Aptamer-Based Sandwich Assay for Measurement of Thymidine Kinase 1 in Serum of Cancerous Patients.

Thymidine kinase 1 (TK1) is traditionally a serum biomarker that is elevated in the early stages of malignancies. The diagnostic and prognostic role of TK1 for screening and monitoring human malignancies has recently been investigated. Anti-human TK1 aptamers were selected through 12 iterative rounds of systematic evolution of ligands by exponential enrichment from a DNA library. The aptamer pool of round 12 was amplified, and the polymerase chain reaction product was cloned on the TA vector. Of the 85 colonies obtained, 52 were identified as positive clones. These aptamers were screened for TK1 with surface plasmon resonance, where apta37 and apta69 showed the highest affinity for TK1. The TK1_apta37 and TK1_apta69 aptamers were used in a sandwich assay platform and successfully detected TK1 in the concentration range of 54-3500 pg mL-1. Clinical samples from 60 cancerous patients were also tested with this assay system and compared using the conventional antibody-based enzyme-linked immunosorbent assay kit. The aptamer sandwich assay demonstrated a dynamic range for TK1 at clinically relevant serum levels, covering subpicogram per milliliter concentrations. The new approach offers a simple and robust method for detecting serum biomarkers that have low and moderate abundance. The results of this study demonstrate the screening capability of the aptamer sandwich assay platform and its potential applicability to the point-of-care testing system.

Selection of DNA aptamers against Human Cardiac Troponin I for colorimetric sensor based dot blot application.

Troponin T and I are ideal markers which are highly sensitive and specific for myocardial injury and have shown better efficacy than earlier markers. Since aptamers are ssDNA or RNA that bind to a wide variety of target molecules, the purpose of this research was to select an aptamer from a 79bp single-stranded DNA (ssDNA) random library that was used to bind the Human Cardiac Troponin I from a synthetic nucleic acids library by systematic evolution of ligands exponential enrichment (Selex) based on several selection and amplification steps. Human Cardiac Troponin I protein was coated onto the surface of streptavidin magnetic beads to extract specific aptamer from a large and diverse random ssDNA initial oligonucleotide library. As a result, several aptamers were selected and further examined for binding affinity and specificity. Finally TnIApt 23 showed beast affinity in nanomolar range (2.69nM) toward the target protein. A simple and rapid colorimetric detection assay for Human Cardiac Troponin I using the novel and specific aptamer-AuNPs conjugates based on dot blot assay was developed. The detection limit for this protein using aptamer-AuNPs-based assay was found to be 5ng/ml.