After 20 min of treatment at this temperature, GOD (final concentration 5 mg mL?1) was added under vigorous stirring and then the mixture was poured into a Plexiglas square frame, 4 x 4.5 cm in size and 5 mm in depth. The preparation was quickly put into a freezer at ?24 ��C and after 16 h brought back to room temperature, thus obtaining a flexible gelatine membrane, which was extensively washed with distilled water. At this point rectangular pieces of the same size (40 mm �� 13 mm) were cut and used for fluorescence measurements. In this way, catalytic membrane comparable for dimension and amount of entrapped GOD were obtained. The gelatine membranes had a lattice structure, which efficiently held the biocatalyst and allowed free diffusion of substrate and reaction products.

When not used, the membranes were stored at 4 ��C in 0.1 M acetate buffer, pH 5.0.2.2.2. Fluorescence measurements2.2.2.1. Intrinsic fluorescence emission measurementsGOD is an oxidase and exhibits at pH 6.5 a very intense UV fluorescence with an emission maximum at 334 nm and two absorption maxima at 224 nm and 278 nm due to tryptophan. GOD is also a typical flavoprotein. GOD from A. niger is a dimmer with two very tightly bound FAD molecules per dimer. As all flavoproteins, GOD shows absorption maxima at about 380 and 450 nm and an intrinsic fluorescence with an emission maximum at about 530 nm, at pH 7.0. As previously reported changes in the fluorescence of free and immobilized GOD have been found during its interaction with glucose, since the oxidized and reduced flavines have been found to exhibit different fluorescences [12, 14, 18].

In this research the emission fluorescence spectra have been collected by means of a spectrofluorimeter (Perkin-Elmer, model LS55) equipped with a Xenon discharge lamp with an emission spectrum ranging from 200 to 800 nm. Sample excitation was performed at 295 Cilengitide nm, while the emission spectrum was recorded in the range 310 �C 400 nm. Spectra have been acquired with entrance and exit slits fixed at 5 nm and with a scan speed of 100 nm s-1. Just to give an example, in Figure 1a the normalized emission fluorescence spectra of free GOD in the presence (2 mM) or in the absence of glucose are reported. Figure 1a shows a fluorescence increase (about 20% for both peak and integral values) when glucose is in the aqueous solution.

In Figure1b the normalized emission fluorescence spectra for GOD entrapped into the gelatine membrane in the presence (20 mM) or in the absence of glucose are reported. Also in this case a fluorescence increase (nearly equal to 20% for both peak and integral values) is evident in the presence of glucose. We have checked that the changes in the fluorescence were not due to GOD diffusion from gelatine to solution. In fact fluorescence spectra were absent after removing the catalytic gelatine membrane.

The optimal buffers, storage conditions, and other procedures to attach biomolecules to glass surfaces, such as microarrays, are beginning to be developed [8,9].Microarrays are traditionally orthogonally-arrayed micron-diameter spots, at micron-spaced distances on microscope slides (typically referred to as substrates), which contain biomolecules that are chemically attached to the surface. To produce the spots, small droplets are applied to the surface using either robotic or manual printing techniques. Microarrays have been used extensively in the past 10 years, especially those containing nucleic acid sequences for gene expression studies [10]. More recently, microarrays containing protein have been developed and used to study protein-protein interactions [11].

Perhaps the most significant characteristic of microarrays, and the reason for their popularity, is their ability to contain thousands of spots per substrate, and therefore, simultaneously accommodate thousands of analyses with a single sample. Thus, in the past few years, efforts to produce microarray biosensors, which serve diagnostic purposes, have been undertaken [12-14]. In particular, combining the sandwich immunoassay with microarray format is a current area of interest [12,13,15].In order to reduce stresses on immobilized antibodies, print buffers with various salts, surfactants, and stabilizers have been developed [9]. In an early protein microarray article [11], antibodies were reconstituted in phosphate-buffered saline (PBS) plus 40% glycerol, and a recent report [16] has indicated that PBS with 20% glycerin (glycerol) produced a superior microarray response signal relative to PBS alone.

The authors speculated that glycerol served as a protein stabilizer by maintaining a hydrated state [16]. We recently developed an Anacetrapib antibody microarray method for the capture and detection of E. coli O157:H7 [17]. It became apparent that the interactions of the biotinylated capture antibodies in PBS/glycerol spots with the streptavidin-coated glass substrate markedly affected the immunoassay, at least in terms of whole bacterial cell detection. Therefore, in this study, evidence for thixotropic-like properties of the glycerol-containing spots is presented, and the implications of these properties on bacterial capture and immunoassay results, within a protein microarray format, are examined.2.?Results and DiscussionIn order to determine background fluorescent signals, the appropriate blank samples were analyzed. Immunoassays performed without bacteria, but treated with reporter antibody, generated fluorescent signals that were less than, or equal to, the localized background AFU (arbitrary fluorescence units; data not shown) measurements.