Development of Novel Biosensor Platforms for Point-Of-Care (POC) Diagnostics

Change log
Prattis, Ioannis 

This thesis presents contributions to the field of point-of-care (POC) testing through the identification of novel biosensing technologies for the detection of stress-related proteins and monitoring of DNA amplification procedures in real-time. The studies presented herein successfully demonstrated proof-of-concept cases using interdigitated microelectrode arrays (IMEAs) and graphene field-effect transistors (GFETs) for POC applications.

IMEAs were utilized to fabricate a conductometric biosensor for label-free, time-point, DNA loop-mediated isothermal amplification (LAMP) detection. The incorporation of Au nanoislands between the electrodes resulted in a notable increase in sensor sensitivity (51.3%, 200%, and 48.5% enhancement for acidic, neutral, and basic solutions, respectively). Moreover, the presence of these nanoislands substantially improved sensor sensitivity during LAMP time-point detection, resulting in a near doubling of current outputs at 200 mV. These findings suggest that IMEA biosensors equipped with nanoislands offer a viable approach for pH sensing applications and end-point detection of LAMP amplification, using label-free, cost- effective, and readily scalable POC testing devices.

Secondly, a flexible GFET biosensor was developed for detecting the neuropeptide Y (NPY) biomarker in artificial sweat with high sensitivity and specificity. The biosensor utilized a polyimide flexible substrate and graphene functionalized with NPY-specific antibodies. NPY is an important biomarker associated with stress levels. The developed biosensor was capable of detecting NPY concentrations as low as 1 pM and exhibited a linear response within the concentration range of 1 pM to 1 μM, with a correlation coefficient of approximately 0.99. This proof-of-concept device represents a potential breakthrough for the development of flexible POC devices for real-time consumer stress monitoring.

The final part of this research was focused on developing GFET-based biosensors for real-time monitoring of LAMP reactions, with an ultimate goal of integrating these biosensors into LAMP-based POC diagnostics. The development of a pH-dependent LAMP master mix was necessary to overcome the challenge of detecting pH changes during the amplification process. The successful development of this master mix provided the foundation for the subsequent assay design and optimization for the detection of the N-acetyltransferase 2 (NAT2) gene. The GFET biosensors were evaluated for their ability to detect changes in the amplification process with a shift in the Dirac point of 150 mV for the 30 min reaction, indicating production of amplified products from the NAT2 template. The biosensors were further evaluated for real-time monitoring of LAMP reaction progress and were capable of detecting amplified products in real-time through detection of changes in pH values, as evidenced by a decrease of about 20% in drain current. This research highlights the potential of GFET-based biosensors for the real-time monitoring of LAMP reactions, which could have significant implications for the development of more efficient and accurate POC diagnostic tools.

Occhipinti, Luigi
Biosensors, flexible, Graphene, Interdigitated, LAMP, PCR, Point-Of-Care, stress
Doctor of Philosophy (PhD)
Awarding Institution
University of Cambridge
EPSRC (2108855)