Christopher Karim Akhgar, Andreas Schwaighofer – TU Wien
21 July 2020
One of the major milestones within the NUTRISHIELD project are the novel laser-based analyzers, allowing fast measurement of clinically relevant parameters in urine and human milk. Before the analyser prototypes will be implemented, comprehensive method development to specify the framework for hardware development is conducted by Technische Universität Wien
Optimization of the transmission path length
The applied analytical method, namely infrared (IR) spectroscopy detects the fundamental vibrations of molecular bonds in a label free and nondestructive manner. Within the NUTRISHIELD project, a novel laser-based method was developed for protein analysis in human breast milk. An important parameter in method development is the path length of the transmission cell. A higher path length leads to a higher specific analyte signal, but causes also more unspecific absorption by the solvent that results in an increased noise level in the final measurements. Consequently, optimization of the path length is a crucial step in method development. A related important parameter of a method is the limit of detection (LOD) which defines the lowest concentration of an analyte that can be reliably detected. Within the NUTRISHIELD project, a novel laser-based IR transmission spectroscopy setup was presented that achieves a LOD better by a factor 10 compared to conventional Fourier transform infrared spectroscopy.
Identification of the optimal analysis wavelength
Within the NUTRISHIELD project, partner Alpes Lasers provides dedicated lasers for the urine and protein analyser prototype. During method development, the characteristic wavelengths for laser production need to identified. In multicomponent analytes such as bodily fluids, the IR spectra may show an overlap of distinctive spectral features. For some substances in e.g. urine, the absorption bands vary with changing pH-value, so this parameter can be used for optimization of the method and determination of the characteristic wavelengths. The criteria for the optimum pH-value are high absorbance of the targeted component in a spectral region of low background noise and minimum spectral overlap with the absorption bands of other components in the analyte. For human breast milk, the IR spectra of the investigated proteins show a strong overlap, which cannot be avoided by wet-chemical sample treatment. In this case the characteristic wavelength for quantification of every component can be achieved by computational methods such as chemometric analysis.
A further important step during method development is the elaboration of a sample preparation routine, if necessary. As outlined above, for urine samples adjustment of the pH-value is necessary. Raw human milk samples need to be homogenized prior to an IR measurement to decrease the average fat globule diameter and to narrow the size distribution. Large fat droplets lead to light scattering which has a negative effect on the quality of the IR spectra. Here, homogenization increases the reproducibility and accuracy of IR measurements of human breast milk.
In conclusion, systematic method development is the basis to assure well-functioning final devices. Within the NUTRISHIELD project, the technical knowledge of Technische Universität Wien complements the expertise of laser engineers, device manufacturers, dieticians, computer scientists and further specialists to achieve optimal results.