Silica Sol–Gel Optical Biosensors: Ultrahigh Enzyme Loading Capacity on Thin Films via Kinetic Doping

Abstract

Easy to use and easy to produce biosensors would have a huge range of applications. To reach this goal many see the incorporation of a protein into a sol-gel network as one of the most viable options. The current most prevalent technique of predoping presents inherent limits on the concentration possible for the resulting thin film. In this study we demonstrate a new process utilizing the newly developed kinetic doping method to load silica sol-gel thin films with cytochrome C (CytC) and horseradish peroxidase (HRP). Both enzymes are shown to successfully load and have a concentration increase over their original loading solution by factors of 1300× and 2600×, respectively. Furthermore, each enzyme once loaded retained the ability to act as a catalyst for the detection of hydrogen peroxide. Ultimately the CytC- and HRP-loaded thin films were found to have enzyme concentrations of 11 ± 1 mM and 6.0 ± 0.4 mM, respectively, a considerable step up from any doping method reported in the past.

Kinetically Doped Silica Sol–Gel Optical Biosensors: Expanding Potential Through Dip-Coating

“… 21 Subsequent experiments employing more stringent procedures that significantly suppress the vibration were able to produce more evenly loaded thin films such as the film shown in Figure 1 B. Compared to that for spin-coating, where the optimal time delay is near zero, 15 the optimal time delay for dip-coating is longer. This can be explained as in spin-coating with its lengthy 70 s spinning process as well as approximately 30 more seconds for the spin coater to slow to a complete halt, which followed by another 10 s to dislodge the thin-film-coated coverslip from the vacuum hold before transferring to the loading solution, the time between the glass coverslip coming into contact with the sol solution and when it is available to be immersed in the loading solution in spin-coating is greater than the time needed for the dip-coating process.…”

“…The amounts of CytC and HRP loaded into the dip-coated films are summarized in Table 1 along with reference values obtained from loaded samples made through spin-coating 15 as well as a post-doped spin-coated sample and a blank coverslip control. Although the average change in absorbance for the dip-coated samples is nearly double that of the spin-coated sample, this is primarily due to the fact that the dip-coated method can produce thin films on both sides of the glass coverslip.…”

“…Recently, our group has reported a new technique, known as kinetic doping, 14 to produce thin films highly loaded with the horseradish peroxidase (HRP) or cytochrome C (CytC) proteins via spin-coating. 15 As opposed to the established techniques, 16 − 18 pre-doping where a guest molecule is included with the sol solution and post-doping where any guest molecule is adsorbed to the surface-accessible areas of the film after the sol–gel chemistry for thin film formation has completed, often after heat annealing, kinetic doping takes advantage of a window of opportunity after casting the film and driving off the vast majority of the ethanol, but before the sol–gel chemistry on the film has progressed to a significant degree. The quick immersion in an aqueous loading solution significantly slows the poly-condensation step of the sol–gel process, extending the window for dopant loading before the film is fully set.…”

Enzyme Loading in Internally-Coated Capillary Tubes Via Kinetic Doping

“…Initially, attempts were made to quantify HRP loading in capillary tubes with the modified Bradford Assay developed by Crosley et al [8]. Briefly, Coomassie Brilliant Blue was pumped into capillaries and allowed to equilibrate.…”

“…Many researchers have investigated the encapsulation of enzymes in silica in order to gain the benefits of immobilization, namely, high reusability and higher chemical and thermal stability [1][2][3][4][5][6][7][8][9][10][11]. Sol-gel-based technologies are especially promising, with their ease of synthesis and high loading capacity [9,12].…”