Evaluation of the applicability of CUsKit with different solutions

The hydrogen peroxide-based disinfectant detection kit test was performed with 5 different solutions: solution A, hydrogen peroxide textile disinfectant; solution B, commercial solution of hydrogen peroxide at 49.5% (w/w) diluted to a concentration of 3% (w/w); solution C, antiseptic solution of hydrogen peroxide at a concentration of 3% (w/w); solution D, multi-surface disinfectant based on hydrogen peroxide at a concentration of 1.49% and the solution E, multi-surface disinfectant without hydrogen peroxide. For this purpose, swabs were used, each swab placed in the Eppendorf tube containing the kit’s indicator solution and after removal, brought into in contact with solutions A to E.

To further refine the study, it was decided to use solution D, called Care Us, supplied by the company Success Gadget, Nanotecnologia e Novos Materiais, Lda. This particular solution contains hydrogen peroxide as the main active ingredient and has been shown to provide antimicrobial activity for 7 days after application (Castro et al. 2022).

Evaluation of CUsKit on porous surfaces

Textile sample (2.0 × 2.0) cm2 used in the CUsKit were provided by a local textile industry (Malhas Sonix, S.A., Barcelos, Portugal) and consists of a single jersey of 137 g/m2, 30/1, 100% cotton, BCI (Better Cotton Initiative), set 28, with optical brightener.

For the 7-day tests with solution D, nine textile samples were used: one sample as a control (no disinfectant applied) and eight samples for each day (day 0 to day 7). The eight textile samples were treated with disinfectant on day 0 and then one sample was analysed in each day by placing it an Eppendorf with the indicator solution (Fig. 2).

Fig. 2

Schematic representation of the use of CUsKit, on porous surfaces

The quantification of hydrogen peroxide was conducted in accordance with the standard IS:7045–1973 (Indian Standards, 1973). The textile samples under study were quantified by means of a titration method.

Evaluation of CUsKit on non-porous surfaces

The presence of disinfectant on non-porous surfaces was investigated using a glass surface. Nine squares (10.0 × 10.0) cm2 were marked on the glass surface, one for the control (no disinfectant applied) and eight for the day after the disinfectant was applied (day 0 to day 7) The eight squares corresponding to days 0 to 7 were treated with the disinfectant on day 0. On each day, a test swab was dipped into the indicator solution and after removing it, it was rotated on the surface corresponding to the day under study, both parallel and perpendicular to its own orientation, for a period of 20 s. The procedure is illustrated in Fig. 3.

Fig. 3

Schematic representation of the use of CUsKit, on non-porous surfaces

Accelerated stability test of indicator solution

The accelerated stability test was conducted in accordance with a methodology delineated by CIPAC (Collaborative International Pesticides Analytical Council), a widely accepted protocol within the cosmetics industry (CIPAC 2000). The formulation is subjected to elevated temperatures for a limited duration. This extreme time–temperature binomial has been shown to simulate the normal aging of a formulation (CIPAC 2000). The 14-day period at 54 °C in this test is equivalent to 2 years at ambient temperature in terms of shelf life (CIPAC 2000).

The objective of this test is to provide actionable guidelines on the performance of the product by analysing its chemical and physical characteristics. It is imperative to note that this test is mandatory to predict the stability of the formulation and its shelf life.

In this study, accelerated stability testing was employed to ensure that the potassium iodide indicator solution remained stable and effective throughout the kit’s 2-year shelf life. The indicator solution is placed in an oven (Memmert) at 54 ± 2 °C for 14 days, which can be equivalent to a shelf life of 2 years (CIPAC 2000). For the present study, approximately 40 mL of the indicator solution was exposed to a temperature of 54 ± 2 °C for a period of 14 days.

Following a 14-day period of accelerated stability testing, the detection kit was used on both surfaces in a manner consistent with the description provided in the preceding section. This was done to ascertain whether the solution retained its capacity to detect the presence of hydrogen peroxide-based disinfectant.

Biological analysesEvaluation of antimicrobial activity on non-porous surfaces

The primary function of the detection kit is to minimise the use of disinfectants. Consequently, it is imperative to ensure that the colour change of the test is indicative of antimicrobial activity of the disinfectant. The procedure for these tests has been adapted from the ISO 18593:2004 standard (ISO 18593, 2004).

Nine zones were demarcated, referring to control and the time after application of the disinfectant (day 0 to day 7). The zones were demarcated with adhesive tape, with each zone having a surface area of 100 cm2 (ISO 18593, 2004). In the control zone, no disinfectant was applied, while in the other zones, the disinfectant was applied in the same way and at the same time (day 0). From day 0 to day 7, samples were taken using a swab moistened in a tube containing approximately 2 mL of phosphate-buffered saline 1 × (PBS 1 ×). The swab was then moved in a parallel and perpendicular direction to the surface, with constant rotation to ensure thorough sampling (ISO 18593, 2004). The swab was then transferred to a Petri dish containing Tryptone Soy Agar (TSA) (purchased from Frilabo), inverted, and left at room temperature for 168 h to analyse the appearance of bacteria, fungi, and yeasts (ISO 18593, 2004). This experiment was replicated in at least three independent experiments. It should be noted that this test was carried out against native microorganisms present on the test surface; no test organisms were seeded on the surface.