Vrednotenje spektralnih lastnosti fluorescenčnih sond ob vezavi na fibrile amiloida β

Kruščič, Anja (avtor), Knez, Damijan (mentor)

Alzheimerjeva bolezen (AB) je ena izmed vodilnih nevrodegenerativnih bolezni, ki prizadene predvsem starejšo populacijo in se kaže s postopnim pešanjem kognitivnih sposobnosti. Za AB je značilno zunajcelično kopičenje plakov amiloida β (Aβ) in znotrajcelično kopičenje proteina tau v obliki nevrofibrilarnih pentelj (NTF). Ker še zmeraj ne poznamo zdravila za to bolezen, je ključnega pomena za dobro obvladovanje in zdravljenje bolezni zgodnja diagnostika AB. Razvoj novih diagnostičnih pristopov gre v smeri in vivo detekcije plakov Aβ in nevrofibrilarnih pentelj v realnem času, kar nam ne omogoča le zaznave prisotnosti bolezni, temveč tudi njeno sprotno spremljanje poslabšanja ali izboljšanja zdravstvenega stanja. V zadnjih letih je opazen velik napredek pri razvoju fluorescenčnih sond v bližnjem infrardečem območju, ki selektivno zaznajo različne biomarkerje AB. Zanje je značilna velika selektivnost, visoka občutljivost ter neinvazivnost. V magistrski nalogi smo osmim fluorescenčnim sondam posneli emisijske spektre v prisotnosti fibril Aβ1–42 in jim določili emisijske maksimume. Pri teh vrednostih smo nato izmerili vezavne afinitete (Kd) sond do Aβ. Najnižje vrednosti Kd sta imeli sondi ALZ239 in ALZ248, kar pomeni, da imata ti dve sondi najmočnejšo afiniteto do fibril Aβ1–42. Posneli smo tudi emisijske spektre sond po inkubaciji z neagregiranim Aβ1–42, fibrilami inzulina in nativnim humanim serumskim albuminom ter govejim serumskim albuminom, s čimer smo preverili nespecifično vezavo sond na druge proteine, ki so lahko prisotni v kompleksnih vzorcih, npr. plazmi. Potrdili smo selektivno in specifično vezavo na fibrile Aβ1–42, saj je bilo povečanje intenzitete fluorescence ob vezavi sond na fibrile Aβ1–42 največje. Pri vezavi sond na fibrile inzulina smo opazili, da se je emisijski maksimum premaknil k višjim valovnim dolžinam, kar nam omogoča razlikovanje med fibrilami Aβ1–42 in fibrilami inzulina. Za kontrolne sonde (ThT, UVE-58 in UVE-79) smo izmerili tudi vezavne afinitete do fibril Aβ1–42, pripravljenih v pufru Tris, ki ima višjo ionsko moč in fiziološki pH v primerjavi z 10 mM HCl, v katerem smo pripravili fibrile Aβ1–42 po ustaljenem postopku. Ugotovili smo, da imajo testirane sonde večjo vezavno afiniteto do fibril pripravljenih v pufru Tris kot do fibril v 10 mM HCl. Sonde UVE-58, UVE-79, ALZ239 ter ALZ248 predstavljajo dobro izhodišče za nadaljnje vrednotenje, ki bi obsegalo zaznavo fibril Aβ na tkivnih možganskih rezinah ter v plazemskih vzorcih.

• Jezik: Slovenski jezik
• Ključne besede: Alzheimerjeva bolezen, amiloid β, fibrile, fluorescenčne sonde, vezavna afiniteta
• Vrsta gradiva: Magistrsko delo/naloga
• Organizacija: FFA - Fakulteta za farmacijo
• Leto izida: 2025
• PID: 20.500.12556/RUL-177264
• Datum objave v RUL: 19.12.2025

Sekundarni jezik

• Jezik: Angleški jezik
• Naslov: Evaluation of fluorescent probes’ spectral properties upon binding to β-amyloid fibrils

Izvleček

Alzheimer’s disease (AD) is one of the leading neurodegenerative diseases, primarily affecting the elderly population, and is characterized by a gradual decline in cognitive functions. A typical hallmark of AD is the extracellular accumulation of amyloid β (Aβ) plaques and intracellular accumulation of tau protein in the form of the neurofibrillary tangles (NTF). Since there is still no cure for this disease, early diagnosis of AD is crucial for effective management and treatment. The development of new diagnostic approaches is moving towards in vivo detection of Aβ plaques and neurofibrillary tangles in real time, which not only enables disease detection but also allows real-time monitoring of disease progression, including deterioration or improvement in the patient’s condition. In recent years, significant progress has been made in the development of near-infrared fluorescent probes that selectively bind to various AD biomarkers. These probes are known for their high selectivity, sensitivity and non-invasiveness. In the master’s thesis, we recorded the emission spectra of eight probes in the presence of Aβ1–42 fibrils and determined their emission maxima. At these wavelengths, we then measured the binding affinities (Kd) of the probes to Aβ1–42 fibrils. The probes ALZ239 and ALZ248 showed the lowest Kd values, indicating the highest affinity for Aβ1–42 fibrils among the tested probes. We also determined the emission spectra for the probes after incubation with non-aggregated Aβ1–42, insulin fibrils and native human serum albumin and bovine serum albumin. This was done to examine potential nonspecific binding of probes to other proteins that might be present in a complex biological sample (e.g. plasma). The results confirmed the selective binding to Aβ1–42 fibrils, as the increase in fluorescence intensity was the greatest when the probes bound to Aβ1–42 fibrils. When probes bound to insulin fibrils, we observed a red shift of the emission maximum, which allows us to distinguish between Aβ1–42 fibrils and insulin fibrils. For the control probes (ThT, UVE-58 and UVE-79) we also measured the binding affinities to Aβ1–42 fibrils prepared in Tris buffer, which has higher ionic strength and physiological pH compared to 10 mM HCl, in which the Aβ1–42 fibrils were prepared according to the standard protocol. We found that the tested probes exhibited higher binding affinity for fibrils prepared in Tris buffer than for fibrils prepared in 10 mM HCl. The probes UVE-58, UVE-79, ALZ239 and ALZ248 therefore represent a good starting point for further evaluation, including detection of Aβ fibrils in brain tissue section and plasma samples.

• Ključne besede: Alzheimer's disease, β-amyloid, fibrils, fluorescence probes, binding affinity