Abstract
An aptasensor with enhanced anti-fouling properties has been developed. As a case study, the aptasensor was designed with specificity for human thrombin. The sensing platform was developed on screen printed electrodes and is composed of a self-assembled monolayer made from a ternary mixture of 15-base thiolated DNA aptamers specific for human thrombin co-immobilised with 1,6-hexanedithiol (HDT) and further passivated with 1-mercapto-6-hexanol (MCH). HDT binds to the surface by two of its thiol groups forming alkyl chain bridges and this architecture protects from non-specific attachment of molecules to the electrode surface. Using Electrochemical Impedance Spectroscopy (EIS), the aptasensor is able to detect human thrombin as variations in charge transfer resistance (Rct) upon protein binding. After exposure to a high concentration of non-specific Bovine Serum Albumin (BSA) solution, no changes in the Rct value were observed, highlighting the bio-fouling resistance of the surface generated. In this paper, we present the optimisation and characterisation of the aptasensor based on the ternary self-assembled monolayer (SAM) layer. We show that anti-fouling properties depend on the type of gold surface used for biosensor construction, which was also confirmed by contact angle measurements. We further studied the ratio between aptamers and HDT, which can determine the specificity and selectivity of the sensing layer. We also report the influence of buffer pH and temperature used for incubation of electrodes with proteins on detection and anti-fouling properties. Finally, the stability of the aptasensor was studied by storage of modified electrodes for up to 28 days in different buffers and atmospheric conditions. Aptasensors based on ternary SAM layers are highly promising for clinical applications for detection of a range of proteins in real biological samples.
Highlights
There is a substantial need for the development of biosensors capable of sensitive and selective detection, that are low cost, easy to use and have the possibility of being integrated into portable devices for point-of-care use with clinical or environmental samples
Based on the procedure of Campuzano et al [35,36], we studied the immobilisation of thrombin DNA
We have demonstrated that antifouling properties of aptasensors can be achieved by means of a ternary self-assembled monolayer (SAM) layer with the same conditions as those reported for DNA hybridisation sensors [35]
Summary
There is a substantial need for the development of biosensors capable of sensitive and selective detection, that are low cost, easy to use and have the possibility of being integrated into portable devices for point-of-care use with clinical or environmental samples. Electrochemical biosensors are some of the most promising platforms with the potential to achieve these goals and have been used to detect a number of analytes These include, e.g., (1) disease biomarkers and pathogenic proteins in serum [1,2], human plasma [3,4] or urine [5]; (2) Nucleic acids such as DNA samples from polymerase chain reaction (PCR) products [6,7] and microRNAs [2,8]; (3) microorganisms in seawater medium [9]. The most common method has been to generate binding resistant layers through manipulation of either the components or the construction of the self-assembled monolayer This can be achieved by co-immobilisation of bioreceptors with alkanethiols of varying lengths of alkyl chains such as
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