## 3 Results and Discussion
### Analytical Figures of Merit  
The analytical merits of the method in the determination of different biothiols were evaluated by preparing standard solutions of Cys, Hcy and GSH at different concentration  
Figure 4: Investigation of interferences from common biomolecules andinorganic electrolytes at physiologically relevant concentration levels. The control sample contains 200 \(\upmu\)M Cys while all other solutions contain 200 \(\upmu\)M Cys and the potentially interfering biomolecule or ion as follows: histidine, glycine, valine, glutamine: 4.0 mM; cysteine: 50 \(\upmu\)M; urea: 2.0 mM; glucose: 5.0 mM; Na2 SO4: 12.0 mM; Na3 PO4: 12.0 mM; NaCl: 150.0 mM). All solutions were diluted 4-fold before analysis. Error bars represent the standard error calculated for triplicate samples. levels and constructing calibration curves of the analytical signal (i.e., mean color intensity in each well) vs. the concentration of Cys, Hcy or GSH. For all the examined biothiols, the calibration functions correlating the analytical signal to the concentration of each biothiol was linear up to 100 uM. The quantification limit (10\(\sigma\)) of the assay was 5 uM for Cys and GSH and 3.5 uM for Hcy. The working concentration range and the quantification limit are lower than the physiological concentration levels of Cys in blood plasma (150-300 uM), which enables method application with minimal sample volume requirements since dilution is necessary to bring the Cys concentrations within the working concentration range of the method. The repeatability, calculated as the relative standard deviation of seven replicate measurements, was below 10% (for solutions containing 50 uM cysteine). The calibration functions, linearity, precision and detection limits (DLs) of the assays are summarized in Table 1.  
Overall, the analytical merits of the assay, as expressed by the working range and the detection limits, are comparable to other published assays [9] and well below the concentration of total biothiols in biological fluids. Nevertheless, other methods that use sophisticated nanomaterial probes or reagents and advanced detectors have achieved even lower detection limits (Table 2). \begin{table}
\begin{tabular}{c c c c c} \hline
**Biothiol** & **Cysteine (Cys)** & **Homocysteine (Hcy)** & **Glutathione (GSH)** \\ \hline Linear range & 5–100 uM & 5–100 uM & 5–100 uM \\ Regression function & y = 1.3x + 5.3, & y = 0.73x + 2.9, & Y = x + 8.6, \\ Quantification limit b & R2 = 0.97 & R2 = 0.99 & R2 = 0.98 \\
5.0 uM & 3.5 uM & 5.0 uM \\ \hline \end{tabular}  
* The concentrations that were used to calculate the calibration curves were 0, 5, 20, 30, 50, 70 and 100 uM for Cys and Glu and 0, 20, 30, 50, 70 and 100 uM for Hcy. The inset graphs show a digitally cropped image (to facilitate the aspect view) of the sensing zones (micro titer plates). The first plate is the control (blank) sample. A circular area occupying 80% of the total well surface was used to acquire the analytical signal, which was recorded as the mean grey area in the RGB color system. b The quantification limit was determined as 10 times the signal to noise ratio.  
\end{table} 
Table 1: Analytical figures of merit of the assay a.