**4. Immobilization of protein monolayers on planar solid supports**

The concept of using self-assembled biomolecules as an elementary units to develop super‐ structures of defined geometry has thus received considerable attention. In this contents, the self-assembly ability of amphiphilic biomolecules such as lipids, to spontaneously organize appears as a suitable concept for the development of membrane models. The concept is clearly illustrated i.e. by Langmuir monolayers, which have been extensively used as mod‐ els to understand the role and the organization of biological membranes [50] and to acquire knowledge about the molecular recognition process [51,52]. Langmuir-Blodgett technology allows to build lamellar lipid stacking by transferring a monomolecular film formed at an air/water interface onto a solid support. When all parameters are optimized, this technique corresponds to one of the most promising for preparing thin films of amphiphilic molecules [6]. The sensitive element produced by LB technology has higher sensitivity and faster re‐ sponse time, can work in room temperature.

The optimal value of the surface pressure to produce the best results depends on the na‐ ture of the monolayer and is often established empirically [53]. However, the LB/LS deposi‐ tion is traditionally carried out in the condensed phase since it is generally believed that the transfer efficiency increases when the monolayer is in a close-packed state. In that con‐ dition the surface pressure is sufficiently high to ensure a strong lateral cohesion in the monolayer, so that the monolayer does not fall apart during the transfer process. Although the optimal surface pressure depends on the nature of the material constituting the film, bi‐ ological amphiphiles can seldom be successfully transferred at surface pressures lower than 10 mN/m and at surface pressures above 40 mN/m, where collapse and film rigidity often pose problems [6].

Moreover, the main advantage of the adsorption of the enzyme onto pre-formed LB films lies in the possible interaction of the enzyme with a hydrophobic or hydrophilic surface de‐ pending on the number of the deposited layers, thus allowing the control of the enzyme en‐ vironment. Likewise, this approach allows the control of the thickness and the homogeneity of the LB films harboring the enzymes. Nevertheless, the release of protein molecules due to the weakness of their association with the surface is often the main reason which explains the poor reproducibility of responses of LB membrane-based sensors. Due to avoid desorp‐ tion, some authors have proposed to covalently immobilize the enzyme on LB film surfaces by the use of cross-linking agents [54].

Electrostatic layer-by-layer assembly was first proposed by Decher in 1990s and proved to be possible to build-up ordered multilayer structures by consecutive adsorption of polyanions and polycations [55]. This film assembly approach has great advantages be‐ cause of the simplicity preparation of ultrathin films with defined composition and uni‐ form thickness in nanoscale in which synergy between distinct materials may be achieved in a straightforward, low-cost manner. With the LbL technique a wide diversity of materials may be employed, and film fabrication is performed under mild conditions, which is particularly important for preserving activity of biomolecules. The fundamental concepts and mechanisms involved in the LbL technique have been detailed in a series of papers [56]. In most cases adsorption in LbL films is governed by electrostatic interac‐ tions between species bearing opposite charges, but secondary interactions have also been shown to be important. The LbL technique is versatile with regard to the substrates that may be used, which include hydrophilic and hydrophobic glass, mica, silicon, met‐ als, quartz, and polymers [57]. In addition, LbL films may be deposited directly onto col‐ loidal suspensions [58].

Several attempts have been made to fabricate hybrid enzyme electrodes with the method [59]. In 1995 this new method was applied to immobilize negatively charged glucose oxidase (GOx) in a polyethyleneimine based multilayer structure [60] and proved to be one of the most perspective methods for preparing amperometric enzyme biosensors. One year later, an oxygen mediated glucose biosensor based on GOx and poly(L-lysine) co-adsorbed onto a negatively charged monolayer of mercaptopropionic acid, deposited on an Au electrode was described [60]. Hodak *et al*. introduced LbL assembly technique to construct reagentless bio‐ sensor with glucose oxidase and ferrocene modified with poly(allylamine). Sun *et al*. [61] and Chen *et al*.[62] fabricated peroxidase and glucose oxidase biosensors with Os-based re‐ dox polymer and enzymes. Also known isreagentless biosensor built of organic dye methyl‐ ene blue with peroxidase [63]. Vossmeyer and co-workers investigated the optical and electrical properties of layer-by-layer self-assembly of gold nanoparticle/alkanedithiol films [64]. Though gold nanoparticles or enzymes have been widely used to form multilayer films by layer-by-layer technology.
