2.3. Control of SPOT Density
Commercially available pre-modified cellulose membrane sheets are typically used, however they do not offer the flexibility of in-house cellulose-functionalized membranes, particularly in reducing SPOT density. We functionalized membranes by esterification using Fmoc-β-Alanine and DIC (Fluka, distributed by Sigma-Aldrich, #38370) as previously reported [4,27]. Small (10 × 15 cm) sheets of Whatman™ cellulose (Whatman™ Chromatography paper Grade 1CHR, GE Healthcare Life Sciences, #3001-878) were typically incubated overnight (or at least for 2 h) with 10 mL of a solution comprising 0.64 g of Fmoc-β-Alanine dissolved in NMP, complemented with 374 µL of DIC and 317 µL of N-methylimidazole (NMI, Fisher Chemicals, Loughborough, UK, #M/4930/PB05). We found that the solution should cover the entire membrane without any bubbles forming in order to obtain consistency in the downstream array generation. Membranes were washed the following day 3 times with NMP for at least 30 s each, before being incubated for 20 min in fresh NMP.
Washed membranes were Fmoc de-protected by incubation (2 times 5 min each) in 20% Piperidine in DMF, followed by wash with NMP and (4 times for 30 s) then twice with EtOH. At this stage membranes were dried and stored at −20 °C or immediately used. Extra care was taken when handling in-house functionalized membranes as they tend to be much softer than the commercially available pre-functionalized ones. We found that they are also very easy to tear when manipulated with pincers. To avoid ring SPOT effects we found that (at least in the case of acetylation-dependent interactions) using 0.3 g of Fmoc-β-Alanine in 10 mL of solution produced the best results without any significant loss of signal.