Y Detecting cancer metastasis and accompanying protein biomarkers at single cell levels using a 3D-printed microfluidic immunoarray

Sharafeldin M., Chen T., UÇAK ÖZKAYA G., Choudhary D., Molinolo A. A., Gutkind J. S., ...More

BIOSENSORS & BIOELECTRONICS, vol.171, 2021 (SCI-Expanded) identifier identifier identifier

  • Publication Type: Article / Article
  • Volume: 171
  • Publication Date: 2021
  • Doi Number: 10.1016/j.bios.2020.112681
  • Journal Indexes: Science Citation Index Expanded (SCI-EXPANDED), Scopus, Academic Search Premier, PASCAL, Aerospace Database, BIOSIS, Biotechnology Research Abstracts, CAB Abstracts, Chemical Abstracts Core, Chimica, Communication Abstracts, Compendex, EMBASE, INSPEC, MEDLINE, Metadex, Veterinary Science Database, Civil Engineering Abstracts
  • Yıldız Technical University Affiliated: Yes


A low-cost microfluidic microarray capable of lysing cells and quantifying proteins released after lysis was designed and 3D-printed. The array lyses cells on-chip in lysis buffer augmented with a 2s pulse of a sonic cell disruptor. Detection of desmoglein 3 (DSG3), a metastatic biomarker for head and neck squamous cell carcinoma (HNSCC), along with two accompanying HNSCC biomarkers from a single cell lysate of oral cancer cell cultures was demonstrated. A lysis chamber and reagent compartments deliver sample and reagents into detection chambers decorated with capture antibodies immobilized onto inner walls coated with a highly swollen 3D chitosan hydrogel film. Sandwich immunoassays are achieved when captured analytes labeled with biotinylated secondary antibodies, which then capture streptavidin-poly [horse radish peroxidase] (Poly-HRP). Subsequent delivery of super-bright femto-luminol with H2O2 generates chemiluminescence captured with a CCD camera. DSG3 is membrane-bound protein in HNSCC cells of invaded lymph nodes, vascular endothelial growth factor-A (VEGF-A), vascular endothelial growth factor-C (VEGF-C) were positive controls overexpressed into the HNSCC culture medium. Beta-tubulin (beta-Tub) was used as a loading control to estimate the number of cells in analyzed samples. Limits of detection (LOD) were 0.10 fg/mL for DSG3, and 0.20 fg/mL for VEGF-A, VEGF-C and beta-Tub. Three orders of magnitude semilogarithmic dynamic ranges were achieved. VEGF-A showed high in-cell expression, but VEGF-C had low levels inside cells. The very low LODs enabled quantifying these proteins released from single cells. Strong correlation between results from on-chip cell lysis, conventional off-line lysis and ELISA confirmed accuracy.