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 (Journal Indexed in SCI) identifier identifier identifier

  • Publication Type: Article / Article
  • Volume: 171
  • Publication Date: 2021
  • Doi Number: 10.1016/j.bios.2020.112681
  • Journal Name: BIOSENSORS & BIOELECTRONICS
  • Journal Indexes: Science Citation Index 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

Abstract

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.