## 2 Materials and Methods
### Methods  
The proposed technology for rapid fabrication of MF chips is a hybrid technology of laser micromachining and xurography. PMMA layers were cut with CO\({}_{2}\) laser, and the bonding layers were cut with Plotter Cutter in 3M double-sided adhesive tape. In order to prevent the sample contamination, the design of the MF chip contained the chamber for sample, which enabled measurements in the closed system environment. The chamber design contained curved edges (Figure 1, middle layer) in order to prevent forming bubbles inside the chip. The proposed design and dimensions of the channels enabled integration of different sensing components in order to get a multifunctional and compact system that enabled simultaneous measurements in the sample. The proposed design presented a proof of concept for the methodology and opened a possibility for additional improvement of the system. However, for the purpose of measurements, the chip design can be simplified to a single channel and dimensions can be additionally miniaturized for more compact systems. Figure 1: The 2D layer layouts of the microfluidic chip. The top layer contains inlet/outlet holes and three holes for the PreSens sensors. The middle layer contains chamber design, and the bottom layer closes the MF chip. The top layer in PMMA contained an inlet and outlet customized for pipetting the sample and three holes with diameters of 2.1 mm for placing the PreSens sensors. The middle layer in PMMA contained the chamber design, which was filled with the sample, and the bottom layer that closed the MF system. The interconnecting layers, 3M double-sided adhesive tapes, were made in the same design as the middle PMMA layer, i.e., containing a chamber. Bonding of layers was rapidly achieved by the cold lamination process. The experimental setup is presented in Figure 2. The MF chip was filled with samples and the SensorPlugs were placed in the special holes at the top layer to provide direct contact between sensors and sample. Optical fibers connected the SensorPlugs and sensors, and the whole setup was connected to a PC and software PreSens Measurement Studio 2 RC. Calibration of pH and O2 sensors was done according to sensors' user manual and the CO2 sensor was used pre-calibrated. A benchtop pH meter was used for measurements of calibration samples with different pH and control measurements. The calibration samples were prepared in the range between 5.7 and 8.5 by titrating 0.1 M HCl in phosphate-buffered saline (PBS). The measured phase and pH values of calibration samples are presented in Table 1.  
Air-saturated and oxygen-free waters were prepared for calibration of the O2 sensor. Air-saturated water was made with an air pump producing bubbles in 100 mL of water during 20 min and the oxygen-free water was made by dissolving 1 g of sodium sulfite (Na2 SO3) and 50 \(\upmu\)L of cobalt nitrate (Co(NO3)2) standard solution (\(\rho\)(Co) = 1000 mg/L; in nitric acid 0.5 mol/L) in 100 mL of water. The calibration values and measured phase for O2 sensor are presented in Table 2. \begin{table}
\begin{tabular}{c c} \hline
**pH** & **Phase** [\({}^{\circ}\)] \\ \hline
5.7 & 52.5810 \\ \hline
6.3 & 48.0080 \\ \hline
6.7 & 42.3190 \\ \hline
8.46 & 21.7290 \\ \hline \end{tabular}
\end{table} 
Table 1: Calibration parameters for pH sensor. Figure 2: Experimental setup. Optical cables are connecting the PreSens sensors with SensorPlugs that are in direct contact with the sample in the MF chip. The PreSens sensors are connected to PC and software PreSens Measurement Studio 2 RC. Thirty healthy volunteers participated in the research, 18 females and 12 males with the age range 12-56. The study participants consisted of students and staff volunteers from the Department of Dentistry, selected in line with the recommendations from the similar studies and salivary collecting protocols [22; 23].