Chapter 5: Summary and Future prospects
B) The ways in which lipoproteins may be modified to act as contrast agents. (Adapted with permission from Cormode et al, 2010)
2.6.3 Nanomaterial based assay platforms for Neuro-diagnostics
Although nano-scale imaging techniques like electron microscopy and atomic force microscopy can detect protein misfolding associated with neuro-degenerative diseases, it is usually possible at very severe stages of the disease (Lyubchenko et al, 2010). Most of the biomarkers for such diseases appear at young age, devoid of any clinical symptoms and hence early detection of these biomarkers is essential.
Nanomaterials with their high sensitivity and specificity have been integrated into biosensors which can detect ultra-low concentrations of these biomarkers in a non- invasive, inexpensive and rapid manner.
2.6.3.1 Gold nanoparticle based assays for early detection of Alzheimer’s disease
Alzheimer’s disease (AD) is the most common form of dementia affecting millions of people worldwide every year. Several hypotheses have been put forward in an attempt to explain the disease mechanism. According to the most accepted hypothesis, amphipathic peptides called amyloid beta (Aβ) polymerize, aggregate, accumulate and deposit in Alzheimer’s affected brain and forms protofibrils and fibrils. These amyloid fibrils are highly neurotoxic inducing neuronal cell death possibly by triggering oxidative stress. Another hypothesis suggests that such oxidative stress can lead to hyper-phosphorylation of a microtubule associated protein called ‘τ’. Hyperphosphorylated τ-protein results in destabilization of microtubules which in turn compromise synaptic connections. Another possible factor in AD pathogenesis is thought to be amyloid derived diffusible ligands (ADDL) which is a synthetic Aβ derivative produced from Aβ monomers. ADDL has been shown to be more toxic than Aβ even at nanomolar concentrations. The initial steps towards developing an assay for AD detection were taken by scientists from Northwestern University, USA. They were able to study the binding kinetics of ADDL and anti-ADDL antibody using silver nanotriangles and localised surface plasmon resonance (LSPR) (Haes et al, 2004). The study was extended further to diagnose ADDL in CSF of patients using a gold nanoparticle based bio-barcode assay. The assay could detect below femtomolar concentrations of ADDL rapidly, thereby
providing the first significant example of nano based diagnostic assay for early detection of AD (Georganopoulou et al, 2005). Nanoparticle based plasmonic sensors for detection of Aβ peptide aggregates can be a potent device for early detection of AD. Gold nanoparticle coated with anti-Aβ antibody have been reported to detect ultra-low concentrations of amyloid-beta peptide across various platforms like surface plasmon resonance, dot blot assay and even by naked eye (Lee et al, 2009; Wang et al, 2012; Sakono et al, 2012). Further the application of anti Aβ labelled quantum dots enabled real time imaging of protein aggregation, oligomerization and fibrillation (Tokuraku et al, 2009). Similar biosensors were developed using gold nanoparticle to detect ultra-low concentrations of other AD biomarkers like τ-protein and acetylcholine esterase (AChE) in CSF using two- photon Rayleigh scattering property and colorimetric assay techniques respectively (Neely et al, 2009; Liu et al, 2012). Multifunctional nanoparticles capable of detecting as well as inhibiting Aβ fibril formation have been designed using gold and iron oxide for potential theranostic applications in AD (Choi et al, 2013; Skaat et al, 2013).
2.6.3.2 Nanomaterial based assays for early detection of Parkinson’s disease Like AD, another neurodegenerative disease primarily affecting the aged called Parkinson’s disease (PD) is also characterised by abnormal protein accumulation.
The deposition of high amounts of alpha-synuclein protein leads to formation of Lewy bodies inside neurons. However, the major cause of PD is thought to be the drastic depletion of neurons in the substantia nigra region of the brain primarily involved in secretion of a catecholamine neurotransmitter called dopamine (DA). Tyrosine hydroxylase, a critical enzyme in catecholamine biosynthesis pathway was monitored by gold-nanoparticle based DNA barcode assay. Its quantification could provide a direct indication to the levels of dopamine and hence detect the onset of PD (An et al, 2013, 2012). Similar attempts at measuring dopamine were made by other groups using different transducer platforms. For example, a single walled carbon nanotube based voltammetric biosensor was designed to simultaneously detect DA and Adenosine (metabolic indicator of neuronal activity) with very low detection limit (34.7μM for adenosine, 7μM for dopamine) (Goyal et al, 2008).
Another team reported a voltammetric biosensor designed to detect three potential PD biomarkers – Uric Acid (UA), DA and Ascorbic Acid (AA). The biosensor was fabricated with ZnO nanowire arrays on 3D grapheme foam and was capable of
detecting all three analytes with high sensitivity and accuracy from clinical samples of PD patients (Yue et al, 2014) (Fig 2.17). Recent attempts to detect PD biomarkers in exhaled breath by using nanotechnology platforms are undergoing clinical trials (ClinicalTrials.gov Identifier: NCT01246336).
Fig 2.17: ZnO nanowires on 3D grapheme foam for detection of Parkinson’s disease (i) Structural analysis of the integrated ZnO NWA/GF. (a) Schematic of the ZnO NWA/GF electrode and detection of UA, DA, and AA. (b-e) SEM images of the ZnO NWAs on the 3D GF at different magnifications. Inset: EDX of the ZnO NWAs. (f) SEM images of the height of the ZnO NWAs, ∼2 μm. Inset: diameter of the ZnO NWAs, ∼40 nm.
(ii) Electrochemical cell. (a) Schematic of three-electrode electrochemical cell setup. The ZnO NWA/GF on the ITO glass served as the working electrode (WE), coupled with a platinum as the counter electrode (CE) and an Ag/AgCl (sat. KCl) as the reference electrode (RE). (b) Photograph of the electrochemical cell used in our experiment. (Adapted with permission from Yue et al, 2014).
The need for a cost effective, rapid and highly sensitive diagnostic platform for early detection of biomarkers related to neurodegenerative diseases prompted the European union to initiate a multi-institute collaborative initiative for development of nanosystems for early diagnosis of neurodegenerative diseases (NADINE) in 2010.