Study of thiazole orange in aptamer-based dye-displacement assays

We screened a series of RNA and DNA aptamers for their ability to serve in the dye displacement assays in which analytes compete with TO dye. We conclude that, while the performance of the TO dye displacement approach is not always predictable, it is still a simple and sensitive assay to detect binding between RNA aptamers and small molecules. In particular, we describe efficient assays for tobramycin and theophylline, with up to 90% displacement of TO observed, and we describe the first aptameric assay for cAMP. Figure An RNA or DNA aptamer against a molecule (circle) binds TO dye, resulting in a fluorescent complex. Presence of free molecule in solution results in the displacement of TO from the complex and a reduction in fluorescence Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Diagonal chromatographic selection of cysteinyl peptides modified with benzoquinones

The derivatization of cysteine-containing peptides with benzoquinone compounds is rapid, quantitative and specific in acidic media. The conversion of cysteines into hydrophobic benzoquinone-adducted residues in peptides is used here to alter the chromatographic properties of cysteinyl peptides during liquid chromatography separation. The benzoquinone derivatization is shown to allow the accurate selection of cysteine-containing peptides of bovine serum albumin tryptic digest by diagonal reversed-phase chromatography, which consists of one primary and a series of secondary identical liquid chromatographic separations, before and after a cysteinyl-targeted modification of the peptides by benzoquinone compounds. Figure Diagonal chromatographic selection of cysteinyl peptides modified with benzoquinones Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Development of a photodiode array biochip using a bipolar semiconductor and its application to detection of human papilloma virus

We describe a DNA microarray system using a bipolar integrated circuit photodiode array (PDA) chip as a new platform for DNA analysis. The PDA chip comprises an 8 × 6 array of photodiodes each with a diameter of 600 μm. Each photodiode element acts both as a support for an immobilizing probe DNA and as a two-dimensional photodetector. The usefulness of the PDA microarray platform is demonstrated by the detection of high-risk subtypes of human papilloma virus (HPV). The polymerase chain reaction (PCR)-amplified biotinylated HPV target DNA was hybridized with the immobilized probe DNA on the photodiode surface, and the chip was incubated in an anti-biotin antibody-conjugated gold nanoparticle solution. The silver enhancement by the gold nanoparticles bound to the biotin of the HPV target DNA precipitates silver metal particles at the chip surfaces, which block light irradiated from above. The resulting drop in output voltage depends on the amount of target DNA present in the sample solution, which allows the specific detection and the quantitative analysis of the complementary target DNA. The PDA chip showed high relative signal ratios of HPV probe DNA hybridized with complementary target DNA, indicating an excellent capability in discriminating HPV subtypes. The detection limit for the HPV target DNA analysis improved from 1.2 nM to 30 pM by changing the silver development time from 5 to 10 min. Moreover, the enhanced silver development promoted by the gold nanoparticles could be applied to a broader range of target DNA concentration by controlling the silver development time. Figure An optical image of the PDA chip and target DNA detection through silver enhancement Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Aptamer-based label-free method for hemin recognition and DNA assay by capillary electrophoresis with chemiluminescence detection

An aptamer-based label-free approach to hemin recognition and DNA assay using capillary electrophoresis with chemiluminescence detection is introduced here. Two guanine-rich DNA aptamers were used as the recognition element and target DNA, respectively. In the presence of potassium ions, the two aptamers folded into the G-quartet structures, binding hemin with high specificity and affinity. Based on the G-quartet–hemin interactions, the ligand molecule was specifically recognized with a K _d ≈ 73 nM, and the target DNA could be detected at 0.1 μM. In phosphate buffer of pH 11.0, hemin catalyzed the H₂O₂-mediated oxidation of luminol to generate strong chemiluminescence signal; thus the target molecule itself served as an indicator for the molecule–aptamer interaction, which made the labeling and/or modification of aptamers or target molecules unnecessary. This label-free method for molecular recognition and DNA detection is therefore simple, easy, and effective. Figure A label-free approach to aptamer-based hemin recognition and DNA detection is introduced, which gives great potential for using a small molecule itself as the indicator for molecular recognition and DNA detection thereby avoiding any labeling or modification step     

Real-time PCR detection of telomerase activity using specific molecular beacon probes

Telomerase is a potentially important biomarker and a prognostic indicator of cancer. Several techniques for assessing telomerase activity, including the telomeric repeat amplification protocol (TRAP) and its modified versions, have been developed. Of these methods, real-time quantitative TRAP (RTQ-TRAP) is considered the most promising. In this work, a novel RTQ-TRAP method is developed in which a telomeric repeats-specific molecular beacon is used. The use of the molecular beacon can improve the specificity of the RTQ-TRAP assay, making the method suitable for studying the overall processivity results and the turnover rate of telomerase. In addition, the real-time, closed-tube protocol used obviates the need for post-amplification procedures, reduces the risk of carryover contamination, and supports high throughput. Its performance in synthetic telomerase products and cell extracts suggests that the developed molecular beacon assay can further enhance the clinical utility of telomerase activity as a biomarker/indicator in cancer diagnosis and prognosis. The method also provides a novel approach to the specific detection of some particular gene sequences to which sequence-specific fluorogenic probes cannot be applied directly. Figure Real-time PCR detection of telomerase activity using specific molecular beacon probes Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Electrochemical preparation of copper–dendrimer nanocomposites: picomolar detection of Cu<Superscript>2+</Superscript> ions

The present work describes, for the first time, in situ electrochemical preparation of dendrimer-encapsulated Cu nanoparticles using a self-assembled monolayer of fourth-generation amine-terminated polyamidoamine (PAMAM) dendrimer as the template. Atomic force microscopy (AFM) and X-ray photoelectron spectroscopy (XPS) studies of the modified surface confirmed the presence of Cu nanoparticles entrapped in dendrimer film. Au electrode modified with a monolayer of the dendrimer enables preconcentration and subsequent voltammetric detection of Cu²⁺ at picomolar concentrations. Further, Cu nanoparticles in the dendrimer monolayer could be electrochemically derivatised to Cu hexacyanoferrate, which exhibits specific crystal planes, unlike the random distribution of crystal planes in bulk-formed Cu hexacyanoferrate, which is another catalytically active material for sensor applications. Figure Electrochemical preparation of copper–dendrimer nanocomposite     

Application of surface chemical analysis tools for characterization of nanoparticles

The important role that surface chemical analysis methods can and should play in the characterization of nanoparticles is described. The types of information that can be obtained from analysis of nanoparticles using Auger electron spectroscopy (AES), X-ray photoelectron spectroscopy (XPS), time-of-flight secondary-ion mass spectrometry (TOF-SIMS), low-energy ion scattering (LEIS), and scanning-probe microscopy (SPM), including scanning tunneling microscopy (STM) and atomic force microscopy (AFM), are briefly summarized. Examples describing the characterization of engineered nanoparticles are provided. Specific analysis considerations and issues associated with using surface-analysis methods for the characterization of nanoparticles are discussed and summarized, with the impact that shape instability, environmentally induced changes, deliberate and accidental coating, etc., have on nanoparticle properties.      

Nuclear magnetic resonance of mass-limited samples using small RF coils

Figure Schematic diagram of a typical arrangement used for hyphenating chemical microseparations (e.g. capillary HPLC, CE, or CEC) with microcoil NMR detection     

Molecularly imprinted sol–gel nanoparticles for mass-sensitive engine oil degradation sensing

Titanate sol–gel layers imprinted with midchain carbonic acids have proven highly useful for detecting engine oil degradation processes owing to selective incorporation of oxidised base oil components. Synthesising the material from TiCl₄ in CCl₄ and precipitating with water leads to imprinted TiO₂ nanoparticles with a diameter of 200–300 nm. Replacing the water by a 1 M ammonium hydroxide solution reduces the average particle size to 50–100 nm with retention of the interaction capabilities. Experiments with the latter solution revealed that the 100-nm particles take up substantially more analyte, indicating a size-dependent phenomenon. As the number of interaction sites within each material is the same, this cannot be a consequence of thermodynamics but must be one of accessibility. The sensor characteristic of water-precipitated particles towards engine oil degradation products shows substantially increased sensitivity and dynamic range compared with the corresponding thin films. Coating quartz crystal microbalances with such nanoparticle materials leads to engine oil degradation sensors owing to incorporation of acidic base oil oxidation products. Interaction studies over a large range of layer thicknesses revealed that both the absolute signal and the steepness of the correlation between the sensor signal and the layer height is 2 times higher for the particles. Figure Generation of molecularly imprinted sol–gel nanoparticles     

Evaluation of two methods for the extraction of antioxidants from medicinal plants

The efficiencies of two traditional extraction methods used in Chinese medicine (the decoction method and the maceration method) were evaluated for the extraction of antioxidants from medicinal plants. A group of medicinal plants possessing nutritious and tonic functions were chosen as model plants. A commonly used extraction method was used as a reference method. The antioxidant capacities and total phenolic contents of the extracts were measured by ferric-reducing antioxidant power and Trolox equivalent antioxidant capacity assays as well as the Folin–Ciocalteu method, respectively. The results obtained indicated that the two traditional extraction methods could effectively extract antioxidants from medicinal plants. These extraction methods can be applied to the analysis and purification of antioxidants in plants, respectively. At home, people can use these methods to extract antioxidants from plants for consumption. In the food industry, these methods could be utilized to prepare crude extracts from plants containing antioxidants for use as food additives. Figure Relation and comparison of extraction efficiencies of two traditional extraction methods with the reference method Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Collision-induced reporter fragmentations for identification of covalently modified peptides

Collision-induced reporter fragmentations of the currently most important covalent peptide modifications as detected by tandem mass spectrometry are summarized. These fragmentations comprise the formation of reporter ions, which are preferentially immonium ions, immonium ion-derived fragments or side chain fragments. In addition, the reporter neutral loss reactions for covalently modified amino acid residues are summarized. For each individual covalent modification which can be recognized by a reporter fragmentation, the accurate mass shift and the gross formula shift of the modified amino acid residue are given. The same set of data is provided for the reporter fragmentations. Finally, an extensive accurate mass and gross formula list is presented as supplementary material, describing mostly regular and modified y₁ and dipeptide a and b ions, which are helpful for identification of the peptide ends of covalently modified peptides. Figure When modified peptides are fragmented by collision-induced dissociation in a tandem mass spectrometer, the modification is either lost as part of a charged fragment, so that a reporter ion for the modification is generated or it is lost as part of a neutral fragment, so that a modification-specific reporter neutral loss is observed in the fragment ion spectrum. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Chien-Wen Hung and Andreas Schlosser contributed equally to this work.     

Enhanced recognition of non-complementary hybridization by single-LNA-modified oligonucleotide probes

Locked nucleic acid (LNA) is a deoxyribonucleotide analogue with an unusual ‘locked’ furanose conformation. LNA-modified oligonucleotide probes have demonstrated an enhanced binding affinity towards their complementary strands; however, their potential to discriminate non-complementary hybridization of mismatches has not been explored. In this study, we investigated the effect of the chemical nature of LNA nucleobases on the hybridization stability and the capability of LNA-modified oligonucleotides to discriminate the LNA:DNA mismatched base pairs. It was observed that LNA modification indeed improves the discrimination capability of oligonucleotides by increasing the melting temperature differences between the complementary duplexes and hybrids containing mismatches. Particularly, LNA purines offer a greater potential to recognize the mismatches than LNA pyrimidines and DNA purines. Real-time PCR experiments further confirmed that LNA modifications at the 3′-end are more effective. The results and conclusions in this study provide useful information for hybridization-based nucleic acid analysis where designing sound oligonucleotide probes is crucial to the success of the analyses.   Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Monitoring surface-assisted biomolecular assembly by means of evanescent-field-coupled waveguide-mode nanobiosensors

Biological self-assembly is a natural process that involves various biomolecules, and finding the missing partner in these interactions is crucial for a specific biological function. Previously, we showed that evanescent-field-coupled waveguide-mode sensor in conjunction with a SiO₂ waveguide, the surfaces which contain cylindrical nanometric holes produced by atomic bombardment, allowed us to detect efficiently the biomolecular interactions. In the present studies, we showed that the assembly of biomolecules can be monitored using the evanescent-field-coupled waveguide-mode biosensor and thus provide a methodology in monitoring assembly process in macromolecular machines while they are assembling. Evanescent-field-coupled waveguide-mode sensor Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Enhanced resonance light scattering properties of gold nanoparticles due to cooperative binding

The interaction of 11-mercaptoundecanoic acid capped gold nanoparticles (MUA-GNPs) with europium ions and aminoacids has been studied by UV-Vis spectrophotometry, fluorescence, confocal fluorescence microscopy, resonance light scattering and TEM. Results demonstrated that hyper-Rayleigh scattering emission occurs upon the addition of lysine to the MUA-GNPs–Eu(III) system, thus providing an inherently sensitive method for lysine determination. The effects of geometrical factors of the gold nanoparticles (aspect ratio, particle size, cluster formation) and the surrounding medium (pH) on this behavior are discussed. The cooperative binding interactions of Eu³⁺ and lysine with gold nanoparticles permitted the discrimination of lysine from other amino acids. The probable mechanism for the spectral changes and the enhanced resonance light scattering observed is outlined. Figure Gold nanoparticle resonance light scattering plasmon enhancement through cooperative binding with europium and lysine     

Surface plasmon resonance study on HIV-1 integrase strand transfer activity

Understanding the molecular mechanism of HIV-1 integrase (IN) activity is critical to find functional inhibitors for an effective AIDS therapy. A robust, fast, and sensitive method for studying IN activity is required. In this work, an assay for real-time label-free monitoring of the IN activity based on surface plasmon resonance was developed. This assay enabled direct monitoring of the integration of a viral doubled-stranded (ds) DNA into the host genome. The strand transfer reaction was detected by using two different DNA targets: supercoiled plasmid (pUC 19) and short palindrome oligonucleotide. The effect of the length of the DNA target on the possibility to monitor the actual process of the strand transfer reaction is discussed. The surface density of integrated ds-DNA was determined. IN binding to the oligonucleotide complexes and model DNA triplexes in the presence of various divalent ions as metal cofactors was investigated as well. The assay developed can serve as an important analytical tool to search for potential strand transfer reaction inhibitors as well as for the study of compounds interfering with the binding of ds long terminal repeats–IN complexes with the host DNA. HIV-1 integrase strand transfer activity was monitored in real time using a multichannel surface plasmon resonance biosensor. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Electrospray mass spectrometry analysis of dendritic branches bearing peripheral fullerene subunits

The electrospray mass spectrometric characterization of neutral dendrons with a carboxylic acid function or a t-butyl ester moiety at the central point and up to eight peripheral C₆₀ subunits has been performed and is described in detail. Molecules bearing a carboxylic acid group at the center turned out to be preferentially ionized by deprotonation, whereas those with a t-butyl ester head group were ionized by reduction of the C₆₀ units in the infusion capillary of the electrospray source. Electronic supplementary material Supplementary material is available for this article at and is accessible for authorized users.     

Aptamers as molecular recognition elements for electrical nanobiosensors

Recent advances in nanotechnology have enabled the development of nanoscale sensors that outperform conventional biosensors. This review summarizes the nanoscale biosensors that use aptamers as molecular recognition elements. The advantages of aptamers over antibodies as sensors are highlighted. These advantages are especially apparent with electrical sensors such as electrochemical sensors or those using field-effect transistors. Figure Feeling proteins with aptamer-functionalized carbon nanotubes     

Biosensor for total cholesterol estimation using N-(2-aminoethyl)-3-aminopropyltrimethoxysilane self-assembled monolayer

Cholesterol oxidase (ChOx), cholesterol esterase (ChEt), and horseradish peroxidase (HRP) have been co-immobilized covalently on a self-assembled monolayer (SAM) of N-(2-aminoethyl)-3-aminopropyltrimethoxysilane (AEAPTS) deposited on an indium–tin–oxide (ITO) glass surface. These enzyme-modified (ChOx-ChEt-HRP/AEAPTS/ITO) biosensing electrodes have been used to estimate cholesteryl oleate from 10 to 500 mg dL⁻¹. The sensitivity, K _m value, and shelf-life of these ChEt-ChOx-HRP/AEAPTS/ITO biosensing electrodes have been found to be 124 nA mg⁻¹ dL, 95.098 mg dL⁻¹ (1.46 mmol L⁻¹), and ten weeks, respectively. The ChEt-ChOx-HRP/AEAPTS/ITO bio-electrodes have been used to estimate total cholesterol in serum samples. Figure Covalent immobilization of enzymes onto AEAPTS/ITO surface using EDC/NHS chemistry Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Surface modification using a novel type I hydrophobin HGFI

Surface wettability conversion with hydrophobins is important for its applications in biodevices. In this work, the application of a type I hydrophobin HGFI in surface wettability conversion on mica, glass, and poly(dimethylsiloxane) (PDMS) was investigated. X-ray photoelectron spectroscopy (XPS) and water-contact-angle (WCA) measurements indicated that HGFI modification could efficiently change the surface wettability. Data also showed that self-assembled HGFI had better stability than type II hydrophobin HFBI. Protein patterning and the following immunoassay illustrated that surface modification with HGFI should be a feasible strategy for biosensor device fabrication. Figure A hydrophobin HGFI has been applied into surface wettability conversion for protein immobilization Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Alternating current anodic stripping voltammetry in the study of cadmium complexation by a reference Suwannee river fulvic acid: a model case with strong electrode adsorption and weak binding

The possibilities of anodic stripping voltammetry (ASV) using an alternating current (AC) scan in the stripping step have been checked through the study of the complexation of cadmium by Suwannee river fulvic acid (SRFA), a reference fulvic acid from the International Humic Substances Society. Because of the strong electrode adsorption of SRFA, AC mode appears to be a good approach to the study when proper selection of the phase angle is made. The goodness of AC mode in ASV has been demonstrated, and the complexation constant of 3.71 ± 0.04 determined is in good agreement with the value of the constant obtained by the reference technique of reverse pulse polarography. Some particularities of SRFA have been observed, among them its homofunctional and strongly heterogeneous behaviour in cadmium complexation and the impossibility of avoiding electrode adsorption problems in ASV measurements at very low metal concentrations. Figure DP anodic stripping and AC anodic stripping voltammograms at −12° and −65° during the titration of a 10⁻⁷ mol L⁻¹ Cd(II) solution with SRFA at pH 7.5 in 0.05 L⁻¹ Tris Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.