Aptamer selection and aptasensor construction for bone density biomarkers

Osteoporosis (OP) is a bone disease involved in dysregulation of one of the bone metabolism arms, formation, or desorption cause a porous bone. Osteocalcin (OC) and beta-crosslap (BC), are the well-known markers for OP, which are connected to bone formation and desorption, respectively. In addition to the OP biomarker, BC is also used as an estrogen replacement therapeutic monitoring. ELISA and other antibody-based detection methods are routinely used for measuring OC and BC. These methods have limitations that include thermostability, sensitivity, sacrificing animals, and cost of production. However, aptamer-based-assays are of interest to overcome these drawbacks and achieve the most specific and robust application. Herein, specific aptamers for OC and BC were selected by the systematic evolution of ligands by exponential enrichment (SELEX) method from the pool of ssDNA library with 60 random sequences. The binding affinity (K_d) of the selected aptamers were evaluated against the respective biomarkers. The high-affinity aptamers of OC and BC showed the K_d values of 59 and 55 nM respectively. A graphene oxide-based aptasensors were fabricated from the high-affinity aptamers, and the detection limits of OC and BC were found to be 0.4 pg/ml and 0.21 pg/ml, respectively. These aptasensors have been tested with OC and BC spiked buffer samples and validated using serum samples collected from osteoporotic rats.