Antibody immobilization on to polystyrene substrate–on-chip immunoassay for horse IgG based on fluorescence.

The researchers developed a simple microfluidic device on a polystyrene substrate to detect horse Immunoglobulin G (IgG) using fluorescent microscopy. The device displays a linear response to horse IgG levels, and findings suggest the method could have broader applications in various diagnostic and biological monitoring exercises.

Development of the Immunoassay Card

• The team constructed a microfluidic immunoassay card using a polystyrene (PS) substrate. The goal was to detect horse Immunoglobulin G (IgG), which was chosen because it’s an inexpensive model analyte.
• The primary antibody was captured onto the PS substrate utilizing a self-assembled monolayer (SAM) of thiol to create a chemical pattern on a gold-coated PS. This design creates the basis for the analyte detection process.
• The construction of the immunosensor chip required creating layers on sheets derived from CO2 laser ablation, a process that helps manufacture the layered design of the chip.

Functionalization and Monitoring of PS Surfaces

• Surface characterization of the functionalized PS was done using three main techniques: contact angle measurement, X-ray photoelectron spectroscopy (XPS), and atomic force microscopy (AFM).
• This characterization took place after each step to ensure correct construction and functionality at each stage of the manufacturing process.

Antibody-Antigen Interaction and Fluorescence Measurement

• The researchers utilized a sandwich immunoassay combined with a fluorescein isothiocyanate (FITC)-conjugated secondary antibody to stimulate antibody-antigen interactions.
• The intensity of the resulting fluorescence – stimulated by the immunoassay interaction – was measured directly on the chip as a mean of determining the concentration of the horse IgG, the target analyte.

Performance of the Immunosensor Chip and Future Applications

• The assay revealed a linear response to horse IgG concentrations ranging between 1 microg/ml and 80 microg/ml, with a high correlation factor (r = 0.971, n = 3).
• The device was able to detect horse IgG at a low concentration limit of 0.71 microg/ml demonstrating high sensitivity.
• This technology holds potential for a wide range of applications beyond detecting horse IgG, including medical diagnostics, microarray detection, and monitoring protein-protein interactions, demonstrating the versatility and potential of this technology.