Impedimetric measurement of DNA–DNA hybridisation using microelectrodes with different radii for detection of methicillin resistant Staphylococcus aureus (MRSA)

Abstract

Due to their electroanalytical advantages, microelectrodes are a very attractive technology for sensing and monitoring applications. One highly important application is measurement of DNA hybridisation to detect a wide range of clinically important phenomena, including single nucleotide polymorphisms (SNPs), mutations and drug resistance genes. The use of electrochemical impedance spectroscopy (EIS) for measurement of DNA hybridisation is well established for large electrodes but as yet remains relatively unexplored for microelectrodes due to difficulties associated with electrode functionalisation and impedimetric response interpretation. To shed light on this, microelectrodes were initially fabricated using photolithography and characterised electrochemically to ensure their responses matched established theory. Electrodes with different radii (50, 25, 15 and 5 µm) were then functionalised with a mixed film of 6-mercapto-1-hexanol and a thiolated single stranded ssDNA capture probe for a specific gene from the antibiotic resistant bacterium MRSA. The complementary oligonucleotide target from the mecA MRSA gene was hybridised with the surface tethered ssDNA probe. The EIS response was evaluated as a function of electrode radius and it was found that charge-transfer (R CT ) was more significantly affected by hybridisation of the mecA gene than the non-linear resistance (R NL ) which is associated with the steady state current. The discrimination of mecA hybridisation improved as electrode radius reduced with the R CT component of the response becoming increasingly dominant for smaller radii. It was possible to utilise these findings to produce a real time measurement of oligonucleotide binding where changes in R CT were evident one minute after nanomolar target addition. These data provide a systematic account of the effect of microelectrode radius on the measurement of hybridisation, providing insight into critical aspects of sensor design and implementation for the measurement of clinically important DNA sequences. The findings open up the possibility of developing rapid, sensitive DNA based measurements using microelectrodes.

Electrochemical Evaluations of Fractal Microelectrodes for Energy Efficient Neurostimulation

“…The Nyquist plots were fitted with an equivalent circuit model (Fig. 4C ) to extract the values of R S , R CT , C dl , the Warburg coefficient W, and the nonlinear resistance related to hemi-spherical diffusion R NL 46 . The estimated values of each parameter from different electrode designs (n = 5 each) indicated that the fractal electrodes have the lowest R S , R CT , W, and R NL , and the highest C dl , which suggests that the fractal design has substantially lower overall resistance due to both Faradaic and non-Faradaic processes (Table 4 and Supplementary Fig.…”

A Microelectrode Array with Reproducible Performance Shows Loss of Consistency Following Functionalization with a Self-Assembled 6-Mercapto-1-hexanol Layer

“…To produce a consistent SAM layer on microelectrodes the results presented so far show that it is necessary to modify the conditions of formation to improve reproducibility of the resulting electrochemical response. There are examples in the literature of dilution of SAM forming solutions [ 34 ] and protocols with abbreviated incubation times compared to the standard sixteen hours [ 26 ]. In this study, it was therefore decided to dilute the SAM forming molecules by a factor of ten thousand from 30 µM to 3 nM with the aim of reducing SAM aggregation and/or the degree of multilayer formation.…”

“…This work was performed on microfabricated arrays as well as glass pulled microelectrodes in order to confirm the effect. In addition, we have previously published on the rapid formation of SAM layers using nanoelectrodes [ 34 ] and on successful formation of a DNA-based SAM for MRSA detection [ 26 ]. In both of these reported studies, increases in the magnitude of the impedance similar to the results presented herein were noted, giving additional confidence beyond the fact that six electrodes on these arrays were tested each time, that the reported R CT increases and their magnitudes were realistic.…”

“…Previous work has demonstrated the development of label-free biomedical assays which utilize traditionally sized “macro” electrodes and there are several well-established approaches and protocols for the functionalization and development of such electrochemical assays [ 22 , 23 , 24 , 25 ]. What is less commonly reported is the development of label-free measurements which utilize microelectrodes, EIS [ 26 , 27 ] and in particular arrays of chemically functionalized microelectrodes for biomedical measurements. This is in part due to the additional complexity of the EIS response for a microelectrode [ 28 ] and also because of the difficulties associated with reliable and reproducible manufacture of microelectrodes and effective SAM formation on microelectrode surfaces [ 29 ].…”

Weaving Off‐The‐Shelf Yarns into Textile Micro Total Analysis Systems (μTAS)

“…[ 1–4 ] These have typically been made using microfabrication technologies. [ 5–8 ] However, these often require complex and expensive fabrication. An alternative approach has been the development of paper based diagnostics, a topic which has garnered much interest in recent years.…”