We report on the fabrication and characterisation of Graphene field-effect transistor (GFET) Biosensors for detecting clusterin, a prominent protein biomarker of Alzheimer’s disease (AD). There are approximately 54 million people currently living with dementia worldwide and this is expected to rise to 130 million by 2050. Although there are over 400 different types of dementia, AD is the most common type, affecting between 50-75% of those diagnosed with dementia. Diagnosis of AD can take up to 2 years currently using MRI, PET, CT scans and memory tests. There is, therefore, an urgent need to develop low-cost, accurate, non-invasive and point-of-care (PoC) sensors for early diagnosis of AD. The GFET sensors we are developing to address this challenge were fabricated on Si/SiO₂ substrate through processes of photolithographic patterning and metal lift-off techniques with evaporated chromium and sputtered gold contacts. Raman Spectroscopy was performed on the devices to determine the quality of the graphene. The GFETs were annealed to improve their performance before the channels were functionalized by immobilising the graphene surface with a linker molecule and anti-clusterin antibody. The detection was achieved through the binding reaction between the antibody and varying concentrations of clusterin antigen from 1pg/mL to 1ng/mL. The GFETs were characterized using 4-probe direct current (DC) electrical measurements which demonstrated a limit of detection of the biosensors to be below 1pg/mL.

Reactive oxygen/nitrogen species (ROS/RNS) have great impact on cellular response to stress, cell proliferation, cell death, cancer, aging or male infertility. Also, in the food industry and for consumers it is very important to monitor quality and freshness of raw meat. Different factors are a sign of meat alteration (e.g. discoloration, rancidity) [1]. One pathway of alteration is the scavenging activity of myoglobin towards RNS (such as peroxynitrite, PON). This paper presents the development of an electrochemical PON sensor using cobalt phthalocyanine (CoPc) as simple, cost effective, highly thermally stable, biomimetic catalyst and the application of this screen-printed carbon electrode (SPCE) based sensor to meat extract samples, using flow injection analysis (FIA). The reduction of peroxynitrite, mediated by CoPc, occurs at a very low potential (≈ 0.1 V vs. Ag/AgCl pseudoreference), as for higher potentials, the mechanism of mediation changes, and the oxidation of PON is observed. The surface of the modified electrode was characterized using SEM, FTIR, Raman and Cyclic Voltametry. Peroxynitrite synthesized solutions were characterized using UV-Vis and ATR-FTIR, and compared to DFT models of relevant molecules/interfering species. The interaction of PON with myoglobin was studied using both UV-Vis and cronoamperometry (0.1 V). Calibration was performed: I_red (nA) = 6.313·C_PON (µM) + 17.469; (R² = 0.9938). The calculated LOD was 2.37 µM and the linear range was 3-180 µM. The performance of the electrode was further improved using the reduction of the CoPc film, at -0.3 V, during 60s. This helped us monitor and quantify how much PON was decomposed, when meat extracts were spiked with PON, in a highly selective, good sensitive and reproducible way.

References

1. Exner, M; Herold, S; Kinetic and mechanistic studies of the peroxynitrite-mediated oxidation of oxymyoglobin and oxyhemoglobin. Chemical research in toxicology. 2000;13(4):287-93.