Biosensing Elements Using Biologically Active Sol Gel Thin Films

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The Beta Switch Program by Sue Heintze

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The potential use of sol-gel-based materials as sensing elements has made these materials the subject of intensive study (27,28). Although it is possible to use monoliths, for biosensing applications, the use of thin films is preferred. The primary reason is that thin films have a reduced diffusion length for the target analyte, and, therefore, there is a greatly reduced response time, because diffusion distance varies with i12. The sol-gel matrix has an interconnected network of pores and presents a tortuous path for diffusion of analytes. For thin films, there is an important interplay among porosity and pore size, diffusion coefficient, and response time (29).

Sol-gel thin films are usually deposited on substrates using either dip coating or spin coating. In dip coating, the thickness can be generally controlled by the withdrawal speed, and it is possible to fabricate coatings with excellent optical transparency. Moreover, it is possible to dip coat onto substrates with a variety of geometries, from planar substrates to optical fibers. If spin coating is used, the thickness can be tailored using the spin rate, and, again, films can have excellent optical transparency.

The fabrication of high-quality sol-gel thin films with functional and active biomolecules is by no means trivial. Generally, biomolecules prefer a near-neutral pH and low-alcohol environment. Under these conditions, the condensation reaction in sol-gel synthesis is accelerated, leading to rapid gelation. A major factor in thin film synthesis is that a reasonable gelation time is required, especially for dip coating. One of the most effective means not only to lengthen the gelation time, but also to lower the viscosity of the sol, is the addition of alcohol. Large and delicate proteins can be particularly sensitive to the presence of alcohol, but apparently some proteins, such as antibodies, still retain their biological function. The fabrication of reproducible thin films with uniform thickness is also necessary if these materials are to be used on a large scale. One advantage of sol-gel coatings is that by matching the coating material with the substrate (e.g., silica coatings on silica substrates), strong adhesion between coating and substrate can be attained.

We have successfully prepared biologically active thin films with immobilized antibodies. The encapsulation of antibodies in sol-gel matrices has been studied extensively (16,30-40) although only limited work has been performed with thin films. The synthesis protocol for thin films is similar to that for monoliths, except for the addition of methanol to the silica sol and buffered protein mixture. The thin films were deposited on glass substrates by dip coat-

Fig. 15. Fluorescence signal as a function of Oregon Green-cortisol concentration for sol-gel silica thin films containing 0.5 or 1.0 |iM anticortisol antibody. The sol-gel-encapsulated antibodies retained their ability to bind antigen, and by increasing the antibody concentration, higher signals can be obtained. (Reproduced from Ref. 41 with permission.)

Fig. 15. Fluorescence signal as a function of Oregon Green-cortisol concentration for sol-gel silica thin films containing 0.5 or 1.0 |iM anticortisol antibody. The sol-gel-encapsulated antibodies retained their ability to bind antigen, and by increasing the antibody concentration, higher signals can be obtained. (Reproduced from Ref. 41 with permission.)

ing. We encapsulated anticortisol antibodies in sol-gel silica thin films and used these materials as sensing elements in an immunoassay for cortisol (41). The antibody-doped thin films were of excellent quality and optical transparency, with a thickness of approx 1 |m for films in the "wet" state, and a thickness of approx 0.5 |m for films in the "dried" state. Although a significant amount of methanol was used (approx 30 vol%) in the dip-coating solution, the immobilized antibodies retained the ability to bind their target antigen (cortisol in this case). In our experiments, antibody-antigen binding was detected optically using fluorescent Oregon Green-labeled cortisol. Oregon Green is a derivative of fluorescein and experiences excitation and emission in the visible region (495 and 527 nm, respectively). As seen in Fig. 15, with a constant antibody concentration in the thin films, the fluorescence signal increased with increasing concentration of labeled antigen. Moreover, by raising the antibody concentration in the thin films, one can obtain higher optical signals.

Note that all experiments were conducted with "wet" films, in which pore sizes remain relatively large. When films were allowed to dry fully, no antibody-antigen binding could be observed. We conducted competitive immuno-assays for cortisol using the anticortisol-doped silica thin films. In these immunoassays, in which labeled and unlabeled antigen compete for a fixed number of antibody molecules, the measured signal varies logarithmically with

Fig. 16. Calibration curves from competitive immunoassays conducted with solgel silica thin films containing anticortisol and with surface-adsorbed anticortisol. With sol-gel encapsulation, we were able to obtain substantially higher signals because of the ability to immobilize a substantially higher number of biomolecules per unit area compared to traditional monolayer surface adsorption. (Reproduced from Ref. 41 with permission.)

Fig. 16. Calibration curves from competitive immunoassays conducted with solgel silica thin films containing anticortisol and with surface-adsorbed anticortisol. With sol-gel encapsulation, we were able to obtain substantially higher signals because of the ability to immobilize a substantially higher number of biomolecules per unit area compared to traditional monolayer surface adsorption. (Reproduced from Ref. 41 with permission.)

unlabeled antigen concentration, and the slope is negative (42,43). The competitive immunoassays with anticortisol sol-gel thin films exhibited the expected behavior; a calibration curve with a negative slope was obtained (Fig. 16). Moreover, with a higher antibody concentration in the thin film, higher signals were achieved.

When immobilizing biomolecules, one distinct advantage of sol-gel encapsulation is the ability to create 3D architectures. With traditional immobilization methods, such as surface adsorption or covalent attachment, monolayer coverage of the biomolecule is usually obtained. With sol-gel encapsulation, however, one can achieve a substantially higher number of biomolecules per unit area because of the 3D nature of the bioactive material. The total number of biomolecules immobilized depends on the thickness of the material. This feature was demonstrated by comparing competitive immunoassay results for anticortisol encapsulated in sol-gel thin films (approx 1 ^m thickness) and surface adsorbed on polystyrene. As seen in Fig. 16, the fluorescence signals from the sol-gel films were as much as 10 times higher than those measured using surface-adsorbed antibody.

Finally, there is a distinct advantage with thin films in terms of reduced response times. The immunoassay with anticortisol thin films (approx 1 ^m thickness) required an incubation of only 20 min, and experiments showed that a plateau in the signal was reached after approx 10 min (data not shown). When analogous immunoassays were performed with anticortisol-doped silica monoliths of 1-mm thickness, the required incubation time for the assay was at least 3 h. For biological assays, therefore, thin films are far more effective than monoliths because limitations owing to analyte diffusion through the porous matrix can be minimized.

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