Construction of a panel of glucose indicator proteins for continuous glucose monitoring

Abstract

The development of implantable glucose sensors for use in diabetes treatment has been pursued for decades. However, enzyme-based glucose sensors often fail in vivo. In our previous work, we engineered a novel glucose indicator protein (GIP) that can sense glucose without relying on any enzymes and cofactors. Nevertheless, this GIP is unsuitable for blood glucose monitoring due to its low dissociation constant. Here, we report a novel approach to creating a new GIP that can be used to monitor blood glucose level. By disrupting pi-pi stacking around GIP's glucose binding site through site-directed mutagenesis, we showed that GIP's dissociation constant can be manipulated from 0.026 mM to 7.86 mM. This approach yielded four GIP mutants. We showed that one of the mutants can be used to detect glucose from 0 to 32 mM, while another mutant can be employed to visualize intracellular glucose (0-200 μM) within living cells through FRET imaging microscopy measurement.

Fluorescence Lifetime Imaging Microscopy of Intracellular Glucose Dynamics

“…[39][40][41][42] Another finding in this study is that, compared to the multiexponential decay of ECFP, 43 AcGFP1 exhibits only monoexponential decay, which makes it suitable for continuous intracellular glucose monitoring through FD-FLIM (Figure 3). The dissociation constant of the sensor protein for glucose was estimated to be 0.13 mM, 10 making it suitable for use in monitoring intracellular glucose concentrations because the mean intracellular glucose concentration is approximately 0.10-0.20 mM in both healthy humans and those with diabetes. 3 We have also found that FRET-FLIM is less affected by light scattering and the local concentration of sensor proteins, 38,[44][45][46][47] making it ideal for three-dimensional (3D) imaging.…”

Reagentless fluorescent biosensors based on proteins for continuous monitoring systems

“…In an attempt to combine the best of both systems, an interesting approach consists in immobilizing the GBP into dialysis tubing and placing this in a luminometer flow-cell; the pore diameter of the tube enables glucose perfusion without protein leaching [20]. This system has been recently tested as a sensor for continuous monitoring [32,33]. The reporting was FRET using two GFP mutants and the K D of the mutant GBP was tuned to glucose blood levels; however, all assays were performed using glucose solutions.…”

Opportunities for bioprocess monitoring using FRET biosensors

“…Figure 2 shows a schematic representation of central metabolism and amino acid biosynthesis, features of metabolism that are relatively conserved across organisms. The targets that can currently be detected include glucose [7,9,[15][16][17][18][19][20], glutamine [7,14,21,22], glutamate [9,14,[23][24][25], tryptophan [11,26], arginine [14,27], histidine [14], leucine [14], lactate [12], citrate [28] and other C 4 carboxylates [8], as well as the associated cofactor ATP [20,29]. Several FRET biosensors have been developed to monitor metabolites within these pathways (Figure 2, red boxes).…”

“…The use of FRET biosensors to directly quantify the intracellular concentration of metabolites has been demonstrated in several types of cells [7,[15][16][17][18][19][20]. Glucose is the most common metabolite to be examined quantitatively, in part because FRET biosensors for glucose have been developed with a very high signal-to-noise ratio.…”