Determination of proteins with UV spectrometry with variable optical pathlength

Uporaba UV-Vis spektrometra za določanje koncentracije proteinov v raztopini je ena izmed temeljnih metod v bioanalitiki. Vse dosedanje metode izkoriščajo Beer-Lambertov zakon za določanje koncentracije na podlagi izmerjene absorbance pri eni valovni dolžini in konstantni optični poti. Zaradi omejitev Beer-Lambertovega zakona je potrebno raztopine redčiti. Redčenje ima lahko velik vpliv na optične lastnosti vzorcev, na točnost rezultatov raziskav ter je časovno potratno. Zato smo razvili novo metodo na podlagi prilagojenega Beer-Lambertovega zakona, pri katerem spreminjamo dolžino optične poti – torej je optična pot variabilna. Za natančno spreminjanje optične poti uporabimo sistem SoloVPE. V sklopu te magistrske naloge primerjamo meritve, kjer se spreminja optična pot. z obstoječimi sistemi, pri katerih je optična pot konstantna. Rezultate, pridobljene z uporabo variabilne optične poti, smo primerjali z rezultati, pridobljenimi s klasičnimi UV-Vis metodami, z UV metodami visoke zmogljivosti ter HPLC metodami. Primerljivost točnosti izmerjenih rezultatov smo preverili na protitelesih, denaturiranih proteinih, strukturnih proteinih, produktih encimskih reakcij in Polisorbatu 80. Prav tako smo preverili točnost meritev pri različnih valovnih dolžinah. Rezultati meritev so pokazali ponovljivost meritev (RSD ≤ 2 % med meritvami enakih vzorcev). Relativna razlika nasproti rezultatom, pridobljenim z že obstoječimi UV-Vis metodami, je praviloma bila ≤ 3 %. Pri visokih koncentracijah se je nov sistem izkazal za bolj točnega (odstopanje od teoretične vrednosti ≤ 5 %, pri obstoječih sistemih ≤ 20 %). Sistem se je izkazal za primerljivega pri analizi bioloških molekul z obstoječimi sistemi za določanje koncentracije proteinov v raztopini. Samo pri določitvi koncentracije produktov encimskih reakcij so bili rezultati neponovljivi. Zato bi bilo smiselno narediti dodatne raziskave in določiti vzroke te neponovljivosti.The use of a UV-Vis spectrometer for determining the concentration of proteins in a solution is one of the fundamental methods in bioanalytics. All previous methods have utilized the Beer-Lambert law to determine concentration based on measured absorbance at a single wavelength and a constant optical path length. Due to the limitations of the Beer-Lambert law, solutions need to be diluted. Dilution can have a significant impact on the optical properties of samples, the accuracy of acquired results, and is time-consuming. Therefore, we have developed a new method based on the modified Beer-Lambert law, in which we vary the optical path length - i.e., the optical path is variable. To precisely vary the optical path, we use the SoloVPE system. As part of this master\u27s thesis, we compare measurements where the optical path is varied, with existing systems where the optical path is constant. The results obtained using the variable optical path are compared with results obtained from classical UV-Vis methods, high-throughput UV methods, and HPLC methods. The comparability of the accuracy of the measured results was verified on antibodies, denatured proteins, structural proteins, enzyme reaction products, and Polysorbate 80. We also verified the accuracy of the measurements at different wavelengths. The measurement results showed reproducibility (RSD ≤ 2% between measurements of the same samples). The relative difference against the results obtained with existing UV-Vis methods was generally ≤ 3%. At high concentrations, the new system was shown to be more accurate (deviation from the theoretical value ≤ 5%, with existing systems ≤ 20%). The system proved to be comparable to existing systems for determining protein concentration in a solution. Only in the determination of the concentration of enzyme reaction products were the results non-reproducible. Therefore, it would be sensible to conduct additional research and determine the causes of the non-reproducibility