Screening nucleotide binding to amino acid-coated supports by surface plasmon resonance and nuclear magnetic resonance

Here, we describe a rapid and efficient screening method using surface plasmon resonance (SPR) and saturation transfer difference–nuclear magnetic resonance (STD-NMR) spectroscopy to yield information regarding the residues involved in nucleotide binding to amino acid-coated supports. The aim of this work was to explore the use of these spectroscopic techniques to study amino acid–nucleotide interactions in order to improve the binding specificity of the amino acid ligands used to purify plasmid DNA. For SPR, we present a strategy that immobilizes arginine and lysine on a surface as model supports, and we analyze binding responses when synthetic homo-deoxyoligonucleotides are injected over the amino acid surface. The binding responses are detectable and reproducible despite the small size of the immobilized amino acids. Using STD-NMR, we performed epitope mapping of homo-deoxyoligonucleotides bound to l-arginine–bisoxyran–Sepharose and l-lysine–Sepharose supports. Polynucleotide binding preferences differed; for example, polyC interacted preferentially through its backbone with the two supports, whereas polyT bound the supports through its thymine moiety. STD-NMR combined with SPR measurements was successfully used to screen amino acid–nucleotide interactions and determine the binding affinities of the complexes.     

Detection of carbohydrate-binding proteins by oligosaccharide-modified polypyrrole interfaces using electrochemical surface plasmon resonance

This paper reports on the use of electrochemical surface plasmon resonance (E-SPR) for the detection of carbohydrate-binding proteins. The generation of an SPR sensor specific to lectins Arachis hypogaea (PNA) and Maackia amurensis (MAA) is based on the electrochemical polymerization of oligosaccharide derivatives functionalized by pyrrole groups. The resulting thin conducting polymer films were characterized using E-SPR and atomic force microscopy (AFM). The specific binding of PNA to polypyrrole-lactosyl and of MAA to polypyrrole-3'-sialyllactosyl films was investigated using SPR. The detection limit was 41 nM for PNA and 83 nM for MAA. Through Scatchard analysis and linear transformation of the SPR sensorgram data, association (k(ass)) and dissociation rate constants (k(diss)) could be determined.     

Detection of ozone gas using gold nanoislands and surface plasmon resonance

Gold nanoislands interact with gaseous ozone to produce a surface plasmon resonance shift, similarly to the interaction of ozone and gold nanoparticles in water. Gold nanoislands are produced by sputtering, which significantly simplifies the synthesis and produces controlled size for the gold nanoislands. The shift of surface plasmon resonance peak was monitored while gold nanoislands were exposed to variable concentration of gaseous ozone. The shift was then correlated with ozone concentration. Our current results indicate sensing gaseous ozone at concentration of as low as 20 μg/L is achievable. Gold nanoislands were reversed to their original wavelength and were able to cycle between the wavelengths as ozone was introduced and removed. Potentially, this system can be useful as a sensor that identifies the presence of ozone at low part-per-billion concentrations of ozone in gaseous media.     

Detection of microcystins in environmental samples using surface plasmon resonance biosensor

An indirect inhibitive surface plasmon resonance (SPR) immunoassay was developed for the microcystins (MCs) detection. The bioconjugate of MC-LR and bovine serum albumin (BSA) was immobilized on a CM5 sensor chip. A serial premixture of MC-LR standards (or samples) and monoclonal antibody (mAb) were injected over the functional sensor surface, and the subsequent specific immunoreaction was monitored on the BIAcore 3000 biosensor and generated a signal with an increasing intensity in response to the decreasing MCs concentration. The developed SPR immunoassay has a wide quantitative range in 1-100 μg L⁻¹. Although not as sensitive as conventional enzyme-linked immunosorbent assay (ELISA), the SPR biosensor offered unique advantages: (1) the sensor chip could be reusable without any significant loss in its binding activity after 50 assay-regeneration cycles, (2) one single assay could be accomplished in 50 min (including 30-min preincubation and 20-min BIAcore analysis), and (3) this method did not require multiple steps. The SPR biosensor was also used to detect MCs in environmental samples, and the results compared well with those obtained by ELISA. We conclude that the SPR biosensor offers outstanding advantages for the MCs detection and may be further developed as a field-portable sensor for real-time monitoring of MCs on site in the near future.     

Detection of lectin-glycan interaction using high resolution surface plasmon resonance

We report the real-time and label-free detection of direct disaccharide binding to a lectin using a differential surface plasmon resonance detection method that allows for measurement of nanomolar concentrations of disaccharides.     

Detection of CD4~+ T-lymphocytes from hemodialyzed patients by surface plasmon resonance

A surface plasmon resonance(SPR) method was presented to discriminate hemodialyzed T-lymphocytes from the normal based on antibody-cell recognition.By dynamic reaction with fixed anti-human CD4 antibody,SPR could offer significant signals to distinguish hemodialyzed patients from the healthy controls within 200 s after the cell injection in respect of either rising speed or maximum binding capacity(p < 0.01).The ratio method is also used to exclude the non-specific adsorption.The percentage of hemodialyzed patients’ CD4~+ T cells against the healthy control is 69±18%.The most attractive of the present method is its ability to detect the intact and label-free lymphocytes,and further to detect the subpopulations,or proteins secreted by the desired lymphocytes subset.     

Detection of bisphenol A using a novel surface plasmon resonance biosensor

We present a compact surface plasmon resonance (SPR) biosensor for the detection of bisphenol A (BpA), an endocrine-disrupting chemical. The biosensor is based on an SPR sensor platform (SPRCD) and the binding inhibition detection format. The detection of BpA in PBS and wastewater was performed at concentrations ranging from 0.05 to 1,000 ng/ml. The limit of detection for BpA in PBS and wastewater was estimated to be 0.08 and 0.14 ng/ml, respectively. It was also demonstrated that the biosensor can be regenerated for repeated use. Results achieved with the SPR biosensor are compared with those obtained using ELISA and HPLC methods.     

Detection of Alzheimer's tau protein using localised surface plasmon resonance-based immunochip

In this study, we present the detection of tau protein, at room temperature, using a multi-spot localised surface plasmon resonance (LSPR)-based immunochip. To the best of our knowledge, this is the first report of an immunochip for tau protein. The detection method includes fabrication of a gold-capped nanoparticle LSPR chip, formation and functionalisation of a self-assembled monolayer (SAM), immobilisation of a suitable linker, effective blocking of non-specific adsorption, immobilisation of a monoclonal anti-tau antibody (tau-mAb), and finally, the optimum conditions for the immuno-reaction between tau-mAb and the antigen were determined. The method has a high performance, enables detection of tau at 10pg/mL, lower than the cut-off value of 195pg/mL (for AD) for tau protein in cerebral spinal fluid (CSF). Further, we demonstrated selectivity of the technique by showing that the introduction of bovine serum albumin (BSA), perhaps the most abundant protein component in serum and CSF, does not interfere with the detection of tau. This method also offers a potential platform for studying tau interactions with other proteins and/or potential drug candidates and could also be easily adapted for detecting phosphorylated tau and other AD biomarkers.     

Detection of DNA hybridisation in a diluted serum matrix by surface plasmon resonance and film bulk acoustic resonators

Nanomolar quantities of single-stranded DNA products ~100 nucleotides long can be detected in diluted 1% serum by surface plasmon resonance (SPR) and film bulk acoustic resonators (FBARs). We have used a novel FBAR sensor in parallel with SPR and obtained promising results with both the acoustic and the optical device. Oligonucleotides and a repellent lipoamide, Lipa-DEA, were allowed to assemble on the sensor chip surfaces for only 15 min by dispensing. Lipa-DEA surrounds the analyte-binding probes on the surface and effectively reduces the non-specific binding of bovine serum albumin and non-complementary strands. In a highly diluted serum matrix, the non-specific binding is, however, a hindrance, and the background response must be reduced. Nanomolar concentrations of short complementary oligos could be detected in buffer, whereas the response was too low to be measured in serum. DNA strands that are approximately 100 base pairs long at concentrations as low as 1-nM could be detected both in buffer and in 1% serum by both SPR and the FBAR resonator.     

Detection of transglucosidase-catalyzed polysaccharide synthesis on a surface in real time using surface plasmon resonance spectroscopy

Biological systems that involve enzyme catalysis at surfaces, particularly strategically important ones that involve insoluble substrates/products such as the cell wall and the starch granule, require analyses beyond classical solution state enzymology. Using a model system, we have demonstrated the real-time measurement of transglucosidase activity on a surface using surface plasmon resonance (SPR) spectroscopy. We monitored the extension of a (partially carboxymethylated) dextran surface with alternansucrase and sucrose as a glycosyl donor. Conditions were used where surface polymer synthesis rates were a function of enzyme concentration and proportional to the extent of enzyme binding to the surface. A method to determine the turnover number of the enzyme on the surface was also developed. The presence of a new amorphous polysaccharide was observed optically, detected by lectin binding and imaged by atomic force microscopy. This surface method will have utility in a wide range of carbohydrate enzyme systems including screens.     

Leaching potential of carbamates and their metabolites and comparison with triazines

This paper reports the results of a laboratory study aimed at defining the leaching potential of the following pesticides and respective metabolites belonging to the families of N-methylcarbamates and triazines: benfuracarb (BF), carbofuran (CF), 3-keto-carbofuran (3KC) and 3-hydroxy-carbofuran (3HC), atrazine (ATR), simazine (SIM), terbuthylazine (TER), deethylatrazine (DEA), deisopropylatrazine (DIA) and desethylterbuthylazine (DET).All tested compounds, but BF, are very mobile in soil. Triazines exhibited a relatively high persistence, especially DEA, with a DT₅₀ of 72 days. On the contrary, all the tested carbamates resulted easily degradable in soil with a partial exception represented by CF, with a DT₅₀ of 12 days.The GUS indices show high leaching potentials for all the tested triazines and CF. The GUS index of 3KC lies in the typical area of transient compounds; those of BF and 3HC clearly exhibited a non-leaching behaviour.In the leachate corresponding to the BF column, the parent compound was found at low concentration while its main metabolite, CF, reached much higher values. Also, when applied as parent compound, CF was determined at high values, whereas its metabolites 3KC and 3HC were never detected in the leachates. As to triazines, in the ATR column, the parent compound was found at high levels in the leachate, where DEA exhibited values more than 4 times higher than DIA. In the SIM column DIA reached levels 8-fold higher than those in the ATR column. TER occurred at levels close to that of ATR in the respective leachate; DET was found at high levels whereas DIA was not detectable.     

Mild and selective ring-cleavage of cyclic carbamates to amino alcohols

N-tert-Butoxycarbonylated 2-oxazolidinones and tetrahydro-2-oxazinones are smoothly cleaved to acyclic N-Boc-amino alcohols on treatment with catalytic amounts of cesium carbonate at room temperature. The versatility of the procedure is demonstrated in a facile cleavage of highly functionalized heterocycles without epimerization and β-elimination.     

Mass spectral characteristics of the sulfur-containing ketoxime carbamates thiofanox and metabolites

Deuterium labeling and high resolution mass spectroscopy have been used to determine the modes of electron impact fragmentation of 3,3-dimethyl-1-methylthio-2-butanone O-[(methylamino)carbonyl]oxime, thiofanox and its metabolites, the sulfoxide and sulfone. The mass spectra of the corresponding oximes were also examined. The carbamates fragmented chiefly by two competing pathways—the loss of CH₃N?C? O or the sulfur-containing moiety. The favored process was dependent on the oxidation state at sulfur. Several unusual rearrangements were noted.     

Detection of tamoxifen metabolites by GC-MSD

Tamoxifen is an antiestrogen used in the adjuvant endocrine therapy of early breast cancer and malignant breast disorders. It is also used in women with anovulatory infertility caused by its stimulating effect on the secretion of the pituitary gonadotrophic hormones. In males it could increase the endogenous production of androgens. Because of these properties tamoxifen may be misused in some sports to treat the androgens suppression caused by the extensive abuse of anabolic androgenic steroids. A method for identification and confirmation of tamoxifen metabolites is described. Hydroxymetoxytamoxifen is detected in urine by gas chromatography and mass spectrometry in a selective ion monitoring method followed by the routine postrun in the screening of anabolic steroids. Once the hydroxymetoxytamoxifen is detected, confirmation of reported metabolites could be performed with a 5973 mass selective detector in the scan mode after solid-phase extraction by cationic exchange. This study also reports an excretion profile for a single dose of tamoxifen equivalent to 40 mg administrated orally to two males volunteers.     

Synthesis of carbamates of amino alcohols of pyrrolizidine and quinazolidine alkaloids

Summary Previously undescribed mono- and dicarbamates of pyrrolizidine and quinazolidine amino alcohols have been obtained by the reaction of mono- and diisocyanates with the corresponding alcohol derivatives of the alkaloids.Institute of the Chemistry of Plant Substances, Academy of Sciences of the Uzbek SSR. Translated from Khimiya Prirodnykh Soedinenii, No. 6, pp. 765–768, November–December, 1975.     

Characterization of surface-confined alpha-synuclein by surface plasmon resonance measurements

Urea-driven denaturation and renaturation of surface-bound alpha-synuclein are monitored by surface plasmon resonance (SPR) spectroscopy. The differential SPR angle shift (Delta Theta(SPR))(Net) enables us to estimate the Gibbs free energy change (DeltaG(o)) for the denaturation of the supported alpha-synuclein. DeltaG(o) for the denaturation of the supported alpha-synuclein, which is indirectly related to its biological activity can be increased significantly by the mixed self-assembled monolayers of 11-mercaptoundecanoic acid and 1,6-hexanedithiol. These SPR measurements of surface-bound biomolecules suggested herein can be further utilized to design effective biological scaffold for biosensor, biocatalyst, and possible diagnosis.     

Modeling protein binding and elution over a chromatographic surface probed by surface plasmon resonance

Surface plasmon resonance (SPR) spectroscopy is used as a scaled-down, analytical, pseudo-chromatography tool for analyzing protein binding and elution over an ion-exchange surface under cyclic sorption conditions. A micrometric-scale adsorption surface was produced by immobilizing a typical ion exchange ligand – diethylaminoethyl (DEAE) – onto commercially available planar gold sensor chip surfaces pre-derivatized with a self-assembled monolayer of 11-mercaptoundecanoic acid with known density. An explicit mathematical formulation is provided for the deconvolution and interpretation of the SPR sensorgrams. An adsorption rate model is proposed to describe the SPR sensorgrams for bovine serum albumin, used here as model protein, when the DEAE surface is subjected to a cyclic series of binding and elution steps. Overall, we demonstrate that the adsorption rate model is capable of quantitatively describing BSA binding and elution for protein titers from dilute conditions up to overloaded conditions and a broad range of salt concentrations.