Real-time QCM-D immunoassay through oriented antibody immobilization using cross-linked hydrogel biointerfaces

Area of Science:

• Biomaterials Science
• Biosensor Technology
• Analytical Chemistry

Background:

• Sepsis biomarker detection is critical for timely clinical intervention.
• Quartz Crystal Microbalance with Dissipation monitoring (QCM-D) offers a sensitive and affordable platform for immunoassays.
• Developing efficient antibody immobilization strategies is key to enhancing biosensor performance.

Purpose of the Study:

• To develop and optimize pre-cross-linked and in situ cross-linked polyethyleneimine-carboxymethylcellulose platforms for antibody immobilization.
• To establish a real-time QCM-D immunoassay for sepsis-related biomarkers with enhanced sensitivity and robustness.
• To investigate methods for improving assay performance, including oriented antibody immobilization and signal amplification.

Main Methods:

• Fabrication of pre-cross-linked and in situ cross-linked polymer platforms using polyethyleneimine and carboxymethylcellulose.
• Development of a QCM-D immunoassay using recombinant human interleukin-1 receptor antagonist (rhIL-1ra) as a model sepsis biomarker.
• Optimization of antibody immobilization using Protein A, Protein G, and anti-IgG Fc ligands on higher-frequency QCM-D crystals.
• Implementation of secondary antibody amplification and assessment of assay repeatability and nonspecific adsorption using human serum albumin.

Main Results:

• Achieved ultrafast biointerface deposition through polysaccharide cross-linking.
• Optimized assay with Protein A oriented immobilization and secondary antibodies yielded a detection limit of 25 ng/mL on 5 MHz crystals.
• Demonstrated robust assay repeatability with no loss in antigen detection over 20 cycles of a 10-minute assay.
• Confirmed minimal and negligible nonspecific adsorption of human serum albumin on the developed platforms.

Conclusions:

• The developed polyethyleneimine-carboxymethylcellulose platforms enable rapid and sensitive QCM-D immunoassay of sepsis biomarkers.
• Oriented antibody immobilization and secondary antibody amplification significantly enhance assay performance.
• The optimized QCM-D immunoassay exhibits excellent sensitivity, repeatability, and low nonspecific binding, suitable for real-time sepsis diagnostics.