IDSP-IA: Immunotriggered Double Stem-Loop Probe-Mediated Isothermal Amplification for Attomolar Protein Quantification

Ultrasensitive quantification of low-abundance proteins is critical for disease full-cycle management but remains a significant challenge. Herein, we present an immunotriggered double stem-loop probe-mediated isothermal amplification (IDSP-IA) assay for highly sensitive and specific protein detection. The IDSP-IA assay consists of three modular components: a monoclonal capture antibody (mAb1)-coated reaction tube for target capture, an aptamer-oligonucleotide switch (Apt-OS) for signal transduction, and a universal double stem-loop-mediated isothermal amplification module. After target protein capture and enrichment of mAb1-coated tubes, Apt-OS converts nonamplifiable protein signals to the nucleic acid triggers, initiating efficient exponential amplification. Benefiting from the modular design, the assay allows flexible adaptation to various proteins by simply replacing antibody/aptamer pairs while keeping amplification components unchanged. We have demonstrated that the IDSP-IA assay achieves a wide dynamic range (5 fg/mL to 50 pg/mL) with a limit of detection (LOD) of 1.3 fg/mL for α-fetoprotein (AFP) and 1.4 fg/mL for interleukin-6 (IL-6) detection without cross-reactivity from other common serum proteins. More importantly, clinical validation using human serum samples showed excellent correlation with routinely used electrochemiluminescence and chemiluminescence immunoassays. These advantages, including high specificity, ultrasensitivity, modular design, and universality, enable the IDSP-IA assay to provide a robust and flexible tool for accurate and attomolar protein biomarker quantification in diagnostic and biomedical settings.