Qualitative filter paper

To synthesize alginate-conjugated folic acid (AF), sodium alginate (1 g, 4.632 mmol) was added to a 30 mL mixture of ethanol/0.6 M HCl and stirred for 18 h at 4 °C. Alginic acid was produced and it was filtered in vacuo using qualitative filter paper (Whatman™), washed several times with alcohol and acetone, transferred to a vial, and dried under vacuum overnight. The dried alginic acid was dissolved in water (30 mL) and 4% TBAOH was added with continuous stirring until the solution reached pH 9. The opaque solution was lyophilized to yield white TBA-alginate.
Folic acid (0.408 g, 0.9264 mmol) mixed with DMSO (10 mL) and CDI (0.1502 g, 0.9264 mmol) was added. The compound was stirred under N₂ gas at 25 °C in the dark for 24 h. Then, TBA-alginate was dissolved in 50 mL DMSO with 1 wt% TBAF. Under continuous stirring, the folic acid/CDI compound was added to the TBA-alginate solution and left to react overnight in the dark at 40 °C. The product was precipitated in ice cold ethanol/methanol (1:1) containing 0.01 M HCl, filtered, and washed with alcohol. The product was neutralized by dissolving in a solution of sodium carbonate, and AF was obtained by lyophilization. The synthesis process was shown in Figure 2. The structure of AF was analyzed by Proton nuclear magnetic resonance (¹H NMR) spectra, Fourier transform infrared (FT-IR) spectra, and UV-vis spectroscopy.

The selected Streptomyces were inoculated in one liter of sterilized soybean liquid culture medium (SLM, 15 g of corn flour, 10 g of glucose, 0.5 g of K₂HPO₄, 0.5 g of NaCl, 0.5 g of MgSO₄, 3 g of beef extract, 10 g of yeast extract, 10 g of soluble starch, 2 g of CaCO₃, pH 7.2–7.4) at 180 rpm for 7 days at 28°C (Hong et al., 2009 (link)). The culture filtrate was extracted with EtOAc at a ratio of 1:1 (v/v). The mixture was filtered through the qualitative filter paper (Whatman no. 1). The organic phase (EtOAc) was separated from the filtered liquid media using a decantation funnel. The extracts were evaporated using a rotary vacuum evaporator (EYELA, N-1300, Japan), and then dissolved in 10% of dimethyl sulfoxide (DMSO) with a final concentration of 20.0 mg/mL. After being sterilized by filtration through a 0.22-μm sterile filter (Millipore, Bedford, MA, United States), the extracts were stored at −4°C.

Wound exudate sampling will be performed by applying a filter paper (Whatman™ qualitative filter paper) on the wound for 15 minutes until it is moist. The filter paper will then be stored in a sterile vial and transferred to the laboratory. The wound fluid sampling will be performed before grafting and at each weekly review.
Skin punch biopsies (4 mm) will be taken from two locations, at the centre of the wound and at the wound edge, after administering adequate local anaesthesia (2 % lidocaine). This procedure will be done prior to grafting and repeated at day 7 post-grafting. The specimens are then placed in a sterile vial containing 4 % paraformaldehyde and transferred to the laboratory.