Pristine TC fabric and the DR, DY, DG, or DB singly dyed T/C fabrics from Sect. 2.2.2 were separately soaked in 1 M NaOH over 1 h to deprotonate the hydroxyl groups of the cotton yarn. The alkalized fabric was then placed in a solution of cyanuric chloride (2,4,6-trichloro-1,3,5-triazine; variable concentration based on amount of thionine used in Table 1) in tetrahydrofuran and incubated for 12 h at 40 °C, after which deionized water and tetrahydrofuran were used to wash out unreacted cyanuric acid and other impurities. The activated fabric was then submerged in a solution of thionine acetate (TA; 0.25 molar equiv of cyanuric chloride) in 80 mL deionized (DI) water at 40 °C for another 12 h. The conjugated fabrics were first dried at 40 °C, and then washed with deionized (DI) water and phosphate buffered saline (PBS; aqueous solution of 340 mM NaCl, 6.8 mM KCl, 20.0 mM Na2HPO4, 1.8 mM KH2PO4) to remove free thionine acetate. The washings were collected for calculating the amount of TA loaded onto the fabrics as follows: to ensure the successful removal of unbound thionine from the washing procedure, the prepared fabrics were placed into 3 mL PBS/cm2 fabric for 1 h with vigorous shaking before being transferred to a cuvette and the absorbance at 603 nm, corresponding to the absorption maximum of thionine acetate, was recorded. When the leaching of TA could no longer be observed within the detection limit of the spectrometer, the fabrics were then dried at 40 °C in an oven and stored in the dark until further use. Different initial concentrations of TA were employed to explore the concentration-dependent bactericidal activity of TA, and the names and relative the amounts of TA used are listed in Table 1.

Concentrations of thionine and cyanuric chloride used in the production of the TA-conjugated TC fabrics

TC-TA% (w.o.f.)Amount of TA (mmol)Amount of TCT (mmol)
0% (TC)00
0.72% (TC-TA1)0.1250.5
1.44% (TC-TA2)0.251
5.76% (TC-TA3)14
11.52% (TC-TA4)28