**2. Langmuir-Blodgett, Langmuir-Schaefer, layer-by-layer assembly multilayers of proteins**

The concept of using biomolecules as an elementary structure to develop self-assembled structures of defined geometry has thus received considerable attention. In this way, the self-assembly ability of amphiphilic biomolecules such as lipids, to spontaneously organize into nanostructures mimicking the living cell membranes, appears as a suitable concept for the development of biomimetic membrane models. The potential of two-dimensional molec‐ ular self-assemblies is clearly illustrated by Langmuir monolayers of lipid-like molecules, which have been extensively used as models to understand the role and the organization of biological membranes and to acquire knowledge about the molecular recognition process [6,8]. Langmuir-Blodgett technology allows building up lamellar lipid stacking by transfer‐ ring a monomolecular film formed at an air/water interface – named Langmuir monolayer or Langmuir film– onto a solid support (Figure 2).

**Figure 2.** Langmuir – Blodgett deposition

transported onto a solid support. When all parameters are optimized, this technique corre‐ sponds to one of the most promising for preparing thin films of amphiphilic molecules. Based on the self-assembled properties of amphiphilic biomolecules at the air/water inter‐ face, LB technology offers the possibility to prepare biomimetic layers suitable for adsorp‐

Proteins are more challenging to prepare for the different microarray than i.e. DNA, and protein functionality is often dependent on the state of proteins. Since enzymes often signifi‐ cantly reduce their activity during immobilization, the optimized adsorption methods seem to be optimal for the retaining of conformational states of proteins on solid surfaces. Among the various immobilization techniques available, adsorption may have a higher commercial potential than other methods because the adsorption process is simpler, less expensive, re‐ tains a high catalytic activity, and most importantly, the support could be repeatedly reused

Enzyme-based biosensors play an important role in various industries, such as food, manu‐ facturing, clinic, and environment. Recently, mediators have been employed in enzymebased biosensors in order to shuttle electrons between the redox enzyme and the electrode surface. Solution-phase mediators may cause electrode contamination and operation incon‐ venience. In order to overcome the above-mentioned drawbacks and improve the perform‐ ances of the biosensors, the immobilization of the mediator with protein on a solid support

However, there is many techniques of biocatalysts immobilization and much research is dedicated to fabricate the biosensing elements, the construction of novel type of biosensor is challenge for new technologies and the key problem is modification of electrode by enzyme

Electrode

Biocatalyst

Conducting copolymer

tive immobilization of bioactive molecules [6].

38 State of the Art in Biosensors - General Aspects

after inactivation of the immobilized enzyme.

using thin film preparation methods.

**Figure 1.** Layered biosensor system

provides a new way to construct reagentless biosensors [7].

Lipid-based phases are particularly attractive because they can be nanostructurally custom‐ ized, for instance, to closely resemble cellular components, or to formulate delivery vehicles for biomolecules and drugs. In the presence of water or aqueous buffers, lipid molecules can self-assemble into a wide range of nanostructures [9]. The intrinsically low degree of non‐ specific adsorptivity of supported membranes makes them interesting as an interface be‐ tween the nonbiological materials on the surface of a sensor or implant and biologically active fluids [10,11]. Potential applications include the acceleration and improvement of medical implant acceptance, programmed drug delivery, production of catalytic interfaces, as a platform to study transmembrane proteins and membrane-active peptides, and as bio‐ sensors [12-14].

Although the LB method does not solve all problems associated with engineering the struc‐ ture of condensed phases, it does provide a level of control over the orientation and place‐ ment of molecules in monolayer and multilayer assemblies that is not otherwise available. When all parameters are optimized, this technique corresponds to one of the most promis‐ ing for preparing thin films of amphiphilic molecules as it enables an accurate control of the thickness, an homogeneous deposition of the monolayer over large areas compared to the dimension of the molecules, as well as the possibility to transfer monolayers on almost any kind of solid substrate. Based on the self-assembled properties of amphiphilic biomolecules at the air/water interface, LB technology offers the possibility to prepare biomimetic layers suitable for immobilization of bio-active molecules [8].

Systems mimicking natural membranes appear promising in the field of bioelectronic devi‐ ces and represent useful models in basic research on membrane behavior in life science. For such purposes, the interest of LB films is now largely recognized, and several enzyme sen‐ sors based on the LB technology have been reported (Table 1).


**Table 1.** Selected biosensor based on thin ordered films

In particular LB films are offering a possibility of obtaining extended two–dimensional πelectron systems [23]. Although, majority of conventional conducting polymers are not solu‐ ble in common solvents, making any LB deposition impossible, one can increase their solubility by attachment of side groups (usually n–alkyl ones) to the main chains. According to this Langmuir-Blodgett, horizontal lifting or other self–assembled method is employed for obtaining molecular films of conducting structures. This type of material is popular in designing of sensor devices. Product in any solid-state sensor, analyte molecules have to dif‐ fuse into and react with the acting sensing component and any product of the reaction must diffuse out. It therefore follows that the thinner the sensing layer is, the less time this will take and thereby speed and reversibility being improved.

Device preparation requires use of facilitative methodologies for the organization of biologi‐ cal components in a particular configuration. Self-assembled monolayer and LB methods have been used for organization of functional elements in two-dimensional or layered struc‐ tures, respectively. These methods offer opportunities for immobilization of functional com‐ ponents into well-organized structures. As a convenient methodology alternate layer-bylayer adsorption has been paid much attention as an emerging methodology.

Recent research has proved the great applicability of the LbL technique not only for prepa‐ ration of bio-related devices but also for producing various device structures, including sen‐ sors [24], photovoltaic devices [25], electrochromic devices [26], fuel cells [27].

Materials that can be used in LbL methods cover a wide range including conventional poly‐ electrolytes, conductive polymers, dendrimers, proteins, nucleic acids, saccharides, virus particles, inorganic colloidal particles, quantum dots, clay plates, nanosheets, nanorods, nanowires, nanotubes, dye aggregates, micelles, vesicles, LB film, and lipid membranes [28]. In most cases, the LbL assembly is carried out based on electrostatic interactions. As illus‐ trated in Figure 3, adsorption of counterionic species at relatively high concentrations leads to excess adsorption of the substances, as a result of charge neutralization and resaturation, finally resulting in charge reversal.

**Figure 3.** Layer-by-Layer assembly [28]

at the air/water interface, LB technology offers the possibility to prepare biomimetic layers

Systems mimicking natural membranes appear promising in the field of bioelectronic devi‐ ces and represent useful models in basic research on membrane behavior in life science. For such purposes, the interest of LB films is now largely recognized, and several enzyme sen‐

Glucose oxidase Lipid - coating LB – two layers film of lipids 3 month [15] Catalase Adsorption LB – one layer film of phospholipids >3 months [16]

copolymers in mixture with linoleic acid

octadecylamine

LbL films of alternate layers of poly(allylamine) hydrochloride and glucose oxidase

LbL multilayer films of alternate charged polysaccharides, chitosan and polyaniline

mercaptopropionic acid

LB layers of octadecyltrimethylammonium and nano-sized Prussian blue clusters

In particular LB films are offering a possibility of obtaining extended two–dimensional πelectron systems [23]. Although, majority of conventional conducting polymers are not solu‐ ble in common solvents, making any LB deposition impossible, one can increase their solubility by attachment of side groups (usually n–alkyl ones) to the main chains. According to this Langmuir-Blodgett, horizontal lifting or other self–assembled method is employed for obtaining molecular films of conducting structures. This type of material is popular in designing of sensor devices. Product in any solid-state sensor, analyte molecules have to dif‐ fuse into and react with the acting sensing component and any product of the reaction must

Cellulase Adsorption LS film of cellulose No data [18]

Adsorption self-assembled monolayer of a 3-

**Thin ordered film Stability References**

>3 months [2]

>4 months [17]

20 days [19]

3 weeks [20]

4 weeks [21]

No data [22]

suitable for immobilization of bio-active molecules [8].

40 State of the Art in Biosensors - General Aspects

**Immobilization method**

**Protein**

β-Galactosidase, glucose oxidase, peroxidase

Glucose oxidase Adsorption

Cholesterol oxidase Adsorption

**Table 1.** Selected biosensor based on thin ordered films

Urease Adsorption and

covalent grafting

sors based on the LB technology have been reported (Table 1).

Laccase Adsorption LB – five layers film of benzothiadiazole

Invertase Adsorption LS – one layer film of phospholipids and

The forces for LbL assembly are not limited to electrostatic interactions alone. Various inter‐ actions including metal-ligand interaction, hydrogen-bonding, charge transfer, supramolec‐ ular inclusion, bio-specific recognition, and stereo-complex formation can be used for LbL assembly [28]. Biocompatibility is the most prominent advantage of the LbL assembly be‐ cause this procedure requires mild conditions for film construction. Most proteins, especial‐ ly those soluble in water, have charged sites on their surface, and so the electrostatic LbL adsorption is useful for the construction of various protein organizations.

In order the aim to develop models mimicking biomembranes usable for applications in the biosensor field, studies of biological activities of membraneous proteins after incorporation in a phospholipidic bilayer were widely investigated [29,30]. Well known is the sensing sys‐ tem built by the incorporation of glutamate dehydrogenase or choline oxidase into fatty acid LB films through an adsorption or an inclusion process which consists of sandwiching the protein molecules between two LB layers [31]. The molecules of glutamate dehydrogenase adsorbed on the surface of behenic acid LB films work as a protective screen against the re‐ arrangement of the multilayers induced by diffusion of the alkaline buffer inside the struc‐ ture; on the contrary, the choline oxidase molecules operate as an accelerating factor of the structural lipidic reorganization induced in the same conditions.

The physiological activity of transmembrane proteins, however may depend on the physico‐ chemical properties of neighboring phospholipids. Such dependence has been demonstrated in the case of, among others, hydroxybutyrate dehydrogenase, Ca2+-ATPase and melibiose‐ permease. Moreover, all integral membrane proteins are surrounded by a layer of phospho‐ lipids, the annular region, which provides the adequate lateral pressure and fluidity to seal the membrane during the changes in the protein during transport events [32].

#### **2.1. Main membrane lipids**

All the amphiphilic molecules are potentially surface active agents and substantially mon‐ olayer-forming materials. It is possible to find a discussion on the range of a large variety of amphiphile compounds able to form insoluble monomolecular films [33]. Due to the synthesis of biomimetic membranes, the most important types of amphiphilic molecules are fatty acids, phospholipids and glycolipids. Cholesterol as a type of steroid extremely abundant in the cell membrane, can also form insoluble monolayers but it is generally more studied mixed with other phospholipids [34-36] in order to its implication in the for‐ mation of lipid microdomains. Figure 4 presents the examples of the principal structures of these different types of lipids. The amphiphilic nature of biological surfactants is re‐ sponsible for their aggregation at the air/water interface. Their affinity for the air/water in‐ terface is determined by the physico-chemical properties of the hydrophilic and hydrophobic parts. The monolayer forming abilities of the amphiphiles is dependent on the balance between these two opposite forces, which are determined by the size of the hydrophobic tail group (i.e. the alkyl chain) and the strength of the hydrophilic head group (i.e. size, polarity, charge, hydration capacity) [6]. If the equilibrium between hy‐ drophilic and hydrophobic part of molecule is disturbed, the material dissolve in the sub‐ phase and is not able to form a stable monolayer.

Most of the lipidic cell membrane components are composed of a zwitterionic head group at pH 7.0 (phospholipids) or contain a highly hydrophilic polar group (glycolipids), and a hy‐ drophobic part which is constituted by two hydrocarbon chains per molecule and drastical‐ ly reduces the water solubility of the complete lipidic membrane molecule. Consequently, many components of cell membranes form insoluble monolayers at the air/water interface since the lipid concentration in the aqueous subphase is negligible, and some of them may be built up into multilayer films by Langmuir-Blodgett deposition.

**Figure 4.** Examples of the main membrane lipids; 1) phospholipids: a, phosphatidylcholine; b, phophatidylserine; 2) glycolipids: c, monogalactosyldiglyceride; 3) sphingolipids: d, sphingomyelin; e, cholesterol
