Development of biosensors for biomarker detection and clinical therapeutic drug monitoring

Introduction: Despite technological advancements and clinical breakthroughs in the field of medicine, there remains a gap in the effective diagnosis and the monitoring of diseases to achieve therapeutic success. Current laboratory analysis methods such as mass spectrometry, HPLC and MRI are complex, have high turnaround time, require expertise, are expensive and restricted to laboratory setting. In this regard biosensors offer multiple advantages over conventional techniques by being highly sensitive, cost-effective, rapid, and reliable testing system brought to the patient out of the lab.

In the above context, the current research focused on early diagnosis of Alzheimer’s Disease (AD) and enabled monitoring of antibiotic levels in critically ill patients using biosensing technology. The overlapping of dementia biomarkers and heterogeneity in symptoms has limited the differential diagnosis of AD using the current diagnostic techniques. Antimicrobial resistance along with severe septicaemia are the emerging global health threats in absence of personalised antibiotic therapy that allows correct dosing to reduce the risk of treatment failure and toxicity.

For the first time, a tetrapolar impedimetric biosensing system for detecting two contrasting sized molecules – Visinin like protein1 (VILIP1, 22 kDa), a novel biomarker for AD detection and the antibiotic drug meropenem (a carbapenem beta lactam drug; 383.46 Da) was developed. The objective was to develop a biosensor capable of capturing the analyte independent of its size. The biosensor was developed to monitor VILIP1 in clinical human samples for the early detection of AD. Separately, another biosensor was developed to monitor meropenem concentration in critically ill patients for correct dosing of the drug to mitigate against the development of antibiotic resistance in clinical settings.

Method: The biosensor was constructed with the layers of a self-assembled monolayer, protein G, and antibody immobilised on a tetra-polar gold microband electrode to capture the antigen (VILIP1/meropenem) and form a stable complex for higher measurable impedance. Electrochemical impedance changes were measured using a Solartron 1260 analyser with SMaRT software. Tandem mass spectrometry and Immunocapture Liquid Chromatography-Mass Spectrometry (LC-MS/MS) along with Western blotting was used to validate the coupling between antibody and antigen Ab-Ag (anti-meropenem-meropenem with Immunocapture Tandem mass spectrometry and anti-VILIP1-VILIP1 using western blotting along with immunocapture mass spectrometry). ELISA was conducted using a commercially available ELISA kit on human CSF samples to detect the presence of AD biomarkers - Amyloid beta and VILIP1.

Results: The biosensor detected meropenem between 1-100 µg/mL in spiked commercial human serum and this was within clinical range (2 -25 µg/mL) with the lowest therapeutically useful concentration at 0.003 µM (1 µg/mL). The lowest level of VILIP1 detected by the biosensor was 10-3 pM (1 fM), much lower than detection with Western blotting (2 nM) or with commercial ELISA kit (1.5 pM). The obtained limit of detection of the biosensor for VILIP1 is 0.5 pM and for meropenem is 38.2 µg/mL. The overall time taken to detect respective markers on a fully developed biosensor was 15 min.

The developed biosensor was capable of detecting VILIP1 between 100 pM – 10 -3 pM which is below the baseline level of 4.14 pM. The biosensor for meropenem could detect meropenem between 1-100 µg/mL which encompassed the clinical range 2-16 µg/mL. The sensing technology successfully recognises two different sized molecules and can be further enhanced and translated for use as a point-of-care device for a range of biomarkers in future.