Ternary sensing surface with DNA-based spacer group : characterization, comparison and optimization

Abstract

In the past decade, outbreaks of new diseases have brought much fear in the people worldwide. Many lives were lost due mainly to the late detection of these diseases as well as the lack of knowledge to prevent or cure them. To allow fast response to curb the spread of such infections, real-time sensors with high sensitivity and selectivity are required. With the advancement in technology and the need for miniaturization, biosensors based on electrochemistry has proven to be a powerful detection method due to the ease-of-use, low instrumentation cost, possible non-labeling and fast target detection. In an electrochemical detection, the sensitivity of the biosensors is mostly affected by the accessibility of the specific target towards the recognition site of the receptors immobilized on the electrode surface. To reduce the steric hindrances of the target molecules to the receptors, in this case the single-stranded DNA (ssDNA) probes, short organic spacer groups are normally used to modulate the ssDNA probe density on the electrode. In this report, a ternary sensing surface optimized using DNA-based spacer group for the detection of Methicillin-resistant Staphylococcus aureus (MRSA) has been created. As compared to commonly-used organic spacer group, such as 2-mercaptoethanol (ME), 3-mercaptop-1-propanol (MP) and 6-mercapto-1-hexanol (MCH), thymine-based spacer groups (T9) displayed a 10-fold improvement of signal-to-noise in discriminating between complementary DNA (cDNA) and non-complementary DNA (NcDNA) hybridization. Analysis from Surface Plasmon Resonance (SPR), Quartz Crystal Microbalance (QCM-D) and electrochemistry showed a sensing surface of excellent selectivity, optimized at a ratio of 1:1 (probe:T9). On this surface, the ssDNA probes are aligned by the T9 spacer groups and thereby capable of maximizing cDNA hybridization and differentiating with non-specific NcDNA binding. Single-mismatch (SMM) detections have shown to be possible at this optimized ratio, with the ability to differentiate between the SMM at different positions. By creating similar sensing surfaces on gold-deposited microelectrodes, an improvement of the S/N by a factor of 8 was observed compared to the detection using planar gold electrodes, showing capabilities of creating a highly selective and sensitive biosensor with microelectrodes. Attempts were also made to create binary sensing surfaces and comparisons were made with using shorter thymine spacer groups (T6) on both planar and microelectrodes. While all DNA-based spacer lengths were capable of modulating probe density and reducing steric hindrances, sensing surfaces created using the longer T9 spacer groups performed better in terms of S/N at their respective optimized ratio. Improvement in detection was observed for neutral PNA strands as compared to negatively-charged DNA strands at the same optimized ratio using microelectrodes. Despite the general idea that a compact and uncharged layer is more desirable to form a highly selective biosensor based on electrochemistry, I have…