Evaluation of probe orientation and effect of the digoxigenin-enzymatic label in a sandwich hybridization format to develop toxic algae biosensors

Biosensors, which use an electrochemical detection of a species by means of a sandwich hybridization assay (SHA) of the target sequence between a capture probe with an attachment label and a signal probe with a detection label, typically array the probes over an electrode surface. This arrangement is such that the modification of the capture probe is distal to the signal probe detection label in the 5′–3′ orientation of the RNA of the target species. This work reports on the proof of concept of a DNA-based device that uses SHA to anchor a target sequence on the surface of a transducer platform for sensing three different toxic algae. The probe orientation, the effect of a double DIG label and the use of a directly HRP-labelled signal probe on the electrochemical sensor performance are evaluated here. Results show that the electrochemical responses obtained for Prymnesium parvum and Gymnodinium catenatum with the thiol (biotin)-modified capture probe distal to the signal probe with a DIG label are comparable to those from Prymnesium parvum and Pseudo-nitzschia australis with the probes in a proximal orientation. Different probe orientations did not lead to significant improvement in signal intensity. A double DIG-enzymatic label and the use of a directly HRP-labelled signal probe did not led to further improvements in biosensor performance.

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► Sandwich hybridization assay-based biosensors specially array probes over a surface.
► Labels of capture and signal probes are typically distal oriented to each other.
► Biosensor response is not strongly dependent upon probe orientation.
► Double enzymatic labels did not improve signal.
► A directly HRP-labelled signal probe did not led to improved biosensor performance.