 / 347, 352 / 348, 352 / 349, 352 / 350, 352 / 351, 352 / 352, and 352 / 353. For illustrative purposes only, the amino acid sequence of 348 / 352-TtGBP-cpYFP is shown in SEQ ID NO: 10.

[0157] A DNA fragment of cpYFP was generated using PCR. Simultaneously, a homologous sequence from the cpYFP terminal was introduced at the 5' end using primers. PCR amplification produced a linearized pCDF-D-glucose-binding protein vector, whose 5' and 3' ends contained sequences completely identical to those at the two ends of cpYFP (15-20 bp). The linearized pCDF-TtGBP and cpYFP fragments underwent homologous recombination using HieffCloneEnzyme. The product was transformed into DH5α, and the transformed DH5α was plated on LB agar plates (streptomycin 100 μg / mL) and incubated overnight at 37°C. Positive clones identified by PCR were subjected to plasmid extraction and sequencing. Sequencing was performed by J. Lee Sequencing.

[0158] After successful sequencing, the recombinant plasmid was transformed into BL21(DE3) to induce expression, and the protein was purified. SDS-PAGE electrophoresis showed a size around 70 kDa. This size is consistent with the size of the pCDF-TtGBP-cpYFP fusion protein containing the His-tag purified label. The results are shown in Figure 1.

[0159] D-glucose response screening was performed using the supernatant of fragmented *E. coli* expressing the TtGBP-cpYFP fusion protein. The detection signal of the fusion fluorescent protein containing 100 mM D-glucose was divided by the detection signal of the fusion fluorescent protein without D-glucose. As shown in Table 1, the detection results showed that optical probes expressing the TtGBP-cpYFP fusion protein in the fragmented supernatant exhibited a D-glucose response greater than 1.2 times: 273 / 279, 273 / 284, 350 / 342, 348 / 343, 349 / 343, 348 / 344, 349 / 344, 349 / 345, 348 / 349, 349 / 349, and 3... The following optical probes were selected: 48 / 350, 349 / 350, 346 / 351, 347 / 351, 347 / 352, 348 / 352, and 350 / 353. Seven probes showed a response to D-glucose exceeding 1.25 times: 273 / 279, 348 / 343, 348 / 344, 349 / 345, 349 / 349, 349 / 350, and 347 / 352.

[0160] Table 1

[0161] Example 3: Expression and detection of cpGFP optical probes at different insertion sites

[0162] Following the method in Example 2, cpYFP was replaced with cpGFP to construct a D-glucose green fluorescent protein probe. As shown in Table 2, the detection results showed that optical probes expressing the TtGBP-cpGFP fusion protein responded to D-glucose more than 1.3 times to optical probes at sites 348 / 344, 349 / 345, 349 / 349, 349 / 350, and 347 / 352, or the corresponding amino acid sites of its family proteins, were inserted.

[0163] Table 2

[0164] Example 4: Expression and detection of cpBFP optical probes at different insertion sites

[0165] Following the method in Example 2, cpYFP was replaced with cpBFP to construct a D-glucose blue fluorescent protein probe. As shown in Table 3, the detection results showed that optical probes expressing the TtGBP-cpBFP fusion protein responded to D-glucose more than 1.3 times to optical probes with insertion sites at 348 / 343, 348 / 344, 349 / 345, and 349 / 349 or corresponding amino acid sites of its family proteins were inserted.

[0166] Table 3

[0167] Example 5: Expression and detection of cpmApple optical probes at different insertion sites

[0168] Following the method in Example 2, cpYFP was replaced with cpmApple to construct a D-glucose red fluorescent protein probe. As shown in Table 4, the detection results show that optical probes expressing TtGBP-cpmApple fusion protein respond more than 1.3 times to D-glucose by replacing cpYFP with cpmApple. Optical probes with insertion sites at 349 / 345 and 349 / 349 sites or corresponding amino acid sites of its family proteins are also included.

[0169] Table 4

[0170] Example 6: Performance of the optical probe

[0171] For the 17 optical probes obtained in Example 2 that responded to D-glucose more than 1.2 times, namely those inserted at sites 273 / 279, 273 / 284, 350 / 342, 348 / 343, 349 / 343, 348 / 344, 349 / 344, 349 / 345, 348 / 349, 349 / 349, 348 / 350, 349 / 350, 346 / 351, 347 / 351, 347 / 352, 348 / 352, and 350 / 353, glucose detection was performed using a concentration gradient (0–100 mM), and the change in the ratio of fluorescence intensity at 420 nm excitation and 528 nm emission to fluorescence intensity at 485 nm excitation and 528 nm emission was detected. Fourteen D-glucose optical probes with insertion sites of 273 / 279, 273 / 284, 350 / 342, 348 / 343, 349 / 343, 348 / 344, 348 / 349, 349 / 349, 348 / 350, 349 / 350, 346 / 351, 347 / 351, 347 / 352, and 350 / 353 had excessively high Kd (binding constant), making them unsuitable for detection due to their inability to be properly fitted. In addition, three D-glucose optical probes with insertion sites of 349 / 344, 349 / 345, and 348 / 352 had Kd (binding constant) values ​​of 1.2 mM, 0.2 mM, and 0.008 mM, respectively.

[0172] Example 7: Expression and detection of mutated cpYFP optical probes

[0173] Optical probe mutants were constructed based on TtGBP-348 / 352-cpYFP. The plasmid pCDF-TtGBP-348 / 352-cpYFP was linearized by PCR, with primers containing the base sequences of the desired mutation sites. Homologous recombination was performed on the obtained PCR product to obtain mutant plasmids for 12 sites: V347, H348, H66, D278, W9, E13, W8, A42, K312 of the D-glucose-sensitive polypeptide, and Y1 of the optically active polypeptide. Sequencing was performed by J.Lee Sequencing. As an example, the amino acid sequence of 348 / 352-TtGBP-V347W / H348S / H66C-cpYFP-Y1A is shown in SEQ ID NO: 11.

[0174] The successfully constructed mutant plasmid was transformed into BL21(DE3) to induce expression. The supernatant from the fragmented *E. coli* expressing the probe protein was used for response screening to D-glucose and other non-specific substrates. The detection signal of the fusion fluorescent protein containing D-glucose or other non-specific substrates was divided by the detection signal of the fusion fluorescent protein without D-glucose. The results are shown in Table 5. The detection results show that the optical probes with a response to D-glucose greater than 3-fold and good specificity are listed below.

[0175] Table 5

[0176] Example 8: Performance of optical probe mutants

[0177] The D-glucose optical probes described in Table 5 of Example 7 were used to detect D-glucose at concentration gradients (0–100 mM). After treating the probes for 10 minutes, the change in the ratio of fluorescence intensity at 420 nm excitation and 528 nm emission was measured to be that at 485 nm excitation and 528 nm emission. The probe titration results are shown in Figure 3, indicating that different mutants have different affinities for D-glucose.

[0178] The D-glucose probes in Table 5 were subjected to specificity testing. Reactivity was measured against D-glucose structural analogs, glucose-1-phosphate, sorbitol, mannitol, sodium lactate, sucrose, pyruvate, ribose, fructose, oxaloacetic acid, ATP, glucose-6-phosphate, citric acid, D-glucose, isocitrate, sodium gluconate, NAD+, NADH, malic acid, NADPH, arabinose, PEP, lactose, maltose, 2-deoxyglucose, and galactose. The results showed good specificity, as shown in Figure 4.

[0179] Example 9: Subcellular organelle localization of optical probes and performance of optical probes within subcellular organelles

[0180] In this embodiment, different localization signal peptides were fused with the optical probe 348 / 352-TtGBP-V347W / H348S / H66C-cpYFP-Y1A to localize the optical probe to different organelles.

[0181] After transfecting 293 cells with optical probe plasmids fused with different localization signal peptides for 36 hours, the cells were washed with PBS and placed in HBSS solution for fluorescence detection using an inverted fluorescence microscope under the FITC channel. The results are shown in Figure 5. The D-glucose optical probes, by fusing with different specific localization signal peptides, can localize to subcellular organelles including the cytoplasm, outer membrane, nucleus, endoplasmic reticulum, mitochondria, and nuclear exclusion. Fluorescence was observed in all different subcellular structures, and the distribution and intensity of the fluorescence varied.

[0182] HEK293 cells were transfected with a cytoplasmic optical probe plasmid for 36 hours. After washing with PBS, the cells were placed in HBSS solution, and the changes in the fluorescence intensity ratio at 420 nm excitation and 528 nm emission (485 nm excitation and 528 nm emission) were detected over a 30-minute period. The results are shown in Figure 6. After adding 5 mM D-glucose, the detection was continued for another 30 minutes. The 420 / 485 ratio gradually increased in the D-glucose-added samples, reaching a maximum of 1.8 times the initial value, while the 420 / 485 ratio remained unchanged in the control group without Oxalate.

[0183] Example 10: High-throughput compound screening based on optical probes in living cells

[0184] In this embodiment, we used HeLa cells with cytoplasmic expression of 348 / 352-TtGBP-V347W / H348S / H66C-cpYFP-Y1A for high-throughput compound screening.

[0185] Transfected 293 cells were washed with PBS, treated with HBSS solution (without D-glucose) for 1 hour, and then treated with 10 μM of the compound for 1 hour. D-glucose was added to each sample. The ratio of fluorescence intensity at 420 nm excitation to 528 nm emission and the ratio of fluorescence intensity at 485 nm excitation to 528 nm emission were recorded using a microplate reader. Samples without any compound treatment were used as controls for standardization. The results are shown in Figure 7. Of the 2000 compounds used, the vast majority had minimal effect on D-glucose uptake by cells. Five compounds increased the cells' ability to take up D-glucose, while nine compounds significantly reduced it.

[0186] Example 11: Quantitative detection of D-glucose in blood using an optical probe

[0187] In this embodiment, purified Kd 48.33 μM 348 / 352-TtGBP-V347W / H348S-cpYFP-Y1A was used to analyze D-glucose in the blood supernatant of mice and humans.

[0188] After mixing 348 / 352-TtGBP-V347W / H348S-cpYFP-Y1A with diluted blood supernatant and treating for 10 minutes, the ratio of fluorescence intensity at 420nm excitation and 528nm emission to fluorescence intensity at 485nm excitation and 528nm emission was detected using an ELISA reader. The results are shown in Figure 8. The D-glucose content in mouse blood was approximately 8 mM, while the D-glucose content in human blood was approximately 4 mM.

[0189] As can be seen from the above embodiments, the D-glucose optical probe provided by the present invention has a relatively small protein molecular weight and is easy to mature. It exhibits large fluorescence dynamic changes, good specificity, and can be expressed in cells through gene manipulation. It can locate and quantify D-glucose in and out of cells in real time and can also perform high-throughput compound screening.

[0190] Other implementation methods

[0191] This specification describes many embodiments. However, it should be understood that various modifications that may be learned by those skilled in the art upon reading this specification without departing from the spirit and scope of the invention should also be included within the scope of the appended claims.

[0192] sequence of this article

Claims

1. An optical probe comprising a D-glucose-sensitive polypeptide and an optically active polypeptide, wherein, The D-glucose-sensitive polypeptide is a TtGBP protein or a variant thereof, and the optically active polypeptide is a fluorescent protein or a variant thereof. The optically active polypeptide is located at one or more sites selected from the following: 266 / 267, 266 / 268, 266 / 269, 266 / 270, 266 / 271, 266 / 272, 266 / 273, 266 / 274, 266 / 275, 266 / 276, 266 / 277, 266 / 278, 266 / 279, 266 / 280, 266 / 281, 266 / 282, 266 / 283, 266 / 284, 266 / 285, 266 / 286, 266 / 287, 266 / 288. 8, 267 / 267, 267 / 268, 267 / 269, 267 / 270, 267 / 271, 267 / 272, 267 / 273, 267 / 274, 267 / 275, 267 / 276, 267 / 277, 267 / 278, 267 / 279, 267 / 280, 267 / 281, 26 7 / 282, 267 / 283, 267 / 284, 267 / 285, 267 / 286, 267 / 287, 267 / 288, 268 / 267, 268 / 268, 268 / 269, 268 / 270, 268 / 271, 268 / 272, 268 / 273, 268 / 274, 268 / 275 268 / 276, 268 / 277, 268 / 278, 268 / 279, 268 / 280, 268 / 281, 268 / 282, 268 / 283, 268 / 284, 268 / 285, 268 / 286, 268 / 287, 268 / 288, 269 / 267, 269 / 268, 269 / 269, 269 / 270, 269 / 271, 269 / 272, 269 / 273, 269 / 274, 269 / 275, 269 / 276, 269 / 277, 269 / 278, 269 / 279, 269 / 280, 269 / 281, 269 / 282, 269 / 283, 269 / 284 269 / 285, 269 / 286, 269 / 287, 269 / 288, 270 / 267, 270 / 268, 270 / 269, 270 / 270, 270 / 271, 270 / 272, 270 / 273, 270 / 274, 270 / 275, 270 / 276, 270 / 277, 270 / 278, 270 / 279, 270 / 280, 270 / 281, 270 / 282, 270 / 283, 270 / 284, 270 / 285, 270 / 286, 270 / 287, 270 / 288, 271 / 267, 271 / 268, 271 / 269, 271 / 270, 271 / 271271 / 272、271 / 273、271 / 274、271 / 275、271 / 276、271 / 277、271 / 278、271 / 279、271 / 280、271 / 281、271 / 282、271 / 283、271 / 284、271 / 285、271 / 286、271 / 287、271 / 288、272 / 267、272 / 268、272 / 269、272 / 270、272 / 271、272 / 272、272 / 273、272 / 274、272 / 275、272 / 276、272 / 277、272 / 278、272 / 279、272 / 280、272 / 281、272 / 282、272 / 283、272 / 284、272 / 285、272 / 286、272 / 287、272 / 288、273 / 267、273 / 268、273 / 269、273 / 270、273 / 271、273 / 272、273 / 273、273 / 274、273 / 275、273 / 276、273 / 277、273 / 278、273 / 279、273 / 280、273 / 281、273 / 282、273 / 283、273 / 284、273 / 285、273 / 286、273 / 287、273 / 288、274 / 267、274 / 268、274 / 269、274 / 270、274 / 271、274 / 272、274 / 273、274 / 274、274 / 275、274 / 276、274 / 277、274 / 278、274 / 279、274 / 280、274 / 281、274 / 282、274 / 283、274 / 284、274 / 285、274 / 286、274 / 287、274 / 288、275 / 267、275 / 268、275 / 269、275 / 270、275 / 271、275 / 272、275 / 273、275 / 274、275 / 275、275 / 276、275 / 277、275 / 278、275 / 279、275 / 280、275 / 281、275 / 282、275 / 283、275 / 284、275 / 285、275 / 286、275 / 287、275 / 288、276 / 267、276 / 268、276 / 269、276 / 270、276 / 271、276 / 272、276 / 273、276 / 274、276 / 275、276 / 276、276 / 277、276 / 278、276 / 279、276 / 280、276 / 281、276 / 282、276 / 283、276 / 284、276 / 285、276 / 286、276 / 287、276 / 288、277 / 267、277 / 268、277 / 269、277 / 270、277 / 271、277 / 272、277 / 273、277 / 274、277 / 275、277 / 276、277 / 277、277 / 278、277 / 279、277 / 280、277 / 281、277 / 282、277 / 283、277 / 284、277 / 285、277 / 286、277 / 287、277 / 288、278 / 267、278 / 268、278 / 269、278 / 270、278 / 271、278 / 272、278 / 273、278 / 274、278 / 275、278 / 276、278 / 277、278 / 278、278 / 279、278 / 280、278 / 281、278 / 282、278 / 283、278 / 284、278 / 285、278 / 286、278 / 287、278 / 288、279 / 267、279 / 268、279 / 269、279 / 270、279 / 271、279 / 272、279 / 273、279 / 274、279 / 275、279 / 276、279 / 277、279 / 278、279 / 279、279 / 280、279 / 281、279 / 282、279 / 283、279 / 284、279 / 285、279 / 286、279 / 287、279 / 288、280 / 267、280 / 268、280 / 269、280 / 270、280 / 271、280 / 272、280 / 273、280 / 274、280 / 275、280 / 276、280 / 277、280 / 278、280 / 279、280 / 280、280 / 281、280 / 282、280 / 283、280 / 284、280 / 285、280 / 286、280 / 287、280 / 288、281 / 267、281 / 268、281 / 269、281 / 270、281 / 271、281 / 272、281 / 273、281 / 274、281 / 275、281 / 276、281 / 277、281 / 278、281 / 279、281 / 280、281 / 281、281 / 282、281 / 283、281 / 284、281 / 285、281 / 286、281 / 287、281 / 288、282 / 267、282 / 268、282 / 269、282 / 270、282 / 271、282 / 272、282 / 273、282 / 274、282 / 275、282 / 276、282 / 277、282 / 278、282 / 279、282 / 280、282 / 281、282 / 282、282 / 283、282 / 284、282 / 285、282 / 286、282 / 287、282 / 288、283 / 267、283 / 268、283 / 269、283 / 270、283 / 271、283 / 272、283 / 273、283 / 274、283 / 275、283 / 276、283 / 277、283 / 278、283 / 279、283 / 280、283 / 281、283 / 282、283 / 283、283 / 284、283 / 285、283 / 286、283 / 287、283 / 288、284 / 267、284 / 268、284 / 269、284 / 270、284 / 271、284 / 272、284 / 273、284 / 274、284 / 275、284 / 276、284 / 277、284 / 278、284 / 279、284 / 280、284 / 281、284 / 282、284 / 283、284 / 284、284 / 285、284 / 286、284 / 287、284 / 288、285 / 267、285 / 268、285 / 269、285 / 270、285 / 271、285 / 272、285 / 273、285 / 274、285 / 275、285 / 276、285 / 277、285 / 278、285 / 279、285 / 280、285 / 281、285 / 282、285 / 283、285 / 284、285 / 285、285 / 286、285 / 287、285 / 288、286 / 267、286 / 268、286 / 269、286 / 270、286 / 271、286 / 272、286 / 273、286 / 274、286 / 275、286 / 276、286 / 277、286 / 278、286 / 279、286 / 280、286 / 281、286 / 282、286 / 283、286 / 284、286 / 285、286 / 286、286 / 287、286 / 288、287 / 267、287 / 268、287 / 269、287 / 270、287 / 271、287 / 272、287 / 273、287 / 274、287 / 275、287 / 276、287 / 277、287 / 278、287 / 279、287 / 280、287 / 281、287 / 282、287 / 283、287 / 284、287 / 285、287 / 286、287 / 287、287 / 288，117 / 118、117 / 119、117 / 120、117 / 121、117 / 122、117 / 123、118 / 118、118 / 119、118 / 120、118 / 121、118 / 122、118 / 123、119 / 118、119 / 119、119 / 120、119 / 121、119 / 122、119 / 123、120 / 118、120 / 119、120 / 120、120 / 121、120 / 122、120 / 123、121 / 118、121 / 119、121 / 120、121 / 121、121 / 122、121 / 123、122 / 118、122 / 119、122 / 120、122 / 121、122 / 122、122 / 123，340 / 341、340 / 342、340 / 343、340 / 344、340 / 345、340 / 346、340 / 347、340 / 348、340 / 349、340 / 350、340 / 351、340 / 352、340 / 353、341 / 341、341 / 342、341 / 343、341 / 344、341 / 345、341 / 346、341 / 347、341 / 348、341 / 349、341 / 350、341 / 351、341 / 352、341 / 353、342 / 341、342 / 342、342 / 343、342 / 344、342 / 345、342 / 346、342 / 347、342 / 348、342 / 349、342 / 350、342 / 351、342 / 352、342 / 353、343 / 341、343 / 342、343 / 343、343 / 344、343 / 345、343 / 346、343 / 347、343 / 348、343 / 349、343 / 350、343 / 351、343 / 352、343 / 353、344 / 341、344 / 342、344 / 343、344 / 344、344 / 345、344 / 346、344 / 347、344 / 348、344 / 349、344 / 350、344 / 351、344 / 352、344 / 353、345 / 341、345 / 342、345 / 343、345 / 344、345 / 345、345 / 346、345 / 347、345 / 348、345 / 349、345 / 350、345 / 351、345 / 352、345 / 353、346 / 341、346 / 342、346 / 343、346 / 344、346 / 345、346 / 346、346 / 347、346 / 348、346 / 349、346 / 350、346 / 351、346 / 352、346 / 353、347 / 341、347 / 342、347 / 343、347 / 344、347 / 345, 347 / 346, 347 / 347, 347 / 348, 347 / 349, 347 / 350, 347 / 351, 347 / 352, 347 / 353, 348 / 341, 348 / 342, 348 / 343, 348 / 344, 348 / 345, 348 / 346, 348 / 347, 348 / 348, 348 / 349, 348 / 350 348 / 351, 348 / 352, 348 / 353, 349 / 341, 349 / 342, 349 / 343, 349 / 344, 349 / 345, 349 / 346, 349 / 347, 349 / 348, 349 / 349, 349 / 350, 349 / 351, 349 / 352, 349 / 353, 350 / 341, 350 / 342, 350 / 343 350 / 344, 350 / 345, 350 / 346, 350 / 347, 350 / 348, 350 / 349, 350 / 350, 350 / 351, 350 / 352, 350 / 353, 351 / 341, 351 / 342, 351 / 343, 351 / 344, 351 / 345, 351 / 346, 351 / 347, 351 / 348, 351 / 34 9, 351 / 350, 351 / 351, 351 / 352, 351 / 353, 352 / 341, 352 / 342, 352 / 343, 352 / 344, 352 / 345, 352 / 346, 352 / 347, 352 / 348, 352 / 349, 352 / 350, 352 / 351, 352 / 352, and 352 / 353, wherein the TtGBP protein has the sequence shown in SEQ ID NO: 1 or a functional fragment thereof, and the variants of the TtGBP protein contain mutations selected from the following sites: W8, W9, A42, H66, K312, V347, H348, and the variants of the fluorescent protein contain a mutation at the Y1 site. Preferably, the optically active polypeptide is selected from any one of the following: cpYFP, ​​cpGFP, cpBFP, and cpmApple.

2. The optical probe as described in claim 1, characterized in that, The fluorescent protein is as shown in any one of SEQ ID NO: 2-9, wherein the Y1 variant of the fluorescent protein is mutated to A or G; Preferably, the fluorescent protein is as shown in any one of SEQ ID NO: 2, 6, 7, 9.

3. The optical probe as described in claim 1, characterized in that, Among the variants of the TtGBP protein, W8 is mutated to R, H, E, A, V, L, F, I, M, C, N, G, K, D, or T; W9 is mutated to R, H, E, A, V, L, F, I, M, C, P, D, N, G, K, Y, S, or T; A42 is mutated to R, E, W, F, M, C, P, N, G, K, Y, or T; H66 is mutated to R, E, A, F, M, C, P, Q, or T; K312 is mutated to R, A, Q, G, H, or S; V347 is mutated to W; and H348 is mutated to T, G, or S. Preferably, the mutations in the variants of the TtGBP protein include mutations selected from any of the following groups: (1) V347W and H348T, (2) V347W and H348G, (3) V347W and H348S, (4) V347W, H348T and H66R, (5) V347W, H348T and H66E, (6) V347W, H348T and H66A, (7) V347W, H348T and H66F, (8) V347W, H348T and H66M, (9) V347W, H348T and H66C, (10) V347W, H348T and H66P, (11) V347W, H348T and H66Q, (12) V3 47W, H348T and H66T, (13)V347W, H348T and W9R, (14)V347W, H348T and W9H, (15)V347W, H348T and W9E, (16)V347W, H348T and W9A, (17)V347W, H348T and W9V, (18)V347 W, H348T and W9L, (19) V347W, H348T and W9F, (20) V347W, H348T and W9I, (21) V347W, H348T and W9M, (22) V347W, H348T and W9C, (23) V347W, H348T and W9P, (24) V347W, H 348T and W9N, (25)V347W, H348T and W9G, (26)V347W, H348T and W9K, (27)V347W, H348T and W9Y, (28)V347W, H348T and W9S, (29)V347W, H348T and W9T, (30)V347W, H348 T and W9D, (31) V347W, H348T and W8R, (32) V347W, H348T and W8H, (33) V347W, H348T and W8E, (34) V347W, H348T and W8A, (35) V347W, H348T and W8V, (36) V347W, H348T and W 8L, (37)V347W, H348T and W8F, (38)V347W, H348T and W8I, (39)V347W, H348T and W8M, (40)V347W, H348T and W8C, (41)V347W, H348T and W8N, (42)V347W, H348T and W8G, (43) V347W, H348T and W8K, (44) V347W, H348T and W8T, (45) V347W, H348T and W8D, (46) V347W, H348T and A42R, (47) V347W, H348T and A42E, (48) V347W, H348T and A42W,(49) V347W, H348T and A42F, (50) V347W, H348T and A42M, (51) V347W, H348T and A42C, (52) V347W, H348T and A42P, (53) V347W, H348T and A42N, (54) V347W, H348T and A42G, (55) V347W, H348T and A42K, (56) V347W, H34 8T and A42Y, (57)V347W, H348T and A42T, (58)V347W, H348T and K312R, (59)V347W, H348T and K312A, (60)V347W, H348T and K312Q, (61)V347W, H348T and K312G, (62)V347W, H348T and K312H, (63)V347W, H348T and K312S.

4. A fusion polypeptide comprising the optical probe of any one of claims 1-3 and other polypeptides, wherein the other polypeptides include a localization sequence, a tag for easy purification, or a tag for an immune response.

5. A nucleic acid molecule comprising a sequence selected from: (1) A multinucleotide sequence encoding the optical probe of any one of claims 1-3 or the fusion polypeptide of claim 4, and (2)(1) complementary sequences.

6. A nucleic acid construct comprising the nucleic acid molecule of claim 5. Preferably, the nucleic acid construct is an expression vector.

7. A host cell, wherein the host cell: (1) Containing, expressing or secreting the optical probe of any one of claims 1-3 or the fusion polypeptide of claim 4; (2) Contains the nucleic acid molecule as described in claim 5; or (3) It includes the nucleic acid construct of claim 6.

8. A method for preparing the optical probe of any one of claims 1-3 or the fusion polypeptide of claim 4, comprising culturing the host cell of claim 7 and isolating the optical probe from the culture.

9. The use of the optical probe of any one of claims 1-3, the fusion peptide of claim 4, the nucleic acid molecule of claim 5, or the nucleic acid construct of claim 6 in a kit for preparing a sample to detect D-glucose, screen compounds, or for intracellular and / or extracellular localization of D-glucose.

10. A test kit comprising... (1) The optical probe according to any one of claims 1-3 or the fusion polypeptide according to claim 4; (2) The nucleic acid molecule according to claim 5; (3) The nucleic acid construct according to claim 6; or (4) The host cell as described in claim 7; The detection kit may optionally also include other reagents required for the detection of D-glucose using an optical probe. Preferably, the test kit further comprises one or more reagents selected from the following: buffer solution, culture medium, and D-glucose standard.

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