**5. Biorecognition elements**

Bioreceptors or biorecognition elements are the key to specification of biosensor technolo‐ gies. They are responsible for binding the analyte of interest to the sensor for the measure‐ ment. These bioreceptors can take many forms and the different bioreceptors that have been used are as numerous as the different analytes that have been monitored using biosensors. However, bioreceptors can generally be classified into a few different major categories (Fig‐ ure 6). These categories include: antibody/antigen, enzymes, nucleic acids/DNA, cellular structures/cells ( i.e. microorganisms), and biomimetics. The specificity of molecular recog‐ nition makes these molecules very attractive as tools for therapeutic diagnostic and other an‐ alytical applications.

#### **5.1. Enzymes**

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

sponse time, can work in room temperature.

46 State of the Art in Biosensors - General Aspects

by the use of cross-linking agents [54].

pose problems [6].

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‐

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

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

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

Enzymes are the most commonly used bio-receptors in bioassays. The analyte can be the en‐ zyme, whose enzymatic activity is determined, or the substrate or the enzyme cofactors. En‐ zymatic assays are mainly based on either inhibition of the enzyme activity or catalysis. According to the fact, variety of enzymes such as organophosphorous hydrolase, alkaline phosphatase, ascorbate oxidase, tyrosinase and acid phosphatase have been employed in de‐ sign of pesticide bioassays and biosensors [65].

**Figure 6.** Classification of bioreceptors

Enzymes are often chosen as bioreceptors based on their specific binding capabilities as well as their catalytic activity. In biocatalytic recognition mechanisms, the detection is amplified by a reaction catalyzed by macromolecules - biocatalysts. With the exception of a small group of catalytic ribonucleic acid molecules, all enzymes are proteins. Some enzymes re‐ quire no chemical groups other than their amino acid residues for activity. Others require an additional cofactor, which may be either one or more inorganic ions (Fe2+, Mg2+, Mn2+, or Zn2+), or a more complex coenzyme. The catalytic activity provided by enzymes allows for much lower limits of detection than would be obtained with common binding techniques. The catalytic activity of enzymes depends upon the integrity of their native protein confor‐ mation. Enzyme-coupled receptors can also be used to modify the recognition mechanisms. In example, the activity of an enzyme can be modulated when a ligand binds at the receptor. This enzymatic activity is often greatly enhanced by an enzyme cascade, which leads to complex reactions in the cell [66].

8

The use of enzymes as the recognition element was very popular in the first generation of biosensor due to their availability. Among various oxidoreductases, glucose oxidase, horse‐ radish peroxidase and alkaline phosphatase have been employed in most biosensor studies [67-69]. Some amperometric based methods use duel enzyme systems such as acetylcholine esterase and choline oxidase. In example, organophosphorous hydrolase catalyzes the hy‐ drolysis of a wide range of organophosphate pesticides, and as a result of its versatility, this enzyme has been incorporated into a number of assays and sensors for the detection of this type of compounds. Additional enzymes can be used to detect other environmental and food contaminants such as nitrate, nitrite, sulfate, phosphate, heavy metals and phenols. Ty‐ rosinase is frequently used to determine phenols, chlorophenols, cyanide, carbamates and atrazine.

Enzymes offer many advantages connected with high sensitivity, possibility of direct visual‐ ization and stability, but there are still some problems, which include multiple assay steps and the possibility of the interference from endogenous enzymes. Many enzyme detection procedures are visual eliminating the need for expensive equipment, but the enzyme stabili‐ ty is still problematic and the ability to maintain enzyme activity for a long time [70].

#### **5.2. Nucleic acids**

Recently, advances in nucleic acid recognition have enhanced the using of DNA biosensors and biochips [71]. In the case of nucleic acid bioreceptors for pathogen detection, the identi‐ fication of analyte's nucleic acid is achieved by matching the complementary base pairs. Since each organism has unique DNA sequences, any self-replicating microorganism can be easily detect [70].

Grabley and coworkers have reported on the use of DNA biosensors for the monitoring of DNA-ligand interactions [72]. Surface plasmon resonance was used to monitor real-time binding of low molecular weight ligands to DNA fragments that were irreversibly bound to the sensor surface *via* coulombic interactions. The detection of specific DNA sequences has been employed for detecting microbial and viral pathogens [73] and food pathogen like *E. coli* [74], *Salmonella sp*.

Recent advances in nucleic acid recognition, like the introduction of Peptide Nucleic Acid (PNA) has opened new opportunities for DNA biosensors. PNA is a synthesized DNA in which the sugar-phosphate backbone is replaced with a pseudopeptide. PNA as a probe molecule has several advantages, i.e. superior hybridization characteristics, detection of sin‐ gle-based mismatches, better stability compared to enzymes [70].

#### **5.3. Antibodies**

8

Enzymes are often chosen as bioreceptors based on their specific binding capabilities as well as their catalytic activity. In biocatalytic recognition mechanisms, the detection is amplified by a reaction catalyzed by macromolecules - biocatalysts. With the exception of a small group of catalytic ribonucleic acid molecules, all enzymes are proteins. Some enzymes re‐ quire no chemical groups other than their amino acid residues for activity. Others require an additional cofactor, which may be either one or more inorganic ions (Fe2+, Mg2+, Mn2+, or Zn2+), or a more complex coenzyme. The catalytic activity provided by enzymes allows for much lower limits of detection than would be obtained with common binding techniques. The catalytic activity of enzymes depends upon the integrity of their native protein confor‐ mation. Enzyme-coupled receptors can also be used to modify the recognition mechanisms. In example, the activity of an enzyme can be modulated when a ligand binds at the receptor. This enzymatic activity is often greatly enhanced by an enzyme cascade, which leads to

**BIORECEPTORS** 

**WHOLE CELLS (cellular system or non-enzymatic proteins) –** 

**i.e. MICROORGANISMS**

48 State of the Art in Biosensors - General Aspects

**Figure 6.** Classification of bioreceptors

complex reactions in the cell [66].

**ENZYMES**

**NUCLEIC ACIDS**

**ANTIBODIES**

**APTAMERS**

**BIOMIMETIC RECEPTORS**

Antibodies are common bioreceptors used in biosensor technologies. Antibodies are biologi‐ cal molecules that exhibit very specific binding capabilities for specific structures. This is very important due to the complex nature of most biological systems. An antibody is a com‐ plex biomolecule, made up of hundreds of individual amino acids arranged in a highly or‐ dered sequence. For an immune response to be produced against a particular molecule, a certain molecular size and complexity are necessary: proteins with molecular weights great‐ er than 5000 Da are generally immunogenic.

The way in which an antigen and its antigen-specific antibody interact is analogues of a lock and key fit [66], by which specific geometrical configurations of a unique key enables it to open a lock.

An antigen-specific antibody fits its special antigen in a highly specific manner, according to that the three-dimensional structures of antigen and antibody molecules are matching [70]. Antibody biosensors are interested wide and interesting group of sensing devices, which in‐ cludes i.e. Surface plasmon resonance [74], fiber-optic biosensor [75], magnetoelastic reso‐ nance sensor [76] and immunosensor [77].

#### **5.4. Aptamers**

Aptamers are folded single stranded DNA or RNA oligonucleotide sequences with the ca‐ pacity to recognize various target molecules. They are generated in the systematic evolution of ligands by exponential enrichment process which was first time reported by Ellington [78] and Tuerk [79]. In this way suitable binding sequences are first isolated from large oli‐ gonucleotide libraries and subsequently amplified. The main application for aptamers is in biosensors. While antibodies are used in ELISA, the similar process for aptamers is called ELONA (enzyme linked oligonucleotide assay). They have many advantages over antibod‐ ies such as easier deposition on sensing surfaces, higher reproducibility, longer shelf life, easier regeneration and a higher resistance to denaturation. As antibodies, they are charac‐ terized by both, their high affinity and specificity to their targets [80].

#### **5.5. Biomimetic receptors**

A receptor which is designed and fabricated and to mimic a bioreceptor is often defined as biomimetic receptor. According to the phenomena several different techniques have been developed over the years for the construction of biomimetic receptors [81,82]. These proce‐ dures include: genetically engineered molecules, artificial membrane fabrication and molec‐ ular imprinting method. The molecular imprinting method has existed as an attractive and accepted tool in developing an artificial recognition agents.

Artificial membrane fabrication for bioreception has been performed for many different ap‐ plications. Stevens *et al.* has developed an artificial membrane by incorporating gangliosides into a matrix of diacetylenic lipids [83]. The lipids were allowed to self-assemble into Lang‐ muir-Blodgett layers and were then photopolymerized *via* ultraviolet irradiation into poly‐ diacetylene membranes. However, molecular imprinting has been used for the construction of a biosensor based on electrochemical detection of morphine [84].

#### **5.6. Cellular bioreceptors**

Cellular structures and cells have been used in the development of biosensors and bio‐ chips. These bioreceptors are either based on biorecognition by an entire cell/microorgan‐ ism or a specific cellular component that is capable of specific binding to certain species. There are presently three major subclasses of this category: a) cellular systems, b) en‐ zymes and c) non-enzymatic proteins. Due to the importance and large number of biosen‐ sors based on enzymes, these have been given their own classification [85]. Microorganisms offer a form of bioreceptor that often allows a whole class of compounds to be monitored. Generally these microorganism biosensors rely on the uptake of certain chemicals into the microorganism for digestion. Often, a class of chemicals is ingested by a microorganism, therefore allowing a class-specific biosensor to be created. Microorgan‐ isms such as bacteria and fungi have been used as indicators of toxicity or for the meas‐ urement of specific substances. For example, cell metabolism (e.g., growth inhibition, cell viability, substrate uptake), cell respiration or bacterial bioluminescence have been used to evaluate the effects of toxic heavy metals [85].

A microbial biosensor has been developed for the monitoring of short-chain fatty acids in milk [86]. *Arthrobacternicotianae* microorganisms were immobilized in a calciumalginate gel on an electrode surface. By monitoring the oxygen consumption of the *Anthrobacter* electro‐ chemically, its respiratory activity could be monitored, thereby providing an indirect means of monitoring fatty acid consumption.
