Simultaneous multi-biomarker detection: streptavidin-biotin mediated high-quality quantum dot immunosensor with...

Developing highly sensitive, high-throughput, and multiplexed detection methods for tumor markers is critical for early cancer diagnosis. Traditional streptavidin-biotin (SA-biotin) quantum dot (QD) immunoassays often struggle to detect multiple biomarkers simultaneously within a single reaction environment. This study aims to address these limitations by designing a highly sensitive, SA-biotin-mediated, dual-color QD fluorescence signal amplification immunosensor for the simultaneous detection of prostate-specific antigen (PSA) and alpha-fetoprotein (AFP) as model analytes within a single microplate well.

The immunosensor utilizes SA-modified red and green QD linked to biotin-PEG12-NHS ester-modified secondary antibodies (Ab₂) to form dual-color QD-SA-bio-Ab₂ probes. This architecture leverages the high-affinity SA-biotin interaction and the long spacer arm of biotin-PEG12-NHS to increase antibody loading and reduce steric hindrance, creating a synergistic signal amplification effect. Under optimized conditions, the sensor demonstrated a wide linear range for both PSA and AFP (0.5 to 1000 ng/mL). The limits of detection (LOD) were 0.25 ng/mL for PSA and 0.32 ng/mL for AFP-approximately 6-fold lower than standard dual-color QD-based immunosensors. The platform's accuracy was validated through recovery tests and clinical serum sample analysis, yielding results competitive with currently prevalent testing technologies.

The proposed dual-color QD-SA-bio-Ab₂ probe provides a universal fluorescent platform that overcomes the multiplexing constraints of traditional assays. By combining high-performance QD with a strategic PEG-spacer architecture, this method offers a novel approach for fluorescence enhancement. It represents a significant step forward in achieving high-sensitivity, high-throughput multiplexed detection in clinical diagnostics.