Sandwich-type complex-activated DNA circuit for highly sensitive and versatile detection of protein biomarkers for monitoring bone health

Background: In recent years, aptamer-based biosensing methods have achieved notable advances in the detection of protein biomarkers associated with various diseases. Despite this progress, their clinical utility remains constrained by their reliance on prior knowledge or precise engineering of aptamer structures, or the necessity for target proteins to possess multiple binding sites with specific spatial arrangements. These requirements restrict the range of protein biomarkers that can be effectively detected, thereby limiting the wide applicability and utility of current aptamer-based biosensing methods.

Results: In this work, we develop a fluorescent biosensing method that utilizes a sandwich-type complex-activated DNA circuit for highly sensitive detection of protein biomarkers. The method begins with the capture of target proteins by aptamer-functionalized Magnetic Beads, followed by the recruitment of initiator probes T labeled with 3-aminophenylboronic acid via boronate-affinity recognition. Subsequently, a DNA circuit featuring a positive feedback mechanism is activated to generate a significantly amplified fluorescence signal. Using osteoporosis-related biomarkers Osteopontin and osteocalcin as representative models, the method achieves detection limits of 0.289 pg/mL and 0.127 pg/mL, respectively, outperforming existing methods and commercially available kits. Furthermore, the method demonstrates high selectivity and satisfactory performance in serum samples, and shows broad potential for adaptation to other protein targets.

Significance: These advantages suggest that this method holds promise as a valuable tool for detecting protein biomarkers related to diseases such as Osteoporosis. Moreover, by eliminating the dependence on prior aptamer structural information and the requirement for multiple independent binding sites on the target protein, this method may provide an effective way for advancing aptamer-based biosensing platforms and diagnostic applications.