Sensors and Actuators B 97 (2004) 81–89 Phosphate binding protein as the biorecognition element in a biosensor for phosphate Lyndon L.E. Salins, Sapna K. Deo, Sylvia Daunert ∗ Departments of Chemistry and Pharmaceutical Sciences, University of Kentucky, Lexington, KY 40506-0055, USA Received 3 September 2002; received in revised form 25 July 2003; accepted 29 July 2003 Abstract This work explores the potential use of a member of the periplasmic family of binding proteins, the phosphate binding protein (PBP), as the biorecognition element in a sensing scheme for the detection of inorganic phosphate (P i ). The selectivity of this protein originates from its natural role which, in Escherichia coli, is to serve as the initial receptor for the highly specific translocation of P i to the cytoplasm. The single polypeptide chain of PBP is folded into two similar domains connected by three short peptide linkages that serve as a hinge. The P i binding site is located deep within the cleft between the two domains. In the presence of the ligand, the two globular domains engulf the former in a hinge-like manner. The resultant conformational change constitutes the basis of the sensor development. A mutant of PBP (MPBP), where an alanine was replaced by a cysteine residue, was prepared by site-directed mutagenesis using the polymerase chain reaction (PCR). The mutant was expressed, from plasmid pSD501, in the periplasmic space of E. coli and purified in a single chromatographic step on a perfusion anion-exchange column. Site-specific labeling was achieved by attaching the fluorophore, N-[2-(1-maleimidyl)ethyl]-7-(diethylamino)coumarin-3-carboxamide (MDCC), to the protein through the sulfhydryl group of the cysteine moiety. Steady-state fluorescence studies of the MPBP–MDCC conjugate showed a change in the intensity of the signal upon addition of P i . Calibration curves for P i were constructed by relating the intensity of the fluorescence signal with the amount of analyte present in the sample. The sensing system was first developed and optimized on a spectrofluorometer using ml volumes of sample. It was then adapted to be used on a microtiter plate arrangement with l sample volumes. The system’s versatility was finally proven by developing a fiber optic fluorescence-based sensor for monitoring P i . In all three cases the detection limits for the analyte were in the sub-M range. It was also demonstrated that the sensing system was selective for phosphate over other structurally-similar anions, paving the way for the design and development of a new family of biosensors utilizing the specific binding properties of periplasmic proteins. © 2003 Elsevier B.V. All rights reserved. Keywords: Phosphate binding protein (PBP); Conformational change; Site-directed mutagenesis; Fluorescence; Fiber optic sensor; Phosphate 1. Introduction The transport of ligands in Gram-negative bacteria like Escherichia coli (E. coli) and Salmonella is mediated by periplasmic binding proteins (PBPs). There are over two dozen such proteins which serve as an uptake sys- tem for sugars, oxyanions, amino acids, and oligopeptides [1,2]. Phosphate uptake in E. coli is mediated by the phosphate-specific transport (PST) system [3–7], which is specific for inorganic phosphate (P i ) [5,7–9]. The synthesis of the PBP, the product of the phoS gene [10], is induced in E. coli under conditions of P i limitation. Mature PBP consists of 321 amino acids with a molec- ular weight of 34,422 Da [11]. In E. coli, there are 20,000 ∗ Corresponding author. Tel.: +1-859-257-7060; fax: +1-859-323-1069. E-mail address: daunert@pop.uky.edu (S. Daunert). molecules of PBP per cell [12]. From kinetic plots and the resin method for assaying binding activity, it has been shown that one protein molecule binds to one molecule of phosphate [13]. PBP can bind to both monobasic (H 2 PO 4 - ) and dibasic (HPO 4 2- ) phosphate [14]. It binds P i rapidly (1.36 × 10 8 M -1 s -1 ) and in a highly specific manner with a K d of 0.1 M. The dissociation rate constant is 21 s -1 at pH 7.0 and at low-ionic strength [15]. PBP consists of two globular domains connected by three short peptide segments that serve as a flexible hinge [16]. In the absence of P i , the two domains remain far apart with the cleft accessible to solvent. The binding site for the ligand is located deep within this cleft. P i forms hydrogen bonds with eleven donor groups and one acceptor group. In the active site of PBP, the eleven donor groups form hydrogen bonds with the four oxygen atoms of P i . The lone acceptor is Asp56 whose carboxylate side-chain interacts with the hydrogen atom of the phosphate molecule [17]. In the bound-form 0925-4005/$ – see front matter © 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.snb.2003.07.019