Optimization of whole-cell bacterial bioreporter immobilization on electrospun cellulose acetate (CA) and polycaprolactone (PCL) fibers for arsenic detection.

Arsenic contamination is a critical global problem, and its widespread environmental detection is becoming a prominent issue. Herein, electrospun fibers of cellulose acetate (CA) and polycaprolactone (PCL) were successfully fabricated and used as the support material for immobilization of arsenic-sensing bacterial bioreporter for the first time. To date, no attempt has been made to immobilize fluorescent whole-cell bioreporter cells on electrospun fibers for arsenic detection. CA and PCL electrospun fibers were fabricated via traditional electrospinning technique and characterized by scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), and contact angle meter. Following immobilization of the bacterial bioreporter cells, the immobilized bacteria were also characterized by viability assay using AlamarBlue. The effects of growth phase and cell concentration on the fluorescence response of fiber-immobilized arsenic bioreporters to arsenic were also investigated. After immobilization of arsenic bioreporters on 10 wt% PCL fiber, 91% of bacterial cells remained viable, while this value was 55.4% for cells immobilized on 12.5 wt% CA fiber. Bioreporter cells in the exponential growth phase were shown to be more sensitive to arsenic compared to aged cells. While both the electropsun PCL- and CA-immobilized bioreporters successfully detected 50 and 100 µg/L of arsenite (As (III)) concentrations, the PCL-immobilized bioreporter showed better fluorescence performance which should be investigated in future studies. This study helps to fill some gaps in the literature and demonstrates the potential for using electrospun fiber-immobilized arsenic whole-cell bioreporter for arsenic detection in water.

Development of rapid dipstick assay for food pathogens, Salmonella, by optimized parameters.

Salmonella is among the very important pathogens threating the human and animal health. Rapid and easy detection of these pathogens is crucial. In this context, antibody (Ab) based lateral flow assays (LFAs) which are simple immunochromatographic point of care test kits were developed by gold nanoparticles (GNPs) as labelling agent for Salmonella detection. For that purpose some critical parameters such as reagent concentrations on the capture zones, conjugate concentrations and ideal membrane type needed for LFAs for whole cell detection were tested for naked eye analysis. Therefore, prepared LFAs were applied to the live and heat inactivated cells when they were used alone or included in different bacterial mixtures. Among the test platforms, membrane 180 (M180) was found as an ideal membrane and 36 nm GNPs showed highly good labelling in the developed LFAs. Diluted conjugates and low concentrations of reagents affected the test signal negatively. Salmonella was detected in different bacterial mixtures, selectively in 4-5 min. The best recognized species by used Ab were S. enteritidis and S. infantis. 5 × 105 S. typhimurium cells were also determined as a limit of detection of this study with mentioned parameters.

Eriochrome Black T-Eu3+ Complex as a Ratiometric Colorimetric and Fluorescent Probe for the Detection of Dipicolinic Acid, a Biomarker of Bacterial Spores.

A novel ratiometric colorimetric and fluorescent dual probe based on Eriochrome Black T (EBT)-Eu3+ complex was designed to detect dipicolinic acid (DPA), a major constituent of bacterial spores, with high sensitivity and selectivity. UV-vis titration experiments demonstrated that EBT and Eu3+ ions formed a 1:1 coordination pair in water. In the presence of Eu3+ ions, the blue solution of EBT changed to magenta, however, upon the addition of DPA, the magenta color changed to blue immediately and characteristic fluorescence emission from DPA-Eu3+ complex was observed. In addition, the sensitivity of the system was further evaluated on Geobacillus stearothermophilus spores and as low as 2.5 × 105 spores were detected.

Evaluation of Staphylococcus aureus DNA aptamer by enzyme-linked aptamer assay and isothermal titration calorimetry.

To monitor the specificity of Staphylococcus aureus aptamer (SA-31) against its target cell, we used enzyme-linked aptamer assay. In the presence of target cell, horseradish peroxidase-conjugated streptavidin bound to biotin-labeled SA-31 showed specific binding to S  aureus among 3 different bacteria with limit of detection of 103 colony-forming unit per milliliter. The apparent Ka was 1.39 µM-1  ± 0.3 µM-1 . The binding of SA-31 to membrane proteins extracted from cell surface was characterized using isothermal titration calorimetry, and the effect of changes in binding temperature and salt concentrations of binding buffer was evaluated based on thermodynamic parameters (Ka , ΔH, and ΔG). Since binding of aptamer to its targets solely depends on its 3-dimensional structure under experimental conditions used in selection process, the change in temperature and ion concentration changed the affinity of SA-31 to its target on surface of bacteria. At 4°C, SA-31 did not show an affinity to its target with poor heat change upon injection of membrane fraction to aptamer solution. However, the apparent association constants of SA-31 slightly varied from Ka  = 1.56 µM-1  ± 0.69 µM-1 at 25°C to Ka  = 1.03 µM-1  ± 0.9 µM-1 at 37°C. At spontaneously occurring exothermic binding reactions, affinities of S aureus aptamer to its target were also 9.44 µM-1  ± 0.38 µM-1 at 50mM, 1.60 µM-1  ± 0.11 µM-1 at 137mM, and 3.28 µM-1  ± 0.46 µM-1 at 200 mM of salt concentration. In this study, it was demonstrated that enzyme-linked aptamer assay and isothermal titration calorimetry were useful tools for studying the fundamental binding mechanism between a DNA aptamer and its target on the outer surface of S aureus.

Fluorescent silica nanoparticles as nano-chemosensors for the sequential detection of Pb2+ ions and bacterial-spore biomarker dipicolinic acid (DPA) in aqueous solution.

Herein, we report fluorescein-labelled silica nanoparticles (FSNP) which serve as fluorescent nano-chemosensors for sequential detection of Pb2+ (which is a toxic heavy metal) and dipicolinic acid (DPA) (which is a distinctive indicator biomarker of bacterial spores) with high sensitivity and selectivity. The fluorescence of FSNP is quenched because of the complex formation between Pb2+ ions and surface amide groups, however, the fluorescence is recovered in contact with DPA, resulting from the association of DPA with surface bound Pb2+ ions. FSNP-Pb2+ complexes show high sensitivity towards DPA with a low detection limit of 850 nM which is approximately seventy times lower than the infectious dosage of bacterial spores (60 µM). Lateral flow test platform was further developed to show the applicability and practicability of our system.

Designing of rapid assay for the detection of RdRp/Orf1ab specific to SARS-CoV-2.

SARS-CoV-2 is still threat and mostly used detection method is real time reverse transcriptase polymerase chain reaction (rRT-PCR) for the open reading frame (Orf1ab), RNA-dependent RNA polymerase (RdRp), nucleocapsid (N) and envelope (E) genes of virus. However, rRT-PCR may have false negative rate for the nucleic acid detection. Since the RdRp/Orf1ab has high sensitivity for the molecular detection, two sandwich models, Model 1A-Model 1B, based on hybridization on lateral flow assay (LFA) were designed here and applied with the synthetic and clinical samples of RdRp/Orf1ab. In this purpose colloidal gold nanoparticles (AuNPs) were used as label. Membranes having different flow rate, three oligonucleotide probe concentrations and running buffers were used. Although synthetic target sequence was recognized by all the LFAs, PCR products obtained from either the synthetic plasmid DNA or oro/nasopharyngeal swabs were detected by Model 1 A using W12 membrane. Designed strip assays detected the RdRp/Orf1ab of the clinical samples as 100% sensitivity and specifity. It means that they might be used for the detection of virus and can be modified for the recognition of mutant genes of virus. These findings also demonstrated the importance of membranes, sandwich models, probe concentrations and sample contents for developing LFAs for viral detection.

Immobilization of fluorescent bacterial bioreporter for arsenic detection.

Whole-cell bacterial biosensors hold great promise as a practical complementary approach for in-field detection of arsenic. Although there are various bacterial bioreporter systems for arsenic detection, fewer studies reported the immobilization of arsenic bioreporters. This study aimed at determining immobilization of specific bacterial bioreporter in agar and alginate biopolymers to measure level of arsenite and/or arsenate. To achieve sensitive detection, immobilization parameters of polymer concentration and cell density were evaluated. Moreover, by changing the culture medium, immobilized bioreporter cells in minimal medium can detect arsenite while they can detect both arsenite and arsenate in phosphate-limited minimal medium. When optimal parameters were applied, agar and alginate immobilized bioreporter systems can detect arsenite and arsenate concentrations of 10 µg/l and 200 µg/l within 5 h and 2 h, respectively. The results showed that the immobilized bacterial bioreporter systems are able to determine the concentrations of the two abundant species of arsenic; arsenite and arsenate, as opposed to other studies which reported only arsenite detection. This is the first study describe agar hydrogel and alginate bead immobilization of fluorescent arsenic bacterial bioreporter that can detect both arsenite and arsenate at the safe drinking water limit. Thus, this study will enable further steps to be taken towards developing sensitive and selective portable devices to assess environmental arsenic contamination and prevent acute arsenic toxicity.

A highly substituted and fluorescent aromatic-fused imidazole derivative that shows enhanced antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA).

A novel highly substituted and fluorescent aromatic-fused imidazole derivative has been synthesized by rational design. This novel fluorescent material acts as an alternative antibacterial agent against Gram positive bacteria strains. It shows superior antibacterial activity (with MIC value of 8 µg/mL) against methicillin-resistant Staphylococcus aureus (MRSA) when compared with standard antibiotic drugs Ampicillin (with MIC value of 128 µg/mL) and Kanamycin (with MIC value of >512 µg/mL). The interaction of this novel compound with the bacterial cell and genomic DNA has also been studied to elucidate antibacterial mode of action. Fluorescence spectroscopy and microscopy studies have proved the intracellular uptake of this special compound. Likewise, UV-vis and fluorescence spectroscopy studies have revealed a significant decrease in the absorption and emission bands of the compound upon its interaction with plasmid and genomic DNA, which is likely due to its DNA intercalation property. Furthermore, these findings have been supported by gel electrophoresis of genomic DNA of S. aureus cells treated with the compound. The results indicate that this novel compound exerts its antibacterial activity by causing DNA damage, suggesting the potential utility of fluorescent probes for real-time diagnosis and treatment of bacterial infections.

Ratiometric fluorescence detection of an anthrax biomarker with Eu3+-chelated chitosan biopolymers.

A novel chitosan-based ratiometric fluorescent probe incorporating an EDTA-Eu3+ complex as the sensing unit and fluorescein dye as the internal standard was designed to detect dipicolinic acid (DPA) as an anthrax biomarker with high sensitivity and selectivity. The fluorescence intensity of fluorescein dye attached to the chitosan backbone remains constant as an internal reference, while the Eu3+ emission increased linearly upon the consecutive addition of DPA. The selectivity studies were performed by adding different competitive aromatic ligands to the sensing environment and no signifacant fluorescence response was observed. The results demonstrated the superior selectivity of the system to DPA. Overall, this novel chitosan-based ratiometric fluorescent probe enables ratiometric and sensitive DPA detection over nanomolar concentrations (as low as 10nM) and displays straightforward selectivity over other competitive aromatic ligands.

Evaluation of heterotrophic and mixotrophic cultivation of novel Micractinium sp. ME05 on vinasse and its scale up for biodiesel production.

Direct disposal of vinasse, a by-product of molasses fermentation plants, threatens environmental health. This study investigated the usage of vinasse as a nutrient source for the heterotrophic and mixotrophic cultivation of novel Micractinium sp. ME05. The 500-mL flask experiments resulted in higher biomass productivities under mixotrophic conditions (0.16 ±â€¯0.01 g L-1 day1) than the heterotrophic conditions (0.13 ±â€¯0.01 g L-1 day1). A 1.7-fold increase in biomass productivity was achieved by scaling up from 500-mL flasks (0.16 ±â€¯0.01 g L-1 day1) to 2-L flasks (0.27 ±â€¯0.019 g L-1 day1). The 5-L bioreactor experiments resulted in a biomass productivity of 0.32 ±â€¯0.2 g L-1 day1 and lipid productivity of 3.4 ±â€¯0.20 g L-1 day-1. This study demonstrated that Micractinium sp. ME05 can be cultivated with vinasse to produce large amounts of biomass. The FAME profile of mixotrophic Micractinium sp. ME05 cells was promising for further biodiesel production. This study highlights the feasibility of industrial by- product-vinasse as the nutrient source for biomass and lipid productions using the novel Micractinium sp. ME05 cells.

Target-specific delivery of doxorubicin to human glioblastoma cell line via ssDNA aptamer.

Targeted drug delivery approaches have been implementing significant therapeutic gain for cancer treatment since last decades. Aptamers are one of the mostly used and highly selective targeting agents for cancer cells. Herein, we address a nano-sized targeted drug delivery approach adorned with A-172 glioblastoma cell-line-specific single stranded DNA (ssDNA) aptamer in which the chemotherapeutic agent Doxorubicin (DOX) had been conjugated. DNA aptamer, GMT-3, was previously selected for specific recognition of glioblastoma and represented many advantageous characteristics for drug targeting purposes. Flow cytometry analysis proved the binding efficiency of the specific aptamer to tumour cell lines. Celltype- specific toxicity of GMT-3:DOX complex was showed by XTT assay and terminated cytotoxic effects were screened for both target cell and a control breast cancer cell line. The result of this contribution demonstrated the potential utility of GMT-3 aptamer-mediated therapeutic drug transportation in the treatment of gliomas specifically. It was concluded that aptamer-mediated drug delivery can be applied successfully for clinical use.

Optimizations needed for lateral flow assay for rapid detection of pathogenic E. coli.

Lateral flow assay (LFA), or the immunochromatographic strip test, is popular to use for rapid and sensitive immunoassays. Gold nanoparticles (GNPs), due to tunable optical characteristics and easy manipulation of size or shape, represent an attractive approach for LFA technology. Since most enterohemorrhagic infections result from water and food contaminations of Escherichia coli O157:H7, selective and rapid detection of this organism in environmental and biological complexes is necessary. In this study, optimized parameters of antibody (Ab)-based LFA for rapid detection of pathogenic E. coli O157:H7 are described. GNPs were used as visualizing agents. The measuring parameters include the Ab concentration on the capture lines, the concentration of gold conjugate, and flow rate. M180 and 36 nm were the ideal membrane and GNP size, respectively, for bacterial detection of LFA. The target, E. coli O157:H7, could be detected with a visual limit of detection of 105 cfu/mL in 3-5 min. Selectivity of the system was very high and the target was recognized by developed strips, regardless of its presence singly or in mixed bacterial samples.

Evaluation of novel thermo-resistant Micractinium and Scenedesmus sp. for efficient biomass and lipid production under different temperature and nutrient regimes.

Despite the vast interest in microalgae as feedstock for biodiesel production, relatively few studies examined their response to diurnal temperature fluctuation. Here, we describe biomass and lipid productivities and fatty acid profiles of thermo-resistant Micractinium sp. and Scenedesmus sp. grown in batch cultures in a laboratory set-up that mimics a typically warm summer day in Central Anatolia with a 16-h light temperature of 30°C and 8-h dark temperature of 16°C (30°C (day)/16°C (night)). Both strains can survive a temperature range of 10-50°C. We found the lipid productivities of Micractinium sp. and Scenedesmus sp. as 30/21mgL(-1)d(-1) and 6/7mgL(-1)d(-1), respectively during the 30°C (day)/16°C (night) cycle. Saturated fatty acid content increased with increasing temperature. Additionally, we cultured Micractinium sp. under Nitrogen (N) and Phosphorus (P) limiting conditions. Highest lipid productivity of 85.4±2mgL(-1)d(-1) was obtained under P-depletion during exponential growth phase. Oleic acid amount also increased eight fold during P-deplete.

Lentil (Lens culinaris Medik).

This chapter describes an efficient Agrobacterium-mediated genetic transformation of lentil by use of cotyledonary node explants, an optimized wounding method, and vacuum infiltration. Transformation protocol was followed by direct regeneration of transgenic shoots and micrografting of the shoots on root stocks to obtain whole-plant regeneration. The most efficient transgene expression on the axil region was obtained when the Agrobacterium KYRT1 strain was used. Gradually increasing selection pressure and repeated removal of regenerated shoots between selection steps increased the number of transgene-expressing shoots greatly. This protocol allowed 2.3 % transformation efficiency and stable transgene expression and transmission which were tracked through three generations.

Thermo-resistant green microalgae for effective biodiesel production: isolation and characterization of unialgal species from geothermal flora of Central Anatolia.

Oil content and composition, biomass productivity and adaptability to different growth conditions are important parameters in selecting a suitable microalgal strain for biodiesel production. Here, we describe isolation and characterization of three green microalgal species from geothermal flora of Central Anatolia. All three isolates, namely, Scenedesmus sp. METUNERGY1402 (Scenedesmus sp. ME02), Hindakia tetrachotoma METUNERGY1403 (H. tetrachotoma ME03) and Micractinium sp. METUNERGY1405 (Micractinium sp. ME05) are adaptable to growth at a wide temperature range (25-50 °C). Micractinium sp. ME05, particularly has superior properties for biodiesel production. Biomass productivity, lipid content and lipid productivity of this isolate are 0.17 g L(-1) d(-1), 22.7% and 0.04 g L(-1) d(-1), respectively. In addition, Micractinium sp. ME05 and Scenedesmus sp. ME03 mainly contain desirable fatty acid methyl esters (i.e. 16:0, 16:1, 18:0 and 18:1) for biodiesel production. All isolates can further be improved via genetic and metabolic engineering strategies.

Portable bioactive paper-based sensor for quantification of pesticides.

A paper-based biosensor was developed for the detection of the degradation products of organophosphorus pesticides. The biosensor quantifies acetylcholine esterase inhibitors in a fast, disposable, cheap, and accurate format. We specifically focused on the use of sugar or protein stabilizer to achieve a biosensor with long shelf-life. The new biosensor detected malathion with a detection limit of 2.5 ppm in 5 min incubation time. The operational stability was confirmed by testing 60 days storage at 4°C when glucose was used as stabilizer.

Antimicrobial aptamers for detection and inhibition of microbial pathogen growth.

Discovery of alternative sources of antimicrobial agents are essential in the ongoing battle against microbial pathogens. Legislative and scientific challenges considerably hinder the discovery and use of new antimicrobial drugs, and new approaches are in urgent demand. On the other hand, rapid, specific and sensitive detection of airborne pathogens is becoming increasingly critical for public health. In this respect affinity oligonucleotides, aptamers, provide unique opportunities for the development of nanotechnological solutions for such medical applications. In recent years, aptamers specifically recognizing microbial cells and viruses showed great potential in a range of analytical and therapeutic applications. This article describes the significant advances in the development of aptamers targeting specific pathogens. Therapeutic application of aptamers as neutralizing agents demonstrates great potential as a future source of antimicrobial agent.

In vitro Selection of DNA Aptamers to Glioblastoma Multiforme.

Aptamer probes for specific recognition of glioblastoma multiforme were generated using a repetitive and broad cell-SELEX-based procedure without negative selection. The 454 sequencing technology was used to monitor SELEX, and bioinformatics tools were used to identify aptamers from high throughput data. A group of aptamers were generated that can bind to target cells specifically with dissociation constants (K(d)) in the nanomolar range. Selected aptamers showed high affinity to different types of glioblastoma cell lines, while showing little or no affinity to other cancer cell lines. The aptamers generated in this study have potential use in different applications, such as probes for diagnosis and devices for targeted drug delivery, as well as tools for molecular marker discovery for glioblastomas.

Label-free detection of telomerase activity using guanine electrochemical oxidation signal.

Telomerase is an important biomarker for cancer cells and its activation in 85% of all cancer types confers a clinical diagnostic value. A label-free electrochemical assay based on guanine oxidation signal to measure telomerase activity is described. This developed technology combined with a disposable sensor, carbon graphite electrode (CGE), and differential pulse voltammetry (DPV) was performed by using PCR amplicons with/without telomeric repeats as the guanine oxidation signal observed at +1.0 V measured after the immobilization of PCR products. Guanine oxidation signal was chosen as a measure of telomerase activity because a substantial increase in the number of guanines was introduced by the action of telomerase which adds hexameric repeats (TTAGGG)n that contain 50% guanine. The developed assay was shown to specifically measure telomerase activity from cell extracts, and elongation rates increased linearly in a concentration dependent manner. Telomerase activity could be detected in cell extracts containing as low as 100 ng/microL of protein. All of the electrochemical measurements were also confirmed with the conventional TRAP-silver staining assay. Rapidity, simplicity, and the label-free nature of the developed assay make it suitable for practical use in quantitative determination of telomerase activity from clinical samples for diagnosis of cancer.

Single-step purification of recombinant Thermus aquaticus DNA polymerase using DNA-aptamer immobilized novel affinity magnetic beads.

A DNA aptamer specific for Thermus aquaticus DNA polymerase (Taq-polymerase) was immobilized on magnetic beads, which were prepared in the presented study. The effect of various parameters including pH, temperaturem and aptamer concentration on the immobilization of 5'-thiol labeled DNA-aptamer onto glutaric dialdhyde activated magnetic beads was evaluated. The binding conditions of Taq-polymerase on the aptamer immobilized magnetic beads were studied using commercial Taq-polymerase to characterize the surface complexation reaction. Efficiency of affinity magnetic beads in the purification of recombinant Taq-polymerase from crude extracts was also evaluated. For this case, the enzyme "recombinant Taq-DNA polymerase" was cloned and expressed using an Amersham E. coli GST-Gene Fusion Expression system. Crude extracts were in contact with affinity magnetic beads for 30 min and were collected by magnetic field application. The purity of the eluted Tag-polymerase from the affinity beads, as determined by HPLC, was 93% with a recovery of 89% in a one-step purification protocol. Apparently, the system was found highly effective as one step for the low-cost purification of Taq-polymerase in bacterial crude extract.