4. Notes

1. Suggested ratios are shown in Table 1. However, other ratios, as well as other precursors, can be used. It should be taken into account that the higher the TMOS proportion, the faster the subsequent polymerization process.

2. Hydrolysis time depends on each case and should be optimized.

3. At basic pH, the condensation is favored. The appropriate basic pH will depend on the enzyme performance (not too high to inactivate it). The buffer composition is not particularly important. However, (NH4)2SO4 should not be used to avoid precipitation.

4. The 50:50 ratio is suggested. However, other ratios can be used.

5. Controls are usually performed without enzyme but using the basic buffer.

6. Sometimes the condensation occurs at faster rates. As a result the sol-gel starts to form before its deposition on the support surface. It is then convenient to carry out this step at 4°C (cold chamber).

7. The volume of mixture deposited on the support surface depends on the particular interest, but it has never to spread out from the surface of interest.

8. Our experience is based on the sol-gel immobilization on screen-printed graphite electrodes. However, the sol-gel immobilization on other supports is also possible.

9. Drying time depends on each case and should be optimized. Desiccators with or without vacuum can also be used. This step can be also performed at room temperature. However, 4°C are preferred to maintain the enzyme activity.

10. Buffer can also be used to rinse the support.

11. Although in Subheading 2.2., item 3 PVA-SbQ with degree of polymerization: 1700 is suggested, other degrees of polymerization can be used.

12. The 50:50 ratio is suggested. However, other ratios can be used. We have observed satisfactory results with the 70:30 ratio.

13. If foam or bubbles are observed after vortex mixing, briefly centrifuge using a benchtop centrifuge.

14. Our experience is based on the immobilization on screen-printed graphite electrodes, at the bottom of Maxisorp microtiter wells and on Ultrabind modified polyethersulfone affinity membranes. However, the PVA-SbQ immobilization on other supports is also possible.

15. Exposure to ultraviolet light for 2 min at 4°C is also possible. However, exposure to neon light for 3 h at 4°C is more convenient, as it provides slower polymerization and higher reproducibility.


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