Sol-gel materials with antibody properties are described. These were constructed by the entrapment of monoclonal anti-atrazine antibodies (Mabs) in SiO 2 sol-gel derived matrixes, which successfully recognized and bound atrazine, a widely used herbicide. The binding properties for atrazine were evaluated by comparing sol-gel entrapped anti-atrazine hybridoma culture fluids with entrapped purified immunoglobulins (IgGs). In the study, 14 C-labeled and unlabeled atrazine, which are stable to hydrolysis or to other chemical decay processes, were used as analytes. Leaching of the antibodies was found to be zero. Stability was tested under various storage conditions and was found to be 100% for at least 2 months at room temperature, compared with a drop of 40% in solution. The response time was found not to differ considerably from that obtained in solution. Other factors tested included reproducibility of binding, dose response, nonspecific physisorption of atrazine to the ceramic matrix, and elution recoveries of atrazine from the doped sol-gel columns. An advantage of the sol-gel methodology is the elimination of the need to purify the IgGs from the Mab hybridoma culture fluids.
A sol-gel-based method for immunoaffinity purification using sol-gel-entrapped antidinitrophenyl (DNP) antibodies (Abs) was developed. Polyclonal antiserum (whole antiserum) and purified immunoglobulines (IgGs, isolated from the whole antiserum), which recognize nanogram quantities of a variety of di-and trinitroaromatic compounds, were entrapped in SiO 2 sol-gel-derived matrixes, and their binding properties were examined with 2,4dinitrophenylhydrazine (DNPH) used as an analyte. Binding properties of the entrapped Abs were determined by the evaluation of the optimal sol-gel composition for entrapment and the optimal conditions for binding and elution of the analyte. We found that a hydrophilic, flexible "wet" gel with a tetramethoxysilane:aqueous ratio of 1:8, enriched with 10% PEG exhibited high binding capacities with low nonspecific binding. Under the tested conditions the sol-gel-entrapped Abs bound the analyte in a dose-dependent, highly reproducible manner (antibody-and antigen-wise), and binding was equally effective with either polyclonal whole antiserum or protein A purified IgGs (eliminating the need to purify IgGs from the whole antiserum). The analyte could easily be eluted at high recoveries (90%) and the Abs were well-retained in the sol-gel matrix and did not leach out even at extreme pH conditions or in organic solvents. The sol-gel immunoaffinity columns exhibited binding capacities that were either significantly higher or did not differ significantly from those of protein A-agarose covalently coupled Abs over a wide range of IgG (0.5-15 µL corresponding to 1-30 µg protein) and analyte amounts (20-320 ng).
We describe recent advances made in our laboratories in the general field of organically and bio-organically doped sol-gel sensors. The developments described are: (a) The first miniaturization of a sol-gel sensor down to the microns scale, with potential applications to near-field optical microscopy, using a fluorescent pH-indicator. (b) The first successful sol-gel encapsulation of purified polyclonal antibodies, and in particular an anti-nitroaromatics immunoglobulin, with which selective sensing of nitroaromatics, an important class of environmental pollutants, was demonstrated, (c) The leaching problem, occasionally encountered in doping procedures, is solved by two methodologies: First, TMOS polymerization at high acidity and low water content was found to result in non-leachable yet reactive matrices, as demonstrated with O2 sensing by excited state pyrene and with H+ sensing by excited state pyranine; and second, doping with molecules capable of forming a covalent bond within the encapsulating cage results in the permanent anchoring of the dopant. Thus, Methyl-Red, a pH indicator, was derivatized with a silylating residue, and a polymerizing TMOS was doped with it forming a pH-shifted indicator. With both methodologies, leachability was practically zero.
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