Glutamine-Binding Protein from Escherichia coli Specifically Binds a Wheat Gliadin Peptide Allowing the Design of a New Porous Silicon-Based Optical Biosensor † Luca De Stefano, ‡ Mauro Rossi, § Maria Staiano, | Gianfranco Mamone, § Antonoietta Parracino, | Lucia Rotiroti, ‡ Ivo Rendina, ‡ Mose ` Rossi, | and Sabato D’Auria* ,| Institute for Microelectronics and Microsystems, CNR, Naples, Italy, Institute of Food Sciences, CNR, Avellino, Italy, and Institute of Protein Biochemistry, CNR, Naples, Italy In this work, the binding of the recombinant glutamine-binding protein (GlnBP) from Escherichia coli to gliadin peptides, toxic for celiac patients, was investigated by mass spectrometry experiments and optical techniques. Mass spectrometry experiments demonstrated that GlnBP binds the following amino acid sequence: XXQPQPQQQQQQQQQQQQL, present only into the toxic prolamines. The binding of GlnBP to gliadin suggested us to design a new optical biosensor based on nanostructured porous silicon (PSi) for the detection of trace amounts of gliadin in food. The GlnBP, which acts as a molecular probe for the gliadin, was covalently linked to the surface of the PSi wafer by a proper passivation process. The GlnBP-gliadin interaction was revealed as a shift in wavelength of the fringes in the reflectivity spectrum of the PSi layer. The GlnBP, covalently bonded to the PSi chip, selectively recognized the toxic peptide. Finally, the sensor response to the protein concentration was measured in the range 2.0-40.0 μg/L and the sensitivity of the sensor was determined. Keywords: gliadin • optical biosensors • porous silicon • celiac disease There is a strong need for integrated and automated bio- analytical systems in medical, food, agricultural, environmental, and defense testing. Now, the main market is shared between blood glucose, pregnancy, antibody-based infectious diseases, and biological warfare agent detection. 1 The interaction be- tween an analyte and a biological recognition system is normally detected in biosensors by the transducer element which converts the molecular event into a measurable effect, such as an electrical or optical signal. Because of its spongelike structure, porous silicon (PSi) is an almost ideal material as a transducer: its surface has a specific area of the order of 200- 500 m 2 cm -3 , so that a very effective interaction with liquid or gaseous substances is assured. 2,3 PSi optical sensors are based on changes of photoluminescence or reflectivity when exposed to the target analytes which substitute the air into the PSi pores. 4 The effect depends on the chemical and physical properties of each analyte, so that the sensor can be used to recognize the pure substances. Because of the sensing mech- anism, these kinds of devices are not able to identify the components of a complex mixture. To enhance the sensor selectivity through specific interactions, some researchers have proposed to chemically or physically modify the PSi surface; the common approach is to create a covalent bond between the porous silicon surface and the biomolecules which specif- ically recognize the unknown analytes. 5-7 Among different probes of biological nature, ligand-binding proteins are par- ticularly good candidates in designing highly specific biosensors for small analytes; in particular, the glutamine-binding protein (GlnBP) from Escherichia coli is a monomeric protein com- posed of 224 amino acid residues (26 kDa) responsible for the first step in the active transport of L-glutamine across the cytoplasm membrane. The GlnBP consists of two similar globular domains, the large domain (residues 1-84 and 186- 224) and the small domain (residues 90-180), linked by two peptides. The deep cleft formed between the two domains contains the ligand-binding site. The GlnBP binds L-glutamine with a dissociation constant Kd of 5 × 10 -9 M 8 as well as poly- glutamine residues. In this study, we show that GlnBP is able to bind with high affinity the amino acid sequences present in the gliadin allowing the design of a new reagentless microsen- sor for the optical interferometric detection of gliadin based on a chemically modified porous silicon nanostructured surface covalently linking the GlnBP. The gliadin by the wheat gluten is mainly responsible of inducing celiac disease, an inflamma- tory disease of the small intestine affecting genetically suscep- tible people. 9 Presently, a strict gluten-free lifelong diet is mandatory for celiac patients for both intestinal mucosal recovery and prevention of complicating conditions such as lymphoma. However, dietary compliance has been shown to † The authors wish to dedicate this work to the memory of Prof. Arturo Leone, Director of the Institute of Food Sciences, CNR, Avellino, Italy. * Addess correspondence to Dr. Sabato D’Auria, Institute of Protein Biochemistry, Consiglio Nazionale delle Ricerche, Via Pietro Castellino 111, 80131 Naples, Italy. Fax, +39-0816132277; e-mail, s.dauria@ibp.cnr.it. ‡ Institute for Microelectronics and Microsystems, CNR. § Institute of Food Sciences, CNR. | Institute of Protein Biochemistry, CNR.