A dual-antibody gold nanoparticle-based lateral flow assay for rapid and selective detection of mesenchymal stem cell stemness

Rapid and reliable identification of stem cells is a critical prerequisite for regenerative medicine and quality control during cell-based therapies. We report herein a lateral flow assay capable of detecting MSCs by dual-marker recognition using CD105 and CD29 surface antibodies. The biosensing platform relies on gold nanoparticle-conjugated CD29 antibodies as signal probes and immobilized CD105 antibodies as capture agents on the test line, allowing selective MSC recognition through sandwich-type immunocomplex formation with direct colorimetric readout. A distinct red band indicated the successful detection of MSCs, while a control line ensured assay validity and reliability. The developed LFA showed a naked eye detection limit as low as 600 cells within 15 min, which agreed with the qualitative data obtained via a smartphone-based Color Grab application. Notably, the device exhibited excellent stability with no cross-reactivity with non-target cells and successfully distinguished MSCs from their differentiated lineages. Besides this, the device retained stability for up to 28 days under room temperature. Further, MSC stemness was independently validated for CD105 and CD29 by flow cytometry (FACS), which requires a minimum of ≥10 000 cells per analysis. In comparison, the developed LFA provided reliable detection at substantially lower cell numbers, highlighting its significance as a low-input, rapid, and point-of-care alternative. This user-friendly platform represents a promising tool for rapid stemness assessment of MSCs towards their quality control and clinical translation in stem cell research and regenerative medicine applications.