Chemically functionalised graphene biosensor for the label-free sensing of exosomes

Exosomes are a subpopulation of vesicles that are expelled from all cells and are speculated to have some role in the development of cancerous tissue. The work in this thesis presents the sensitive and specific electrical detection of exosomes in solution using a graphene field effect transistor (gFET) biosensor: non-covalently functionalised using an intermediate linker and conjugated with anti-CD63 antibodies. The intermediate linker chosen was a pyrene based molecule, which pi-stacks to the graphene surface without disruption, or introduction of high defect density, to the graphene sp2 hybridised structure unlike covalent functionalisation. The gFET biosensor could act as an cheap, wafer-scalable alternative to more conventional labelling techniques and the current state-of-the-art optical techniques that use surface plasmon resonance (SPR) on functionalised gold substrates, which have so far detected down to 1000 exosomes/μL. By exposing only part of the graphene surface to charged species using a microfluidic channel, regions of different doping levels arose in the same graphene film. This manifested as an additional minimum, which shifted to higher gate voltage (Vg) in the transfer characteristic and saturated over 30 minutes when graphene was exposed to exosomes. The saturation was attributed to an equilibrium between exosomes binding and unbinding to anti-CD63 antibodies. As exosomes exhibit negative charge when in phosphate buffered saline (PBS) at pH 7, the accumulation of positive charge in the exposed graphene as they come close to its surface causes a shift to higher Vg by the electrostatic field effect. Therefore the exposed graphene becomes p-doped relative to when exposed only to PBS. The value of the shift after 30 minutes was also concentration dependent with sensitivity down to a concentration of 0.1 μg/mL exosomes (5000 exosomes/μL). By using two negative controls: an isotype control antibody and a non-specific protein target, the gFET sensor was also shown to be specific to CD63 in exosome membranes. Both back-gated and electrolyte top-gated gFETs were used for exosome detection whereby capacitative effects as well as electrostatic effects contributed to the sensing mechanism for the electrolyte top-gated gFET.Open Acces

Electronic and electrochemical viral detection for point-of-care use: A systematic review

From PLOS via Jisc Publications RouterHistory: collection 2021, received 2021-07-05, accepted 2021-09-15, epub 2021-09-30Publication status: PublishedFunder: EPSRC Graphene NowNano CDT; Grant(s): EP/L01548X/1Funder: Dame Kathleen Ollerenshaw FellowshipDetecting viruses, which have significant impact on health and the economy, is essential for controlling and combating viral infections. In recent years there has been a focus towards simpler and faster detection methods, specifically through the use of electronic-based detection at the point-of-care. Point-of-care sensors play a particularly important role in the detection of viruses. Tests can be performed in the field or in resource limited regions in a simple manner and short time frame, allowing for rapid treatment. Electronic based detection allows for speed and quantitative detection not otherwise possible at the point-of-care. Such approaches are largely based upon voltammetry, electrochemical impedance spectroscopy, field effect transistors, and similar electrical techniques. Here, we systematically review electronic and electrochemical point-of-care sensors for the detection of human viral pathogens. Using the reported limits of detection and assay times we compare approaches both by detection method and by the target analyte of interest. Compared to recent scoping and narrative reviews, this systematic review which follows established best practice for evidence synthesis adds substantial new evidence on 1) performance and 2) limitations, needed for sensor uptake in the clinical arena. 104 relevant studies were identified by conducting a search of current literature using 7 databases, only including original research articles detecting human viruses and reporting a limit of detection. Detection units were converted to nanomolars where possible in order to compare performance across devices. This approach allows us to identify field effect transistors as having the fastest median response time, and as being the most sensitive, some achieving single-molecule detection. In general, we found that antigens are the quickest targets to detect. We also observe however, that reports are highly variable in their chosen metrics of interest. We suggest that this lack of systematisation across studies may be a major bottleneck in sensor development and translation. Where appropriate, we use the findings of the systematic review to give recommendations for best reporting practice

Diagnosis methods for COVID-19: A systematic review

At the end of 2019, the coronavirus appeared and spread extremely rapidly, causing millions of infections and deaths worldwide, and becoming a global pandemic. For this reason, it became urgent and essential to find adequate tests for an accurate and fast diagnosis of this disease. In the present study, a systematic review was performed in order to provide an overview of the COVID-19 diagnosis methods and tests already available, as well as their evolution in recent months. For this purpose, the Science Direct, PubMed, and Scopus databases were used to collect the data and three authors independently screened the references, extracted the main information, and assessed the quality of the included studies. After the analysis of the collected data, 34 studies reporting new methods to diagnose COVID-19 were selected. Although RT-PCR is the gold-standard method for COVID-19 diagnosis, it cannot fulfill all the requirements of this pandemic, being limited by the need for highly specialized equipment and personnel to perform the assays, as well as the long time to get the test results. To fulfill the limitations of this method, other alternatives, including biological and imaging analysis methods, also became commonly reported. The comparison of the different diagnosis tests allowed to understand the importance and potential of combining different techniques, not only to improve diagnosis but also for a further understanding of the virus, the disease, and their implications in humans