3.5. Self-Assembled Monolayers (SAMs)

SAMs are self-assembled monolayers of organic compounds which are formed on the surface by chemisorption [69]. SAMs are composed of three parts. The first one—the head group—is the end of the molecule containing a functional group such as thiol or disulfide, which is connected to the platform. The second one—the backbone—is an aliphatic chain or an aromatic oligomer. This part is responsible for molecular ordering. Finally, the third one, known as the terminal group, is accountable for the chemistry of the constructed layer and makes it possible for the enzyme to be attached [70]. It should be mentioned that this method is usually characterized by numerous difficult procedures of modification, as well as expensive reagents.

The most commonly used SAMs-modified materials for glucose biosensor application are unpatterned gold electrodes. Acting according to Shervedami et al., the polycrystalline gold working electrode was modified by a water/ethanol solution of 20 mM MPA (3-mercaptopropionic acid), in which it was placed for 12 h, and then activated by 5 mM NHS and 0.2 mM EDC. Next, the GOx was immobilized for 1.5 h to fabricate the Au-MPA-GOx SAMs electrode [71]. The prepared electrode was used to determine the glucose content in 0.1 M PBS solution with the addition of parabenzopinone (PBQ) mediator by EIS (electrochemical impedance spectroscopy). The linear relation of 1/R_ct and glucose concentration was found to be within the range of 0–10.00 mM.

Acting according to Zhong et al. [72], the material was composed of two silane layers of a 2d-network of (3 mercaptopropyl)-trimethoxysilane MPS and self-assembled gold nanoparticles, as well as an enzyme. The gold electrode was immersed in an ethanol solution of 40 mM MPS for 3 h. Next, the electrode was immersed in a 0.01 M NaOH solution for two hours in order to form a hydrolyzed and condensed monolayer. In another step, the second layer was created by dipping it back in MPS overnight. After that, the sample was dipped in the AuNPs solution for 10 h. In the end, the GOx enzyme was immobilized at 4 °C overnight. The addition of the second layer of MPS resulted in an increase in the surface area as well as an increase in enzyme loading. As a result, higher sensitivity and stability were achieved.

Another example of a glucose biosensor in which SAMs were used is enzymatic electrospun nanofibers decorated with AuNPs [73]. The process of electrode fabrication is shown in Figure 13a. Firstly, the gold electrode was incubated in an ethanol solution of 10 mM 4-ATP (4-aminothiophenol) for 12 h for the formation of the 4-ATP SAM modified electrode. Secondly, the electrospinning process and the preparation of an electrospun solution took place. The mixture was composed of PVA (poly(vinyl alcohol)), PEI (poly(ethyleneimine)) and a glucose oxidase. The concentration of polymer was equal to 12 wt% with a mass ratio of 3:1 PVA/PEI and 15 mg/mL GOx per mL of a polymer. The nanofibers were directly formed on the surface of the Au electrode during electrospinning. Thirdly, the process of NFs cross-linking by glutaraldehyde vapors was initiated. In the end, the electrode was immersed in the AuNPs solution. The SEM images of the PVA/PEI NFs and PVA/PEI NFs/AuNPs surfaces are shown in Figure 13b,c. As can be seen in Figure 13d, the impedance increases with glucose addition in the range of 0–1.00 mM, and is caused by the accumulation of reaction products at the electrode surface. For the 4-ATP/PVA/PEI/AuNPs electrode without an enzyme, no significant response was obtained. It is possible that the abovementioned modification combines two immobilization methods—not only SAMs but also the entrapment of GOx in the polymer matrix.

(a) Scheme of the fabrication process of the biosensor; (b) SEM image of PVA/PEI NFs; (c) SEM image of PVA/PEI NFs/AuNPs; (d) Nyquist plots of impedance spectra for the ATP/PVA/PEI/AuNPs/GOx electrode with glucose addition in phosphate buffer solution. Reprinted with permission from [73]; Copyright 2017, Elsevier.

The comparison of the performance of various electrodes modified using different types of immobilization methods is shown in Table 2. The case studies were carried out using cyclic voltammetry, chronoamperometry and electrochemical impedance spectroscopy techniques. In order to compare results, sensor parameters such as sensitivity, linear range and detection limit were listed. The lowest limit of detection equals 0.1 nM and was achieved for the GOD/Fc/Au/SLG/GCE electrode modified using a covalent linking method, and the 2dMPS-AuNPs-GOx electrode in which SAMs was used for enzyme immobilization. The highest sensitivity, accompanied by a wide linear range, was reached by the electrode fabricated via the covalent bonding method—M3(GOx)/Au-TiND (25.74 µA cm⁻² mM⁻¹) and entrapment method—CHIT(GOx)/AuLr-TiND (23.47 µA cm⁻² mM⁻¹).

The comparison of the performance of electrodes reported in the literature for different types of immobilization method (IM).

CB—Covalent bonding, A—Adsorption, CL—Cross-linking, E—Entrapment, S—Self-assembled monolayers.