Development of quantitative Detection Strategies for potential Cancer Biomarkers from Extracellular Vesicles using Lateral Flow Assay
Author
Baumgarte, Simon
Term
4. term
Education
Publication year
2019
Submitted on
2019-08-31
Pages
144
Abstract
Dette speciale undersøger, om PTEN—et tumorsuppressor‑protein, der kan fungere som biomarkør—kan måles i ekstracellulære vesikler (små partikler, som celler frigiver) ved hjælp af lateral flow‑tests (papirstrimler som graviditetstests). Flere mærknings‑ og detektionsstrategier blev sammenlignet for at finde den mest følsomme metode. I et AlphaLISA‑format (beslægtet med den velkendte ELISA‑teknik) gav målingen af PTEN den højeste følsomhed. Når formatet blev overført til teststrimler, faldt følsomheden cirka 25‑fold og svarede til en simpel visuel aflæsning med guld‑nanostjerner (stjerneformede guldnanopartikler, der giver et synligt signal). Guld‑nanostjerner blev også koblet til et Raman‑aktivt molekyle for at muliggøre SERS (surface‑enhanced Raman scattering), men det øgede ikke følsomheden væsentligt. Samlet set var Raman‑baseret detektion på niveau med visuel og fluorescens‑baseret aflæsning til kvantitativ måling af PTEN. Foreløbige forsøg med Raman‑aktive guld‑nanostjerner sammen med en bærbar læserprototype viste potentiale for yderligere optimering, men var en smule mindre følsomme end Europium‑doterede AlphaLISA‑acceptorkugler, som kræver en dedikeret strimmel‑scanner. En praktisk fordel ved guld‑nanostjerner er, at resultater også kan vurderes med det blotte øje, hvilket muliggør PTEN‑detektion uden instrumenter ved omtrent samme følsomhed. Spike‑and‑recovery‑forsøg på strimmelbaseret AlphaLISA afslørede stærke matrixeffekter (forstyrrelser fra andre komponenter i prøven), hvilket understreger behovet for omhyggelig kalibrering samt oprensning eller berigelse af prøverne. I disse studier gav det standard kemiluminescens‑baserede AlphaLISA‑format igen de mest præcise resultater, men kræver mere kompleks udstyr. Specialet beskriver også forskellige konjugeringskemier mellem antistoffer og nanopartikler samt karakteriseringsmetoder til at bekræfte vellykket kobling, og det giver et udgangspunkt for at opbygge og vurdere en række mærker til lateral flow‑tests. Fremtidigt arbejde bør validere Raman‑baseret detektion som den mest alsidige løsning identificeret her og udvide testningen fra in vitro‑prøver til kliniske prøver med yderligere optimering og kalibrering. Resultaterne er lovende og støtter en fremtidig praktisk implementering.
This thesis evaluates whether PTEN—a tumor suppressor protein that can serve as a biomarker—is detectable in extracellular vesicles (small particles released by cells) using lateral flow assays (paper test strips similar to pregnancy tests). Several labeling and read‑out strategies were compared to identify the most sensitive approach. In the AlphaLISA format (related to the common ELISA technique), PTEN was measured with the highest sensitivity. However, when the assay was moved onto lateral flow strips, sensitivity decreased by about 25‑fold and matched the performance of a simple visual read‑out using gold nanostars (star‑shaped gold nanoparticles that produce a visible signal). Gold nanostars were also combined with a Raman‑active molecule to enable SERS (surface‑enhanced Raman scattering) detection, but this did not substantially improve sensitivity. Overall, Raman‑based detection was comparable to visual and fluorescence‑based read‑outs for quantitative PTEN measurement. Preliminary tests with Raman‑active gold nanostars, combined with a portable reader prototype, demonstrated potential for further optimization, though they were slightly less sensitive than Europium‑doped AlphaLISA acceptor beads, which require a dedicated strip‑scanning device. A practical advantage of gold nanostars is that results can also be judged by eye, enabling PTEN detection without instruments at roughly similar sensitivity. Spike‑and‑recovery experiments on strip‑based AlphaLISA revealed strong matrix effects (interference from other sample components), underscoring the need for careful calibration and for sample purification or enrichment. In these studies, the standard chemiluminescence AlphaLISA format again delivered the most precise results, but at the cost of more complex instrumentation. The thesis also explores antibody–nanoparticle conjugation chemistries and characterization methods to verify successful coupling, providing a starting point for building and assessing a range of labels for lateral flow assays. Future work should validate Raman‑based detection as the most versatile option identified here and extend testing from in vitro samples to clinical specimens, with further optimization and calibration. The results are promising and support continued development toward practical implementation.
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