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Selection of Highly Specific Aptamers by Graphene Oxide-SELEX to Ultrasensitive Label-Free Impedimetric Biosensor Development for Glyphosate Detection
... The GO-SELEX is a distortion-free selection approach for aptamers incapacitating the limitations of immobilization-based SELEX methods. [19,20] Aptamer-target interaction and its detection at nanomolar scales with minimum signal amplification are prerequisites for the use in a biosensor at the grassroots level. Quartz crystal microbalance (QCM) has been employed for sensitive and labelfree detection of different analytes at low nanomolar scales. ...
... Further, counter-selection was carried out in SELEX round 10, and a dip in percentage recovery is expected in this round as many non-specific sequences are removed [16] [19] . The different counter ions used in the study eliminated any cross-reacting aptamer sequence. ...
Heavy metal arsenic is a water pollutant that affects millions of lives worldwide. A novel aptamer candidate for specific and sensitive arsenic detection was identified using Graphene Oxide‐SELEX (GO‐SELEX). Eleven rounds of GO‐SELEX were performed to screen As(III) specific sequences. The selected aptamer sequences were evaluated for their binding affinity. The dissociation constant of the best aptamer candidate, As‐06 was estimated by fluorescence recovery upon target addition, and it was found to be 8.15 nM. A QCM‐based biosensing platform was designed based on the target‐triggered release of aptamer from the QCM electrode. An rGO‐SWCNT nanocomposite was adsorbed on the gold surface, and the single‐stranded probe was stacked on the rGO‐CNT layer. Upon addition of the target to the solution, a concentration‐dependent release of the ssDNA probe was observed and recorded as the change in the electrode frequency. The developed QCM sensor showed a dynamic linear range from 10 nM to 100 nM and a low detection limit of 8.6 nM. The sensor exhibited excellent selectivity when challenged with common interfering anions and cations.
... Table SIX represents recently developed electrochemical (bio) sensors for the detection of GLY. 37,39,[43][44][45][46][47][48][49][50][51][52][53][54][55] Although low detection limits were reached by using these reported (bio)sensors, most of them required to apply exhausted experimental steps that included the use of intensive chemical agents or heat applications. The molecularly imprinted polymers (MIPs) based electrochemical sensor platforms [44][45][46] were fabricated by following the experimental steps including synthesizing monomer and GLY-specific polymers, modification of these polymers at selected electrode surfaces, the interaction process and the electrochemical measurements. ...
The development of monitoring tools for the detection of glyphosate (GLY) is an attractive topic since its consumption is one of the debated worldwide issue. Herein, a bionanocomposite modified disposable electrochemical biosensor platform was developed for sensitive and selective detection of GLY. For this purpose, carboxymethyl cellulose (CMC) and hydroxyapatite nanoparticles (HaNP) were modified at the surface of disposable pencil graphite electrodes (PGEs). After the fabrication of CMC/HaNP-PGEs, GLY specific DNA aptamer (DNA APT) that had G-quadruplex structure was immobilized at the surface of CMC/HaNP-PGEs. The specific interaction between DNA APT and GLY was performed at the electrode surface. Before/after each modification/immobilization/interaction step, anodic current (Ia) value obtained by cyclic voltammetry (CV) measurements performed in 2.00 mM K3[Fe(CN)6]/K4[Fe(CN)6] (1:1) and 0.10 M KCl was recorded, and the changes at the average Ia values were evaluated in terms of the optimization of experimental parameters. The LOD and LOQ values were calculated as 0.04 and 0.13 µg/mL, respectively. The selectivity of the developed aptasensor was tested against 2,4-dichlorophenoxyacetic acid (2,4-D), glufosinate (GFS), and (aminomethyl) phosphonic acid (AMPA). The applicability of the aptasensor was shown using water samples. This novel aptasensor platform is a prototype for future hand-held devices developed for GLY monitoring.
... However, it is a well-known fact that GO is a universal substance that can be used not only for producing aptamers but also for developing biosensors based on aptamers because GO has various characteristics including high mechanical strength and easy fabrication [14]. Moreover, the fact that GO can elevate its electrical conductivity through a reduction process and has a fluorescence-quenching effect also enables GO to be utilized in various types of biosensors with aptamers, including electrical and optical sensors [15][16][17]. To be specific, among the various aptamer-based GO-sensor studies, the most actively studied field is a sensor targeting thrombin [18][19][20][21]. ...
Nucleic acid aptamer-based research has focused on achieving the highest performance for bioassays. However, there are limitations in evaluating the affinity for the target analytes in these nucleic acid aptamer-based bioassays. In this study, we mainly propose graphene oxide (GO)-based electrical and optical analyses to efficiently evaluate the affinity between an aptamer and its target. We found that an aptamer-coupled GO-based chip with an electrical resistance induced by a field-effect transistor, with aptamers as low as 100 pM, can detect the target, thrombin, at yields as low as 250 pM within five minutes. In the optical approach, the fluorescent dye-linked aptamer, as low as 100 nM, was efficiently used with GO, enabling the sensitive detection of thrombin at yields as low as 5 nM. The cantilever type of mechanical analysis also demonstrated the intuitive aptamer–thrombin reaction in the signal using dBm units. Finally, a comparison of electrical and optical sensors’ characteristics was introduced in the attachment and detachment of aptamer to propose an efficient analysis that can be utilized for various aptamer-based research fields.
Since humans first started farming, agriculture has developed quickly and has significantly impacted civilization. The ability to feed a growing population and the wonderful benefits of this revolution in food production also brought to consumers today unknown concerns associated with food borne illness. As a result, regulatory bodies responsible for food safety face challenges not just responding to existing problems but also articulating an idea of food safety that accounts for potential dangers in the future.Food is a fundamental requirement for our existence, growth, survival, and preserving healthy bodily functions. Both producers and consumers are looking for new food sources with high nutritional value because of increasing food demand. Food products may never be 100% safe. Additionally, ensuring the complete safety of a food or ingredient is impossible. However, the dangers associated with any food can be kept to an absolute minimum by taking the necessary safeguards throughout the development, fabrication into goods, processing, final preparation, and distribution. For every conceivable variable or contaminant, it is impossible to test 100% of all foods. Additionally, the food producer has little influence over consumer overcompensation or nutritional misuse. This chapter aims to focus on recent advances in innovative analytical tools with special reference to the assessment of food safety. These tools leverage cutting-edge technologies, such as artificial intelligence, blockchain, sensor systems, and rapid diagnostic techniques, to enhance the accuracy, speed, and reliability of food safety assessments. By harnessing these tools, food producers, regulatory authorities, and consumers can make more informed decisions, mitigate risks, and safeguard public health.
The existence of multiple pesticide residues in fruits and vegetables constitutes a direct peril to living organisms. Therefore, it is crucial to develop a low-cost screening method for determining organophosphate pesticides (OPPs) in food samples. This study describes the solvothermal synthesis of a ternary composite comprising multi-walled carbon nanotubes (MWCNT), zirconium oxide, and a zirconium-metal-organic framework (Zr-MOF). The ternary composite was characterised using XRD, FESEM, FTIR, and BET. The ternary composite provides a large surface area (1158 m2/g) compared with the pristine Zr-MOF (868 m2/g). The composite-modified glassy carbon electrode was used to determine nine pesticides, including organophosphate (malathion, dimethoate, chlorpyrifos, monocrotophos, and glyphosate) and non-organophosphate (thiophanate methyl, carbendazim, atrazine, and 2,4, D). In particular, various chemical combinations of OPPs were selected, such as S-P=S, P=S, P=O, and non-OPPs such as C=S (with sulphur), and without sulphur. The sensor results show that the sensor selectivity is high for OPPs containing both phosphorus and sulphur molecules. The low detection limit of the sensor was 2.02, 2.8, 2.5, 1.11, and 4.21 nM for malathion, chlorpyrifos, dimethoate, monocrotophos, and glyphosate, respectively. The electrode exhibited significant chemical stability (93%) after 100 cycles, good repeatability, and a long shelf life. The sensor is reliable for qualitative real-time applications.
Investigating the interactions between small, charged molecules and aptamers using surface plasmon resonance (SPR) is limited by the inherent low response of small molecules and difficulties with nonspecific electrostatic interactions between the aptamer, analyte, and sensor surface. However, aptamers are increasingly being used in sensors for small molecule detection in critical areas like healthcare and environmental safety. The ability to probe these interactions through simple, direct SPR assays would be greatly beneficial and allow for the development of improved sensors without the need for complicated signal enhancement. However, these assays are nearly nonexistent in the current literature and are instead surpassed by sandwich or competitive binding techniques, which require additional sample preparation and reagents. In this work, we develop a method to characterize the interaction between the charged small molecule serotonin (176 Da) and an aptamer with SPR using streptavidin-biotin capture and a high-ionic-strength buffer. Additionally, other methods, such as serotonin immobilization and thiol-coupling of the aptamer, were investigated for comparison. These techniques give insight into working with small molecules and allow for quickly adapting a binding affinity assay into a direct SPR sensor.
An aptamer-based electrochemical biosensor was designed and fabricated for methamphetamine (METH) analysis. Several aptamer sequences developed by the GO-SELEX method were immobilized onto gold electrodes (GE) via gold-thiol affinity. Surface characterizations were performed by electrochemical methods including cyclic voltammetry (CV), differential pulse voltammetry (DPV), and electrochemical impedance spectrometry (EIS). GE/Apta-4/METH aptasensor was selected for further studies since the best results were obtained with the Apta-4 sequence in terms of analytical parameters. Accordingly, it exhibited good selectivity for METH analysis with a wide linear range of 0.1-50 ng/mL and a low limit of detection (LOD) which was found to be 0.467 ng/mL. The proposed aptasensor platform displayed good selectivity for the detection of METH in synthetic urine samples spiked with METH.
Systematic evolution of ligands by exponential enrichment (SELEX) is a method that is generally used for developing aptamers, which have arisen the promising alternatives for antibodies. However, conventional SELEX methods have limitations, such as a limited selection of target molecules, time-consuming and complex fabrication processes, and labor-intensive processes, which result in low selection yields. Here, we used (i) graphene oxide (GO)-coated magnetic nanoparticles in the selection process for separation and label-free detection and (ii) a multilayered microfluidic device manufactured using a three-dimensionally printed mold that is equipped with automated control valves to achieve precise fluid flows. The developed on-chip aptamer selection device and GO-coated magnetic nanoparticles were used to screen aptamer candidates for adenosine in eight cycles of the selection process within approximately 2 h for each cycle. Based on results from isothermal titration calorimetry, an aptamer with a dissociation constant of 18.6 ± 1.5 μM was selected. Therefore, the on-chip platform based on GO-coated magnetic nanoparticles provides a novel label-free screening technology for biosensors and micro/nanobiotechnology for achieving high-quality aptamers.
A stable and magnetic graphene oxide (GO) foam-polyethyleneimine-iron nanoparticle (GO-PEI-FeNPs) composite has been fabricated for removal of endocrine disruptors-bisphenol A, progesterone and norethisterone-from aqueous solution. The foam with porous and hierarchical structures was synthesized by reduction of graphene oxide layers coupled with co-precipitation of iron under a hydrothermal system using polyethyleneimine as a cross linker. The presence of magnetic iron nanoparticles facilitates the separation process after decontamination. The foam was fully characterized by surface and structural scanning electron microscopy, Fourier transform infrared spectroscopy, Raman spectroscopy and X-ray photoelectron spectroscopy. The foam exhibits a high adsorption capacity, and the maximum adsorption percentages are 68%, 49% and 80% for bisphenol A, progesterone and norethisterone, respectively. The adsorption process of bisphenol A is explained according to the Langmuir model, whereas the Freundlich model was used for progesterone and norethisterone adsorption.
Three-dimensional (3D) reduced graphene oxide (rGO) modified by polyethyleneimine (PEI) was prepared and functionalized by fluorophore-labeled dexamethasone-aptamer (Flu-DEX-apt) via π-π stacking interaction. The rGO/PEI/Flu-DEX-apt was used as a selective membrane for dexamethasone hormone removal from water. The prepared rGO/PEI/Flu-DEX-apt membranes were stable, insoluble, and easily removable from liquid media. The membrane was characterized by Raman spectroscopy, scanning electron spectroscopy, and FTIR spectroscopy. The rGO/PEI/Flu-DEX-apt membrane showed high sensitivity and specificity toward the dexamethasone hormone in the presence of other steroid hormone analogs, such as progesterone, estrone, estradiol, and 19-norethindrone. The fluorescence and UV-visible spectroscopy were used to confirm the membranes performance and the quantification of hormones removal. The resulting data clearly show that the graphene oxide concentration influence the aptamers and analytes interaction (π-π stacking interaction). It was found that by varying the graphene oxide concentration yields to different porosities of rGO/PEI/Flu-DEX-apt membranes affects the adsorption recovery rate, as well as the specificity and selectivity toward the dexamethasone hormone.
Headed for developing minimalistic strategies to produce graphene@metal hybrids for electronics on a larger scale, we discovered that graphene oxide (GO)-metal oxide (MO) hybrids are formed spontaneously in water at room temperature in the presence of nothing else than graphene oxide itself and metal ions. Our observations show metal oxide nanoparticles decorating the surface of graphene oxide with particle diameter in the range of 10–40 nm after only 1 h of mixing. Their load ranged from 0.2% to 6.3% depending on the nature of the selected metal. To show the generality of the reactivity of GO with different ions in standard conditions, we prepared common hybrids with GO and tin, iron, zinc, aluminum and magnesium. By means of carbon-13 solid-state nuclear magnetic resonance using magic angle spinning, we have found that graphene oxide is also moderately reduced at the same time. Our method is powerful and unique because it avoids the use of chemicals and heat to promote the coprecipitation and the reduction of GO. This advantage allows synthesizing GO@MO hybrids with higher structural integrity and purity with a tunable level of oxidization, in a faster and greener way.
Farmland biodiversity is an important characteristic when assessing sustainability of agricultural practices and is of major international concern. Scientific data indicate that agricultural intensification and pesticide use are among the main drivers of biodiversity loss. The analysed data and experiences do not support statements that herbicide-resistant crops provide consistently better yields than conventional crops or reduce herbicide amounts. They rather show that the adoption of herbicide-resistant crops impacts agronomy, agricultural practice, and weed management and contributes to biodiversity loss in several ways: (i) many studies show that glyphosate-based herbicides, which were commonly regarded as less harmful, are toxic to a range of aquatic organisms and adversely affect the soil and intestinal microflora and plant disease resistance; the increased use of 2,4-D or dicamba, linked to new herbicide-resistant crops, causes special concerns. (ii) The adoption of herbicide-resistant crops has reduced crop rotation and favoured weed management that is solely based on the use of herbicides. (iii) Continuous herbicide resistance cropping and the intensive use of glyphosate over the last 20 years have led to the appearance of at least 34 glyphosate-resistant weed species worldwide. Although recommended for many years, farmers did not counter resistance development in weeds by integrated weed management, but continued to rely on herbicides as sole measure. Despite occurrence of widespread resistance in weeds to other herbicides, industry rather develops transgenic crops with additional herbicide resistance genes. (iv) Agricultural management based on broad-spectrum herbicides as in herbicide-resistant crops further decreases diversity and abundance of wild plants and impacts arthropod fauna and other farmland animals. Taken together, adverse impacts of herbicide-resistant crops on biodiversity, when widely adopted, should be expected and are indeed very hard to avoid. For that reason, and in order to comply with international agreements to protect and enhance biodiversity, agriculture needs to focus on practices that are more environmentally friendly, including an overall reduction in pesticide use. (Pesticides are used for agricultural as well non-agricultural purposes. Most commonly they are used as plant protection products and regarded as a synonym for it and so also in this text.)
A high specificity aptamer-ligand biorecognition and binding system to monitor of dexamethasone (DXN) was developed. The detection principle was based on a label-free electrochemical aptasensor. The selection of the aptamer was successfully performed by the systematic evolution of ligands through exponential enrichment technique (SELEX). From a random library of 1.08 × 1015 single-stranded DNA, an aptamer designated as DEX04 showed a highest affinity with a dissociation constant of 18.35 nM. It also showed a good conformational change when binding with DXN. In addition, the aptamer DEX04 did not show any cross-reactivity with other commonly used hormones. An impedimetric aptasensor for DXN was then developed by immobilizing DEX04 on a gold electrode. The binding upon to DXN was monitored by following the change in the charge transfer resistance (Rct) of the [Fe(CN)6]4−/3− redox couple. The aptasensor exhibited a linear range from 2.5 to 100 nM with a detection limit of 2.12 nM. When applied aptasensor to test in water samples, it showed good recovery percentages. The new DXN aptamer can be employed in other biosensing applications for food control and the diagnosis of some diseases in medicine as a cost-effective, sensitive and rapid detection method.
Glyphosate-based herbicides are the dominant products used internationally for control of vegetation in planted forests. Few international, scientific syntheses on glyphosate, specific to its use in planted forests, are publically available. We provide an international overview of the current use of glyphosate-based herbicides in planted forests and the associated risks. Glyphosate is used infrequently in planted forests and at rates not exceeding 4 kg ha1. It is used within legal label recommendations and applied by trained applicators. While the highest risk of human exposure to glyphosate is during manual operational application, when applied according to label recommendations the risk of exposure to levels that exceed accepted toxicity standards is low. A review of the literature on the direct and indirect risks of operationally applied glyphosate-based herbicides indicated no significant adverse effects to terrestrial and aquatic fauna. While additional research in some areas is required, such as the use of glyphosate-based products in forests outside of North America, and the potential indirect effects of glyphosate stored in sediments, most of the priority questions have been addressed by scientific investigations. Based on the extensive available scientific evidence we conclude that glyphosate-based herbicides, as typically employed in planted forest management, do not pose a significant risk to humans and the terrestrial and aquatic environments.
Glyphosate is one of the most widely used herbicides in the world. Its safety for both human health and aquatic biomes is a subject of wide debate. There are limits to glyphosate’s presence in bodies of water, and it is usually detected through complex analytical procedures. In this work, the presence of glyphosate is detected directly through optical interrogation of aqueous solution. For this purpose, silver nanoparticles were produced by pulsed laser ablation in liquids. Limits of detection of 0.9 mg/L and 3.2 mg/L were obtained with UV-Vis extinction and Surface Enhanced Raman spectroscopies, respectively. The sensing mechanism was evaluated in the presence of potential interferents as well as with commercial glyphosate-based herbicides.
The broad-spectrum herbicide glyphosate (common trade name "Roundup") was first sold to farmers in 1974. Since the late 1970s, the volume of glyphosate-based herbicides (GBHs) applied has increased approximately 100-fold. Further increases in the volume applied are likely due to more and higher rates of application in response to the widespread emergence of glyphosate-resistant weeds and new, pre-harvest, dessicant use patterns. GBHs were developed to replace or reduce reliance on herbicides causing well-documented problems associated with drift and crop damage, slipping efficacy, and human health risks. Initial industry toxicity testing suggested that GBHs posed relatively low risks to non-target species, including mammals, leading regulatory authorities worldwide to set high acceptable exposure limits. To accommodate changes in GBH use patterns associated with genetically engineered, herbicide-tolerant crops, regulators have dramatically increased tolerance levels in maize, oilseed (soybeans and canola), and alfalfa crops and related livestock feeds. Animal and epidemiology studies published in the last decade, however, point to the need for a fresh look at glyphosate toxicity. Furthermore, the World Health Organization's International Agency for Research on Cancer recently concluded that glyphosate is "probably carcinogenic to humans." In response to changing GBH use patterns and advances in scientific understanding of their potential hazards, we have produced a Statement of Concern drawing on emerging science relevant to the safety of GBHs. Our Statement of Concern considers current published literature describing GBH uses, mechanisms of action, toxicity in laboratory animals, and epidemiological studies. It also examines the derivation of current human safety standards. We conclude that: (1) GBHs are the most heavily applied herbicide in the world and usage continues to rise; (2) Worldwide, GBHs often contaminate drinking water sources, precipitation, and air, especially in agricultural regions; (3) The half-life of glyphosate in water and soil is longer than previously recognized; (4) Glyphosate and its metabolites are widely present in the global soybean supply; (5) Human exposures to GBHs are rising; (6) Glyphosate is now authoritatively classified as a probable human carcinogen; (7) Regulatory estimates of tolerable daily intakes for glyphosate in the United States and European Union are based on outdated science. We offer a series of recommendations related to the need for new investments in epidemiological studies, biomonitoring, and toxicology studies that draw on the principles of endocrinology to determine whether the effects of GBHs are due to endocrine disrupting activities. We suggest that common commercial formulations of GBHs should be prioritized for inclusion in government-led toxicology testing programs such as the U.S. National Toxicology Program, as well as for biomonitoring as conducted by the U.S. Centers for Disease Control and Prevention.
Energetic technologies, nanoelectronics, biomedicine including gene therapy, cell imaging or tissue engineering are only few from all possible applications for graphene, the thinnest known carbon configuration and a basic element for other more complicated, better discovered and widely used nanostructures such as graphite, fullerenes and carbon nanotubes. The number of researches concerning graphene applications is rising every day which proves the great interest in its unique structure and properties. Ideal pristine graphene sheet presents a flat membrane of unlimited size with no imperfections while in practice we get different flakes with irregular edges and structural defects which influence the reactivity. Nanomaterials from graphene family differ in size, shape, layer number, lateral dimension, surface chemistry and defect density causing the existence of graphene samples with various influence on biological systems. Whether graphene induces cellular stress and activates apoptosis, or on the contrary facilitates growth and differentiation of the cells depends on its structure, chemical modifications and the growth process. A certain number of in vitro studies has indicated cytotoxic effects of graphene while the other show that it is safe. The diversity of the samples and methods of the production make it impossible to establish clearly the biological impact of graphene.
Rising progesterone (P4) levels in humans due to its overconsumption through hormonal therapy, food products or drinking water can lead to many negative health effects. Thus, the simple and accurate assessment of P4 in both environmental and clinical samples is highly important to protect the public health. In this work, we present the selection, identification and characterization of ssDNA aptamers with high binding affinity to progesterone (P4). The aptamers were selected in vitro from a ssDNA library of 1.8×1015 oligonucleotides showing a dissociation constants (KD) in the low nanomolar range. The dissociation constant of the best aptamer, designated as P4G13, was estimated to be 17 nM using electrochemical impedance spectroscopy (EIS) as well as fluorometric assay. Moreover, the aptamer P4G13 did not show cross-reactivity to analogues similar to progesterone such as 17b-estradiol (E2) and norethisterone (NET). An impedimetric aptasensor for progesterone was then fabricated based on the conformational change of P4G13 aptamer, immobilized on the gold electrode by self-assembly, upon binding to P4 which results in an increase in the electron transfer resistance. An aptamer-complementary DNA (cDNA) oligonucleotides were tested to maximize the signal gain of the aptasensor after binding with progesterone. A significant signal enhancement was observed when the aptamer hybridized with a short complementary sequence at specific site was used instead of the pure aptamer. This signal gain is likely due to the more significant conformational change of the aptamer-cDNA than the pure aptamer upon binding with P4 as confirmed by circular dichroism (CD) spectroscopy. The developed aptasensor exhibited a linear range for the concentrations of P4 from 10 ng/ml to 60 ng/ml with a detection limit of 0.90 ng/mL. Moreover, the aptasensor was applied in spiked tap water samples showing good recovery percentages. The new selected progesterone aptamers can be exploited in further biosensing applications for environmental, clinical and medical diagnostic purposes.
The broad-spectrum herbicide glyphosate has become the largest-selling crop-protection product worldwide. The increased use of glyphosate is associated with the appearance of a growing number of tolerant or resistant weeds, with socio-environmental consequences apart from the loss of productivity. In 2002, a glyphosate-resistant biotype of johnsongrass (Sorghum halepense (L.)) appeared in Argentina and now covers at least 10,000 ha. This paper analyzes the driving forces behind the emergence and spread of this weed and also examines management responses and their implications.Preventive strategies against glyphosate-resistant johnsongrass fail because of the institutional setting. Reactive measures, however, transfer the risks to the society and the environment through the introduction of novel genetically modified crops that allow the use of yet more herbicide. This in turn reinforces the emergence of herbicide-resistant weeds, constituting a new phenomenon of intensification, the “transgenic treadmill”.
The abbreviated name, ‘mfold web server’, describes a number of closely related software applications available on the World
Wide Web (WWW) for the prediction of the secondary structure of single stranded nucleic acids. The objective of this web server
is to provide easy access to RNA and DNA folding and hybridization software to the scientific community at large. By making
use of universally available web GUIs (Graphical User Interfaces), the server circumvents the problem of portability of this
software. Detailed output, in the form of structure plots with or without reliability information, single strand frequency
plots and ‘energy dot plots’, are available for the folding of single sequences. A variety of ‘bulk’ servers give less information,
but in a shorter time and for up to hundreds of sequences at once. The portal for the mfold web server is http://www.bioinfo.rpi.edu/applications/mfold. This URL will be referred to as ‘MFOLDROOT’.
We present a profile–profile multiple alignment strategy that uses database searching to collect homologues for each sequence
in a given set, in order to enrich their available evolutionary information for the alignment. For each of the alignment sequences,
the putative homologous sequences that score above a pre-defined threshold are incorporated into a position-specific pre-alignment
profile. The enriched position-specific profile is used for standard progressive alignment, thereby more accurately describing
the characteristic features of the given sequence set. We show that owing to the incorporation of the pre-alignment information
into a standard progressive multiple alignment routine, the alignment quality between distant sequences increases significantly
and outperforms state-of-the-art methods, such as T-COFFEE and MUSCLE. We also show that although entirely sequence-based,
our novel strategy is better at aligning distant sequences when compared with a recent contact-based alignment method. Therefore,
our pre-alignment profile strategy should be advantageous for applications that rely on high alignment accuracy such as local
structure prediction, comparative modelling and threading.
Serotonin is one of the important neurotransmitters in human nervous system and associated with central nervous system diseases. Herein, we have prepared a novel electrochemical aptasensor for rapid and sensitive detection of serotonin by using the pre-designed and prepared DNA aptamers. In the absence of serotonin, the electron transfer rate on the aptasensor was faster than that in the presence of serotonin due to the hairpin structure of the aptamer was loose and MB could be closer to the electrode surface. While in the presence of serotonin, the hairpin structure of the aptamer was extended and MB was far away from the electrode surface. The effect of MB labeled sites on analytical performances of the proposed aptasensors was discussed by comparing sensitivity of the aptasensors that MB labeled in the intermediate of the aptamer with that MB labeled at the 3′ end of the aptamer. It was found that sensitivity of the intermediate-labeled aptasensor was much higher than the terminal-labeled aptasensor due to the specific conformational changes before and after aptamer binding to serotonin. The developed aptasensors exhibits a rapid electrochemical response and high sensitivity for the determination of serotonin. Under the optimal experimental conditions, the linear range for serotonin concentrations by the intermediate-labeled aptasensor was 1 pM–10 nM with a detection limit of 0.017 fM (S/N = 3). Moreover, the proposed aptasensor is reusable and shows good reproducibility and selectivity for the detection of serotonin in 100-fold diluted rat cerebrospinal fluid, suggesting a good application prospect in the detection of serotonin in real samples.
Glyphosate (Gly) is a broad-spectrum herbicide and currently one of the most studied pesticides. New Gly-related data are published daily worldwide. Despite the large number of publications, there is no published scientometric revision that presently addresses this issue systematically. We aimed to scientometrically analyze the publication patterns of main topics related to Gly research. Web of Science data was obtained searching the topic “Glyphosate” (10,069 publications). Toxicology was the most influential area, and a subset was delimited containing the categories “Environmental Sciences”, “Toxicology” and “Ecology” (2077 publications). The datasets were analyzed using Citespace. The publications number presented a high correlation with the Gross Domestic Product (GDP) in both datasets. USA was the leader of general publication about Gly, followed by Brazil, Canada and China. USA, Canada, Argentina, China and Brazil were the main countries in Gly toxicology. This subset was related with data of the GDP spending on Research & Developing and with the number of researchers by country. Thus, we ranked the main countries interested in the Gly and its toxicology and that invest their economic and human resources in these researches. Based on a keyword analysis by CiteSpace of the Gly toxicology, it was highlighted the “glyphosate-induced habitat alteration”, that reflected the concern about Gly impact on agricultural and natural ecosystems. The researchers are also focused in studies involving AMPA (aminomethylphosphonic acid), the main Gly degradation product, the genotoxicity, herbicides mixture and in its presence in drinking water. More researches about Gly genotoxicity and carcinogenicity to humans are needed and more studies to compile the results of independent researches, such as meta-analytical reviews. Our study can support decisions and future efforts about Gly impacts and use, since more sustainable agriculture with less environment impact is important to the maintenance of ecosystem services and consequently the human health.
In recent years, multilayered graphite oxide and graphene oxide (GO) has attracted considerable attention in fields such as physics, chemistry, biology and materials sciences in general, because they are important building blocks and promising routes towards the large-scale production of graphene, the ‘’wonder material’’. This review provides an exhaustive survey of the synthetic methods developed during the last 160 years for the preparation of GO and focuses especially on the work done more recently. The numerous state-of-the-art synthetic methods (>50) are reviewed and regrouped under eight (8) major categories: those which use oxidative (i) chemical, (ii) electrochemical and (iii) microbial exfoliation methods for graphite, for (iv) 3D-carbon structures and (v) 2D-graphene. Other routes such (vi) chemical vapor deposition (CVD) methods for hydrocarbon, (vii) hydrothermal methods for carbohydrate and finally, (viii) thermal decomposition methods for organic matter rich in carbon are also covered. Emphasis is placed on the molecular structure of graphene oxide, the synthetic methods, the impurities, the properties, the mechanistical insights, the reagents, the different carbon precursors, its purification, the exfoliation process and finally, its fractionation by size. This review will be of valuable guide (>200 references) for synthetic and materials scientists/engineers in the field of graphene oxide production.
Sarcosine is an amino acid derivative, which is considered as a key metabolite in various metabolic processes. Therefore, simple and sensitive detection methods are needed for further understanding its metabolic role and diagnostic value. In this study, we developed a novel method that meets the need for practical and sensitive detection in a complex medium mimicking urine conditions. For this aim, we selected sarcosine-specific DNA aptamers using graphene oxide-assisted systemic evolution of ligands by exponential enrichment (GO-SELEX). The candidate aptamers were labeled with 6-carboxyfluorescein (6-FAM) at their 5′ ends. Two aptamers, namely 9S and 13S produced a significant fluorescence signal upon sarcosine binding. Both aptamers enabled a sensitive analysis with a detection limit of 0.5 pM. The linear detection ranged between 5 pM and 50 μM for 9S aptamer, while 13S aptamer enabled a wider linear detection range between 5 pM and 500 μM. The aptamer-based assay allowed rapid detection with no need for chemical derivatization of sarcosine and sophisticated instruments. Moreover, the aptamer-based assay was free of interference from urea and human serum albumin.
A reliable and sensitive method was developed for simultaneous determination of glyphosate and glufosinate in various food products by liquid chromatography-tandem mass spectrometry. Based on extraction, derivatization with 9-fluorenylmethylchloroformate and purification on solid phase extraction column, quantification was done by using isotopic-labeled analytes as internal standard and calibration in matrix. Good selectivity and sensitivity were achieved with a limit of quantification of 5 μg/kg. The recoveries of these two pesticides ranged from 91% to 114% with inter-day and relative standard deviation of 3.8-6.1% in five matrices of cereal group spiked at 5, 10, and 20 μg/kg. An accuracy profile was performed for method validation, demonstrating the accuracy and precision of the method for the studied food groups. The verification results in expanded food groups indicated extensive applicability for the analysis of glyphosate and glufosinate. Finally, the developed method was applied to analyze 136 food samples including milk-based baby foods from the French Agency for Food, Environmental and Occupational Health & Safety. Glyphosate residues were detected in two breakfast cereal samples (6.0 and 34 μg/kg). Glufosinate residues were found in a sample of boiled potatoes (9.8 μg/kg). No residues were detected in the other samples, including milk-based baby foods with limits of detection ranging from 1 to 2 μg/kg. The method has been applied for routine national monitoring of glyphosate and glufosinate in various foods.
In order to evaluate the pollution caused due to glyphosate (Glyp) in soils and sludge, it is important to establish a set of standard determination techniques. In this work, the previously reported HPLC analytical method for determination of Glyp has been improved in order to be applied for all kinds of soils/sludge. The soil/sludge samples were extracted using sodium phosphate and trisodium citrate aqueous solutions. The extract was adjusted to pH 9 and contaminations were removed by washing with n-hexane. The method developed in this work further involves derivatization with 9-fluorenylmethylchloroformate (FMOC-Cl) followed by high performance liquid chromatography (HPLC) coupled with fluorescence detection. The method was validated in three soil (red soil, black soil and paddy soil) and two sludge samples (lake and river sludge) from China and verified in six laboratories. A good linear relationship (correlation coefficients ≥0.999) was observed within the range of 0.005–0.5 mg/L. The limit of detection (LOD) and the limit of quantitation (LOQ) were determined to be 0.01 mg/kg and 0.04 mg/kg, respectively. The precision and accuracy were satisfactory with the relative standard deviation (RSD) lower than 15% and the mean recovery values ranging from 75% to 110% (n = 6), that spiked at three levels (0.1, 0.5 and 1.0 mg/kg).
N-(Phosphonomethyl)glycine (glyphosate), known by the trade name Roundup®, is a broad spectrum systemic herbicide used to kill several types of grass weed. The hazard potential of Roundup® is unclear, which is a serious issue within the European Union; however, after an intense debate, the EU Commission extended its approval of glyphosate use until the end of 2017.
A persistent need exists for rapid, inexpensive, and sensitive detection of glyphosate and (aminomethyl)phosphonic acid (AMPA), the hydrolysis product of glyphosate. This article presents reliable and easily performed (spectro)electrochemical measurements (e.g., electrogenerated chemiluminescence (ECL) and fast amperometry) for identifying glyphosate and AMPA on the basis of [Ru(bpy)3]²⁺. The limit of detection of both methods is also determined in this study. The main feature of the (spectro)electrochemical methods is screen-printed electrodes (SPE) that are made from either gold or multi-walled carbon nanotubes (MWCNTs), optionally decorated with nano-ZnO. Nano-ZnO can significantly enhance the ECL signal to result in a detection limit lower than 1 μmol/L for glyphosate. In addition, these methods are cheaper, faster, and more sensitive than, for example, spectroscopic tests.
The International Agency for Research on Cancer (IARC) published a monograph in 2015 concluding that glyphosate is “probably carcinogenic to humans” (Group 2A) based on limited evidence in humans and sufficient evidence in experimental animals. It was also concluded that there was strong evidence of genotoxicity and oxidative stress. Four Expert Panels have been convened for the purpose of conducting a detailed critique of the evidence in light of IARC’s assessment and to review all relevant information pertaining to glyphosate exposure, animal carcinogenicity, genotoxicity, and epidemiologic studies. Two of the Panels (animal bioassay and genetic toxicology) also provided a critique of the IARC position with respect to conclusions made in these areas. The incidences of neoplasms in the animal bioassays were found not to be associated with glyphosate exposure on the basis that they lacked statistical strength, were inconsistent across studies, lacked dose-response relationships, were not associated with preneoplasia, and/or were not plausible from a mechanistic perspective. The overall weight of evidence from the genetic toxicology data supports a conclusion that glyphosate (including GBFs and AMPA) does not pose a genotoxic hazard and therefore, should not be considered support for the classification of glyphosate as a genotoxic carcinogen. The assessment of the epidemiological data found that the data do not support a causal relationship between glyphosate exposure and non-Hodgkin’s lymphoma while the data were judged to be too sparse to assess a potential relationship between glyphosate exposure and multiple myeloma. As a result, following the review of the totality of the evidence, the Panels concluded that the data do not support IARC’s conclusion that glyphosate is a “probable human carcinogen” and, consistent with previous regulatory assessments, further concluded that glyphosate is unlikely to pose a carcinogenic risk to humans.
A sensitive fluorescence detection method for glyphosate (GLY) was established based on immune reaction. Firstly, carbon dots labeled antibody (lgG-CDs) which were able to specifically identify glyphosate were prepared with the environmental friendly carbon dots (CDs) and glyphosate antibody (lgG). lgG-CDs could be used to in situ visualize the distribution of glyphosate in plant tissues. In order to eliminate the effects of excess lgG-CDs on the determination of GLY, antigen magnetic beads Fe3O4-GLY based on magnetic nanoparticles Fe3O4 and glyphosate were constructed and utilized to couple with the excess lgG-CDs. After magnetic separation to remove antigen magnetic beads, there was a liner relationship between the fluorescence intensity of lgG-CDs and the logarithmic concentration of glyphosate in the range of 0.01~80 μg/mL with a detection limit of 8 ng/mL. The method was used for the detection of glyphosate in Pearl River water, tea and soil samples with satisfactory recovery ratio between 87.4%~103.7%.
A simple method, incorporating protein-precipitation/organic backwashing and liquid chromatography-tandem mass spectrometry (LC-MS-MS), has been successfully developed for the simultaneous analysis of four highly water-soluble and less volatile herbicides (paraquat, diquat, glufosinate and glyphosate) in ante- and postmortem blood, urine and gastric content samples. Respective isotopically labeled analogs of these analytes were adopted as internal standards. Acetonitrile and dichloromethane were used for protein precipitation and organic solvent backwashing, respectively, followed by injecting the upper aqueous phase into the LC-MS-MS system. Chromatographic separation was achieved using an Agilent Zorbax SB-Aq analytical column, with gradient elution of 15 mM heptafluorobutyric acid and acetonitrile. Mass spectrometric analysis was performed under electrospray ionization in positive-ion multiple reaction monitoring mode. The precursor ions and the two transition ions (m/z) adopted for each of these four analytes were paraquat (185; 169 and 115), diquat (183; 157 and 78), glufosinate (182; 136 and 119) and glyphosate (170; 88 and 60), respectively. Analyte-free blood and urine samples, fortified with the analytes of interest, were used for method development/validation and yielded acceptable recoveries of the analytes; interday and intraday precision and accuracy data; calibration linearity and limits of detection and quantitation. This method was successfully incorporated into an overall analytical scheme, designed for the analysis of a broad range of compounds present in postmortem samples, helpful to medical examiners' efforts to determine victims' causes of death.
Graphene oxide is regarded as a major precursor for graphene-based materials. The development of graphene oxide derivatives with new functionalities requires a thorough understanding of its chemical reactivity, especially for canonical synthetic methods such as the Diels-Alder cycloaddition. We successfully extended the Diels-Alder reaction to graphene oxide as a source of diene using maleic anhydride as a dienophile, thereby outlining the presence of cis diene present on graphene oxide framework which is a fundamental element in the understanding of its exact structure and chemical nature. On the basis of high resolution 13C-SS-NMR spectra, we show evidence for the formation of new sp3 carbon centers covalently bonded to graphene oxide following hydrolysis of the reaction product. We use density functional theory calculations to show that the presence of cis dihydroxyl and C vacancy on the surface of graphene oxide is promoting the reaction to significant negative reaction enthalpies.
The detection of glyphosate (Glyp) in water is of increasing importance because of its potential danger to environmental and health. In this study, a simple and label free colorimetric method for Glyp detection is proposed based on the inhibition of peroxidase-like activity of Cu2+. Cu2+ possesses the peroxidase-like activity that could catalyze the oxidation of 3,3′,5,5′-tetramethylbenzidine (TMB) to oxidized TMB (oxTMB) in the presence of H2O2, resulting in a color change of the solution. However, the peroxidase-like activity of Cu2+ could be strongly hindered by Glyp due to the formation of Glyp-Cu2+ complexes. The color change of the solution or the absorbance change of oxTMB is utilized to monitor the concentration of Glyp. The linear range of the proposed method is 2 to 200 μM and the detection limit is 1 μM. Furthermore, as low as 10 μM Glyp can be clearly distinguished by naked eyes. Also this proposed method is successfully applied for screening Glyp in real samples and shows great promise for environmental monitoring owing to its simplicity, rapidness and high selectivity.
The various types of biosensors such as enzyme-based, tissue-based, immunosensors, DNA biosensors, thermal and piezoelectric biosensors have been deliberated here to highlight their indispensable applications in multitudinous fields.
Some of the popular fields implementing the use of biosensors are food industry to keep a check on its quality and safety, to help distinguish between the natural and artificial; in the fermentation industry and in the saccharification process to detect precise glucose concentrations; in metabolic engineering to enable in vivo monitoring of cellular metabolism. Biosensors and their role in medical science including early stage detection of human interleukin-10 causing heart diseases, rapid detection of human papilloma virus, etc. are important aspects. Fluorescent biosensors play a vital role in drug discovery and in cancer. Biosensor applications are prevalent in the plant biology sector to find out the missing links required in metabolic processes. Other applications are involved in defence, clinical sector, and for marine applications.
There is a need for simple, fast, efficient, and sensitive methods of analysis for glyphosate and it's degradate, aminomethylphosphonic acid (AMPA), in diverse matrices such as water, plant materials, and soil to facilitate environmental research needed to address the continuing concerns related to increasing glyphosate use. A variety of water based solutions have been used to extract the chemicals from different matrices. Many methods require extensive sample preparation, including derivatization and clean up, prior to analysis by a variety of detection techniques. This review summarizes methods used during the past 15 years for analysis of glyphosate and AMPA in water, plant materials, and soil. The simplest methods use aqueous extraction of glyphosate and AMPA from plant materials and soil, no derivatization, solid phase extraction (SPE) columns for clean up, guard columns for separation, and confirmation of the analytes by mass spectrometry and quantitation using isotope-labeled internal standards. They have levels of detection (LODs) below regulatory limits in the North America. These methods are discussed in more detail in the review.
Glyphosate is a broad-spectrum herbicide frequently used world widely in agricultural and non-agricultural areas to control unwanted plants. Health risk of chronic and subchronic exposure of glyphosate on animals and humans has received increasing attention in recent years. The aim of this study was to evaluate the effects of glyphosate on the immunoglobulin M (IgM), complement C3 (C3), and lysozyme (LYZ) in the kidney of common carp exposed to 52.08 or 104.15 mg L−1 of glyphosate for 168 h. The results showed that the transcriptions of IgM, C3, or LYZ were altered due to glyphosate-exposure, for example, IgM and C3 initially increased at 24 h later it decreased (except for a increase of C3 in higher dose group at 24 h) while the expression of G-type LYZ were not affected at 24 h, then increased at 72 h, but decreased at the end of test, however C-type LYZ expression was initially up-regulated (24–72 h) but down-regulated at the end of exposure (168 h). However, glyphosate-exposure generally decreased the contents of IgM and C3 or inhibited LYZ activity in the kidney of common carp. In addition, glyphosate-exposure also caused remarkable histopathological damage, mainly including vacuolization of the renal parenchyma and intumescence of the renal tubule in fish kidney. The results of this study indicate that glyphosate causes immunotoxicity on common carp via suppressing the expressions of IgM, C3, and LYZ and also via damaging the fish kidney.
A high-affinity ssDNA aptamer that specifically binds to T-2 toxin was generated by the systemic evolution of ligands by exponential enrichment (SELEX) procedure assisted by graphene oxide (GO). After ten rounds of selection against T-2 toxin, a highly enriched ssDNA pool was sequenced, and the representative aptamers were subjected to binding assays to evaluate their affinity and specificity. Circular dichroism spectroscopy was also utilized to study the inherent interaction of T-2 toxin and the preferred aptamer Seq. 16, which demonstrated a low dissociation constant (Kd) of 20.8±3.1 nM and excellent selectivity for T-2 toxin. The results indicate that GO-SELEX technology is appropriate for the screening of aptamers recognizing small molecule, such as T-2 toxin, which is of great significance for SELEX methods of generating ligands against small molecules. Further research on use of the identified aptamer Seq. 16 in the development of biosensors for T-2 toxin detection in food and feed samples is in progress.
A highly sensitive and label-free electrochemical impedance spectroscopy (EIS) aptasensor for the detection of adenosine was fabricated by co-assembling thiolated aptamer, dithiothreitol (DTT) and 6-mercaptohexanol (MCH) on gold electrode surface, forming Au/aptamer-DTT/MCH. The interfacial electron transfer resistance (Ret) of the aptasensor using [Fe(CN)6](3-/4-) as the probe increased with adenosine concentration, and the change in Ret (∆Ret) against the logarithm of adenosine concentration was linear over the range from 0.05pM to 17pM with a detection limit of 0.02pM. Compared to that of aptasensors fabricated with MCH or DTT alone as the backfiller, the detection limit was improved dramatically (LOD was 0.03nM and 0.2pM for Au/aptamer/MCH and Au/aptamer-DTT, respectively), which was attributed primarily to the coupling of the cyclic- and linear -configuration backfillers. The coupling showed remarkably higher resistance to nonspecific adsorption, leading to low background noise and high response signal. The aptasensor reported herein is applicable for the detection of other kinds of aptamer-binding chemicals and biomolecules.
Listeria monocytogenes is a pathogenic bacterium capable of causing severe disease leading to high hospitalization and case fatality rates. Here, we report the use of Systemic Evolution of Ligands by Exponential Enrichment (SELEX) to screen DNA aptamers that recognize L. monocytogenes with high affinity and specificity. Whole L. monocytogenes were incubated with a library of single-stranded DNA (ssDNA) aptamers. The aptamers that bound to L. monocytogenes were isolated using centrifugation, PCR amplified, and purified to yield an enriched ssDNA pool suitable for further rounds of selection. Aptamers with a dissociation constant (Kd) of 48.74 ± 3.11 nM were isolated after eight rounds of selection. An aptamer-based fluorescent bioassay was used to examine the binding ability of the selected aptamers to L. monocytogenes. To demonstrate the potential use of the aptamers in the quantitative determination of L. monocytogenes, a sandwich-type structure fluorescent bioassay with the aptamer A 15 was developed. The assay had a limit of detection (LOD) of 75 cfu/mL and a wide linear range from 102 to 107 with a correlation coefficient of 0.9937. We believe that the design of aptamer-based molecular probes that do not require the labour-intensive steps of isolating and purifying complex markers or targets represents a potentially useful method in the rapid screening and detection of foodborne pathogens.
Aptamers are artificial nucleic acid ligands that can be generated against amino acids, drugs, proteins and other molecules. They are isolated from complex libraries of synthetic nucleic acids by an iterative process of adsorption, recovery and amplification. This review described the in vitro process to obtain aptamers (SELEX). It mentions the main characteristics of these molecules (i.e. affinity, specificity and stability). Moreover, it discusses advantages over antibodies. It reports potential applications of aptamers in analytical and diagnostic assays as biocomponents of biosensors (aptasensors) and allosteric ribozymes (aptazymes).
This article presents a simple, fast and low cost UV–vis spectrophotometric method to quantify glyphosate.
This method can be used to perform adsorption isotherms on soils and metal oxides. It comprises a derivatization step and further measurement of the absorbance at 265 nm. The trueness of the results is validated using Ultra Performance Liquid Chromatography with tandem mass spectrometry detection (UPLC-MS/MS) as a reference method. The proposed spectrophotometric method is able to quantify glyphosate in the concentration range from 0.084 to 21.8 mg L− 1 . This range is suitable to construct reliable adsorption isotherms. Examples of adsorption isotherms on goethite at pH 4.5 and a soil sample at pH 4.5, 6.0 and 8.0 are given. Interferences caused by dissolved organic matter can be corrected at least up to an organic matter concentration of 12 mg L− 1
A simple aptasensor for sensitive and selective detection of acetamiprid has been developed based on electrochemical impedance spectroscopy (EIS). To improve sensitivity of the aptasensor, gold nanoparticles (AuNPs) were electrodeposited on the bare gold electrode surface by cycle voltammetry (CV), which was employed as a platform for aptamer immobilization. With the addition of acetamiprid, the formation of acetamiprid-aptamer complex on the AuNPs-deposited electrode surface resulted in an increase of electron transfer resistance (R(et)). The change of R(et) strongly depends on acetamiprid concentration, which is applied for acetamiprid quantification. A wide linear range was obtained from 5 to 600nM with a low detection limit of 1nM. The control experiments performed by employing the pesticides that may coexist or have similar structure with acetamiprid demonstrate that the aptasensor has only specific recognition to acetamiprid, resulting in high selectivity of the aptasensor. The dissociation constant, K(d) of 23.41nM for acetamiprid-aptamer complex has been determined from the differential capacitance (C(d)) by assuming a Langmuir isotherm, which indicates strong interaction between acetamiprid and aptamer, further proving high selectivity of the aptasensor. Besides, the applicability of the developed aptasensor has been successfully evaluated by determining acetamiprid in the real samples, wastewater and tomatoes.
The efficiency of current microcystin detection methods has been hampered by the low detection limits required in drinking water and that routine detection is restricted to a few of the congeners with high degree of undesired cross-reactivity. Here, we report the development of novel microcystin-targeting molecules and their application in microcystin detection. We have selected DNA aptamers from a diverse random library that exhibit high affinity and specificity to microcystin-LR, -YR, and -LA. We obtained aptamers that bind to all chosen congeners with high affinity with K(D) ranging from 28 to 60 nM. More importantly, we also obtained aptamers that are selective among the different congeners, with selectivity from 3-folds difference in binding affinity to total discrimination (K(D) of 50 nM versus nonspecific binding). Electrochemical aptasensors constructed with the selected aptamers were able to achieve sensitive and congener-specific microcystin detection with detection limit as low as 10 pM.
After 25 years of sales, glyphosate (N-(phosphonomethyl)glycine) is the world's biggest selling and fastest growing agrochemical. Glyphosate has many strengths, particularly its systemicity and ability to control perennial weeds, which have facilitated its role as an essential tool in modern agriculture. Surveys of recent scientific and patent literature point to several key areas of current and future interest in glyphosate and its use. Biotechnology topics have been the most popular, but there is still much interest in the molecule per se, particularly in manufacturing processes. The mode of action and physiology of glyphosate are common subjects. More applied aspects concern the development of formulations with new adjuvants, or mixtures with other herbicides, to enhance performance. An increasing number of papers are reporting on its environmental fate and on agronomic techniques designed to improve the efficiency of application and minimise its environmental impact. Lack of weed resistance is a key feature of glyphosate despite its widespread use. However, differential levels of tolerance between species have led to changes in weed succession. There is a need to develop integrated weed management systems in which glyphosate still plays a key role. Glyphosate is an exceedingly effective herbicide and presents a considerable challenge to the development of new herbicides for sustainable cropping systems and situations in which cost-effective and environmentally considerate weed control is required.© 2000 Society of Chemical Industry
Auf Graphenoxid (GO) beruht ein empfindliches und selektives Verfahren zum DNA- und Proteinnachweis. Durch die Wechselwirkung von GO mit einer farbstoffmarkierten Einzelstrang-DNA wird die Fluoreszenz des Farbstoffs gelöscht. Sind Ziel-DNAs oder Zielproteine vorhanden, binden sie an die farbstoffmarkierte DNA und spalten sie von GO ab, sodass sie erneut fluoresziert (siehe Bild).
A simple method was developed to determine the residues of glyphosate, N-(phosphonomethyl)glycine, in soil. The residues were
extracted from soil matrices with 2 M NH4OH, derivatized by trifluoroacetic anhydride and trifluoroethanol, and, then, determined by gas chromatography with a nitrogen-phosphorus
detector (GC-NPD). The results were further confirmed by gas chromatography-mass spectrometry (GC-MS). The limit of detection
was estimated to be 9 × 10−12 g, and the minimum determination concentration of glyphosate in the samples was 0.01 mg/kg. The ranges for the average recoveries
and relative standard deviation (RSD) of the method were 84.4–94.0% and 8.1–13.7%, respectively, in agreement with the directives
for method validation in residue analysis. The proposed method was successfully employed for the determination of glyphosate
residue levels and dissipation retes in the soil from an apple orchard.
Atrazine effects on the microbial food web were assessed in mesocosms designed to simulate a southeastern tidal creek ecosystem. The test chambers simulate the major features unique to estuarine systems, such as tidal flow and salinity changes. Microbial communities were colonized on artificial substrates and analyzed for productivity, biomass, and community composition. Atrazine was found to reduce chlorophyll a, phototrophic carbon assimilation and phototrophic biovolume in the 40 and 160 μg l−1 treatments. Significant reductions in phototrophic variables occurred within 24 h of pesticide exposure and no recovery was detected after 9 days. Dissolved oxygen content in the mesocosms was not significantly affected by atrazine. Atrazine induced changes in composition of the algal community. Most taxa were reduced in abundance by atrazine exposure; however, cyanobacterial taxa generally became more abundant. Bacterial abundance was significantly elevated in the highest dose after 24 h, but decreased in a dose–response manner after 48 h. There was no significant effect on small ciliates; however, large ciliates and small flagellates increased significantly in number after 48 h and remained elevated after 9 days. Atrazine at 160 μg l−1 significantly reduced large flagellate density throughout the experiment. These results suggest that exposure of the estuarine microbial food web to agricultural pesticides can lead to both functional and structural changes, which may potentially impact higher trophic levels.
Nucleic-acid aptamers have attracted intense interest and found wide applications in a range of areas. In this review, we summarize recent advances in the development of aptamer-based biosensors and bioassay methods, most of which have employed electrochemical, optical and mass-sensitive analytical techniques. Aptamers exhibit many advantages as recognition elements in biosensing when compared to traditional antibodies. They are small in size, chemically stable and cost effective. More importantly, aptamers offer remarkable flexibility and convenience in the design of their structures, which has led to novel biosensors that have exhibited high sensitivity and selectivity. Recently, the combination of aptamers with novel nanomaterials has significantly improved the performance of aptamer-based sensors, which we also review in this article. In view of the unprecedented advantages brought by aptamers, we expect aptamer-based biosensors to find broad applications in biomedical diagnostics, environmental monitoring and homeland security.
An impedance spectroscopic study of the interaction between thiol-modified Au electrodes and Saccharomyces cerevisiae of strain EBY44 revealed that the cells formed an integral part of the interface, modulating the capacitive properties until a complete monolayer was obtained, whereas the charge transfer resistance ( R ct) to the redox process of [Fe(CN)6] 3-/4- showed a linear relationship to the number of cells even beyond the monolayer coverage. R ct showed strong pH dependence upon increasing the pH of the utilized buffer to 7.2. Upon addition of S. cerevisiae cells at pH 7.2, the obtained value of R ct showed over 560% increase with respect to the value obtained on the same thiol-modified electrode without cells. It was demonstrated that real-time monitoring of S. cerevisiae proliferation, with frequency-normalized imaginary admittance (real capacitance) as the indicator, was possible using a miniaturized culture system, ECIS Cultureware, with integrated planar cysteamine-modified Au microelectrodes. A monolayer coverage was reached after 20-28 h of cultivation, observed as an approximately 15% decrease in the real capacitance of the system.
With over 2,000 publications, including about 250 reviews, resulting from a SciFinder search in just a two year period (2009–2010), the field of aptamer research has continuously generated lots of interest in the scientific community. Aptamers, first reported by three groups independently in 1990,1–3 are the artificial single-stranded DNA or RNA sequences (more recently, peptides) that fold into secondary and tertiary structures making them bind to certain targets with extremely high specificity. Owing to the high specific affinity of an aptamer to its target molecule (small molecules, proteins and even entire cells), it is thought to resemble chemical antibodies, with the dissociation constants ranging from nanomolar to picomolar level. Aptamers have a number of unique features which make them a more effective choice than antibodies. First, aptamers can be screened via in vitro process against a synthetic library, making it possible to target any molecules (from small inorganic ions to intact cells), overcoming the limit of having to use cell lines or animals, as is necessary for antibodies. Second, aptamers, once selected, can undergo subsequent amplification through polymerase chain reaction to produce a large quantity with high purity. Third, the simple chemical structure of aptamer makes it easily amendable to further modifications with functional groups according to different purposes. Finally, aptamers are much more stable than antibodies, making them suitable in applications requiring harsh conditions (e.g., high temperature or extreme pH).
The applications of aptamers remain very dynamic, with increasing explorations in the fields of biosensing, diagnostics and therapeutics (some aptamer-based applications are illustrated in Figure 1). There have been a numbers of excellent reviews in recent years with different emphases.4–8 Herein, as the first review of aptamers on Analytical Chemistry, we attempt to cover major progresses in bioanalytical applications of aptamers in the past 2 years.
Figure 1
The widespread use of aptamers for numerous analytical and biological applications. Bioanalytical applications of aptamers are highlighted in red.
DNA and RNA microarrays have become an important analytical technique in the understanding and characterisation of genomes and transcriptomes. A recent development in this field of combinatorial chemistry has concentrated on using artificial DNA/RNA sequences--aptamers--as the screening ligand. In reviewing this technology, the article covers the topic, following a general introduction, under the headings: generating an aptamer library, aptamer library complexity, aspects of nucleotide chemistry, constant region primer design, and the SELEX protocol for exposing an aptamer library to the desired target.
The in vitro selection of nucleic acid libraries has driven the discovery of RNA and DNA receptors (aptamers) and catalysts with tailor-made functional properties. Functional nucleic acids emerging from selections have been observed to possess an unusually high degree of secondary structure. In this study, we experimentally examined the relationship between the degree of secondary structure in a nucleic acid library and its ability to yield aptamers that bind protein targets. We designed a patterned nucleic acid library (denoted R*Y*) to enhance the formation of stem-loop structures without imposing any specific sequence or secondary structural requirement. This patterned library was predicted computationally to contain a significantly higher average folding energy compared to a standard, unpatterned N(60) library of the same length. We performed three different iterated selections for protein binding using patterned and unpatterned libraries competing in the same solution. In all three cases, the patterned R*Y* library was enriched relative to the unpatterned library over the course of the 9- to 10-round selection. Characterization of individual aptamer clones emerging from the three selections revealed that the highest affinity aptamer assayed arose from the patterned library for two protein targets, while in the third case, the highest affinity aptamers from the patterned and random libraries exhibited comparable affinity. We identified the binding motif requirements for the most active aptamers generated against two of the targets. The two binding motifs are 3.4- and 27-fold more likely to occur in the R*Y* library than in the N(60) library. Collectively, our findings suggest that researchers performing selections for nucleic acid aptamers and catalysts should consider patterned libraries rather than commonly used N(m) libraries to increase both the likelihood of isolating functional molecules and the potential activities of the resulting molecules.
An ion chromatography system for the determination of glyphosate was described. Ion chromatograph was carried out by suppressed conductivity detection (DX-100). The eluent contained 9 mmol l-1 Na2CO3 and 4 mmol l-1 NaOH. The detection limit was 0.042 microgram ml-1 (S/N = 3). The relative standard deviation was 1.99% and the correlation coefficient of the calibration curve for area was 0.9995. The linear range was 0.042-100 micrograms ml-1. Common inorganic ion and organic acids did not interfere. The recovery was 96.4-103.2%. The method was simple, rapid, reliable and inexpensive.
A novel, sensitive, linker-assisted enzyme-linked immunosorbent assay (L'ELISA) was compared to on-line solid-phase extraction (SPE) with high-performance liquid chromatography/mass spectrometry (HPLC/MS) for the analysis of glyphosate in surface water and groundwater samples. The L'ELISA used succinic anhydride to derivatize glyphosate, which mimics the epitotic attachment of glyphosate to horseradish peroxidase hapten. Thus, L'ELISA recognized the derivatized glyphosate more effectively (detection limit of 0.1 microg/L) and with increased sensitivity (10-100 times) over conventional ELISA and showed the potential for other applications. The precision and accuracy of L'ELISA then was compared with on-line SPE/HPLC/MS, which detected glyphosate and its degradate derivatized with 9-fluorenylmethyl chloroformate using negative-ion electrospray (detection limit 0.1 microg/ L, relative standard deviation +/- 15%). Derivatization efficiency and matrix effects were minimized by adding an isotope-labeled glyphosate (2-13C15N). The accuracy of L'EUSA gave a false positive rate of 18% between 0.1 and 1.0 microg/L and a false positive rate of only 1% above 1.0 microg/L The relative standard deviation was +/- 20%. The correlation of L'ELISA and HPLC/MS for 66 surface water and groundwater samples was 0.97 with a slope of 1.28, with many detections of glyphosate and its degradate in surface water but not in groundwater.
A competitive direct enzyme-linked immunosorbent assay (ELISA) and high-pressure liquid chromatographic (HPLC) methods were compared in terms of accuracy and precision for the detection and quantification of glyphosate-spiked Nanopure, tap, and river waters. The ELISA had a detection limit of 0.6 ng mL(-)(1) and a linear working range of 1-25 ng mL(-)(1), whereas the HPLC method had a detection limit of 50 ng mL(-)(1) and a linear working range of 100-10000 ng mL(-)(l). No statistically significant differences (95% confidence interval) were found between the ELISA and HPLC analysis of the three water matrixes. The coefficients of variation obtained with the ELISA in tap water were between 10 and 19%, whereas the coefficients of variation for the HPLC analysis were between 7 and 15%. The use of ELISA for the analysis of glyphosate in water is a cost-effective and reliable method capable of meeting water quality guidelines established for Europe and North America.
An analytical method based on on-line solid-phase extraction-liquid chromatography coupled to electrospray tandem mass spectrometry (SPE-LC-ESI-MS/MS) for the determination of glyphosate and aminomethylphosphonic acid (AMPA) residues has been applied to the analysis of water samples within a Round Robin Study. The method had been previously validated in a variety of water samples and it fulfilled all the parameters of precision, accuracy, sensitivity and unequivocal confirmation. The results within the study that we participated were highly satisfactory in all cases with the only exception of glyphosate in groundwater samples, where surprisingly recoveries around 15% were obtained despite the use of isotope-labeled glyphosate as internal standard (I.S.). A slight modification has been introduced in the method, simply consisting of the acidification of sample with hydrochloric acid (HCl) to pH 1. Then, the sample is neutralized and immediately derivatized with 9-fluorenylmethylchloroformate (FMOC) before LC-MS/MS determination. Round Robin Study samples were reanalyzed using this approach, and the recoveries increased up to 98%. A possible explanation might be the slow kinetic interaction between glyphosate and some components of the matrix. These components might act as chelating agents, making glyphosate unavailable for derivatization and therefore for analysis. Several water samples collected at the Mediterranean area of Spain, and previously analyzed and being found to contain glyphosate, were also reanalyzed using this approach, obtaining higher concentrations (between 2 and 14 times) in most of cases.