Meral Yüce

Dual-excitation upconverting nanoparticle and quantum dot aptasensor for multiplexed food pathogen detection.

In this report, a dual-excitation sensing method was developed using aptamer-functionalized quantum dots and upconverting nanoparticles, exhibiting Stokes and anti-Stokes type excitation profiles, respectively. Conjugation of the aptamer-functionalized luminescent nanoparticles with the magnetic beads, comprising short DNA sequences that were partially complementary to the aptamer sequences, enabled facile separation of the analyte-free conjugates for fluorescent measurement. UV-Visible spectroscopy, Circular Dichroism spectroscopy, Dynamic Light Scattering and Polyacrylamide Gel Electrophoresis techniques were used to characterize the aptamer probes developed. The target-specific luminescent conjugates were applied for multiplex detection of model food pathogens, Salmonella typhimurium, and Staphylococcus aureus, in which the fluorescent emission spectra were obtained under UV excitation at 325nm for quantum dots and NIR excitation at 980nm for upconverting nanoparticles, respectively. The dual-excitation strategy was aimed to minimize cross-talk between the luminescent signals for multiplexed detection, and yielded limit of detection values of 16 and 28cfumL(-1) for Staphylococcus aureus, and Salmonella typhimurium, respectively. By employing a greater number of quantum dots and upconverting nanoparticles with non-overlapping fluorescent emissions, the proposed methodology might be exploited further to detect several analytes, simultaneously.

Employment of nanomaterials in polymerase chain reaction: insight into the impacts and putative operating mechanisms of nano-additives in PCR

The unique ability to rapidly amplify low copy number DNA has made in vitro Polymerase Chain Reaction one of the most fundamental techniques in modern biology. In order to harness this technique to its full potential, certain obstacles such as nonspecific by-products, low yield and complexity of GC rich and long genomic DNA amplification need to be surmounted. As in vitro PCR does not have any regulatory mechanisms unlike its counterpart in vivo DNA replication machinery, scientists often use a number of additives like glycerol, betaine, dimethyl sulphoxide and formamide in order to achieve the perfection of in vivo systems. In the last two decades nanotechnology has provided excellent solutions to many classical problems in various scientific fields including biotechnology and recently the PCR technique has begun to benefit from this so called “Nano Era”. In this review, the impacts of several nanomaterials on PCR efficiency, specificity and fidelity are described in accordance with the recent literature. Putative interaction mechanisms between nanomaterials and primary PCR components are also addressed in a comprehensive manner.

Molecular organization and comparative analysis of chromosome 5B of the wild wheat ancestor Triticum dicoccoides

Wild emmer wheat, Triticum turgidum ssp. dicoccoides is the wild relative of Triticum turgidum, the progenitor of durum and bread wheat, and maintains a rich allelic diversity among its wild populations. The lack of adequate genetic and genomic resources, however, restricts its exploitation in wheat improvement. Here, we report next-generation sequencing of the flow-sorted chromosome 5B of T. dicoccoides to shed light into its genome structure, function and organization by exploring the repetitive elements, protein-encoding genes and putative microRNA and tRNA coding sequences. Comparative analyses with its counterparts in modern and wild wheats suggest clues into the B-genome evolution. Syntenic relationships of chromosome 5B with the model grasses can facilitate further efforts for fine-mapping of traits of interest. Mapping of 5B sequences onto the root transcriptomes of two additional T. dicoccoides genotypes, with contrasting drought tolerances, revealed several thousands of single nucleotide polymorphisms, of which 584 shared polymorphisms on 228 transcripts were specific to the drought-tolerant genotype. To our knowledge, this study presents the largest genomics resource currently available for T. dicoccoides, which, we believe, will encourage the exploitation of its genetic and genomic potential for wheat improvement to meet the increasing demand to feed the world.

How to make nanobiosensors: surface modification and characterisation of nanomaterials for biosensing applications

This report aims to provide the audience with a guideline for construction and characterisation of nanobiosensors that are based on widely used affinity probes including antibodies and aptamers and nanomaterials such as carbon-based nanomaterials, plasmonic nanomaterials and luminescent nanomaterials. The affinity probes and major methodologies that have been extensively used to make nanobiosensors, such as thiol–metal interactions, avidin–biotin interaction, π-interactions and EDC–NHS chemistry, were described with the most recent examples from the literature. Characterisation techniques that have been practised to validate nanoparticle surface modification with antibodies and aptamers, including gel electrophoresis, ultraviolet-visible spectrophotometry, dynamic light scattering and circular dichroism were described with examples. This report mainly covers the reports published between 2014 and 2017.

Comparative metabolite profiling of drought stress in roots and leaves of seven Triticeae species

BackgroundDrought is a lifestyle disease. Plant metabolomics has been exercised for understanding the fine-tuning of the potential pathways to surmount the adverse effects of drought stress. A broad spectrum of morphological and metabolic responses from seven Triticeae species including wild types with different drought tolerance/susceptibility level was investigated under control and water scarcity conditions.ResultsSignificant morphological parameters measured were root length, surface area, average root diameter and overall root development. Principal Component Analysis, Partial Least-Squares-Discriminant Analysis and Hierarchical Cluster Analysis were applied to the metabolomic data obtained by Gas Chromatography-Mass Spectrometry technique in order to determine the important metabolites of the drought tolerance across seven different Triticeae species. The metabolites showing significant accumulation under the drought stress were considered as the key metabolites and correlated with potential biochemical pathways, enzymes or gene locations for a better understanding of the tolerance mechanisms. In all tested species, 45 significantly active metabolites with possible roles in drought stress were identified. Twenty-one metabolites out of forty-five including sugars, amino acids, organic acids and low molecular weight compounds increased in both leaf and root samples of TR39477, IG132864 and Bolal under the drought stress, contrasting to TTD-22, Tosunbey, Ligustica and Meyeri samples. Three metabolites including succinate, aspartate and trehalose were selected for further genome analysis due to their increased levels in TR39477, IG132864, and Bolal upon drought stress treatment as well as their significant role in energy producing biochemical pathways.ConclusionThese results demonstrated that the genotypes with high drought tolerance skills, especially wild emmer wheat, have a great potential to be a genetic model system for experiments aiming to validate metabolomics–genomics networks.

Characterization of a dual biotin tag for improved single stranded dna production

Generation of single-stranded DNA plays a key role in many biotechnology applications including production of aptamers, single strand conformation polymorphism, nuclease S1 mapping, pyrosequencing, genosensors, probe preparation and labelling, subtractive hybridization as well as nucleic acid sensing and microarrays. Several methods are available in the literature to produce single-stranded DNA from double-stranded DNA templates, such as extraction of the sense strand from denaturing gels, asymmetric PCR, use of streptavidin–biotin interaction, and some alternative methods, including enzymatic digestion of the negative strand by either lambda exonuclease or T7 Gene 6 exonuclease. In this report, a detailed characterization of a dual biotin tag method to generate single-stranded DNA from the random oligonucleotide library is presented. Unlike the traditional streptavidin–biotin method that uses single biotin tagged molecules during separation, this novel technique is based on a dual biotin molecule covalently attached to the 5′ end of the negative strand. The improved technique takes less than one hour as a consequence of the eliminated alkali treatment step, which makes this procedure the shortest procedure described in the literature so far for single-stranded DNA production. The method can achieve a single-stranded DNA yield around 75% from the corresponding DNA template in Tris–HCl buffer. A number of parameters, such as the effect of different elution buffers and heat treatments, spontaneous release of streptavidin from the magnetic bead surface, loss of beads during consecutive washes, aggregation of the beads, were investigated to reveal the optimal conditions for single-stranded DNA production. FTIR, DLS, SEM, and electrophoresis techniques were used for characterization studies.

Bioconjugated nanomaterials for monitoring food contamination

Abstract Maintaining food safety and hygiene standards is top priority and challenge for farmers, food industries, governments, and food technologists working in the food supply chain. Pesticides, toxins, veterinary drug residues, foodborne pathogens, and many other harmful chemicals that may be present in a vast array of food products, due to various stages of their production such as packaging and transport, constitute a global health problem that requires powerful and innovative technologies allowing constant and accurate detection of food products from production to consumption. Recent progress in generation of specific synthetic oligonucleotides against food contaminants has provided a new insight into the current sensor technologies, where these functional synthetic oligonucleotides, so-called aptamers, have been successfully combined with nanomaterials for rapid and cost-effective detection of several substances related to the food contamination, such as antibiotics, mycotoxins, heavy metals, carcinogenic dyes, pesticides, pathogens, and other plastic products used for food packaging. Unique characteristics of aptamers over antibodies, such as in vitro selection, chemical and thermal stability, small size and ease of labeling have laid the solid foundation for exploring aptamers further in multiplexed food monitoring systems. In this chapter, we review the application of aptamer-conjugated nanomaterials in food safety surveillance as well as the conventional techniques used for food safety monitoring in order to provide a comprehensive and comparative approach.