**1. Introduction**

Self-assembled molecular monolayers (SAMs) are surface monolayers that spontaneously bind to metal surfaces on which, for example, the unique metal-S bonding between metal and thiols offers a versatile pathway to tailor interfacial properties for electrochemical and bioelectrochemical applications [1–4]. Thiol SAMs on metal surfaces are core targets to provide an understanding of self-organization and interfacial interactions at the molecular level in biological systems [5,6]. Bigelow and associates were the first to demonstrate well-oriented SAMs adsorbed on a platinum wire [7]. However, SAMs did not attract much attention until Nuzzo and associates discovered disulfide monolayers on gold substrates in solution, as a phenomenon different from conventional Langmuir–Blodgett (LB) films [8]. Besides gold and platinum substrates, SAMs can also form on the surfaces of other metals including silver, copper, palladium, and mercury [9–14]. Gold is, however, the most extensively investigated because of its chemical inertness, relatively easy handling, and wide potential window, suitable for a range of electrochemical studies [1,15].

In parallel, the nature of the surface Au-S bond has been under intense focus over a number of years [1,2,4] and was recently overviewed [16,17]. In contrast to molecular Au(I)-S(-I) complexes, the "aurophilic" effect arising from collective interactions among surface Au atoms leads to displacement

of the 6s Au electrons out of chemical reach and the filled 5d electrons taking over in Au-S bonding. The Au-S bond on Au surfaces is thus an intriguing example of (very) strong, aurophilically controlled van der Waals binding in an Au(0)-•S(0) gold(0)-thiyl bond.

Electron transfer (ET) reactions between an electrode surface and an oxidoreductase are one of the most important topics in bioelectrocatalysis [18–22]. For example, oxidoreductases possess redox/catalytic center(s) that catalyze the oxidation of fuels on a bioanode (e.g., glucose, fructose, lactate, and sulfite) [20,23–25], which can be assembled with a biocathode undergoing biocatalytic reduction reactions, typically dioxygen reduction, in enzymatic biofuel cells (EBFCs), allowing biopower generation [26–29]. The bioelectrochemical ET processes are classified into direct ET (DET) and mediated ET (MET) [19,20,30]. The MET-type system utilizes external and artificial redox mediators to shuttle the electrons between the electrode and the oxidoreductase, especially if the redox center(s) are buried deep inside the protein structure [31,32]. In DET-type systems, redox enzymes are able to communicate directly with the electrode surface if the redox cofactors/centers are spatially close to the electrode surfaces (generally less than 2 nm), facilitating electron tunneling [33]. DET is thus a simpler mechanism by eliminating the need for external redox mediators, and it is therefore amenable to more detailed mechanistic analysis.

Oxidoreductase immobilization is crucial to improving electrode reusability and stability [19]. To achieve efficient DET, it is important to consider the detailed surface characteristics of both enzyme and electrode for favorable enzyme orientation, leading to minimized electron tunneling distance. A wide range of carbon or metallic supports have been employed for effective enzyme immobilization [25,28,34–37]. Physical adsorption and covalent bonding are most commonly used. SAMs have been introduced into bioelectrocatalysis to serve as a bridge for gentle protein/enzyme immobilization on gold or other metal surfaces [36,38,39]. SAM structures are determined by the Au-S bond, the surface structure of the metal surfaces, lateral interactions, as well as the solvent and electrolytes [6]. Use of SAMs avoids the direct contact of enzyme and solid surfaces [40], mimicking the microenvironment in biological membranes. SAMs exhibit a variety of functional hydrophilic or hydrophobic terminal groups, such as carboxyl, hydroxyl, amino, and alkyl groups [41]. Frequently used alkanethiol and thiophenol molecules are summarized in Figure 1. In addition, the DET kinetics can also be governed by tuning the SAM molecular chain length, as the ET rate is strongly controlled by the tunneling distance [42–44]. As an emerging approach, protein engineering provides oxidoreductases directly with thiol residues, leading to controlled orientations either by direct protein thiol binding or by thioether bond formation with unsaturated maleimide [45].

Current density and enzyme loading can be promoted using nanostructured materials [3,44,46–48]. Among these, metallic nanomaterials exhibit excellent electronic conductivity and large surface area, with promising potential in improving the catalytic response and stability of redox enzymes [25]. Nanoporous gold (NPG), prepared via de-alloying Au alloys or electrodeposition, with three-dimensional porous architecture and a relatively uniform pore size, is a particular candidate for immobilizing enzymes in DET [25,28,32,49,50]. Moreover, gold nanoparticles (AuNPs) featuring a spherical nanostructure and large surface areas have been widely studied in bioelectrocatalysis [51–54]. The combination of SAMs and nanostructured gold offers new opportunities in bioelectrochemistry and has been extensively reviewed [21,32,55], but only a few reviews cover SAMs on planar or porous gold electrodes for controlling enzyme orientation [3,19]. DET based on carbon electrode materials (carbon nanotubes, graphene-based materials) is another major parallel sector, beyond the scope of the present focused review but recently reviewed elsewhere [18,56–58].

In this review, we review recent studies of SAMs in the DET-type bioelectrocatalysis of both atomically planar and nanostructured gold electrode surfaces. Preparations of SAMs coupled with characterization techniques, such as electrochemical, microscopic, and spectroscopic methods, are first overviewed. The use of structurally versatile SAMs and suitable electrode nanostructure supports achieving well-defined orientation for DET is highlighted next. The redox proteins and enzymes to be discussed are organized as (i) heme-containing proteins, i.e., cytochromes (cyts), fructose dehydrogenase (FDH), cellobiose dehydrogenase (CDH), glucose dehydrogenase (GDH), and sulfite oxidase (SOx); (ii) blue copper proteins, i.e., azurin, copper nitrite reductase (CuNiR), bilirubin oxidase (BOD), and laccase (Lac); (iii) [FeS]-cluster hydrogenases, i.e., [FeFe]-, [NiFe]-, and [NiFeSe]-hydrogenase. This classification is warranted primarily by the different nature of the core ET cofactor, but also with the specific secondary and tertiary structures of the protein that envelopes the metallic or non-metallic catalytic sites. Conclusions and further perspectives are offered and discussed in the final section.


**Figure 1.** Frequently used alkanethiols and thiophenols in the formation of self-assembled molecular monolayers (SAMs) with alkyl, amino, hydroxyl, and carboxyl terminal groups.
