Colorimetric detection of nucleic acid signature of shiga toxin producing Escherichia coli using gold nanoparticles

Enterohemorrhagic E. coil (EHEC) serotype O157:H7 is one of the major pathogens, responsible for the severe disease outbreaks. EHEC causes diseases in humans through production of shiga-like toxin leading to bloody diarrhea. The toxin is encoded by stx2 gene in E. coli. The current methodology for detection of EHEC relies on fluorogenic-substrate based culture media or nucleic acid amplification based Real-Time Polymerase Chain Reaction assays that are either time consuming or need expensive instrumentation. In this study, the optical properties of gold nanoparticles (GNPs) have been exploited for detection of nucleic acid of Escherichia coli O157:H7. The stx2 gene representing EHEC signature has been targeted using the gold nanoparticle probes. Gold nanoparticles (GNPs) of 20 +/- 0.2 nm were synthesised by citrate reduction method and characterised by spectroscopy and transmission electron microscopy. The GNPs were functionalised with 19 and 22 bp of thiolated single stranded DNA complementary to target highly conserved 149 bp region of stx2 gene. Transmission Electron Microscopy revealed the hybridization, aggregation and reduction in the interparticle distances of the GNP probes in the presence of target DNA. The aggregation and the spectral shift in the plasmon band observed with 10(6) copies of target DNA indicates feasibility of a simple and quick colorimetric 'spot and read' test in contrast to amplification based detection methods.     

Highly selective colorimetric detection of hydrochloric acid using unlabeled gold nanoparticles and an oxidizing agent

We report a colorimetric system for the detection of HCl in aqueous environments using unlabeled gold nanoparticle (AuNP) probes. This nonaggregation-based detection system relies on the ability of chloro species to cause rapid leaching of AuNPs in an aqueous dispersion containing a strong oxidizing agent, such as HNO(3) or H(2)O(2). The leaching process leads to remarkable damping of the surface plasmon resonance peak of the AuNP dispersion. This method works only with AuNPs of a particular size (～30 nm diameter). It is highly selective for HCl over several common mineral acids, salts, and anions. This simple and cost-effective sensing system provides rapid and simple detection of HCl at concentrations as low as 500 ppm (far below the hazard limit) in natural water systems.     

Tumor detection strategy using ZnO light-emitting nanoprobes

Traditional methods of detecting cancer cells, such as fluorescence, have their limits and can hardly be used for identification during tumor resection. Here we report an alternative tumor detection technology using ZnO nanorods bonded to antibodies as cancer cell probes. Our experiment shows that antibodies toward epidermal growth factor receptor (EGFR) can be connected to ZnO nanorods and to EGFR receptors of SCC (squamous cell carcinoma). The cancer cell can be recognized by the naked eye or an optical microscope with the help of purple light emission from ZnO/EGFR antibody probes. On the other hand, for cells with less EGFR expression, in our case Hs68, no purple light was observed as the probes were washed off. From the photoluminescent spectra, the peak intensity ratio between the purple light (from ZnO at the wavelength 377 nm) and the green band (from the autofluorescence of cells) is much higher with the presence in SCC, as compared with Hs68. The ZnO/EGFR antibody probes have the potential to be applied to surgery for real-time tumor cell identification. The cancer cells will be excised with the help of purple light emission.     

Chip-based scanometric detection of mercuric ion using DNA-functionalized gold nanoparticles

We have developed a chip-based scanometric method for the detection of mercuric ion (Hg (2+)). This method takes advantage of the cooperative binding and catalytic properties of DNA-functionalized gold nanoparticles and the selective binding of a thymine-thymine mismatch for Hg (2+). The limit of detection of this assay in buffer and environmentally relevant samples (lake water) is 10 nM (2 ppb) Hg (2+), which is the U.S. Environmental Protection Agency (EPA) limit of [Hg (2+)] for drinkable water and 1 order of magnitude lower than previous colorimetric assays. This assay is capable of discriminating Hg (2+) from 15 other environmentally relevant metal ions. The method is attractive for potential point-of-use applications due to its high throughput, convenient readout, and portability.     

Immobilization of 1,2-naphthoquinone-4-sulfonic acid on gold electrode: application for cysteamine detection using Michael addition

Fabrication and electrochemical characterization of adsorbed 1,2-naphthoquinone-4-sulfonic acid sodium (Nq)-modified gold electrode is described in a wide pH range (3.00–9.00) in 0.1 M phosphate buffer solution using cyclic voltammetry (CV), differential pulse voltammetry (DPV), and double-step potential chronoamperometry methods. The resulting Nq thin film-modified gold electrode (Nq/Au) was tested successfully to recognize cysteamine in an aqueous solution. It is found that cysteamine participates in Michael addition reaction with adsorbed Nq on gold electrode to form the corresponding thioquinone derivative. The reoxidation of the adduct at a potential of about 650 mV less positive than cysteamine at the surface of the bare Au electrode leads to an increase in the oxidative current, which is proportional to the concentration of cysteamine. The calibration plot for cysteamine was found to be linear in the ranges of 6.0 × 10⁻⁵–4.5 × 10⁻³ M and 8.0 × 10⁻⁶–5.5 × 10⁻⁴ by CV and DPV, respectively.     

Ratiometric coumarin-neutral red (CONER) nanoprobe for detection of hydroxyl radicals

Excessive production of reactive oxygen species can lead to alteration of cellular functions responsible for many diseases including cardiovascular diseases, neurodegenerative diseases, cancer, and aging. Hydroxyl radical is a short-lived radical which is considered very aggressive due to its high reactivity toward biological molecules. In this study, a COumarin-NEutral Red (CONER) nanoprobe was developed for detection of hydroxyl radical based on the ratiometric fluorescence signal between 7-hydroxy coumarin 3-carboxylic acid and neutral red dyes. Biocompatible poly lactide-co-glycolide (PLGA) nanoparticles containing encapsulated neutral red were produced using a coumarin 3-carboxylic acid conjugated poly(sodium N-undecylenyl-Nε-lysinate) (C3C-poly-Nε-SUK) as moiety reactive to hydroxyl radicals. The response of the CONER nanoprobe was dependent on various parameters such as reaction time and nanoparticle concentration. The probe was selective for hydroxyl radicals as compared with other reactive oxygen species including O(2)(?-), H(2)O(2), (1)O(2), and OCl(-). Furthermore, the CONER nanoprobe was used to detect hydroxyl radicals in vitro using viable breast cancer cells exposed to oxidative stress. The results suggest that this nanoprobe represents a promising approach for detection of hydroxyl radicals in biological systems.     

Constructing LBL-assembled functional bio-architecture using gold nanorods for lactate detection

Layer-by-layer assembly technique was employed to fabricate functional bio-architecture on 3D gold nanorods suitable for working electrode for lactate detection. Successive immobilization of horseradish peroxidase and lactate oxidase on the nanorods was carried out by direct adsorption and specific interaction. The bio-architecture constructed using 3D gold nanorods electrode shows higher sensitivity compared to 2D flat gold electrode due to an increased amount of accessible enzymes and a high transduction capacity. This functional structure system was applied to the determination of l-lactate concentration. The CV results showed a linear response at the 0.1 mM level of l-lactate with a sensitivity of 1.02 μA/mM.     

Colorimetric‐based detection of Ureaplasma urealyticum using gold nanoparticles

Ureaplasma urealyticum (uu) is one of the most common agents of urogenital infections and is associated with complications such as infertility, spontaneous abortion and other sexually transmitted diseases. Here, a DNA sensor based on oligonucleotide target‐specific gold nanoparticles (AuNPs) was developed, in which the dispersed and aggregated states of oligonucleotide‐functionalised AuNPs were optimised for the colorimetric detection of a polymerase chain reaction (PCR) amplicon of U. urealyticum DNA. A non‐cross‐linking approach utilising a single Au‐nanoprobe specific of the urease gene was utilised and the effect of a PCR product concentration gradient evaluated. Results from both visual and spectral analyses showed that target–Au‐nanoprobe hybrids were stable against aggregation after adding the inducer. Furthermore, when a non‐target PCR product was used, the peak position shifted and salt‐induced aggregation occurred. The assay''s limit of detection of the assay was 10 ng with a dynamic range of 10–60 ng. This procedure provides a rapid, facile and low‐cost detection format, compared to methods currently used for the identification of U. urealyticum.Inspec keywords: patient diagnosis, diseases, enzymes, nanosensors, microorganisms, molecular biophysics, DNA, nanoparticles, aggregation, cellular biophysics, colorimetry, genetics, gold, nanomedicineOther keywords: urogenital infections, infertility, spontaneous abortion, sexually transmitted diseases, DNA sensor, oligonucleotide target‐specific gold nanoparticles, oligonucleotide‐functionalised AuNPs, colorimetric detection, polymerase chain reaction amplicon, noncross‐linking approach, single Au‐nanoprobe specific, urease gene, visual analyses, spectral analyses, target–Au‐nanoprobe hybrids, nontarget PCR product, salt‐induced aggregation, rapid cost detection format, facile cost detection format, low‐cost detection format, PCR product concentration, Ureaplasma urealyticum DNA, Au     

Fast colorimetric detection of copper ions using L-cysteine functionalized gold nanoparticles

This communication reports an efficient visual detection method of Cu2+ by L-cysteine functionalized gold nanoparticles in aqueous solution. Upon exposure to Cu2+, the gold nanoparticle solution changed from red to blue, in response to surface plasmon absorption of dispersed and aggregated nanoparticles. This colorimetric sensor allows a rapid quantitative assay of Cu2+ down to the concentration range of 10(-5) M. Recognition of Cu2+ and formation of the aggregates are proposed to occur via a 2 : 1 sandwich complex between L-cysteine and Cu2+.     

Naked eye detection of glucose in urine using glucose oxidase immobilized gold nanoparticles

Colloidal gold is extensively used for molecular sensing because of the flexibilities it offers in terms of modification of the gold nanoparticle surface with a variety of functional groups using thiol chemistry. We describe a simple assay that allows the visual detection of glucose in aqueous samples and demonstrates its applicability by estimating glucose in urine. To enable the glucose detection, we functionalized the thiol capped gold nanoparticles with glucose oxidase, the enzyme specific to β-D glucose, using carbodiimide chemistry. The visible color change of the GOD-functionalized gold nanoparticles from red to blue on interaction with glucose is the principle applied here for the sensing of urine glucose level. The solution turns blue when the glucose concentration exceeds 100 μg/mL. The approach depicted here seems to be important, particularly in third world countries where high tech diagnostics aids are inaccessible to the bulk of the population.     

Voltammetric detection of mercury and copper in seawater using a gold microwire electrode

A procedure is presented by which mercury and copper are determined simultaneously in seawater and dilute acid (0.01 M HCl) by anodic stripping voltammetry using gold microwire electrodes. It was found that anion (halide) adsorption is the cause for a gradual decrease in the height and potential of the mercury peak. The effect is eliminated by including an anion desorption step in the analysis at -0.8 V prior to each scan. This step was found to greatly improve the stability of the scans and enabled the use of background subtraction. Advantages of the microwire electrodes were a low roughness of the surface, without a need for pretreatment, and a very small diffusion layer (2 microm with stirring). Under the optimized voltammetric conditions, the detection limits were 6 pM mercury and 25 pM copper using 300-s deposition. These values are well below those reported previously for other electrodes including rotating disk electrodes. Measurements of the influence of the major anions I-, Br-, Cl-, SO4(2-), F-, HCO3-, and B(OH)4 on the response for mercury showed that bromide and chloride are predominantly responsible for the underpotential deposition mechanism of mercury in seawater. The method was applied to coastal water samples from Liverpool Bay.     

Nucleic acid and nucleotide-mediated synthesis of inorganic nanoparticles

Since the advent of practical methods for achieving DNA metallization, the use of nucleic acids as templates for the synthesis of inorganic nanoparticles (NPs) has become an active area of study. It is now widely recognized that nucleic acids have the ability to control the growth and morphology of inorganic NPs. These biopolymers are particularly appealing as templating agents as their ease of synthesis in conjunction with the possibility of screening nucleotide composition, sequence and length, provides the means to modulate the physico-chemical properties of the resulting NPs. Several synthetic procedures leading to NPs with interesting photophysical properties as well as studies aimed at rationalizing the mechanism of nucleic acid-templated NP synthesis are now being reported. This progress article will outline the current understanding of the nucleic acid-templated process and provides an up to date reference in this nascent field.     

Amperometric detection of nucleic acid at femtomolar levels with a nucleic acid/electrochemical activator bilayer on gold electrode

Cationic redox polymers containing osmium-bipyridine complexes strongly interact with anionic enzymes, such as glucose oxidase and peroxidases, and electrochemically "activate" the enzymes. On the basis of these observations, attempts were made to develop an ultrasensitive nucleic acid biosensor. A mixed monolayer of single-stranded oligonucleotide capture probe and 16-mercaptohexadecanoic acid was formed on a gold electrode through self-assembly. Following hybridization with a complementary nucleic acid and glucose oxidase labeled oligonucleotide detection probe, a cationic redox polymer (electrochemical activator) overcoating was applied to the electrode through layer-by-layer electrostatic self-assembly. The formation of an anionic-cationic bilayer brought the glucose oxidase in electrical contact with the redox polymer, making the bilayer an electrocatalyst for the oxidation of glucose. Thus, nucleic acid molecules were quantified amperometrically at femtomolar levels. The effect of experimental variables on the amperometric response was investigated and optimized to maximize the sensitivity and speed up the assay time. A detection limit of 1.0 fmol/L in 1.0-microL droplets and a linear current-concentration relationship up to 800 fmol/L were attained following a 30-min hybridization. The biosensor was applied to the detection of the 16S gene in a mixture of Escherichia coli 16S + 32S rRNA and a full-length rat housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), of a RT-PCR product.     

Sensitive detection of mercury (II) ion using water-soluble captopril-stabilized fluorescent gold nanoparticles

In our work, a simple, facile, and green method was developed for the synthesis of water-soluble and well-dispersed fluorescent gold nanoparticles (Au NPs) within 5 min, using captopril as a capping agent. The as-prepared Au NPs showed strong emission at 414 nm, with a quantum yield of 5.5%. The fluorescence of the Au NPs can be strongly quenched by mercury (II) ion (Hg^2 +) due to the stronger interactions between thiolates (RS^?) and Hg^2 +. It was applied to the detection of Hg^2 + in water samples in the linear ranges of 0.033–0.133 μM and 0.167–2.500 μM, with a detection limit of 0.017 μM. Therefore, the as-prepared Au NPs can meet the requirement for monitoring Hg^2 + in environmental samples.     

Toggled RNA aptamers against aminoglycosides allowing facile detection of antibiotics using gold nanoparticle assays

We have used systematic evolution of ligands by exponential enrichment (SELEX) to isolate RNA aptamers against aminoglycoside antibiotics. The SELEX rounds were toggled against four pairs of aminoglycosides with the goal of isolating reagents that recognize conserved structural features. The resulting aptamers bind both of their selection targets with nanomolar affinities. They also bind the less structurally related targets, although they show clear specificity for this class of antibiotics. We show that this lack of aminoglycoside specificity is a common property of aptamers previously selected against single compounds and described as "specific". Broad target specificity aptamers would be ideal for sensors detecting the entire class of aminoglycosides. We have used ligand-induced aggregation of gold-nanoparticles coated with our aptamers as a rapid and sensitive assay for these compounds. In contrast to DNA aptamers, unmodified RNA aptamers cannot be used as the recognition ligand in this assay, whereas 2'-fluoro-pyrimidine derivatives work reliably. We discuss the possible application of these reagents as sensors for drug residues and the challenges for understanding the structural basis of aminoglycoside-aptamer recognition highlighted by the SELEX results.     

Cathodic detection of H2O2 using iodide-modified gold electrode in alkaline media

Oxidative chemisorption and cathodic stripping reductive desorption of iodide have been studied at a smooth polycrystalline gold (Au (poly)) electrode. Potential-dependent surface coverage of iodide has been controlled on the basis of its reductive desoprtion in 0.1 M KOH alkaline media and its quantitative oxidation to aqueous iodates in acidic media. The Au (poly) electrode surface catalyzes the decomposition of H2O2 to O2. Specific adsorption of iodide on the Au electrode inhibits fully the catalytic decomposition and electrochemical oxidation of H2O2 as well as the adsorption of unknown impurities and the oxidative degradation of the electrode surface by H2O2. A quantitative characterization/detection of H2O2 at the iodide-modified Au (poly) electrode in the alkaline media has, thus, been achieved. Performance of the electrode toward the detection of H2O2 with respect to response time and sensitivity as well as operational stability has been evaluated. It has a sensitivity of 0.272 mA cm(-2) mM(-1) in amperometric measurements with a detection limit of 1.0 x 10(-5) M H2O2, and the response time to achieve 95% of the steady-state current is <20 s. The effect of O2 in the air-saturated solution can be minimized by subtracting the additional current for the O2 reduction. Experimental measurements were based upon cyclic voltametric and amperometric techniques.     

DNA sequence detection using selective fluorescence quenching of tagged oligonucleotide probes by gold nanoparticles

Simple, fast, economical, and sensitive detection of specific DNA sequences is crucial to pathogen detection and biomedical research. We have designed a novel fluorescent assay for DNA hybridization based on the electrostatic properties of DNA. We exploit the ability to create conditions where single-stranded DNA adsorbs on negatively charged gold nanoparticles while double-stranded DNA does not. Dye-tagged probe sequences have their fluorescence efficiently quenched when they are mixed with gold nanoparticles unless they hybridize with components of the analyte. Subfemtomole amounts of untagged target are detected in minutes using commercially available materials. Target sequences in complex mixtures of DNA and single-base mismatches in DNA sequences are easily detected.     

Outliers detection using an iterative strategy for semi‐supervised learning

As a direct consequence of production systems' digitalization, high‐frequency and high‐dimensional data has become more easily available. In terms of data analysis, latent structures‐based methods are often employed when analyzing multivariate and complex data. However, these methods are designed for supervised learning problems when sufficient labeled data are available. Particularly for fast production rates, quality characteristics data tend to be scarcer than available process data generated through multiple sensors and automated data collection schemes. One way to overcome the problem of scarce outputs is to employ semi‐supervised learning methods, which use both labeled and unlabeled data. It has been shown that it is advantageous to use a semi‐supervised approach in case of labeled data and unlabeled data coming from the same distribution. In real applications, there is a chance that unlabeled data contain outliers or even a drift in the process, which will affect the performance of the semi‐supervised methods. The research question addressed in this work is how to detect outliers in the unlabeled data set using the scarce labeled data set. An iterative strategy is proposed using a combined Hotelling's T² and Q statistics and applied using a semi‐supervised principal component regression (SS‐PCR) approach on both simulated and real data sets.