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.