Vrednotenje vezave fluorescenčnih sond na fibrile inzulina in amiloida β

Bolta, Katja (avtor), Knez, Damijan (mentor)

Alzheimerjeva bolezen predstavlja velik zdravstveni problem, ki prizadene v večji meri starejšo populacijo in vodi v progresivno in ireverzibilno oslabitev funkcijskih in vedenjskih odzivov. Bolezen se povezuje s prekomernim zunajceličnim kopičenjem plakov amiloida β in znotrajceličnim kopičenjem nevrofibrilarnih pentelj proteina tau. Ker se patogeneza v telesu začne 20–30 let pred prvimi kliničnimi znaki, se raziskujejo novi pristopi diagnostike, ki bi omogočali hitrejšo in natančnejšo diagnozo. V ta namen se zadnja desetletja razvijajo fluorescenčne sonde za detekcijo proteinskih agregatov, ki bi emitirale v bližnjem delu infrardečega spektra in bi z uporabo raziskav in vivo predstavljale veliko prednost pred obstoječimi tehnikami diagnostike. Za sonde so postavljene številne zahteve, ki otežijo razvoj fluorescenčnih sond, med drugim se zahtevajo visoka selektivnost in specifičnost za ciljne biološke označevalce ter emisija pri valovnih dolžinah, višjih od 650 nm, s čimer se prepreči razpršitev svetlobe in zagotovi penetracija v globoka tkiva. Pri meritvah fluorescence se je treba zavedati številnih faktorjev interference, med katere spadata avtofluorescenca in dušenje fluorescence. V sklopu magistrskega dela smo devetim fluorescenčnim sondam določili emisijske spektre v prisotnosti fibrilov amiloida β1-42, da bi ugotovili, ali izkazujejo ustrezno valovno dolžino emisije in zadostno jakost signala, ter v prisotnosti inzulina, neagregiranega amiloida β1-42, govejega in humanega serumskega albumina, s čimer smo želeli izključiti nespecifično vezavo prob ter potrditi njihovo selektivnost. Po preučitvi emisijskih spektrov smo ugotovili, da večina spojin izkazuje selektivno vezavo na fibrile amiloida β1-42, saj so le ob vezavi nanj izkazale močno ojačitev signala emisije fluorescence, vendar valovne dolžine emisije fluorescence žal ne ustrezajo v literaturi zahtevanim vrednostim, ki so značilne za dobre fluorescenčne sonde. Pridobljeni emisijski spektri so nam začrtali valovne dolžine, okoli katerih smo v nadaljevanju merili afiniteto vezave fluorescenčnih sond do fibrilov amiloida β1-42 in pri izbranih sondah do fibrilov inzulina. Afiniteto vezave smo merili prek neposrednega povišanja fluorescence sond, ki je posledica vezave v vezavna mesta peptidov, pri čemer so nekatere izmed spojin imele visoko afiniteto do fibrilov amiloida β1-42 in nizko afiniteto do fibrilov inzulina, kar bi lahko v kompleksnih vzorcih omogočalo razlikovanje med strukturno različnimi fibrili.

• Jezik: Slovenski jezik
• Ključne besede: fibrili, amiloid β, inzulin, sonde, fluorescenca, afiniteta vezave
• Vrsta gradiva: Magistrsko delo/naloga
• Organizacija: FFA - Fakulteta za farmacijo
• Leto izida: 2024
• PID: 20.500.12556/RUL-160577
• Datum objave v RUL: 31.08.2024

Sekundarni jezik

• Jezik: Angleški jezik
• Naslov: Analysis of fluorescent probe binding to insulin and β-amyloid fibrils

Izvleček

Alzheimer's disease is a major health problem that primarily affects the elderly population, leading to progressive and irreversible impairment of functional and behavioral responses. The disease is associated with excessive extracellular accumulation of β-amyloid plaques and intracellular accumulation of neurofibrillary tangles composed of tau protein. Since pathogenesis begins in the body 20–30 years before the first clinical signs appear, new diagnostic approaches are being explored to enable a faster and more accurate diagnosis. To this end, fluorescent probes that emit in the near-infrared range have been developed in the recent decades and, if used in vivo, would represent a major advantage over existing methods. There are a number of requirements for probes that complicate the development of fluorescent probes. These include the requirement for high selectivity for the target biomarkers and emission at wavelengths above 650 nm to prevent light scattering and ensure penetration into deep tissue. When measuring fluorescence, it is important to consider a number of confounding factors, including autofluorescence and quenching. In our master's thesis, we determined the emission spectra of nine fluorescent probes in the presence of β1-42-amyloid fibrils to determine whether they have the appropriate emission wavelength and sufficient signal intensity, and in the presence of insulin, non-aggregated β1 42-amyloid, bovine and human serum albumin to rule out non-specific binding of the probes and confirm selectivity. After examining the emission spectra, we found that most of the compounds exhibited binding selectivity towards β1-42-amyloid fibrils, as they showed a strong enhancement of the fluorescence emission only when bound to them, but unfortunately the wavelengths of fluorescence emission did not match those deemed suitable according to literature data. The emission spectra obtained provided us with the wavelengths at which we next measured the binding affinity of the fluorescent probes to β1-42-amyloid fibrils and, for selected probes, to insulin fibrils. Binding affinity was measured by the direct increase in fluorescence of the fluorescent probes resulting from binding to the peptide binding sites, with some of the compounds showing high affinity for β1-42-amyloid fibrils and low affinity for insulin fibrils with sufficient reliability, which could ultimately allow distinguishing between structurally different fibrils in complex samples.

• Ključne besede: fibrils, β-amyloid, insulin, probes, fluorescence, binding affinity