Preliminary experiments
Preliminary experiments were carried out employing the gelatin
coated glassy carbon electrode to determine the optimal conditions
for trypsin activity determination, prior screen printed electrodes
application. The developed protocol involved the following steps:
incubation of the gelatin coated electrode in trypsin solution for
different time intervals, rinsing with phosphate buffer, transferring
the electrode into the electrolysis cell containing the buffer solution,
and recording the square wave voltammetric current response
to 1,2-benzoquinone. Following this procedure, current increase
dependence on trypsin activity was investigated under various
experimental conditions. Their effect and the effect of their interactions
were evaluated applying factorial design of experiments. The
considered factors supposed to affect the response variable were
the 3 level factor (0.5 mM, 1 mM, and 5 mM) analyte concentration
(Factor A), the 2 level factor (6% and 8%) gelatin solution concentration
(Factor B), and the 3 level factor (8 L, 10 L, and 15 L) volume
of the gelatin solution deposited on the electrode surface (Factor
C). The number of treatments and replicates was 18 and 2 respectively,
and the total number of observations was 36. The statistical
significance was determined to be 95% (˛ = 0.05). The hypothesis
of interest (H0: null hypothesis, and HA: alternative hypothesis) for
the factors effects on the response variable are presented below:
H0: Analyte concentration = 0; H0: Gelatin concentration = 0; H0:
Gelatin volume = 0.
HA: Analyte concentration / = 0; H0: Gelatin concentration / = 0;
H0: Gelatin volume / = 0.
The factors incubation time and trypsin concentration were kept
constant: 10 min and 100 g mL−1 respectively.Table 1 demonstrates
the results of the factorial design of experiments analysis. It
was found that the p-values of all of the considered factors are lower
than the value of ˛ = 0.05, thus they affect the response variable and
the alternative hypothesis should be accepted as true.