pH‐insensitive glucose indicators

Abstract

There is an urgent need for developing a biosensor that can real-time and noninvasively determine glucose concentration within living cells. In our previous study, we have engineered a glucose indicator protein (GIP) that can provide continuous glucose monitoring through a conformation change-induced Fo¨rster resonance-energy transfer measurement. Because of the pH-sensitivity of the fluorescent proteins used in the GIP construction, the GIP made from these fluorescent proteins is less tolerant to a pH change, especially to the acidic environment. It has been well documented that intracellular pH does not always remain the same, and it fluctuates in metabolism and other cellular activities and also differs between cellular compartments. To address these issues, we developed a GIP that can tolerate to pH change. This GIP was constructed by flanking a glucose binding protein with a cyan fluorescent protein and a pH-insensitive yellow fluorescent protein. Our experimental results indicated that the new GIP is more tolerant to pH change. The glucose response of this new GIP kept almost unchanged from pH 7.3 to 5.3, suggesting its capability of tolerating to acidic environment. This capability is desirable for intracellular glucose measurement.

A glucose sensor protein for continuous glucose monitoring

“…We have previously constructed a glucose indicator protein(GIP)for continuous glucose monitoring through ratiometric FRET measurement(Garrett et al 2008; Ye and Schultz 2003). GIP was developed using a GBP with very strong affinity for glucose.…”

“…Another modification that we made to improve the stability and sensitivity of the glucose sensor protein is to use a new FRET pair in place of the enhanced cyan fluorescent protein (ECFP) and enhanced yellow fluorescent protein (EYFP)FRET pair used in our early study (Garrett et al 2008). As pointed out by a number of studies, the crosstalk or bleed-through between ECFP and EYFP can remarkably interfere with quantitative analysis performed using ECFP/EYFP pair-based FRET sensor(Erickson et al 2003; Gordon et al 1998; Rizzo et al 2004).…”

“…To meet these needs, we engineered a new glucose responsive protein (Garrett et al 2008; Ye and Schultz 2003). The glucose sensing ability of the protein was established by introducing a FRET (fluorescence resonance energy transfer) signal transduction mechanism directly into a glucose binding protein (GBP) in such a manner that the binding of glucose to the protein generates FRET signals for CGM through ratiometric FRET measurement (Garrett et al 2008; Ye and Schultz 2003).…”

“…The glucose sensing ability of the protein was established by introducing a FRET (fluorescence resonance energy transfer) signal transduction mechanism directly into a glucose binding protein (GBP) in such a manner that the binding of glucose to the protein generates FRET signals for CGM through ratiometric FRET measurement (Garrett et al 2008; Ye and Schultz 2003). To further adjust the glucose response of the protein to a range that covers glucose levels in body fluids, we modified the protein structure through site-directed mutagenesis ( Jin, et.…”

Reagentless fluorescent biosensors based on proteins for continuous monitoring systems

“…The pioneering work developed by Ye and Schultz [20] using GFP and yellow fluorescence protein (YFP) already indicated that GFP was not sufficiently suitable because of the excitation wavelength. Subsequently, the enhanced cyan fluorescence protein (ECFP) and enhanced yellow fluorescence protein (EYFP) seemed more appropriate [21]. During the initial studies of this alternative, GFP were located at the ends of the two GBP lobes.…”