Abstract
Immobilisation of aptameric ligands on solid stationary supports for effective binding of target molecules requires understanding of the relationship between aptamer-polymer interactions and the conditions governing the mass transfer of the binding process. Herein, key process parameters affecting the molecular anchoring of a thrombin-binding aptamer (TBA) onto polymethacrylate monolith pore surface, and the binding characteristics of the resulting macroporous aptasensor were investigated. Molecular dynamics (MD) simulations of the TBA-thrombin binding indicated enhanced Guanine 4 (G4) structural stability of TBA upon interaction with thrombin in an ionic environment. Fourier-transform infrared spectroscopy and thermogravimetric analyses were used to characterise the available functional groups and thermo-molecular stability of the immobilised polymer generated with Schiff-base activation and immobilisation scheme. The initial degradation temperature of the polymethacrylate stationary support increased with each step of the Schiff-base process: poly(Ethylene glycol Dimethacrylate-co-Glycidyl methacrylate) or poly(EDMA-co-GMA) [196.0 °C (±1.8)]; poly(EDMA-co-GMA)-Ethylenediamine [235.9 °C (±6.1)]; poly(EDMA-co-GMA)-Ethylenediamine-Glutaraldehyde [255.4 °C (±2.7)]; and aptamer-modified monolith [273.7 °C (±2.5)]. These initial temperature increments reflected in the associated endothermic energies were determined with differential scanning calorimetry. The aptameric ligand density obtained after immobilisation was 480 pmol/μL. Increase in pH and ionic concentration affected the surface charge distribution and the binding characteristics of the aptamer-modified disk-monoliths, resulting in the optimum binding pH and ionic concentration of 8.0 and 5 mM Mg2+, respectively. These results are critical in understanding and setting parametric constraints indispensable to develop and enhance the performance of aptasensors.
Highlights
Biosensors utilise specific bioprobes to detect and analyse target molecules[1,2,3]
Aptamers are developed through a robust iterative process known as Systematic Evolution of Ligands by Exponential enrichment (SELEX)[11,12,13], which inherently endow them with laudable attributes like better binding strength and specificity compared to antibodies[14]
Molecular interactions between aptamers and their targets are affected by physicochemical conditions of the binding environment, including ionic concentration, pH, type and characteristics of the support matrix, aptamer modifications, and temperature[18,19]
Summary
Biosensors utilise specific bioprobes to detect and analyse target molecules[1,2,3]. They have garnered considerable attention in research and applications for medical diagnosis and prognosis, as well as the detection of environmental contaminants such as pesticides and heavy metals[4,5]. The development and application of antibodies as bioaffinity probes, are often challenged by ethical issues, short shelf-life and high production costs, along with a myriad of additional factors such as binding specificity, bioavailability, immunogenicity and thermal stability[7,8,9] On this front, aptamers have garnered widespread attention towards alleviating these challenges to a considerable extent. Aptamers are developed through a robust iterative process known as Systematic Evolution of Ligands by Exponential enrichment (SELEX)[11,12,13], which inherently endow them with laudable attributes like better binding strength and specificity compared to antibodies[14] They possess other benefits concerning manufacturing, biophysical and biochemical attributes, including a large array of target space, low production costs, thermal and chemical stability, low to negligible ethical issues, prolonged shelf-life/reusability and uncomplicated pre-/post biomodification mechanisms[15,16,17]. The present work reports the molecular anchoring of a thrombin binding aptameric ligand on a disk polymethacrylate monolith, including molecular dynamic simulation and analysis of its physicochemical characterisations
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