Integrated Single-Cell Array-Electrochemical Platform for Label-Free, Nondestructive Longitudinal Tracking of Osteogenic Differentiation.

Longitudinal, label-free tracking of stem cell differentiation is hindered by destructive end point assays and variability from cell migration and uncontrolled contacts. Here, we introduce an integrated single-cell array-electrochemical platform that standardizes the microenvironment while enabling repeated, nondestructive readouts on the same culture. Human bone marrow-derived mesenchymal stem cells (hBMSCs) were seeded onto a conductive single-cell array of polydopamine microdots microcontact-printed on poly(vinyl alcohol)-modified indium tin oxide (PVA-ITO). A thin methylcellulose overlay preserved viability and differentiation while limiting migration. Integrated with the electrochemical detection platform, the same substrate serves as the working electrode, enabling nondestructive electrochemical quantification of the osteogenic marker alkaline phosphatase (ALP) via conversion of l-ascorbic acid 2-phosphate (AAP) to electroactive ascorbic acid (AA). This integration enables standardized, label-free time-course measurements and is readily extensible to other enzymatic reporters and lineage programs.