Rizzotto, F. | bioRxiv Science

bioRxiv2023

Electrochemical biosensor based on NAD(P)H-dependent Quinone Reductase for rapid and efficient detection of vitamin K3

Vitamin K refers to a group of vitamins that play an important role in blood coagulation and regulation of bone and vascular metabolism. However, vitamin K3 may give severe side effects in animal and humans when improperly added to food and feed due to its toxicity. Here, an electrochemical biosensor, based on the YaiB NADPH-dependent quinone reductase from Lactococcus lactis (YaiB), was developed to achieve rapid and redox probe-free detection of vitamin K3. First, we demonstrated the ability of the carbon electrode to distinguish between 1,4-benzoquinone and hydroquinone. Then, we engineered YaiB to work as a bioreceptor immobilized at the electrode and we demonstrated its sensitivity and specificity to reduce vitamin K3. Finally, to demonstrate the practical potential of the biosensor, we tested it directly in spiked milk samples, achieving 15-minute quantification of the vitamin K3. The limit of detection was 0.18M and 0.86 M in buffer and milk, respectively.

biophysics↗

bioRxiv2022

Low-cost gold-leaf electrode as a platform for Escherichia coli immuno-detection

Gold electrodes are one of most prevalent substrates in electrochemical biosensors because they can be easily and highly efficiently functionalized with thiolated biomolecules. However, conventional methods to fabricate gold electrodes are costly, time consuming and require onerous equipment. Here, an affordable method for rapid fabrication of an electrochemical immune-sensor for Escherichia coli detection is presented. The gold electrode was generated using 24-karat gold leaves and low-cost polyvinyl chloride adhesive sheets covered with an insulating PTFE layer. The gold-leaf electrode (GLE) was patterned using laser ablation and characterized by cyclic voltammetry, electrochemical impedance spectroscopy, scanning electronic microscopy, contact angle and 3D profiling. The GLEs were modified by a self-assembled mercaptopropionic monolayer, followed by surface activation to allow binding of the specific anti-E. coli antibody via carbodiimide linking. The biosensor showed a detection limit of 2 CFU/ml and a linear dynamic range of 10 - 107 CFU/ml for E. coli cells. No false positive signals were obtained from control bacteria. The obtained results demonstrated suitability of GLE for use in biosensors with high reliability and reproducibility. It is foreseeable that our work will inspire design of point-of-need biosensors broadly applicable in low-resource settings.

microbiology↗