Final Report Summary - NANOSMARTS (Smart nondimensional biosensors for detection of tumor cells and cytotoxic amyloids intermediates)
The project was to develop: (A) biconjugated silicon nanoparticles (NPs) as fluorescent probes to be applied in diagnostic procedures related to tumour biology; and (B) dual-emission band fluorescent biosensors for protein conformational investigations of neurodegenerative disease.
A. In collaboration with group in Santiago de Compostela (Spain) we synthesised silicon NPs and studied their properties. The very low fluorescence quantum yields and UV excitation limited their suitability. We therefore switched to semiconductor quantum dots (QDs) with much better fluorescence properties (quantum yield, broad excitation, tunable emission). To synthesise water-soluble NPs we covered hydrophobic QDs with an amphiphilic polymer, mainly poly(isobutylenealt-maleic acid) dodecyl amide, containing two main building blocks: hydrophobic dodecyl chains compatible with the QD surface, and hydrophilic carboxyl groups exposed to the aqueous solution.
The carboxyl groups also served as anchors for molecules of interest (ligands). By controlling the number of binding sites per NP, unwanted crosslinking effects leading to aggregation were avoided.
In collaboration with Dr. Wolfgang Parak we functionalized QDs with poly(ethylene glycol) (PEG) molecules. The use of PEG with 2 amino groups led to monamino QDs, which were used for labelling the epidermial growth factor receptor (EGFR), overexpressed in many types of human cancer. We covalently labelled the EGFR by fusion with an ACP tag, to which a probe, e.g. a QD was transferred enzymatically from a coenzyme A (CoA) conjugate. Such modified QDs served as specific, versatile markers of the EGFR.
B. The second part of the project involved the development of dual-emission band fluorescent biosensors for conformational studies of neurodegenerative diseases, specifically Parkinson's disease (PD). PD is characterised by the deposition in midbrain neurons of aggregates composed primarily of the protein a-synuclein (AS). The cytopathology is likely due to (1) AS binding to membranes of different organelles and (2) a gain of neurotoxic function by soluble, oligomeric intermediates in the aggregation pathway. Few methods are sensitive to oligomeric states of AS and there is also little understanding of how the binding of AS to cellular membranes may lead to neuronal dysfunction and death.
We created environment-sensitive dual-emission ESIPT (Excited state intramolecular protein transfer) probes, with which we labelled AS covalently for:
(1) the systematic study of the influence of charge, phase, curvature, defects and unsaturation on AS binding to membranes. We modelled these properties in lipid vesicles and used AS labeled with the ESIPT probe MFE. The interaction of AS with membranes is fast and reversible and occurs by different mechanisms to neutral and negatively charged membranes. Binding to negatively charged membranes is much faster and stronger and much less sensitive to other membrane properties. These findings provide new insights into the relation between membrane physical properties and AS binding affinity and dynamics defining the localisation of AS in vivo and its role in the pathophysiology of PD.
(2) the continuous monitoring of AS aggregation. In collaboration with colleagues at the University of Buenos Aires we discovered the existence of the family of AS aggregation intermediates we have denoted as 'acunas'. The detection of these supramolecular transient species was enabled by use of another ESIPT probe, MFC. We established the structural identity of acunas by sampling the reactions at various times by AFM, guided by the temporal course of the ESIPT dual band T*/N* ratio. Cryo-electron tomography was also applied. We propose that the acuna family possess unique structural features promoting interactions with other proteins and structures, thereby constitute (the) toxic species leading to neuronal dysfunction and loss.
A. In collaboration with group in Santiago de Compostela (Spain) we synthesised silicon NPs and studied their properties. The very low fluorescence quantum yields and UV excitation limited their suitability. We therefore switched to semiconductor quantum dots (QDs) with much better fluorescence properties (quantum yield, broad excitation, tunable emission). To synthesise water-soluble NPs we covered hydrophobic QDs with an amphiphilic polymer, mainly poly(isobutylenealt-maleic acid) dodecyl amide, containing two main building blocks: hydrophobic dodecyl chains compatible with the QD surface, and hydrophilic carboxyl groups exposed to the aqueous solution.
The carboxyl groups also served as anchors for molecules of interest (ligands). By controlling the number of binding sites per NP, unwanted crosslinking effects leading to aggregation were avoided.
In collaboration with Dr. Wolfgang Parak we functionalized QDs with poly(ethylene glycol) (PEG) molecules. The use of PEG with 2 amino groups led to monamino QDs, which were used for labelling the epidermial growth factor receptor (EGFR), overexpressed in many types of human cancer. We covalently labelled the EGFR by fusion with an ACP tag, to which a probe, e.g. a QD was transferred enzymatically from a coenzyme A (CoA) conjugate. Such modified QDs served as specific, versatile markers of the EGFR.
B. The second part of the project involved the development of dual-emission band fluorescent biosensors for conformational studies of neurodegenerative diseases, specifically Parkinson's disease (PD). PD is characterised by the deposition in midbrain neurons of aggregates composed primarily of the protein a-synuclein (AS). The cytopathology is likely due to (1) AS binding to membranes of different organelles and (2) a gain of neurotoxic function by soluble, oligomeric intermediates in the aggregation pathway. Few methods are sensitive to oligomeric states of AS and there is also little understanding of how the binding of AS to cellular membranes may lead to neuronal dysfunction and death.
We created environment-sensitive dual-emission ESIPT (Excited state intramolecular protein transfer) probes, with which we labelled AS covalently for:
(1) the systematic study of the influence of charge, phase, curvature, defects and unsaturation on AS binding to membranes. We modelled these properties in lipid vesicles and used AS labeled with the ESIPT probe MFE. The interaction of AS with membranes is fast and reversible and occurs by different mechanisms to neutral and negatively charged membranes. Binding to negatively charged membranes is much faster and stronger and much less sensitive to other membrane properties. These findings provide new insights into the relation between membrane physical properties and AS binding affinity and dynamics defining the localisation of AS in vivo and its role in the pathophysiology of PD.
(2) the continuous monitoring of AS aggregation. In collaboration with colleagues at the University of Buenos Aires we discovered the existence of the family of AS aggregation intermediates we have denoted as 'acunas'. The detection of these supramolecular transient species was enabled by use of another ESIPT probe, MFC. We established the structural identity of acunas by sampling the reactions at various times by AFM, guided by the temporal course of the ESIPT dual band T*/N* ratio. Cryo-electron tomography was also applied. We propose that the acuna family possess unique structural features promoting interactions with other proteins and structures, thereby constitute (the) toxic species leading to neuronal dysfunction and loss.