TY - JOUR
T1 - Glucose sensor for low-cost lifetime-based sensing using a genetically engineered protein
AU - Tolosa, Leah
AU - Gryczynski, Ignacy
AU - Eichhorn, Lisa R.
AU - Dattelbaum, Jonathan D.
AU - Castellano, Felix N.
AU - Rao, Govind
AU - Lakowicz, Joseph R.
N1 - Funding Information:
This work was supported by a grant from the National Institutes of Health to G.R. (RR-10955) and from the National Center for Research Resources to J.R.L. (RR-08119). Unrestricted matching funds from Genentech, Merck, and Pfizer to G.R. are gratefully acknowledged. Dr. W. Boos, Universitat Konstanz, generously provided us the E. coli NM303.
PY - 1999/2/1
Y1 - 1999/2/1
N2 - We describe a glucose sensor based on a mutant glucose/galactose binding protein (GGBP) and phase-modulation fluorometry. The GGBP from Escherichia coli was mutated to contain a single cysteine residue at position 26. When labeled with a sulfhydryl-reactive probe 2-(4'- iodoacetamidoanilino)naphthalene-6-sulfonic acid, the labeled protein displayed a twofold decrease in intensity in response to glucose, with a dissociation constant near 1 μM glucose. The ANS-labeled protein displayed only a modest change in lifetime, precluding lifetime-based sensing of glucose. A modulation sensor was created by combining ANS26-GGBP with a long- lifetime ruthenium (Ru) metal-ligand complex on the surface of the cuvette. Binding of glucose changed the relative intensity of ANS26-GGBP and the Ru complex, resulting in a dramatic change in modulation at a low frequency of 2.1 MHz. Modulation measurements at 2.1 MHz were shown to accurately determine the glucose concentration. These results suggest an approach to glucose sensing with simple devices.
AB - We describe a glucose sensor based on a mutant glucose/galactose binding protein (GGBP) and phase-modulation fluorometry. The GGBP from Escherichia coli was mutated to contain a single cysteine residue at position 26. When labeled with a sulfhydryl-reactive probe 2-(4'- iodoacetamidoanilino)naphthalene-6-sulfonic acid, the labeled protein displayed a twofold decrease in intensity in response to glucose, with a dissociation constant near 1 μM glucose. The ANS-labeled protein displayed only a modest change in lifetime, precluding lifetime-based sensing of glucose. A modulation sensor was created by combining ANS26-GGBP with a long- lifetime ruthenium (Ru) metal-ligand complex on the surface of the cuvette. Binding of glucose changed the relative intensity of ANS26-GGBP and the Ru complex, resulting in a dramatic change in modulation at a low frequency of 2.1 MHz. Modulation measurements at 2.1 MHz were shown to accurately determine the glucose concentration. These results suggest an approach to glucose sensing with simple devices.
UR - http://www.scopus.com/inward/record.url?scp=0033081688&partnerID=8YFLogxK
U2 - 10.1006/abio.1998.2974
DO - 10.1006/abio.1998.2974
M3 - Article
C2 - 9918662
AN - SCOPUS:0033081688
SN - 0003-2697
VL - 267
SP - 114
EP - 120
JO - Analytical Biochemistry
JF - Analytical Biochemistry
IS - 1
ER -