BIO-RECEPTOR BASED METHODS (BIO-ASSAYS) FOR FOOD

An escalating demand exists for simple, quick and reliable screening methods in food and environment monitoring. Although several chemical methods such as gas chromatography (GC), high performance liquid chromatography (HPLC), GC-MS etc. have been introduced for trace level detection of analytes, bio-analytical methods holds an advantage in that they are faster and cheaper. In bio-analysis, enzymes, antibodies, aptamers, peptides, microbial, plant and animal cells are employed as bio-receptor molecules. Advances in biochemistry, molecular biology, and immunochemistry have expanded the range of biological recognition elements. To design the biosensors, bio-receptor molecules are interfaced with various transducing surfaces. By employing these receptor molecules quick detection tools such as dipstick and protein microarrays have been introduced. **Introduction** The importance of environmental and food analysis has become increasingly evident during the last decade for many purposes, such as the diagnosis of pathogens, toxins, pollutants etc. The most commonly used methods for the detection of harmful chemical residues are Gas Chromatography (GC), High Pressure Liquid Chormatography (HPLC), GC-Mass Spectroscopy (GCMS), LC-MS/MS etc. These methods are highly sensitive and reliable, however, costly and time-consuming because they usually require complicated sample preparation steps. The development of new methods for rapid and inexpensive determination of analytes is therefore required. Bioassays are a potential alternative to the expensive chemical methods, as they are reliable, accurate, rapid, inexpensive and simple. Bioreceptors can be divided into three distinct groups: biocatalytic, bioaffinity, and microbe/cell-based systems and play a key role in bioassays as they recognize the target analyte. Biocatalysis- based bioassays depend on the use of enzymes to moderate a biochemical reaction. Bioaffinity-based biosensors rely on the use of proteins or DNA to recognize and bind a particular target. Microbial / cell based biosensors use microorganisms as the biological recognition element. These cell based systems generally involve the measurement of microbial respiration, or its inhibition, using the analyte of interest. Compared to enzyme-based approaches, microorganism-based bioassays / biosensors are relatively inexpensive to construct and can operate over a wide range of pH and temperature. **Enzymes** Enzymes are globular proteins that serve as catalysts. Their folded conformation creates an area known as the active site. The nature and arrangement of amino acids in the active site make it specific for only one type of substrate. Once the enzyme has bound its substrate (much like a key in a lock), a reaction occurs to modify the substrate in some way. The modified substrate is then released as a „product“ and the enzyme is free to catalyse another reaction. Enzymes are the most commonly used bio-receptors in bioassays. The analyte can be the enzyme, whose enzymatic activity is determined, or the substrate or the enzyme cofactors. Enzymatic assays are mainly based on either inhibition of the enzyme activity or catalysis. For example variety of enzymes such as organophosphorous hydrolase (OPH), alkaline phosphatase, ascorbate oxidase, tyrosinase and acid phosphatase have been employed in design of pesticide bioassays and biosensors. Cholinesterases, acetylcholine esterase (AChE) and butyrylcholinesterase (BuChE), have been widely used in bioassays due to their stability and sensitivity and are used in bioassays against pesticides and insecticides based on the pollutants inhibiting action. The reaction is monitored by measuring the color intensity in spectrophotometer, by measuring acid production in potentiometric systems or direct oxidation of thiocholine on the electrode surface in amperometric detection systems (Kandimalla and Ju, 2006). Some amperometric based methods use duel enzyme systems such as AChE and choline oxidase. OPH catalyzes the hydrolysis of a wide range of organophosphate (OP) pesticides, and as a result of its versatility, this enzyme has been incorporated into a number of assays and sensors for the detection of OP compounds and nerve destroying agents. Additional enzymes can be used to detect other environmental and food contaminants such as nitrate, nitrite, sulfate, phosphate, heavy metals and phenols. Tyrosinase is frequently used to determine phenols, chlorophenols, cyanide, carbamates and atrazine. **Aptamers** Aptamers are oligonucleic acid (DNA or RNA) sequences that bind a non-nucleic acid targets with high specificity and affinity. Aptamers are generally produced through an in vitro evolutionary process called „systematic evolution of ligands by exponential en-richment“ (SELEX) (Hamula, 2006). In many cases RNA libraries yield aptamers with higher binding affinities than DNA libraries due to the ability of RNA to take on a wider variety of conformations than DNA. In 1990, the discovery of aptamers by Tuerk and Gold and subsequently by Ellington and Szostak spawned significant interest and were entered into therapeutic and diagnostic applications and emerged as a valuable research tools. Aptamers have been selected for a wide array of targets including proteins, carbohydrates, lipids or small molecules. Aptamers may mimic antibodies in a number of applications, such as flow cytometry, ELISA, cell sorting, fluorescence microscopy, western blotting, and biosensors or chips. Aptamers have a number of advantages compared to antibodies as they are smaller in size, their sensitivity can be increased, the possibility for in vitro production, and thus lack of immunization and animal hosts. The binding affinity, specificity and stability of aptamers can be improved by rational design or molecular evolution techniques (Kandimalla and Ju, 2004). Due to specificity and ease in labeling with fluorescence, radio isotope, or modification of nucleic acid sequence, aptamers are excellent receptor candidates for bioassays and biosensors. One of the best approaches is the development of molecular aptamer beacon (MAB), with which real time target recognition and quantification is possible. In ELISA, immunoglobulins are the primary recognition agents; using the same principle, enzyme linked oligonucleotide assay (ELONA) can be prepared using aptamers as the recognition agents. MABs are similar to the molecular beacons (MBs). Like MBs the 5 and 3 ends in MABs can also be tagged or conformationally changed and/or a distance increase between quencher and fluorophore, leading to a detectable signal. The fluorescence signal increased or quenched is directly proportional to the target concentration. Li et al., (2002) reported an anti-thrombin aptamer beacon capable of recognizing thrombin, with a detection limit of 112 pM in a homogeneous solution. **Polypeptides / Protein scaffold** Natural biopolymers such as proteins and DNAs frequently have helical conformations that contribute to the three-dimensional ordered structure and the specific function of the biopolymer. As a part of the cell signaling pathways and enzymatic reactions, conformational alterations of biomolecules are very important in biological systems. Likewise the artificially and specifically folded polypeptide structures are other attractive receptor molecules in biosensing and screening systems. These are robust and easily synthesized by well-established methods and because of the huge chemical diversity that can be generated in a short time with modest synthetic cost and effort. The amino acid sequence can be varied over a wide range to provide access to a variety of scaffold (binding domain) structures, and highly specific recognition sites and reporter groups can be introduced post-synthetically by orthogonal (controlled synthesis) strategies on the solid phase. Fluorescent probes, chromophores, oligonucleotides, sugars, lipids etc can be introduced in different configurations, turning into a very attractive and general tool for the development of a wide range of ligand receptor systems. Enander et al., (2002) reported a folded ligand modified helix-loophelix polypeptide scaffold for the detection of human carbonic anhydrase II (HCAII), which catalyses the reversible hydration of carbon dioxide. The fluorescence of the peptide modified with a benzenesulfonamide (inhibitor of HCAII) derivative and a fluorescent probe was monitored as a function of HCAII concentration and the dissociation constant (Kd). The fluorescence of polypeptide increases in the presence of HCAII, due to a change in the molecular environment of the dansyl group upon binding with HCAII, whereas the probe is partially quenched in the absence of the HCAII. **Antibodies** Antibodies have made a substantial contribution towards the advancement of diagnostic assays and have become indispensable in most diagnostic tests that are used routinely today. Antibodies are capable of exhibiting very specific binding capabilities for desired structures. The past few years have seen an increase towards greater use of antibody based assays such as enzymelinked immunosorbent assay (ELISA) in the field of food and environmental monitoring (Fránek et al., 2006). The efficiency and accuracy of the immunoassay (IA) is dependant on the stability and affinity of the employed antibody (Tetin and Stroupe 2004). Antibodies are highly complex and made up of hundreds of individual amino acids arranged in a highly ordered sequence. The immune system of mammals and other vertebrates is able to recognize an extremely large number of different molecules and respond to immunogenic stimuli by secreting specific antibodies into the blood stream. When animals are immunized with a strong immunogen for a relatively long period of time their blood serum will contain high concentrations of highly specific polyclonal antibodies. Introduction of hybridomas for monoclonal antibody production in the mid 1970s revolutionized immunochemistry and brought antibody-based techniques to a higher level of specificity, sensitivity and of key importance, manufacturability. The recent progress in construction of recombinant antibodies leads to exciting new fields of antibody design. In addition, plants and crop species in particular, have the potential to enable extremely cost-effective and efficient production of antibodies (plantibodies). Phage display technology has proved to be robust and is able to produce the recombinant antibody fragments, for example single-chain antibody variable region fragment (ScFv) and fragment antigen binding region (Fab) (variable domains plus the two of the constant domains). Some IAs work in homogeneous phases (in solution or gels), whereas the majority are heterogeneous assays, involving the adhesion of antibodies and/or antigens to a solid surface. The surfaces used in the heterogeneous assays are usually the walls of microtiter plates with 96 or more wells. Some assays are competitive, in which labeled analyte competes for the binding sites of antibodies with analyte in samples, where the decrease in 17 bound (or increase in free) labeled antigen is measured. Noncompetitive (sandwich) IAs, which are based on two antibodies to different epitopes, are known to work well for high-molecular-weight analytes. Compared with competitive immunoassays they have many advantages, for example improved speed, sensitivity, and specificity. The first IA used iodine radioisotopes (125I or 131I), however enzymatic labels are now more common such as, horse radish peroxidase, ß-D-glucoseoxidase and alkaline phosphatase. Enzymatic labels are eco-friendly whereas radioisotopes are toxic and have a short halflife. Fluorescent materials such as fluorescene isothiocyanate (FITC) etc are employed as labels in fluorescence based assays. Chiefly, IAs provides a rapid and cost effective analysis for the determination of a variety of environmental contaminants. In addition IA provides high sensitivity and specificity due to the powerful catalytic ability of enzyme and the extraordinary discriminatory capabilities of antibodies. IA methods are fast and relatively easy, compared to conventional GC/MS and HPLC as they don’t require a multi-step cleanup process. One disadvantage is that ELISA is usually performed in a laboratory using microtitre plates and, thus are not suitable for on-site monitoring. Polyclonal and monoclonal antibodies have been successfully developed in the Department of Analytical Biotechnology, Veterinary Research Institute (VRI). Their application in ELISA and immunosensor development has been directed especially towards phenoxyacetic acid herbicides, s-triazine herbicides, sulfonylurea herbicides, polychlorinated biphenyls, surfactants (linear alkylbenzene sulphonates) and toxic metabolites (nonylphenol), and selected veterinary drugs (namely nitrofurans and sulfonamides). More information and technical discussion can be found in papers by Fránek, 1998 and Fránek and Hruska, 2005. **Whole cells** For the biological assay of antibiotics, cup plate bioassay was widely used. In this method the added antibiotic inhibits the growth of microorganisms, seeded in the agar. The resulting inhibition zone (in the agar) is directly proportional to the logarithm of the concentration of antibiotic. In recent years, luminescent bacteriabased bioassays have been introduced for the detection of various compounds such as drugs or toxins. Depending upon the analyte action on the microbial cell, luminescence may increase or decrease. The major advantage with whole cell bioassay is that they can act as a bag of enzymes, whereas protein conformation of the enzyme is more stable, as they are in a native environment. At the same time this technique can reduce the purification costs and the ease of encapsulation. Recombinant luminescent bacteria are generally used in environmental pollutants analysis e.g dioxines. Stability of the cell can be increased by entrapping it in a suitable matrix (i.e sol-gel etc), which allows for the repeated use of whole cells. Plant cells (tissues) have also been employed in bioassays, for the determination or organophosprorous pesticides. A study of cucumber tissue scrapings (oscorbate oxidase) were interfaced with an oxygen electrode. In the presence of pesticide, the reduced enzyme activity was measured amperometricllay. The output voltage of the biosensor is proportional to the pesticide concentration (Rekha et al., 2000). The physiology of the some animal cell lines changes in the presence of ligands / pathogens / viruses. The physiological changes in presence of viruses in African green monkey kidney cell lines were correlated using Bioelectric Recognition Assay (Kintzios et al. 2004). Recombinant Mouse hepatoma cells have been employed for the direct detection of halogenated and polycyclic aromatic hydrocarbons such as dioxines. In this system, the induction of firefly luciferase takes place in presence of halogenated and polycyclic aromatic hydrocarbons (Ziccardi et al., 2000) Author: Vivek Babu Kandimalla, Nagamani Kandimalla, Milan Fránek Veterinary Research Institute, Hudcova 70, Brno 621 00, Czech Republic, e-mail: franek@vri.cz References: 1. Enander K. et al., 2002, J. Org. Chem.67: 3120-3123. 2. Fránek M., 1998, In Biosensors for direct monitoring of environmental pollutants in field, Kluwer publishers, Netherlands, 115-126. 3. Fránek M. and Hruska K., 2005, Vet. Med. 50: 1-10. 4. Fránek et al., 2006, Anal. Chem. 78: 1559-1567. 5. Hamula C. L. A. et al., 2006, Trends Anal. Chem. 25: 681-691. 6. Kandimalla V. B. and Ju H. X. 2004, Anal. Lett. 37: 2215–2233. 7. Kandimalla V. B. et al., 2006, Crit. Rev. Anal. Chem. 36:73 –106. 8. Kandimalla V. B. and Ju H. X. 2006, Chem. Eur. J. 12: 1074-080. 9. Kintzios S. et al. 2004, Biosens. Bioelectron. 20: 907– 916. 10. Li JJ. et al. 2002, Biochem. Biophys. Res. Commun. 292: 31-40. 11. Rekha K et al., 2000, Biosens. Bioelectron. 15: 499–502. 12. Tetin S.Y. and Stroupe S. D. 2004, Curr. Pharmaceu. Biotechnol. 5: 9-16. 13. Ziccardi M. H. et al., 2000, Toxicological Sci. 54:183–193.

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