Jo V Rushworth, Asif Ahmed and Paul A Millner
Biosensor performance and readout are critically dependent upon sensor surface characteristics. It is vital that key steps in sensor construction, such as base layer polymer/Self-Assembled Monolayer (SAM) deposition and bioreceptor tethering, are controlled tightly in order to achieve high sensitivity, specificity and reproducibility. Here, we present a rapid, semi-quantitative method by which key biosensor surface features can be characterised using chemiluminescence. This technique, which we have termed midland blotting, permits the detection of biosensor surface components through the attachment of a HorseRadish Peroxidase (HRP)-conjugated reagent to the target of interest. Upon addition of luminol-based substrate, HRP generates a reagent which emits light when it decays. The light signal is proportional to the bound HRP on the sensor surface. We show here that midland blotting allows the measurement and validation of various important surface features including: (1) availability of functional groups on the polymer or SAM layer; (2) bioreceptor tethering and (3) analyte binding. Midland blotting is rapid, cost-effective and allows for much faster optimisation of biosensor surface design. This method also provides a simple way of troubleshooting and can explain sensor performance when combined with readout data. In this report, we have focussed on midland blotting for electrochemical immunosensors as a proof of concept, but this technique is readily applicable to all biosensor systems.
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