Use of a Cyanine Dye as a Reporter Probe in Reagentless Maltose Sensors Based on E. coli Maltose Binding Protein

ABSTRACT

Reagentless bio‐sensing proteins represent a promising technology being developed for sensitive chemical detection and analysis. The majority of these sensor proteins incorporate environmentally sensitive UV‐excited fluorescent dyes as reporter probes. In this report, we describe conjugation of the commercially available visible wavelength‐excitable polymethine cyanine dye Cy3 (λ_max abs = 556 nm; λ_max em = 567 nm) to several single‐cysteine variants of maltose binding protein (MBP), followed by evaluation of each of these labeled proteins as potential reagentless sensors for maltose. Four of six MBP variants labeled with the Cy3 probe functioned successfully in a homogenous reagentless sensing mode, and yielded maltose dissociation constants ranging from 74 µM to 3.8 mM. Cy3 and other cyanine dye derivatives may be useful as long‐wavelength excitable reporter groups in other reagentless sensor designs.

Keywords:

• Maltose binding protein
• Reagentless sensors
• Fluorescence
• Biosensor
• Cyanine dye
• Reporter probe

ACKNOWLEDGMENTS

The authors thank Prof. Homme Hellinga (Duke University) for providing the plasmid with the MBP‐HIS‐tagged gene sequence utilized. ILM was supported by a National Research Council (NRC) fellowship through the Naval Research Laboratory. This research is supported by the office of Naval Research. The views, opinions, and/or findings described in this report are those of the authors and should not be construed as official Department of the Navy positions, policies, or decisions.

Notes

Note: NA—Not applicable.

^aDistance from the S atom of cysteine in mutated MBP structure to the linking O atom of the α1‐4 disaccharide linkage in maltose.

^bChange in fluorescence as a percent from 0 maltose concentration to saturation.