Construction and interrogation of enzyme microarrays using scanning electrochemical microscopy – optimisation of adsorption and determination of enzymatic activity

Abstract

Scanning electrochemical microscopy (SECM) has been used to image and study the catalytic activity of horseradish peroxidase (HRP) immobilised in a patterned fashion onto glass slides. Microarrays of HRP islands could be deposited on amino-modified glass slides using glutaraldehyde crosslinking combined with the SECM being used as a micro-deposition device. The enzymatic activity of the immobilised enzyme on the surface was in the presence of its substrate observed to give rise to substantial positive feedback between the slide and the SECM microelectrode tip. Conversely when either blank slides - or slides coated with HRP which had been subsequently thermally denatured were utilised, these showed negative feedback effects. Various conditions such as enzyme concentration, incubation time and substrate concentration were systematically varied to optimise sensitivity. Regular arrays of HRP could be assembled and when imaged, displayed lower limits of detection of 1.2 × 10(-12) mol ml(-1) of benzoquinone.

Detection and Quantification of Sulfonamide Antibiotic Residues in Milk Using Scanning Electrochemical Microscopy

“…Although the feedback mode of SECM allows higher lateral resolution, the sample generator/tip collector (GC) mode is more sensitive due to negligible background currents detected at the tip, thus increasing the signal to noise ratio 18, 20. Therefore, in the proposed scheme, the substrate HQ was catalytically converted by the enzyme HRP in the presence of H 2 O 2 to BQ, which was monitored with the positioned carbon fiber tip in the GC mode of SECM as depicted in Figure 1b.…”

“…The BQ/HQ couple exhibits a quasi‐reversible redox behavior and negligible electrode fouling 32, 47, Although different electrode materials were previously used for SECM experiments monitoring BQ reduction 18, 20, 32, in contrast to Pt tips 48 coreduction of molecular oxygen as well as of H 2 O 2 at carbon fiber electrodes is not possible at the applied potentials. By comparing cyclic voltammograms of 1 mM BQ in the absence and presence of 1 mM H 2 O 2 (data not shown), reduction of H 2 O 2 was observed at potentials more negative than −200 mV (vs. Ag/AgCl/3 M Cl − ) at a Pt tip, while no effect was observed with a carbon fiber microelectrode.…”

“…SECM has been successfully used to investigate biological systems on electrified interfaces such as enzymes 16–20, cells 21–23, and DNA hybridization events 24–27, as well as immuno‐complexes 14, 28–36. However, the evaluation and quantification of low molecular weight analytes is still challenging.…”

Monitoring of the Surface Modification of Nanoparticlesby Electrochemical Measurements Using Scanning Electrochemical Microscope

“…In one work [87] for the deactivation of other regions on the HRP micropatterns the authors used the reactive species generated on the electrode as the tip of SECM held at 1. Another application of HRP in patterning experiments has been described by Roberts et al [88]. In this case the immobilisation in a patterned fashion onto glass slides is presented.…”

“…In this case the immobilisation in a patterned fashion onto glass slides is presented. Microarray of HRP islands was deposited on amino-modified glass slides using glutaraldehyde crosslinking combined with the SECM and this was used as a micro-deposition device [88].…”