Circular Permutated PQQ‐Glucose Dehydrogenase as an Ultrasensitive Electrochemical Biosensor

Abstract

Protein biosensors play an increasingly important role as reporters for research and clinical applications. Here we present an approach for the construction of fully integrated but modular electrochemical biosensors based on the principal component of glucose monitors PQQ‐glucose dehydrogenase (PQQ‐GDH). We designed allosterically regulated circular permutated variants of PQQ‐GDH that show large (>10‐fold) changes in enzymatic activity following intramolecular scaffolding of the newly generated N‐ and C termini by ligand binding domain/ligand complexes. The developed biosensors demonstrated sub‐nanomolar affinities for small molecules and proteins in colorimetric and electrochemical assays. For instance, the concentration of Cyclosporine A could be measured in 1 μL of undiluted blood with the same accuracy as the leading diagnostic technique that uses 50 times more sample. We further used this biosensor to construct highly porous gold bioelectrodes capable of robustly detecting concentrations of Cyclosporine A as low as 20 pM and retained functionality in samples containing at least 60 % human serum.

Highly Porous Gold Electrodes – Preparation and Characterization

“…The produced Au electrodes with the high porosity were applied for assembling electrochemical biosensors, biofuel cells, and signal‐triggered biomolecule release [68,79,80] . All studied systems demonstrated reasonable stability, which was limited by the stability of the biomolecules immobilized on the porous Au surfaces (not by stability of the Au support itself).…”

“…The developed procedure for modification of the Au‐SPEs resulting in the dramatic increase of their real (electrochemically active) surface is highly important for many bioelectrochemical applications where the high current density is needed, particularly in ultra‐small‐size biofuel cells powering microelectronic devices [81] . The use of the highly porous Au‐SPEs is also beneficial for enzyme‐based biosensing systems with very high sensitivity (in a pM concentration range) [68] …”

“…Commercially available Au screen-printed electrodes, SPE, (particularly from DropSens Metrohm Company) are often used in various bioelectrochemical applications (biosensors, biofuel cells, etc.). Further modification of these electrodes using the Dynamic Hydrogen Bubble Template (DHBT) method is rather new (only three recent publications are available [68,79,80] ). We wanted to study the Au-SPE applicability for their modification using DHBT method, aiming at substantial increase of their real surface, keeping in mind their future use as a platform for bioelectrochemical application.…”

“…[81] The use of the highly porous Au-SPEs is also beneficial for enzyme-based biosensing systems with very high sensitivity (in a pM concentration range). [68] Experimental Section…”

“…[61] Among many other examples of roughened electrode surfaces, [62][63][64][65] microporous Au electrodes developed recently using the DHBT method [13,66] have found various bioelectrochemical applications, especially in biosensors. [67][68][69][70] These electrodes are particularly attractive for their use in bioelectrochemical systems due to the increased load of biomolecules (e. g., biocatalytic enzymes) at the enlarged surface area and easy diffusional access of reactive species (e. g., enzyme cofactors, substrates or analytes) through broadly open microcavities. The use of the Au conductive support is also convenient for biomolecule immobilization using a thiol selfassembling method for creating a linker to bind biomolecules.…”