Abstract
This proof-of-principle study demonstrates the feasibility of a leaky waveguide (LW) aptasensor, where aptamers were immobilised in a mesoporous chitosan waveguiding film for the detection of thrombin. This work has demonstrated that aptamers immobilised in hydrogels retain their affinity and selectivity towards their target and thus can be used as bioreceptors. The use of antibodies as bioreceptors for sensing thrombin is not viable because it is a serine protease, which will cleave the antibodies. Currently used assays based on clotting time and chromogenic/fluorogenic substrates have limited potential for thrombin measurement in whole blood. Using the initial binding rate over the first 5 min, the limit of detection of our LW aptasensor for thrombin was ∼22 nM. The sensor was tested with spiked serum samples, giving a reading of 46.1 ± 4.6 nM for a sample containing 50 nM thrombin. Our proposed sensor combines the robustness and low cost of aptamers as molecular recognition elements with the simple fabrication process of the chitosan-based leaky waveguide, making LW aptasensors highly attractive for applications in point-of-care diagnostics and healthcare monitoring.
Highlights
Thrombin is a serine protease which will cleave antibodies used as bioreceptors for sensing this protein, and immunoassays are performed for the inactive thrombin/antithrombin complex.[11,12]
We recently reported a leaky waveguide (LW) comprising of mesoporous chitosan films, which were spin coated following which the drying time was controlled before rehydration.[32]
We used a well characterised 15-mer thrombin-binding aptamer (TBA) and commonly used immobilisation chemistry based on streptavidin–biotin in combination with glutaraldehyde as a model system to investigate if the aptamer exhibits the affinity and selectivity toward its target in buffer and serum when immobilised in a mesoporous chitosan LW
Summary
Thrombin is a serine protease which will cleave antibodies used as bioreceptors for sensing this protein, and immunoassays are performed for the inactive thrombin/antithrombin complex.[11,12] Aptamers are beneficial over antibodies because they offer several practical benefits including higher stability, lower cost, purely synthetic production, invariant properties and simpler chemical modifications.[13,14,15] New aptamers against virtually any targets may be identified rapidly6048 | Analyst, 2019, 144, 6048–6054Paper using techniques such as systematic evolution of ligands by exponential enrichment (SELEX),[16,17] which is an in vitro selection method reducing animal use in the development of bioreceptors. We used a well characterised 15-mer thrombin-binding aptamer (TBA) and commonly used immobilisation chemistry based on streptavidin–biotin in combination with glutaraldehyde as a model system to investigate if the aptamer exhibits the affinity and selectivity toward its target in buffer and serum when immobilised in a mesoporous chitosan LW. A buffer wash was performed and the shift in resonance angle to 100 nM thrombin, which was flowed over the LW for 3 h, was measured to determine the optimum concentration of the oligonucelotide.
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