Jaytry Mehta

Recent advances in recognition elements of food and environmental biosensors: A review

A sensitive monitoring of contaminants in food and environment, such as chemical compounds, toxins and pathogens, is essential to assess and avoid risks for both, human and environmental health. To accomplish this, there is a high need for sensitive, robust and cost-effective biosensors that make real time and in situ monitoring possible. Due to their high sensitivity, selectivity and versatility, affinity-based biosensors are interesting for monitoring contaminants in food and environment. Antibodies have long been the most popular affinity-based recognition elements, however recently a lot of research effort has been dedicated to the development of novel recognition elements with improved characteristics, like specificity, stability and cost-efficiency. This review discusses three of these innovative affinity-based recognition elements, namely, phages, nucleic acids and molecular imprinted polymers and gives an overview of biosensors for food and environmental applications where these novel affinity-based recognition elements are applied.

In vitro selection and characterization of DNA aptamers recognizing chloramphenicol.

Chloramphenicol (Cam), although an effective antibiotic, has lost favour due to some fatal side effects. Thus there is an urgent need for rapid and sensitive methods to detect residues in food, feed and environment. We engineered DNA aptamers that recognize Cam as their target, by conducting in vitro selections. Aptamers are nucleic acid recognition elements that are highly specific and sensitive towards their targets and can be synthetically produced in an animal-friendly manner, making them ethical innovative alternatives to antibodies. None of the isolated aptamers in this study shared sequence homology or structural similarities with each other, indicating that specific Cam recognition could be achieved by various DNA sequences under the selection conditions used. Analyzing the binding affinities of the sequences, demonstrated that dissociation constants (K(d)) in the extremely low micromolar range, which were lower than those previously reported for Cam-specific RNA aptamers, were achieved. The two best aptamers had G rich (>35%) nucleotide regions, an attribute distinguishing them from the rest and apparently responsible for their high selectivity and affinity (K(d)∼0.8 and 1μM respectively). These aptamers open up possibilities to allow easy detection of Cam via aptamer-based biosensors.

Aptasensing of chloramphenicol in the presence of its analogues : reaching the maximum residue limit

A novel, label-free folding induced aptamer-based electrochemical biosensor for the detection of chloramphenicol (CAP) in the presence of its analogues has been developed. CAP is a broad-spectrum antibiotic that has lost its favor due to its serious adverse toxic effects on human health. Aptamers are artificial nucleic acid ligands (ssDNA or RNA) able to specifically recognize a target such as CAP. In this article, the aptamers are fixed onto a gold electrode surface by a self-assembly approach. In the presence of CAP, the unfolded ssDNA on the electrode surface changes to a hairpin structure, bringing the target molecules close to the surface and triggering electron transfer. Detection limits were determined to be 1.6 × 10(-9) mol L(-1). In addition, thiamphenicol (TAP) and florfenicol (FF), antibiotics with a structure similar to CAP, did not influence the performance of the aptasensor, suggesting a good selectivity of the CAP-aptasensor. Its simplicity and low detection limit (because of the home-selected aptamers) suggest that the electrochemical aptasensor is suitable for practical use in the detection of CAP in milk samples.

Selection and characterization of PCB-binding DNA aptamers.

Polychlorinated biphenyls (PCBs) are persistent organic pollutants (POPs) that resist natural degradation and bioaccumulate in nature. Combined with their toxicity, this leads them to cause cancer and other health hazards. Thus, there is a vital need for rapid and sensitive methods to detect PCB residues in food and in the environment. In this study, PCB-binding DNA aptamers were developed using PCB72 and PCB106 as targets for aptamer selection. Aptamers are synthetic DNA recognition elements which form unique conformations that enable them to bind specifically to their targets. Using in vitro selection techniques and fluorometry, an aptamer that binds with nanomolar affinity to both the PCBs has been developed. It displayed high selectivity to the original target congeners and limited affinity toward other PCB congeners (105, 118, 153, and 169), suggesting general specificity for the basic PCB skeleton with varying affinities for different congeners. This aptamer provides a basis for constructing an affordable, sensitive, and high-throughput assay for the detection of PCBs in food and environmental samples and offers a promising alternative to existing methods of PCB quantitation. This study therefore advances aptamer technology by targeting one of the highly sought-after POPs, for the first time ever recorded.

cDNA phage display as a novel tool to screen for cellular targets of chemical compounds.

cDNA phage display is frequently used in drug development to screen for cellular target of drugs. However, in toxicology, cDNA phage display remains unexplored, although it has large potential in this field. In this study, cDNA phage display is demonstrated as a novel tool to screen for interactions between chemical compounds and cellular targets. The knowledge of these target interactions is valuable to have a more complete understanding of the mechanisms of action of chemical compounds. Bisphenol A (BPA) was selected as a model compound for this study. By selection of the cellular proteins that bind BPA with cDNA phage display, it was possible to identify a known cellular target of BPA, tubulin alpha and a possible novel cellular target of BPA, transforming acidic coiled-coil containing protein 3. Both these cellular proteins are involved in the mechanism of cell division. The disruption of cell division is a known non-genomic effect of BPA. Non-genomic effects are not mediated by differences in gene expression and therefore important mechanistic information might be missed with the widely used differential gene expression techniques for mode of action research. This cDNA phage display technique can provide important additional information about the interaction of chemical compounds with cellular targets that mediates these non-genomic actions and therefore gives complementary information to toxicogenomic studies to obtain a more complete understanding of the mechanism of action of chemical compounds.

The identification of cellular targets of 17β estradiol using a lytic (T7) cDNA phage display approach.

To unravel the mechanism of action of chemical compounds, it is crucial to know their cellular targets. A novel in vitro tool that can be used as a fast, simple and cost effective alternative is cDNA phage display. This tool is used in our study to select cellular targets of 17β estradiol (E2). It was possible to select two potential cellular targets of E2 out of the T7 Select™ Human Breast cDNA phage library. The selected cellular targets, autophagy/beclin-1 regulator 1 (beclin 1) and ATP synthase F(0) subunit 6 (ATP6) have so far been unknown as binding proteins of E2. To confirm the E2 binding properties of these selected proteins, surface plasmon resonance (SPR) was used. With SPR the K(d) values were determined to be 0.178±0.031 and 0.401±0.142 nM for the ATP6 phage and beclin 1 phage, respectively. These K(d) values in the low nM range verify that the selected cellular proteins are indeed binding proteins for E2. The selection and identification of these two potential cellular targets of E2, can enhance our current understanding of its mechanism of action. This illustrates the potential of lytic (T7) cDNA phage display in toxicology, to provide important information about cellular targets of chemical compounds.

Aptamer-Based Molecular Recognition of Lysergamine, Metergoline and Small Ergot Alkaloids

Ergot alkaloids are mycotoxins produced by fungi of the genus Claviceps, which infect cereal crops and grasses. The uptake of ergot alkaloid contaminated cereal products can be lethal to humans and animals. For food safety assessment, analytical techniques are currently used to determine the presence of ergot alkaloids in food and feed samples. However, the number of samples which can be analyzed is limited, due to the cost of the equipment and the need for skilled personnel. In order to compensate for the lack of rapid tests for the detection of ergot alkaloids, the aim of this study was to develop a specific recognition element for ergot alkaloids, which could be further applied to produce a colorimetric reaction in the presence of these toxins. As recognition elements, single-stranded DNA ligands were selected by using an iterative selection procedure named SELEX, i.e., Systematic Evolution of Ligands by EXponential enrichment. After several selection cycles, the resulting aptamers were cloned and sequenced. A surface plasmon resonance analysis enabled determination of the dissociation constants of the complexes of aptamers and lysergamine. Dissociation constants in the nanomolar range were obtained with three selected aptamers. One of the selected aptamers, having a dissociation constant of 44 nM, was linked to gold nanoparticles and it was possible to produce a colorimetric reaction in the presence of lysergamine. This system could also be applied to small ergot alkaloids in an ergot contaminated flour sample.

The Use of Phages and Aptamers as Alternatives to Antibodies in Medical and Food Diagnostics

Jaytry Mehta1,2, Bieke Van Dorst1,2, Lisa Devriese2 Elsa Rouah-Martin1,2, Karen Bekaert2, Klaartje Somers3, Veerle Somers3, Marie-Louise Scippo4, Ronny Blust1 and Johan Robbens1,2 1University of Antwerp, Department of Biology, Laboratory of Ecophysiology, Biochemistry and Toxicology, Groenenborgerlaan 171, 2020 Antwerp 2Institute for Agricultural and Fisheries research (ILVO), Ankerstraat 1, 8400 Oostende 3Hasselt University, Biomedical Research Institute, B-3590 Diepenbeek 4University of Liege, Food Sciences Department, B-4000 Liege Belgium

Selection of PCB binding phages as potential biorecognition elements for food and environmental monitoring

In order to assess and avoid risks for both human and environmental health, there is a high need for cost-effective and sensitive detection systems suitable for the monitoring of chemical contaminants in food and the environment. Due to their high sensitivity and selectivity, affinity-based detection systems are interesting for monitoring tools. Antibodies have long been the most popular affinity-based recognition elements, however, recently different novel affinity-based recognition elements with improved characteristics, like specificity, stability and cost-efficiency, have gained attention. In the present study, one type of such novel affinity-based recognition elements, namely landscape phages were evaluated as substitute antibodies for detection of PCB. These phages with affinity for PCBs were selected from a landscape phage library, in which each phage displays an 8-mer peptide at all 4000 major coat proteins pVIII. Landscape phages have potential as biorecognition elements, because of their excellent characteristics, such as high affinity and specificity, fast, cheap and animal-friendly manufacturing process with low batch-to-batch variations and superior stability, which allow them to operate as elements of detector systems in complex environmental and food matrices. Phages with a high specificity for PCB106 were isolated from the landscape phage library. Using surface plasmon resonance (SPR) it was shown that the selected phages bind specifically PCB106 and do not bind PCB72 and PCB118. The dissociation constants (Kd) of the phage binding with PCB106 were in μM range. After further improvement of their binding activity, the specific PCB-binding phages can be used as biorecognition elements for food and environmental monitoring.

Phage display as a method for discovering cellular targets of small molecules

Phage display can be used for the discovery of cellular targets of small molecules in order to unravel their mechanism of action, which is important in the drug discovery field to assess biological effects of drugs at the molecular level and to investigate pharmacokinetic characteristics of drugs in clinical use. The potential of phage display in the drug discovery field is shown by a lot of successful cellular target identifications of drug-like small molecules in the last decade. More recently, phage display was also introduced in environmental science to predict risks of small molecules, like nickel, 17β estradiol and bisphenol A on both environmental and human health, wherefore knowledge about the mechanism of action and cellular targets is essential. This paper discusses some important aspects of the phage display approach for the discovery of cellular targets of small molecules. The different phage display libraries and immobilization strategies used for the discovery of cellular target of small molecules are described. In general, the phage display approach is very useful in drug discovery and environmental science as a fast and cost-effective in vitro tool to determine cellular targets of small molecules, which increases our understanding of the mechanisms of action of small molecules.