Measuring Bone Biomarker Alkaline Phosphatase with Wafer-Scale Nanowell Array Electrodes

Biosensors that can analyze a single drop of biological fluid can overcome limitations such as extraction volume from humans or animals, ethical problems, time, and cost. In this work, we have developed a highly sensitive electrochemical (EC) biosensor based on a nanowell array (NWA) for the detection of alkaline phosphatase (ALP), a serum indicator of bone formation. The size of the electrode is 2 × 1 mm² and has over 10 million nanowells (400 nm diameter) arranged uniformly on the electrode surface. For detecting ALP, anti-ALP was immobilized and oriented on the NWA surface using a self-assembled monolayer and protein G. EC impedance spectroscopy (EIS) was used to determine the amount of ALP in 10 μL of sample. The impedance was calibrated with ALP concentration. The NWA has a linear dynamic range from 1 pg/mL to 100 ng/mL with a limit of detection (LOD) at 12 pg/mL. We used the sensor to measure the ALP in real mouse serum from 4, 10, and 20 weeks old mice and compared the results to the standard photometric assay. This work demonstrates the potential of EC NWA sensors to analyze a single drop of a real body fluid sample and to be developed for broad applications.