The immobilization of redox proteins or enzymes onto conductive surfaces has application in the analysis of biological processes, the fabrication of biosensors, and in the development of green technologies and biochemical synthetic approaches. This review evaluates the methods through which redox proteins can be attached to electrode surfaces in a “wired” configuration, that is, one that facilitates direct electron transfer. The feasibility of simple electroactive adsorption onto a range of electrode surfaces is illustrated, with a highlight on the recent advances that have been achieved in biotechnological device construction using carbon materials and metal oxides. The covalent crosslinking strategies commonly used for the modification and biofunctionalization of electrode surfaces are also evaluated. Recent innovations in harnessing chemical biology methods for electrically wiring redox biology to surfaces are emphasized.

Alternating current (AC) voltammetric techniques are experimentally powerful as they enable Faradaic current to be isolated from non-Faradaic contributions. To find the best global fit between experimental voltammetric data and simulations based on reaction models requires searching a substantial parameter space at high resolution. In this paper we estimate parameters from purely sinusoidal voltammetry (PSV) experiments, investigating the redox reactions of a surface-confined ferrocene derivative. The advantage of PSV is that a complete experiment can be simulated relatively rapidly, compared to other AC voltammetric techniques. In one example involving thermodynamic dispersion, a PSV parameter inference effort requiring 7,500,000 simulations was completed in 7 hours whereas the same process for our previously used technique, ramped Fourier transform AC voltammetry (ramped FTACV) would have taken four days. Using both synthetic and experimental data with a surface confined diazonium substituted ferrocene derivative, it is shown that the PSV technique can be used to recover the key chemical and physical parameters. By applying techniques from Bayesian inference and Markov chain Monte-Carlo methods, the confidence, distribution and degree of correlation of the recovered parameters was visualised and quantified.

Site-selective chemical methods for protein bioconjugation have revolutionized the fields of cell and chemical biology through the development of novel protein/enzyme probes bearing fluorescent, spectroscopic, or even toxic cargos. Herein, we report two new methods for the bioconjugation of α-oxo aldehyde handles within proteins using small molecule aniline and/or phenol probes. The “α-oxo-Mannich” and “catalyst-free aldol” ligations both compete for the electrophilic α-oxo aldehyde, which displays pH divergent reactivity proceeding through the “Mannich” pathway at acidic pH to afford bifunctionalized bioconjugates, and the “catalyst-free aldol” pathway at neutral pH to afford monofunctionalized bioconjugates. We explore the substrate scope and utility of both of these bioconjugations in the construction of neoglycoproteins, in the process formulating a mechanistic rationale for how both pathways intersect with each other at different reaction pH’s.

We report a diazonium electro-grafting method for the covalent modification of conducting surfaces with aldehyde-reactive hydroxylamine functionalities that facilitate the wiring of redox-active (bio)molecules to electrode surfaces. Hydroxylamine near-monolayer formation is achieved via a phthalimide-protection and hydrazine-deprotection strategy that overcomes the multilayer formation that typically complicates diazonium surface modification. This surface modification strategy is characterized using electrochemistry (electrochemical impedance spectroscopy and cyclic voltammetry), X-ray photoelectron spectroscopy and quartz crystal microbalance with dissipation monitoring. Thus-modified glassy carbon, boron-doped diamond and gold surfaces are all shown to ligate to small molecule aldehydes, yielding surface coverages of 150-170, 40 and 100 pmol cm-2 , respectively. Bio-conjugation is demonstrated via the coupling of a dilute (50 µM) solution of periodate-oxidized horseradish peroxidase enzyme to a functionalized gold surface under bio-compatible conditions (H 2 O solvent, pH 4.5, 25 °C).

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A laboratory experiment introducing the concept of chemical bioconjugation of proteins to undergraduate students in a therapeutically relevant context was developed. Initially, students installed an aldehyde functionality into a protein via the oxidation of the N-terminal threonine residue of the cholera toxin subunit B (CTB) protein, which was followed by subsequent modification via hydrazone addition under mild conditions with a chromophore bearing a distinct UV–vis-absorption peak. Students determined the yield of the reaction to be ca. 11% by HPLC coupled to UV–vis spectroscopy and developed key skills such as preparation of stock solutions, chemical manipulation of proteins, and analysis via HPLC. The reported experiment can be readily adapted for use with other proteins and may contribute to enhancing constructive alignment in interdisciplinary degree programs at the chemistry–biology interface.

We present catalyst-free “green” site-selective protein bioconjugations that utilise aldol condensations and are compatible with click chemistries, and construct a nanobody-derived bioconjugate capable of selectively labelling prostate cancer cells.

Aryl diazonium cations are versatile bioconjugation reagents due to their reactivity towards electron‐rich aryl residues and secondary amines, but historically their usage has been hampered by both their short lifespan in aqueous solution and the harsh conditions required to generate them in situ. Triazabutadienes address many of these issues as they are stable enough to endure multiple‐step chemical syntheses and can persist for several hours in aqueous solution, yet upon UV‐exposure rapidly release aryl diazonium cations under biologically‐relevant conditions. This paper describes the synthesis of a novel maleimide‐functionalized triazabutadiene suitable for site‐selectively installing aryl diazonium cations into proteins at neutral pH; we show reaction with this molecule and a surface‐cysteine of a thiol disulfide oxidoreductase. Through photoactivation of the site‐selectively installed triazabutadiene motifs, we generate aryl diazonium functionality, which we further derivatize via azo‐bond formation to electron‐rich aryl species, showcasing the potential utility of this strategy for the generation of photoswitches or protein‐drug conjugates.