Periodic Reporting for period 1 - CINMAB (Chiral Plasmons in Protein-Nanoparticle Hybrid Materials for Application as Biosensors)
Reporting period: 2016-10-20 to 2018-10-19
Impact on society: Untreatable conditions arising from age-related neurodegenerative diseases are matter of huge concern. The increasing socioeconomic impact of these diseases has boosted research interests on the fundamental aspects of these disorders. The early diagnosis as well as treatment of these diseases is of prime importance in tackling these social challenges. Currently, almost 16% of the European population is over the age of 65 years, and this figure is expected to reach 25% by 2030. Over 6.3 million people around the world are affected by PD and related disorders among which 1.2 million are in Europe. Approximately €130 billion per annum is spent on people with dementia across Europe, highlighting the socioeconomic impact of such diseases. Therefore, these diseases are considered as one of the leading medical and societal challenges faced by the European society, demanding early diagnosis and therapy. Considering the relevance of this topic, the project was focused on the use of plasmonic NPs for the detection of amyloid fibrils in PD and prion diseases.
Overall objective: The major objective of the project was to develop a plasmonic platform for the detection of neurodegenerative diseases. The identification of protein fibrils, which are the hallmark of these diseases, is pivotal in disease diagnosis and development of therapeutic strategies. Our objective was to develop a methodology for the specific detection of amyloid fibrils using chiral effects in plasmonic NPs. The formation of amyloid fibrils based on α-synuclein (aggregation of which causes PD) was probed using gold nanorods (Au NRs). Au NRs showed no apparent interaction with monomeric proteins but effective adsorption onto fibril structures via noncovalent interactions. The technique was successfully applied for the detection of fibrils in PD-affected human brain samples.
The main objective of the project was to differentiate the stable monomeric state of the protein from its aggregated fibrillar state using Au NRs as substrates (Scheme 1). Initial investigations were carried out on recombinant α-synuclein. To separate solutions of Au NRs, α-synuclein monomers as well as aggregates were added. No Extinction and CD spectral changes were observed even after addition of higher concentration (1µM) of protein monomers (inset, Fig. 1a,b) indicating a weak protein–NR interaction. In contrast, a gradual red-shift and broadening of the localized surface plasmon resonance (LSPR) along with a bisignate circular dichroism (CD) signal at the LSPR was observed in presence of protein fibrils. The CD signal exhibited a time-dependent red-shift in the peak position accompanied by an increase in intensity (Fig. 1a,b). The red-shift in extinction indicates preferential tip-to-tip assembly of NRs whereas the CD signals at the LSPR is indicative of a 3D chiral arrangement of the Au NRs directed by helical structure of the template.
Better insight into the nature of assembly was obtained through transmission electron microscopic (TEM) imaging of the nanocomposites. While the TEM images of protein monomer-Au NRs mixture showed no specific patterns (Fig. 2a), solutions containing protein fibrils exhibited hybrid structures with NRs arranged following the direction of the fibrils (Fig. 2b). Cryogenic EM tomography experiments accomplished a clear double-helical structure for the NR assembly (Fig. 2c). High positive (+30 mV) and negative (-30 mV) zeta potential values observed for NRs and fibrils, respectively suggested the NR-protein fibril interactions to be likely electrostatic. Theoretical modelling results were in good agreement with the experimental data establishing that the observed optical activity arises from dipolar coupling between adjacent NRs. Intense chiroptical activity of Au NRs allowed the detection of amyloid fibrils down to nanomolar concentrations.
Detection of Parkinson’s disease in human brain homogenates
With an ultimate aim of developing a biodetection platform for potential applications, experiments were designed on brain homogenates to identify the presence of protein fibrils in human brain samples (samples collected postmortem). Brain samples collected from PD-affected patients were homogenized in a suitable buffer. Healthy brain homogenates were used as controls. Addition of purified brain samples to Au NRs resulted in a red-shift in the longitudinal LSPR in the case of PD-affected samples but least effect from healthy controls (Fig. 3a). Interestingly, intense bisignate CD signals were recorded from the PD sample. However, experiments with purified healthy controls showed only weak CD signals exhibiting a clear distinction between PD-affected samples and healthy controls (Fig. 3b).
Extension of the technique for the detection of infectious prion fibrils
To establish a wider applicability of the proposed technique, investigations on infectious prion protein from bank vole was carried out. Addition of monomeric PrP to Au NRs showed no spectral changes ruling out any protein interaction with Au NRs. In contrast, addition of prion fibrils resulted in the red-shift and broadening of the Au NR longitudinal plasmon band indicating tip to tip assembly of NRs (Fig. 4a). Intense CD signals with a negative couplet at the LSPR indicated the helical assembly of NRs on the fibril surface (Fig. 4b). These experiments established a new technique for the detection of infectious prion fibrils.