20.500.12556/RUL-187017 MERITEV FLUORESCENCE PRI METODI QPCR FLUORESCENCE MEASUREMENT IN THE QPCR METHOD Kvantitativni PCR (qPCR) omogoča sprotno sledenje pomnoževanju DNK z merjenjem fluorescence interkalacijskega barvila, kot je SYBR Green I, vendar so komercialni qPCR-sistemi praviloma obsežne namizne naprave, neprimerne za terenske aplikacije. V okviru širšega projekta razvoja prenosnega (POC) qPCR-sistema je bila v diplomskem delu zasnovana, izdelana in ovrednotena osnovna fluorescenčna merilna veja tovrstnega sistema, katere namen je potrditi izvedljivost principa moduliranega vzbujanja z LED in fazno-občutljive detekcije z lock-in ojačevalnikom SIGNAL RECOVERY 7265 ter ugotoviti, do katerih vrednosti je zaznavanje z izbrano postavitvijo možno. Sistem sestavljajo svetlobni vir LED z valovno dolžino 490 nm, krmiljen z lastnim gonilnikom na osnovi modificirane Howlandove tokovne črpalke, linearni optični sistem z vzbujevalnim in emisijskim interferenčnim filtrom ter dvema plano-konveksnima lečama, silicijeva PIN-fotodioda TEMD5510FX01 ter dvostopenjski transimpedančni ojačevalnik. Mehanska izvedba sistema temelji na 3D-tiskanem ohišju in aluminijasti profilni konstrukciji. Končne meritve so bile izvedene s strojno sinhronizacijo funkcijskega generatorja in lock-in ojačevalnika: sinhronizacijski izhod generatorja HP 33220A je bil povezan na referenčni vhod TTL lock-in ojačevalnika SIGNAL RECOVERY 7265. Modulacijska frekvenca je bila nastavljena na 5,02 kHz, s čimer je bila meritev odmaknjena od točne vrednosti 5,00 kHz, ki predstavlja 100. harmonik omrežne frekvence 50 Hz. Po zaklepu se je fazni kot ustalil približno pri −109°. Meritve so bile izvedene pri občutljivostih LIA 50, 20, 10, 5 in 2 μV pri koncentracijah fluoresceina 10 μM, 100 μM in 1 mM ter pri količinah dodane raztopine 0,5, 1, 1,5 in 2 μL. Za slepi vzorec je bila v 5 μL mineralnega olja namesto fluoresceinske raztopine dodana enaka količina PBS. Pri občutljivosti 50 μV je bilo mogoče brez preobremenitve izmeriti celoten koncentracijski razpon, medtem ko so nižja merilna območja pri višjih koncentracijah prešla v preobremenitev. Za oceno meje zaznavnosti je bilo pri občutljivosti 2 μV opravljenih deset meritev slepega vzorca z 0,5 µL PBS. Povprečje je znašalo 82,3 nV, vzorčni standardni odklon 8,76 nV, prag R̄₀ + 3,3s₀ pa 111,2 nV. Najnižja preizkušena koncentracija, ki je še presegla ta prag in dala stabilen odčitek, je bila 2,5 µM; pri 1,25 µM je bil odziv pod pragom. Meja zaznavnosti je zato za uporabljeni diskretni niz koncentracij umeščena med 1,25 in 2,5 µM pri čemer 2,5 µM predstavlja najnižjo zanesljivo zaznano koncentracijo. Rezultati predstavljajo karakterizacijo fluorescenčne merilne veje, namenjene prihodnji integraciji v qPCR-sistem, in ne validacije celotne biokemijske qPCR-reakcije. Quantitative PCR (qPCR) enables real-time monitoring of DNA amplification by measuring the fluorescence of an intercalating dye such as SYBR Green I, yet commercial qPCR systems are typically bulky benchtop instruments unsuitable for point-of-care applications. As part of a larger project developing a point-of-care (POC) qPCR system, this thesis presents the design, construction, and evaluation of the basic fluorescence measurement branch of such a system, aiming to verify the feasibility of modulated LED excitation combined with phase-sensitive detection via a SIGNAL RECOVERY 7265 lock-in amplifier and to determine the detectable range of the chosen setup. The system consists of a 490 nm LED, driven by a custom driver based on a modified Howland current pump, a linear optical path with excitation and emission interference filters and two plano-convex lenses, a silicon PIN photodiode (TEMD5510FX01), and a two-stage transimpedance amplifier. The mechanical design is based on 3D-printed housing parts mounted on an aluminum profile frame. The final measurements were performed with hardware synchronization between the function generator and the lock-in amplifier: the HP 33220A Sync Out was connected to the SIGNAL RECOVERY 7265 TTL In. The modulation frequency was set to 5.02 kHz, shifting the measurement away from exactly 5.00 kHz, which corresponds to the 100th harmonic of the 50-Hz mains frequency. After locking, the measured phase settled at approximately −109°. Measurements were performed at LIA sensitivity settings of 50, 20, 10, 5, and 2 μV for fluorescein concentrations of 10 μM, 100 μM, and 1 mM and added solution volumes of 0.5, 1, 1.5, and 2 μL. For the blank, the same volume of PBS was added to 5 μL of mineral oil instead of fluorescein solution. The 50-μV setting allowed the complete concentration range to be measured without overload, whereas the lower measurement ranges overloaded at the higher concentrations. For limit-of-detection assessment, ten blank measurements were collected at the 2-μV setting using 0.5 µL PBS. The mean blank response was 82.3 nV, the sample standard deviation was 8.76 nV, and the response threshold R̄₀ + 3.3s₀ was 111.2 nV. The lowest tested concentration that exceeded this threshold and still produced a stable reading was 2.5 µM while the 1.25-µM response was below the threshold. Thus, for the tested discrete concentration series, the detection limit lies between 1.25 and 2.5 μM, with 2.5 μM being the lowest reliably detected concentration. These results characterize the fluorescence measurement branch intended for future integration into a qPCR system; they do not constitute validation of the complete biochemical qPCR reaction. fluorescenca qPCR fluorescein SYBR Green I lock-in ojačevalnik fazno-občutljiva detekcija transimpedančni ojačevalnik fluorescence qPCR fluorescein SYBR Green I lock-in amplifier phase-sensitive detection transimpedance amplifier true false false Slovenski jezik Angleški jezik Diplomsko delo/naloga 2026-09-08 11:10:03 2026-09-08 11:10:18 2026-09-15 11:16:55 0000-00-00 00:00:00 2026 0 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 62892 291127043 Snajder_Jakob_-_MERITEV_FLUORESCENCE_PRI_METODI_QPCR.pdf Snajder_Jakob_-_MERITEV_FLUORESCENCE_PRI_METODI_QPCR.pdf 1 3E0623295D103C37770ACAA50CC78F26 1ef3889d5d0d35c3f707024ff0f3c2a7530d5d6a8f2eea24653ae76d4ab99a81 1a87750d-ab65-11f1-8bc5-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=246444 Fakulteta za elektrotehniko 0 0 0