Minimally invasive diagnosis of Alzheimer’s disease by detecting microRNA using a quartz crystal resonator

In 2014, there were 850,000 people living with dementia in the UK, creating an economic burden of £26.3 billion a year. 62% of dementia patients are diagnosed with Alzheimer’s Disease (AD). AD is a slow progressing disease with three phases: a long prodromal stage followed by mild cognitive impairment and then late AD. The prodromal phase of AD is on average 30 years. So, when the first symptoms become apparent, the pathology in the brain is extensive. If Alzheimer’s could be diagnosed early, when the pathological load is lower, this may improve the chances of finding a disease modifying therapy.

For diagnosis during the prodromal stage to be viable, patients would need to be diagnosed through mass screening, with the most appropriate diagnosis method being biomarker detection in peripheral blood. There are an increasing number of articles in literature researching the use of non coding RNA sequences called microRNA (miRNA) as biomarkers for AD. However, their viability in diagnosing prodromal AD is unknown and the current miRNA detection method, the polymerase chain reaction (PCR), is time consuming, expensive and requires experienced personnel, making it unsuitable for use in mass screening. Therefore, the aim of the thesis was to investigate miRNA as a prodromal biomarker with a new detection method to determine the usability of miRNA as a prodromal AD biomarker.

AD is characterised by the build up of amyloid β and hyper phosphorylated tau in the brain. The movement of tau through the brain is divided into 6 Braak stages. Chapter 4 aimed to determine the point at which miRNAs deregulate in AD. To achieve this, post mortem brain tissue was obtained through all 6 Braak stages. The RNA from the post-mortem brain samples were isolated and the change in miRNA levels were determined using PCR and the ΔΔct method. After comparing miRNA to a spike in control the 4 miRNAs tested showed no significant change in miRNA levels through the progression of AD.

One of the first signs of AD is the activation of astrocytes in the brain. The aim of Chapter 5 was to determine the effect of the deregulated miRNAs on astrocytes. An astrocytoma cell line was activated using lipopolysaccharides and TNF α then transfected so specific miRNA were over expressed. The concentration of metabolites, cytokines and growth factors were measured in the cell supernatant. Results showed mir 210 regulated G CSF and mir 223 regulated glutamate consumption.

Finally, the feasibility of rapid detection of miRNA was investigated using a quartz crystal microblance (QCM). The QCM, assembled within a custom built microfluidic flow cell, was driven at its fundamental resonance frequency. Shifts in the third Fourier harmonic current and the resonance frequency were measured for a range of concentrations of a single stranded DNA (ssDNA). The results show feasibility for rapid quantitative detection of ssDNA to 60 ng/mL, in an easy to use label free assay. Amplification of the signal was seen with the addition of electrochemical potential and the use of particles.

Results looking at the deregulation of miRNA in the temporal cortex showed no significant change, therefore further work is needed looking at alternative miRNA sequences. The results with ssDNA suggest that quartz crystal resonator has the potential for rapid, specific and quantitative miRNA detection. However, amplification strategies would help to achieve the clinically relevant limit of detection in peripheral blood.