Signal enhancement in capillary electrophoresis by using a sleeve cell arrangement for optical detection

This study comparts signal enhancement and efficiency in CZK employing three modes of detection: the sleeve cell (a simple method for creating a region of extended path length for absorption detection), the bubble cell (extended light path capillaries), and on-column detection in 75 μm i.d. capillary. Flow profile in the sleeve cell was monitored under a microscope. An abrupt change in capillary diameter in the sleeve cell region (from 50 μm to 220 μm) did not produce extensive band broadening. The sleeve-cell detection arrangement delivered a 3.5 fold increase in corrected peak area when compared with an oil-column detection in 75 μm i.d. column.     

Amperometric method for rapid detection of Escherichia coli by flow injection analysis using a bismuth nano-film modified glassy carbon electrode

A new method for rapid detection of Escherichia coli (E. coli) was developed by flow injection analysis (FIA) using bismuth nano-film modified glassy carbon electrode (BiNFE) in this paper. The method depended on a good marker β-d-glucuronidase which is found in E. coli strains. β-d-Glucuronidase was produced by the induction of methyl-β-d-glucuronide sodium (MetGlu), then released from E. coli cells through the permeabilization of cell membrane caused by polymyxin B nonapeptide and lysozyme. The released β-d-glucuronidase could catalyze the hydrolysis of the substrate 4-nitrophenyl β-d-glucuronide (PNPG) in the culture medium to produce 4-nitrophenol. Since 4-nitrophenol is electroactive and its quantity is proportional to the concentration of E. coli, E. coli could be determined by electroanalysis of 4-nitrophenol. The BiNFE was fabricated by an electrodeposition of metallic bismuth onto a glassy carbon electrode, which showed a high sensitivity in determination of 4-nitrophenol when used in conjunction with FIA system. Experimental results showed that the amplified response current of 4-nitrophenol obtained at the BiNFE was linear with the concentration of E. coli ranging from 1.5 × 10² to 1.0 × 10⁶ cfu/ml, the detection limit of this method for E. coli is 100 cfu/ml, and the complete assay was performed in 3 h.     

Developing a genetically encoded green fluorescent protein mutant for sensitive light-up fluorescent sensing and cellular imaging of Hg(II)

Hg(II) is well-known for quenching fluorescence in a distance dependent manner. Nevertheless, when we exposed the fluorophore of a green fluorescent protein (GFP) toward Hg(II), through H148C mutation, the GFP fluorescence could be “lighted up” by Hg(II) down to sub-nM level. The detection linear range is 0.5–3.0 nM for protein solutions at 8.0 nM. The GFPH148C protein displayed a promising selectivity toward Hg(II) and also the cellular imaging capacity. Spectra measurements suggested that the ground-state redistribution of protein contributed to the fluorescence enhancement, which was found not limited to Hg(II), and thus presented an opening for building a pool of GFP-based chemosensors toward other heavy metal ions.     

Rapid detection of Escherichia coli by flow injection analysis coupled with amperometric method using an IrO2–Pd chemically modified electrode

An amperometric method for the rapid detection of Escherichia coli (E. coli) by flow injection analysis (FIA) using an IrO₂–Pd chemically modified electrode (CME) was developed in this paper. The method is based on a good marker β-d-galactosidase which was found in E. coli strains. β-d-galactosidase was produced by the induction of isopropyl β-d-thiogalactopyranoside (IPTG) and released from E. coli cells through the permeabilization of both polymyxin B nonapeptide and lysozyme to E. coli cells wall. The released β-d-galactosidase could catalyze the hydrolysis of the substrate p-aminophenyl β-d-galactopyranoside (PAPG) in the culture medium to produce 4-aminophenol which was proportional to the concentration of E. coli. Hence, E. coli could be detected by the determination of 4-aminophenol. An IrO₂–Pd CME, which showed high sensitivity in determination of 4-aminophenol, was prepared as the electro-detector in FIA. The amplified response current of 4-aminophenol obtained at the IrO₂–Pd CME was linear with the concentration of E. coli ranging from 2.0 × 10² to 1.0 × 10⁶ cfu/mL, the detection limit of this method to E. coli was 150 cfu/mL and the complete assay could be performed in 3 h.     

Discovery of a monomeric green fluorescent protein sensor for chloride by structure-guided bioinformatics

Chloride is an essential anion for all forms of life. Beyond electrolyte balance, an increasing body of evidence points to new roles for chloride in normal physiology and disease. Over the last two decades, this understanding has been advanced by chloride-sensitive fluorescent proteins for imaging applications in living cells. To our surprise, these sensors have primarily been engineered from the green fluorescent protein (GFP) found in the jellyfish Aequorea victoria. However, the GFP family has a rich sequence space that could already encode for new sensors with desired properties, thereby minimizing protein engineering efforts and accelerating biological applications. To efficiently sample this space, we present and validate a stepwise bioinformatics strategy focused first on the chloride binding pocket and second on a monomeric oligomerization state. Using this, we identified GFPxm163 from GFPxm found in the jellyfish Aequorea macrodactyla. In vitro characterization shows that the binding of chloride as well as bromide, iodide, and nitrate rapidly tunes the ground state chromophore equilibrium from the phenolate to the phenol state generating a pH-dependent, turn-off fluorescence response. Furthermore, live-cell fluorescence microscopy reveals that GFPxm163 provides a reversible, yet indirect readout of chloride transport via iodide exchange. With this demonstration, we anticipate that the pairing of bioinformatics with protein engineering methods will provide an efficient methodology to discover and design new chloride-sensitive fluorescent proteins for cellular applications.

We developed a workflow to identify and apply GFPxm163 as a new green fluorescent protein-based sensor for chloride.     

Size characterization of green fluorescent protein inclusion bodies in E. coli using asymmetrical flow field-flow fractionation-multi-angle light scattering

The goal of this study was to investigate the applicability of asymmetrical flow field-flow fractionation-multi-angle light scattering (AsFlFFF-MALS) for size analysis of green fluorescent protein inclusion bodies (GFPIBs). The size distributions of GFPIBs prepared by various culture conditions were determined. For GFPIBs prepared at 37 degrees C the peak maximum hydrodynamic diameter (d(H)) first increased and then decreased with the increase of the induction times in the presence of 0.1 and 2 mM isopropyl-beta-D-thiogalactoside (IPTG). For GFPIBs prepared at 30 degrees C the peak maximum d(H) was constant at about 700 nm irrespectively of the induction times and IPTG concentrations.     

New trends in bioanalytical tools for the detection of genetically modified organisms: an update

Despite the controversies surrounding genetically modified organisms (GMOs), the production of GM crops is increasing, especially in developing countries. Thanks to new technologies involving genetic engineering and unprecedented access to genomic resources, the next decade will certainly see exponential growth in GMO production. Indeed, EU regulations based on the precautionary principle require any food containing more than 0.9% GM content to be labeled as such. The implementation of these regulations necessitates sampling protocols, the availability of certified reference materials and analytical methodologies that allow the accurate determination of the content of GMOs. In order to qualify for the validation process, a method should fulfil some criteria, defined as “acceptance criteria” by the European Network of GMO Laboratories (ENGL). Several methods have recently been developed for GMO detection and quantitation, mostly based on polymerase chain reaction (PCR) technology. PCR (including its different formats, e.g., double competitive PCR and real-time PCR) remains the technique of choice, thanks to its ability to detect even small amounts of transgenes in raw materials and processed foods. Other approaches relying on DNA detection are based on quartz crystal microbalance piezoelectric biosensors, dry reagent dipstick-type sensors and surface plasmon resonance sensors. The application of visible/near-infrared (vis/NIR) spectroscopy or mass spectrometry combined with chemometrics techniques has also been envisaged as a powerful GMO detection tool. Furthermore, in order to cope with the multiplicity of GMOs released onto the market, the new challenge is the development of routine detection systems for the simultaneous detection of numerous GMOs, including unknown GMOs.     

Metal-ion-dependent GFP emission in vivo by combining a circularly permutated green fluorescent protein with an engineered metal-ion-binding coiled-coil

Coordination of metal ions significantly contributes to protein structures and functions. Here we constructed a fusion protein, consisting of a de novo designed, metal-ion-binding, trimeric coiled-coil and a circularly permutated green fluorescent protein (cpGFP), where the fluorescent emission from cpGFP was induced by metal ion coordination to the coiled-coil. A circularly permutated GFP, (191)cpGFP(190), was constructed by connecting the original N- and C-termini of GFP(UV) by a GGSGG linker and cleaving it between Asp(190) and Gly(191). The metal-ion-binding coiled-coil, IZ-HH, was designed to have three alpha-helical structures, with 12 His residues in the hydrophobic core of the coiled-coil structure. IZ-HH exhibited an unfolded structure, whereas it formed the trimeric coiled-coil structure in the presence of divalent metal ions, such as Cu(2+), Ni(2+), or Zn(2+). The fusion protein (191)cpGFP(190)-IZ-HH was constructed, in which (191)cpGFP(190) was inserted between the second and third alpha-helices of IZ-HH. Escherichia coli cells, expressing (191)cpGFP(190)-IZ-HH, exhibited strong fluorescence when the Cu(2+) and Zn(2+) ions were present in the medium, indicating that they passed through the cell membrane and induced the proper folding of the (191)cpGFP(190) domain. This strategy, in which protein function is regulated by a metal-ion-responsive coiled-coil, should be applicable to the design of various metal-ion-responsive, nonnatural proteins that work both in vitro and in vivo.     

Validation of an immunoassay for detection and quantitation of a genetically modified soybean in food and food fractions using reference materials: interlaboratory study

An immunoassay for detection of a specific genetically modified soybean (Roundup-Ready) was validated on dried soybean powder in an interlaboratory study. Different percentages of genetically modified soybeans in nonmodified soybean matrix were evaluated in a blind study. Thirty-eight laboratories from 13 countries participated. The immunoassay was evaluated for 2 endpoints: (1) To give a semiquantitative result, i.e., determination of a given sample above or below a given threshold, or (2) to compute a quantitative result, i.e., percentage of genetically modified soybeans in the sample. Semiquantitative results showed that a given sample which contained <2% genetically modified soybeans was identified as below 2% with a 99% confidence level. Quantitative use of the assay resulted in a repeatability (r) and reproducibility (R) that were computed to be RSDr = 7% and RSDR = 10%, respectively, for a sample containing 2% genetically modified soybeans. Application of this method depends on availability of appropriate reference materials for a specific food matrix. Only matrix-matched reference materials can be used for analysis of food or food fractions.     

Quantification of green fluorescent protein in cellular supernatant by capillary electrophoresis with laser-induced fluorescence detection for measurement of cell death

A common method for quantifying cell death is measuring the concentration of lactate dehydrogenase (LDH) released by cells as their membranes become unstable. In cells expressing green fluorescent protein (GFP), degradation of the cell membrane also results in the release of GFP into the surrounding supernatant. In this study, we used capillary electrophoresis with laser-induced fluorescence detection to measure the levels of GFP in supernatants of UBIGFP/BL6 primary macrophages that had been infected with Salmonella typhimurium, treated with staurosporine, or exposed to H₂O₂, all known inducers of cell death. We also used a standard LDH assay to measure the release of LDH into supernatants. We observed the rate of cell death quantified by release of GFP and LDH into supernatant to be essentially identical, demonstrating that GFP release is at least as good as an indicator of macrophage cell death as the established LDH release method.     

Simplified determination of bacterial contamination by Escherichia coli using a flow injection system with piezoelectric detection

A rapid and automated method was developed for the determination of bacterial contamination and using Escherichia coli as a model microorganism. The method involves the use of a sensor connected to a flow injection (FI) system. The sample is introduced through a flow injection system into a piezoelectric quartz crystal (PQC) flow-cell. The resulting change of the resonance frequency is related to the bacterial contamination in the sample. The parameters associated with the flow system and the conditions for introducing the sample culture were optimized. Calibration curves are linear in the range from 3.2?×?10⁷ to 3.2?×?10⁹ cfu per mL^-1, with a correlation coefficient of 0.997. The reproducibility was between 3.1 and 7.6%, and the detection limit is 1.1?×?10⁷ cfu per mL^-1. The method allowed the determination of bacterial contamination in residual water and in samples of milk and chicken stock within 5 h, while the conventional plate count method requires 24 to 48 h. The results obtained by these two methods are in good agreement.

Simulation of large-scale production of a soluble recombinant protein expressed in Escherichia coli using an intein-mediated purification system

Inteins are self-cleavable proteins that under reducing conditions can be cleaved from a recombinant target protein. Industrially, an intein-based system could potentially reduce production costs of recombinant proteins by facilitating a highly selective affinity purification using an inexpensive substrate such as chitin. In this study, SuperPro Designer was used to simulate the large-scale recovery of a soluble recombinant protein expressed in Escherichia coli using an intein-mediated purification process based on the commercially available IMPACT system. The intein process was also compared with a conventional process simulated by SuperPro. The intein purification process initially simulated was significantly more expensive than the conventional process, primarily owing to the properties of the chitin resin and high reducing-agent (dithiothreitol [DTT]) raw material cost. The intein process was sensitive to the chitin resin binding capacity, cleavage efficiency of the intein fusion protein, the size of the target protein relative to the intein tag, and DTT costs. An optimized intein purification process considerably reduced costs by simulating an improved chitin resin and alternative reducing agents. Thus, to realize the full potential of intein purification processes, research is needed to improve the properties of chitin resin and to find alternative, inexpensive raw materials.     

Chemically modified dansyl probes: a fluorescent diagnostic for ion and proton detection in solution and in polymers

[reaction: see text] The fluorescence emission intensity of the dansyl group is significantly diminished upon appending an ethyldimethylamino group to the N1 nitrogen substituent. Addition of acids and metal ions (i.e., Zn(2+)) to solutions of trimethylethylenediamine naphthalene sulfonamide (trinsyl) 2 produces a >25-fold increase in fluorescence intensity. Trinsyl probe 2 has been used as a diagnostic for the diffusion of protons and metal ions in a network polymer as well as an optical reporter for the glass transition temperature.     

Direct monitoring of the expression of the green fluorescent protein-extracellular signal-regulated kinase 2 fusion protein in transfected cells using capillary electrophoresis with laser-induced fluorescence detection

Proper subcellular localization of the extracellular signal-regulated kinases (ERKs) is important in regulating physiological functions such as proliferation and differentiation in the pheochromocytoma cell line (PC12 cells). Thus, a direct visualization method is necessary to observe ERK localization within the cell or in crude cellular extracts. In this paper, a determination method was established for the detection of ERK2 localization in PC12 cells using green fluorescent protein (GFP) and capillary electrophoresis with laser-induced fluorescence (LIF). GFP as a reporter or labeling tag for gene expression in biochemistry and cell biology was used for the detection of ERK2 localization in PC12 cells. PC12 cells were transfected with GFP-ERK2 plasmid construct that was inserted into a variant GFP gene (enhanced green fluorescent protein), and successfully expressed GFP-ERK2 fusion proteins. GFP-ERK2 fusion proteins were detected within 5 min by CE analysis using an uncoated fused-silica capillary with LIF. Optimum conditions for GFP-ERK2 fusion proteins detection were 100 mM 3-(cyclohexylamino)-1-propanesulfonic acid buffer containing 100 mM sodium dodecylsulfate, pH 11, running at 20 degrees C. This result offers new opportunity in screening for the determination of localization of intracellular components, protein-protein interactions and kinase activity within the cells.     

Porous silicon surfaces: a candidate substrate for reverse protein arrays in cancer biomarker detection

This paper introduces a new substrate for reverse-phase protein microarray applications based on macroporous silicon. A key feature of the microarray substrate is the vastly surface enlarging properties of the porous silicon, which simultaneously offers highly confined microarray spots. The proof of principle of the reverse array concept was demonstrated in the detection of different levels of cyclin E, a possible cancer biomarker candidate which regulates G1-S transition and correlates with poor prognosis in different types of human cancers. The substrate properties were studied performing analysis of total cyclin E expression in human colon cancer cell lines Hct116 and SW480. The absence of unspecific binding and good microarray quality was demonstrated. In order to verify the performance of the 3-D textured macroporous surface for complex biological samples, lysates of the human tissue spiked to different levels with cell extract overproducing cyclin E (Hct116) were arrayed on the chip surface. The samples were spotted in a noncontact mode in 100 pL droplets with spots sizes ranged between 50 and 70 mum and spot-to-spot center distances 100 mum, allowing microarray spot densities up to 14 000 spots per cm(2). The different sample types of increasing complexities did not have any impact on the spot intensities recorded and the protein spots showed good homogeneity and reproducibility over the recorded microarrays. The data demonstrate the potential use of macroporous silicon as a substrate for quantitative determination of a cancer biomarker cyclin E in tissue lysates.     

Impedimetric detection of trinitrotoluene by using a glassy carbon electrode modified with a gold nanoparticle@fullerene composite and an aptamer-imprinted polydopamine

Microchimica Acta - This study introduces a method based on a combination of aptasensing and molecular imprinting for highly sensitive detection of 2,4,6-trinitrotoluene (TNT). An amino-aptamer...     

Measurement of radon in air by α track method enhancement of detection sensitivity using a lamp

A new α track method is proposed for the measurement of radon (²²²Rn) concentration in environmental levels. This involves collecting radon daughters on the surface of pilot lamp and detecting α-particles emitted from the nuclides (²¹⁸Po and²¹⁴Po) by a detector (LR 115). The detection sensitivity of this method is 6 times greater than that of the conventional α track method.     

Escherichia coli single-stranded DNA-binding protein,a molecular tool for improved sequence quality in pyrosequencing

Pyrosequencing is a four-enzyme bioluminometric DNA sequencing technique based on a DNA sequencing by synthesis principle. Currently, the technique is limited to analysis of short DNA sequences exemplified by single-nucleotide polymorphism analysis. In order to expand the field for pyrosequencing, the read length needs to be improved and efforts have been made to purify reaction components as well as add single-stranded DNA-binding protein (SSB) to the pyrosequencing reaction. In this study, we have performed a systematic effort to analyze the effects of SSB by comparing the pyrosequencing result of 103 independent complementary DNA (cDNA) clones. More detailed information about the cause of low quality sequences on templates with different characteristics was achieved by thorough analysis of the pyrograms. Also, real-time biosensor analysis was performed on individual cDNA clones for investigation of primer annealing and SSB binding on these templates. Results from these studies indicate that templates with high performance in pyrosequencing without SSB possess efficient primer annealing and low SSB affinity. Alternative strategies to improve the performance in pyrosequencing by increasing the primer-annealing efficiency have also been evaluated.