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Title: Developing ratiometric biosensing platforms for the detection of substrates using protein bound upconverting phosphors
Author: Burgess, Letitia
ISNI:       0000 0004 7971 1111
Awarding Body: University of Manchester
Current Institution: University of Manchester
Date of Award: 2018
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Current biological sensing methods exploit fluorescent organic dyes and quantum dots as the optical probe. They suffer from several drawbacks including UV-Visible excitation, competitive autofluorescence and biotoxicity. This limits their effectiveness in vivo. As an alternative, lanthanide doped upconverting nanophosphors (UCNP) have emerged as a new class of materials for use in optical sensing; they exhibit high photo and chemical stability and utilise near infrared excitation. Current methods of sensing biologically important substrates using upconverting nanoparticles use Luminescent Resonance Energy Transfer (LRET), which is distance dependent. Enzymes are proteins that catalyse biological reactions and they have unrivalled sensitivity and selectivity for their substrates. However to date UCNP-enzyme based biosensors have received relatively little interest in the literature. Chapter 1 summarises the theory behind UCNPs and their current bioapplications. Chapter 2 describes the synthesis of UCNPs via a one-pot hydro(solvo)thermal method. This method is used to produce a series of UCNPs based on a NaYF4 core doped with either Yb/Er or Yb/Tm with a variety of different capping thiol and maleimide ligands. Surface modification of the industry-donated UCPs (donated by Phosphor Technology Limited) to introduce hydrophilicity and functional groups for conjugation was achieved through silica capping. All of the UCNP syntheses and ligand modifications are reproducible, can be applied to UCNPs of different cores and have been fully characterised by a variety of analytical methods. Chapter 3 proved that LRET could occur from a UCP (donor) to a dye (acceptor) resulting in the ratiometric quenching of the UCP emission bands. Then covalent attachment methodology, in order to covalently attach the dyes to the surface of the UCPs, was developed. Chapter 4 demonstrated the ability of the UCPs to ratiometrically sense six chromophoric biomolecules, with a non-linear relationship between spectral overlap and Stern-Volmer constant observed. Chapter 5 screened all the thiol and maleimide capped UCNPs synthesised in Chapter 2 for their bioconjugation ability using GFP as the proof-of-principle protein and the maleimide capping ligands were found to be the better ligands for biomolecule attachment. In Chapter 6, a robust method for the attachment of enzymes to the surface of Gd2SO4:Yb,Er and Gd2SO4:Yb,Tm phosphors through thiol-maleimide coupling was developed. Six different biomolecules were attached to the UCPs and fully characterised. The activity of the enzymes covalently bound to the UCP surfaces was tested before the ratiometric detection of enzyme turnover, using 980 nm excitation of the UCP was investigated. The PETNR(FMN)475 nanoparticle system was proven to act a ratiometric biosensor for NADPH, sodium dithionite, oxygen and ketoisophorone. This is the first known example of a covalently bound UCP-enzyme system acting as a ratiometric biosensor.
Supervisor: Natrajan, Louise ; Hay, Sam ; Jones, Alex Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available