Determination of drug–serum protein interactions via fluorescence polarization measurements

New fast methods for the determination of pharmacokinetic behaviour of potential drug candidates are receiving increasing interest. We present a new homogeneous method for the determination of drug binding and drug competition for human serum albumin and α₁-acid glycoprotein that is amenable to high-throughput-screening. It is based on selective fluorescent probes and the measurement of fluorescence polarization. This leads to decreased interference with fluorescent drugs as compared with previously published methods based on similar probes and the measurement of fluorescence intensity. The binding of highly fluorescent drugs that still interfere with the probes can be measured by simply titrating the drugs in a two-component system with the serum protein. The assay may also be used to discover strongly binding protein ligands that are interesting for drug-targeting strategies. Additionally, binding data could be obtained from larger libraries of compounds for in silico predictive pharmacokinetics. Figure Fluorescence polarization displacement titration of dansylsarcosine (3D-structure as insert) bound to human serum albumin (HSA) by naproxene Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Several concerns about the primer design in the universal molecular beacon real-time PCR assay and its application in HBV DNA detection

A universal hepatitis B virus (HBV) DNA detection kit is appealing for the worldwide diagnosis and monitoring of the treatment of different mutant types of hepatitis B virus. A sensitive and reproducible real-time PCR assay based on the universal molecular beacon (U-MB) technique was developed for the detection of HBV DNA in serum. The U-MB probe used in the assay has no interaction with the HBV DNA sequence. The U-MB technique not only reduced the cost of HBV detection but also had the potential for the development of a universal detection kit for different mutant HBV types and other DNA systems. To demonstrate its clinical utility, 90 serum samples were analyzed using the U-MB real-time PCR method. In the experiments we found that several crucial factors needed to be considered in the primer design, such as the avoidance of formation of severe primer–dimer and primer self-hairpin structure. With the optimized primer sets, satisfactory results were obtained for all the tested samples. We concluded that this assay would be an excellent candidate for a universal HBV DNA detection method. Principle of the U-MB real-time PCR method for HBV DNAdetection     

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.     

On-line NMR detection of microgram quantities of heparin-derived oligosaccharides and their structure elucidation by microcoil NMR

The isolation and purification of sufficient quantities of heparin-derived oligosaccharides for characterization by NMR is a tedious and time-consuming process. In addition, the structural complexity and microheterogeneity of heparin makes its characterization a challenging task. The improved mass-sensitivity of microcoil NMR probe technology makes this technique well suited for characterization of mass-limited heparin-derived oligosaccharides. Although microcoil probes have poorer concentration sensitivity than conventional NMR probes, this limitation can be overcome by coupling capillary isotachophoresis (cITP) with on-line microcoil NMR detection (cITP-NMR). Strategies to improve the sensitivity of on-line NMR detection through changes in probe design and in the cITP-NMR experimental protocol are discussed. These improvements in sensitivity allow acquisition of cITP-NMR survey spectra facilitating tentative identification of unknown oligosaccharides. Complete structure elucidation for microgram quantities of the purified material can be carried out through acquisition of 2D NMR spectra using a CapNMR microcoil probe. Survey NMR spectrum obtained by cITP-NMR using a second-generation probe (the microcoil of which is shown) facilitates tentative identification of unknown oligosaccharides (e.g., the heparin-derived tetrasaccharide illustrated)     

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     

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     

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.     

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.     

G-rich oligonucleotide-functionalized gold nanoparticle aggregation

Guanine-rich DNA sequences commonly form helical quadruplex structures via Hoogsteen hydrogen bonds. The aggregation behavior of the nanoparticles, which are functionalized with four-guanine-terminated 27-base sequences at a nanoparticle-to-DNA ratio of 1:60, is investigated. To some extent, the guanine-quadruplex structures between the gold nanoparticles (GNPs) promote nanoparticle aggregation. However, the coordination site of the metal ion on the nanoparticle surface is partially passivated: the stability of guanine-rich DNA-GNPs is slightly lower than that of the usual DNA-GNPs, and the metal-ion specificity of nanoparticle assembly is substantially decreased. Thus, a mechanism for the aggregation of guanine-rich sequence-modified GNPs is proposed. It is possible to obtain a stable guanine-rich sequence-functionalized nanoparticle solution at high ionic strength by regulating guanine-rich DNA sequences. The controllability of guanine-rich sequence-modified nanoparticles makes the secondary structure of DNA a potentially useful candidate for DNA analysis and disease diagnostics. Figure Proposed mechanism for the aggregation of G-rich sequence-functionalized GNP Electronic supplementary material The online version of this article (doi: ) contains supplementary material, which is available to authorized users.     

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.     

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     

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.     

Alteration of biological samples in speciation analysis of metalloproteins

For investigations of metalloproteins by speciation analysis, the integrity of the protein–metal complexes before and during separation is crucial. Knowledge about potential alterations of the samples is thus essential to avoid misinterpretations of the analytical results. Chromatographic element profiles of different cytosolic samples from animal tissues were measured repeatedly to estimate the sample stability. The dependence of the signals on the dwell time of the sample in an autosampling device at 4 °C for a period of 10 h was observed. Alterations in the element content of different metal-containing fractions were quantified by means of recovery values. Some metalloprotein fractions (e.g. ≈27-kDa arsenic, ≈27-kDa iron and different zinc fractions) were stable or only minor alterations were observed and for their investigation an autosampling device is therefore suitable. However, most of the other metalloprotein fractions, especially nickel-containing proteins, showed major alterations: these samples should therefore be analysed immediately after preparation or directly after thawing. Figure Chromatographic manganese-profiles of 11 repeated SEC-ICP-MS-separations of rat brain cytosol. The first sample at time 0 h was the run immediately started after thawing of the prepared cytosol; the other samples were measured hourly, taken from the same sample vial. In addition to the time axis the estimated molecular mass axis is plotted Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Fourier transform ion cyclotron resonance mass spectrometry of covalent adducts of proteins and 4-hydroxy-2-nonenal, a reactive end-product of lipid peroxidation

Covalent adduction of the model protein apomyoglobin by 4-hydroxy-2-nonenal, a reactive end-product of lipid peroxidation, was characterized by nanoelectrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (FTICR). The high mass resolving power and mass measurement accuracy of the instrument facilitated a detailed compositional analysis of the complex reaction product without the need for deconvolution and transformation to clearly show the pattern of adduction and component molecular weights. Our study has also demonstrated the value of electron capture dissociation over collision-induced dissociation for the tandem mass spectrometric determination of site modification for the 4-hydroxy-2-nonenal adduct of oxidized insulin B chain as an example. Figure FTICR allowed characterization of 4-hydroxy-2-nonenal (HNE)-modified apomyoglobin (an expanded spectrum of the +15 charge state is shown)     

Characterizing the interaction between aptamers and human IgE by use of surface plasmon resonance

Human immunoglobulin E (hIgE) is such an important protein, because of its involvement in allergic disease, that it is of significance to study the interactions between it and its recognizing elements. In this report an analytical strategy based on surface plasmon resonance (SPR) was developed to probe the pattern of interaction between hIgE and its recognizing molecules, including aptamers and antibodies. The affinity constants of hIgE for the antibody and the aptamer were compared first; the aptamer has more affinity than the antibody for human IgE. To study their pattern of interaction, three different binding approaches, including adding the antibody and the streptavidin-coupled aptamer to the sensing surface, were designed. The results showed that hIgE captured on the sensing surface could form a multivalent complex with the aptamer. An ELISA-like assay using the aptamer as both capture and detection probes was then developed. This work highlights an SPR method for characterizing the interaction between the protein and aptamers that is useful for study of biomolecular interaction patterns and binding properties. Figure Schematic diagram of the use of surface plasmon resonance for detection of the pattern of interaction of human IgE with its DNA aptamer and antibody     

Novel gel-based rapid test for non-instrumental detection of ochratoxin A in beer

A rapid easy-to-use immunoassay was optimised for the non-instrumental detection of ochratoxin A (OTA) in beer. The analytical method involves preconcentration on the immunoaffinity layer inside a column followed by direct competitive ELISA detection in the same layer. The visual cut-off value, i.e. the lowest OTA concentration resulting in no colour development, was 0.2 μg L^-1. Assay validation was performed using samples spiked with OTA. Thirty-seven naturally contaminated samples were screened with the gel-based method developed and no false-negative results were obtained. The method described offers a simple, rapid and cost-effective screening tool, thus contributing to better health protection of consumers. Figure Gel-based immunoassay of spiked beer samples.     

Fast and sensitive DNA analysis using changes in the FRET signals of molecular beacons in a PDMS microfluidic channel

A new DNA hybridization analytical method using a microfluidic channel and a molecular beacon-based probe (MB-probe) is described. A stem-loop DNA oligonucleotide labeled with two fluorophores at the 5′ and 3′ termini (a donor dye, TET, and an acceptor dye, TAMRA, respectively) was used to carry out a fast and sensitive DNA analysis. The MB-probe utilized the specificity and selectivity of the DNA hairpin-type probe DNA to detect a specific target DNA of interest. The quenching of the fluorescence resonance energy transfer (FRET) signal between the two fluorophores, caused by the sequence-specific hybridization of the MB-probe and the target DNA, was used to detect a DNA hybridization reaction in a poly(dimethylsiloxane) (PDMS) microfluidic channel. The azoospermia gene, DYS 209, was used as the target DNA to demonstrate the applicability of the method. A simple syringe pumping system was used for quick and accurate analysis. The laminar flow along the channel could be easily controlled by the 3-D channel structure and flow speed. By injecting the MB-probe and target DNA solutions into a zigzag-shaped PDMS microfluidic channel, it was possible to detect their sequence-specific hybridization. Surface-enhanced Raman spectroscopy (SERS) was also used to provide complementary evidence of the DNA hybridization. Our data show that this technique is a promising real-time detection method for label-free DNA targets in the solution phase. Figure FRET-based DNA hybridization detection using a molecular beacon in a zigzag-shaped PDMS microfluidic channel     

Profiling of 3,4-methylenedioxymethamphetamine by means of high-performance liquid chromatography

An impurity-profiling method for 3,4-methylenedioxymethamphetamine (MDMA) is presented. The impurities of interest were extracted by solid-phase extraction (SPE) on Bakerbond C₁₈ spe columns from a weakly alkaline solution (pH 8.5). Development of the extraction conditions covered selection of the buffer for dissolution of the sample and the volume of the eluent used to elute the impurities. An important part of the studies was to optimise the separation conditions, and the simplex method was used for this purpose. Cluster analysis was applied for comparison of samples and its grouping. The developed method was based on the areas of 33 selected peaks corresponding to MDMA impurities. All examined samples were correctly classified into clusters corresponding to the synthetic route.      

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.     

Simultaneous use of multiple fluorescent reporter dyes for glucose sensing in aqueous solution

The simultaneous use of several fluorescent reporter dyes in a multicomponent boronic acid-based glucose sensing system is reported. In one application, two dyes with widely different emission wavelengths are used to report changes in glucose concentration. A third glucose-insensitive dye was then added to act as a reference dye and provide for a ratiometric correction to the two reporter dye signals. The inclusion of such a reference dye reduces errors arising from sources such as fluctuations in lamp intensity and sample dilution. The simultaneous use of multiple fluorescent reporter dyes