BackgroundThere are no blood-based molecular biomarkers of temporal lobe epilepsy (TLE) to support clinical diagnosis. MicroRNAs are short noncoding RNAs with strong biomarker potential due to their cell-specific expression, mechanistic links to brain excitability, and stable detection in biofluids. Altered levels of circulating microRNAs have been reported in human epilepsy, but most studies collected samples from one clinical site, used a single profiling platform or conducted minimal validation.MethodUsing a case-control design, we collected plasma samples from video-electroencephalogram-monitored adult TLE patients at epilepsy specialist centers in two countries, performed genome-wide PCR-based and RNA sequencing during the discovery phase and validated findings in a large (>250) cohort of samples that included patients with psychogenic non-epileptic seizures (PNES).FindingsAfter profiling and validation, we identified miR-27a-3p, miR-328-3p and miR-654-3p with biomarker potential. Plasma levels of these microRNAs were also changed in a mouse model of TLE but were not different to healthy controls in PNES patients. We determined copy number of the three microRNAs in plasma and demonstrate their rapid detection using an electrochemical RNA microfluidic disk as a prototype point-of-care device. Analysis of the microRNAs within the exosome-enriched fraction provided high diagnostic accuracy while Argonaute-bound miR-328-3p selectively increased in patient samples after seizures. In situ hybridization localized miR-27a-3p and miR-328-3p within neurons in human brain and bioinformatics predicted targets linked to growth factor signaling and apoptosis.InterpretationThis study demonstrates the biomarker potential of circulating microRNAs for epilepsy diagnosis and mechanistic links to underlying pathomechanisms.

The need for practical biomarkers for early diagnosis of Alzheimer’s disease (AD) remains largely unmet. Here we investigated the use of blood-based microRNAs as prognostic biomarkers for AD and their application in a novel electrochemical microfluidic device for microRNA detection. MicroRNA transcriptome was profiled in plasma from patients with mild cognitive impairment (MCI) and AD. MicroRNAs Let-7b and microRNA-206 were validated at elevated levels in MCI and AD, respectively. MicroRNA-206 displayed a strong correlation with cognitive decline and memory deficits. Longitudinal follow-ups over five years identified microRNA-206 increases preceding the onset of dementia. MicroRNA-206 was increased in unprocessed plasma of AD and MCI subjects, detected by our microfluidic device. While increased Let-7b levels in plasma may be used to identify patients with MCI, changes in plasma levels of microRNA-206 may be used to predict cognitive decline and progression towards dementia at an MCI stage. MicroRNA quantification via a microfluidic device could provide a practical cost-effective tool for the stratification of patients with MCI according to risk of developing AD.

Diagnosis of seizure disorders such as epilepsy currently relies on clinical examination and electroencephalogram recordings and is associated with substantial mis-diagnosis. The miRNA, miR-134 (MIR134 in humans), has been found to be elevated in brain tissue after experimental status epilepticus and in human epilepsy cells and their detection in biofluids may serve as unique biomarkers. miRNAs from unprocessed human plasma and human cerebrospinal fluid samples were used in a novel electrochemical detection based on electrocatalytic platinum nanoparticles inside a centrifugal microfluidic device where the sandwich assay is formed using an event triggered release system, suitable for the rapid point-of-care detection of low abundance biomarkers of disease. The device has the advantage of controlling the rotation speed of the centrifugal device to pump nanoliter volumes of fluid at a set time and manipulate the transfer of liquids within the device. The centrifugal platform improves reaction rates and yields by proposing efficient mixing strategies to overcome diffusionlimited processes and improve mass transport rates, resulting in reduced hybridization times with a limit of detection of 1 pM target concentration. Plasma and cerebrospinal fluid samples (unprocessed) from patients with epilepsy or who experienced status epilepticus were tested and the catalytic response obtained was in range of the calibration plot. This study demonstrates a rapid and simple detection for epilepsy biomarkers in biofluid.Epilepsy is a common neurological disease characterised by an enduring predisposition to recurrent seizures 1, 2 . Seizures can also occur in patients without epilepsy as a result of a systemic disturbance (e.g. infection). Prolonged seizures, termed status epilepticus, are a neurological emergency and non-convulsive SE is extremely hard to diagnosis due to the not specific EEG pattern in NCSE meaning they are frequently misdiagnosed. Diagnosis of both epilepsy and status epilepticus is sometimes challenging and often relies heavily on clinical examination and history alone. The primary tool used for diagnosis of seizure disorders is the electroencephalogram (EEG). While invaluable, EEG is costly and technically demanding 3 . Moreover, many patients with epilepsy have a normal EEG recording while patients without epilepsy can have apparently abnormal EEG findings. As a result there are

Oligonucleotide hybridization to a complementary sequence that is covalently attached to an electrochemically active conducting polymer (ECP) coating the working electrode of an electrochemical cell causes an increase in reaction impedance for the ferro-ferricyanide redox couple. We demonstrate the use of this effect to measure, in real time, the progress of DNA polymerase chain reaction (PCR) amplification of a minor component of a DNA extract. The forward primer is attached to the ECP. The solution contains other PCR components and the redox couple. Each cycle of amplification gives an easily measurable impedance increase. Target concentration can be estimated by cycle count to reach a threshold impedance. As proof of principle, we demonstrate an electrochemical real-time quantitative PCR (e-PCR) measurement in the total DNA extracted from chicken blood of an 844 base pair region of the mitochondrial Cytochrome c oxidase gene, present at ∼1 ppm of total DNA. We show that the detection and semiquantitation of as few as 2 copies/μL of target can be achieved within less than 10 PCR cycles.

Elevated levels of transfer RNA (tRNA) fragments were recently identified in plasma samples from people with epilepsy in advance of a seizure, indicting a potential novel class of circulating biomarker. Current methods for detection and quantitation of tRNA fragments (tRFs) include northern blotting, RNA sequencing or custom Taqman-based PCR assays. The development of a simple, at home or clinic-based test, would benefit from a simple and reliable method to detect the tRFs using small volumes of biofluids. Here we describe an electrochemical direct detection method based on electrocatalytic platinum nanoparticles to detect 3 specific tRFs: 5’AlaTGC, 5'GlyGCC, and 5'GluCTC. Using synthetic tRF mimics we showed this system was linear over 9 orders of magnitude with sub-attomolar limits of detection. Specificity was tested using naturally occurring mismatched tRF mimics. Finally, we quantified tRF levels in patient plasma and showed that our detection system recapitulates results obtained by qPCR. We have designed a tRF detection system with high sensitivity and specificity capable of quantifying tRFs in low volumes of plasma using benchtop apparatus. This is an important step in the development of a point-of-care device for quantifying tRFs in whole blood.