dissertation
Sandra Oloketuyi (Author), Ario De Marco (Mentor)

Abstract

Early detection and monitoring of toxic microalgae remains a challenge that will be necessary to overcome for understanding harmful algal bloom events and their consequent ecological and environmental impacts. Although there are diagnostic techniques based on microscopy, fluorescence in situ hybridization (FISH), DNA microarrays, real-time PCR, sandwich hybridization assays employed for the detection of harmful algal species, such techniques are not suitable for field monitoring and especially for the quantification of Alexandrium minutum due to abundance of cryptic species that cannot be differentiated based on morphology or genomic traits. These approaches are also time-consuming and laborious, hence there is need for alternative simple, rapid and cost-effective methods. In this study, our aim was to develop different nanobody-based capture strategies for the detection of A. minutum. Nanobodies were successfully displayed on the surface of Escherichia coli to facilitate detection of A. minutum. The method is simple and cost effective as no antibody purification step is necessary. As an alternative, A. minutum was quantified by exploiting the peroxidase activity of a G-quadruplex DNAzyme covalently bound to nanobodies and by means of a label free electrochemical immunosensor which exploit nanobodies bound via an irreversible SpyTag-SpyCatcher system as the specific immunocapture element. Such immunosensor was prepared by immobilizing the SpyTagged anti-A. minutum nanobody (SpyTagged C1) by means of a selfassembled monolayer (SAMs) of L- cysteine (L-Cys) displayed onto a gold nanoparticle-modified glassy carbon electrode. Electrochemical Impedance Spectroscopy (EIS) was performed to measure the electrical response of the circuit as the function of A. minutum immunocaptured cells. The results of EIS studies confirmed that the sensing chip detected A. minutum selectively, exhibited a wide dynamic range spanning from 103 to 109 cell L-1 and a limit of detection of 3.1× 103 cell L-1. The immunosensor data can be recorded and then analyzed with a portable potentiostat. Such device can be interfaced with a smart phone, a condition suitable for the rapid A. minutum quantification in situ.

Keywords

Alexandrium minutum;nanobodies;bioreagents;immunosensor;toxic microalgae;

Data

Language: English
Year of publishing:
Typology: 2.08 - Doctoral Dissertation
Organization: UNG FPŠ - Graduate School
Publisher: [S. Folarin Oloketuyi]
UDC: 577
COBISS: 19986179 Link will open in a new window
Views: 3242
Downloads: 227
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Other data

Secondary language: Slovenian
Secondary title: FUNKCIONALIZIRANA NANOTELESCA KOT DIAGNOSTIČNI REAGENTI ZA DOLOČANJE TOKSIČNIH MIKROALG
Secondary abstract: Zgodnje odkrivanje in monitoring strupenih mikroalg ostaja izziv, ki ga bo treba premagati za boljše razumevanje cvetenja alg in posledičnih ekoloških in okoljskih vplivov. Za odkrivanje škodljivih vrst alg obstajajo številne diagnostične metode, kot na primer mikroskopija, fluorescenčna hibridizacija in situ (FISH), mikromreže DNA, PCR v realnem času in hibridizacijske metode. Vendar te tehnike niso primerne za monitoring na terenu. Zlasti pri mikroalgi Alexandrium minutum te tehnike tudi ne omogočajo ustrezne kvantifikacije, saj obstajajo številne kriptične vrste, ki jih ni mogoče razlikovati na podlagi morfoloških ali genetskih lastnosti. Ker so te metode tudi zamudne in tehnično zahtevne, je še kako dobrodošel razvoj alternativnih metod, ki bi bile preproste, hitre in stroškovno učinkovite. V doktorskem raziskovalnem delu smo želeli razviti različne detekcijske metode na osnovi nanotelesc za odkrivanje mikroalge A. minutum. Nanotelesca za specifično detekcijo A. minutum smo uspešno izrazili na površini bakterije Escherichia coli. Ta metoda je preprosta in stroškovno učinkovita, saj čiščenje protiteles ni potrebno. Zatem smo razvili postopek za količinsko ovrednotenje A. minutum, kjer smo spremljali peroksidazno aktivnosti G-kvadrupleksnega DNA-zyma, ki je bil kovalentno vezan na nanotelesca. Peroksidazno aktivnost smo ugotavljali s pomočjo elektrokemičnega imunosenzorja brez oznake (label-free), na katerega so bila nanotelesca, kot specifičen imunodetekcijski element, vezana preko ireverzibilnega sistema SpyTag-SpyCatcher. Imunosenzor smo pripravili tako, da smo nanotelesca proti A. minutum, označena z molekulo Spy (SpyTagged C1), imobilizirali na enoplastne sloje (SAMs) L-cisteina (L-Cys), ki so bili nanešeni na ogljikovo elektrodo, modificirano z zlatimi nano-delci. Merjenje električnega odziva tokokroga kot funkcije vezave celic A. minutum smo merili z elektrokemijsko impedančno spektroskopijo (EIS). Rezultati merjenja EIS so potrdili, da je senzorski čip selektivno zaznal prisotnost A. minutum, da je imel širok dinamični razpon od 103 do 109 celic L-1 ter da je bila meja zaznavnosti 3,1 × 103 celice L-1. Podatke z imunosenzorja lahko zabeležimo in nato analiziramo s prenosnim potenciostatom. Takšno napravo je mogoče povezati s pametnim telefonom, kar omogoča hitro in učinkovito spremljanje A. minutum in situ.
Secondary keywords: Alexandrium minutum;nanotelesa;bioreagenti;imunosenzorji;disertacije;
URN: URN:SI:UNG
Type (COBISS): Doctoral dissertation
Thesis comment: Univ. v Novi Gorici, Fak. za podiplomski študij
Pages: XV, 119 str.
ID: 11743870