Determination of the Distance Between the Cytochrome and Dehydrogenase Domains of Immobilized Cellobiose Dehydrogenase by Using Surface Plasmon Resonance with a Center of Mass Based Model.

Changes in the tertiary conformation of adsorbed biomolecules can induce detectable shifts (Δθr) in the surface plasmon resonance (SPR) angle. Here it is shown how to calculate the corresponding shifts in the adsorbate's center of mass (Δzavg) along the sensing surface normal from the measured Δθr. The novel developed model was used for determining the mean distance between the cytochrome (CYT) and flavodehydrogenase (DH) domains of the enzyme cellobiose dehydrogenase (CDH) isolated from the fungi Neurospora crassa, Corynascus thermophilus, and Myriococcum thermophilum as a function of pH, [Ca2+], and substrate concentration. SPR confirmed the results from earlier electrochemical and SAXS studies stating that the closed conformation, where the two domains are in close vicinity, is stabilized by a lower pH and an increased [Ca2+]. Interestingly, an increasing substrate concentration in the absence of any electron acceptors stabilizes the open conformation as the electrostatic repulsion due to the reaped electrons pushes the DH and CYT domains apart. The accuracy of distance determination was limited mostly by the random fluctuations between replicate measurements, and it was possible to detect movements <1 nm of the domains with respect to each other. The results agreed with calculations using already established models treating conformational changes as contraction or expansion of the thickness of the adsorbate layer (tprotein). Although the models yielded equivalent results, in this case, the Δzavg-based method also works in situations, where the adsorbate's mass is not evenly distributed within the layer.

Surface Plasmon Resonance for Measuring Exocytosis from Populations of PC12 Cells: Mechanisms of Signal Formation and Assessment of Analytical Capabilities.

Because of cell to cell variation, it is difficult to obtain statistically significant data on the frequency of exocytosis events (Rexocytosis, t-1 m-2) with traditional single cell electrophysiological or fluorescence microscopy based methods. Here we take the first steps toward a rapid cost-effective surface plasmon resonance (SPR) based method for measuring the Rexocytosis for populations of PC12 cells. The conditions for culturing confluent monolayers on the sensor slides were optimized, and neurotransmitter exocytosis was evoked by injecting solutions with elevated [K+]. Exocytosis caused a shift of the resonance angle (Δθr) that was linearly proportional to Rexocytosis. The Δθr was mainly due to elevated concentration of secretory vesicles close to the cell membrane. The increased vesicle concentration thus acted as a proxy for the Rexocytosis that could not be measured directly. The Δθr was calibrated for Rexocytosis using single cell amperometry on parallel cell cultures. The cell populations were large enough for variation in responses between sensor slides to only reflect actual differences in biological condition. The applicability for drug screening is demonstrated by studying the effects of EGTA, reserpine, and prolonged stimulation by K+.

Determining Number Concentrations and Diameters of Polystyrene Particles by Measuring the Effective Refractive Index of Colloids Using Surface Plasmon Resonance.

The capabilities of surface plasmon resonance (SPR) for characterization of colloidal particles were evaluated for 100, 300, and 460 nm nominal diameter polystyrene (PS) latexes. First the accuracy of measuring the effective refractive index (neff) of turbid colloids using SPR was quantified. It was concluded that for submicrometer sized PS particles the accuracy is limited by the reproducibility between replicate injections of samples. An SPR method was developed for obtaining the particle mean diameter (dpart) and the particle number concentration (cp) by fitting the measured neff of polystyrene (PS) colloids diluted in series with theoretical values calculated using the coherent scattering theory (CST). The dpart and cp determined using SPR agreed with reference values obtained from size distributions measured by scanning electron microscopy (SEM), and the mass concentrations stated by the manufacturer. The 100 nm particles adsorbed on the sensing surface, which hampered the analysis. Once the adsorption problem has been overcome, the developed SPR method has potential to become a versatile tool for characterization of colloidal particles. In particular, SPR could form the basis of rapid and accurate methods for measuring the cp of submicrometer particles in dispersion.

Carbon nanotube fiber microelectrodes show a higher resistance to dopamine fouling.

We have compared the properties and resistance to DA fouling of a carbon nanotube fiber (CNTF) microelectrode to a traditional carbon fiber (CF) microelectrode. These two materials show comparable electrochemical activities for outer-sphere and inner-sphere redox reactions. Although the CNTF might have a higher intrinsic RC constant, thus limiting its high-frequency behavior, the CNTF shows a significantly higher durability than the CF in terms of electrode stability. During constant oxidation of 100 µM DA, the signal measured by the CNTF microelectrode shows a 2-h window over which no decrease in current is observed. Under the same conditions, the current obtained at the CF microelectrode decreases by almost 50%. A model of the fouling process, assuming the formation of growing patches of insulator on the surface, has been compared to the data. This model is found to be in good agreement with our results and indicates a growth rate of the patches in the 0.1-2 nm s(-1) range.

Characteristics of third-generation glucose biosensors based on Corynascus thermophilus cellobiose dehydrogenase immobilized on commercially available screen-printed electrodes working under physiological conditions.

In this article, we describe a third-generation amperometric glucose biosensor working under physiological conditions. This glucose biosensor consists of a recently discovered cellobiose dehydrogenase from the ascomycete Corynascus thermophilus (CtCDH) immobilized on different commercially available screen-printed electrodes made of carbon (SPCEs), carboxyl-functionalized single-walled carbon nanotubes (SPCE-SWCNTs), or multiwalled carbon nanotubes (SPCE-MWCNTs) by simple physical adsorption or a combination of adsorption followed by cross-linking using poly(ethyleneglycol) (400) diglycidyl ether (PEGDGE) or glutaraldehyde (GA). The CtCDH-based third-generation glucose biosensor has a linear range between 0.025 and 30 mM and a detection limit of 10 µM glucose. Biosensors based on SWCNTs showed a higher sensitivity and catalytic response than the ones functionalized with MWCNTs and the SPCEs. A drastic increase in response was observed for all three electrodes when the adsorbed enzyme was cross-linked with PEGDGE or GA. The operational stability of the biosensor was tested for 7 h by repeated injections of 50 mM glucose, and only a slight decrease in the electrochemical response was found. The selectivity of the CtCDH-based biosensor was tested on other potentially interfering carbohydrates such as mannose, galactose, sucrose, and fucose that might be present in blood. No significant analytical response from any of these compounds was observed.

High performance magneto-fluorescent nanoparticles assembled from terbium and gadolinium 1,3-diketones.

Polyelectrolyte-coated nanoparticles consisting of terbium and gadolinium complexes with calix[4]arene tetra-diketone ligand were first synthesized. The antenna effect of the ligand on Tb(III) green luminescence and the presence of water molecules in the coordination sphere of Gd(III) bring strong luminescent and magnetic performance to the core-shell nanoparticles. The size and the core-shell morphology of the colloids were studied using transmission electron microscopy and dynamic light scattering. The correlation between photophysical and magnetic properties of the nanoparticles and their core composition was highlighted. The core composition was optimized for the longitudinal relaxivity to be greater than that of the commercial magnetic resonance imaging (MRI) contrast agents together with high level of Tb(III)-centered luminescence. The tuning of both magnetic and luminescent output of nanoparticles is obtained via the simple variation of lanthanide chelates concentrations in the initial synthetic solution. The exposure of the pheochromocytoma 12 (PC 12) tumor cells and periphery human blood lymphocytes to nanoparticles results in negligible effect on cell viability, decreased platelet aggregation and bright coloring, indicating the nanoparticles as promising candidates for dual magneto-fluorescent bioimaging.

Application of surface plasmon resonance for the detection of carbohydrates, glycoconjugates, and measurement of the carbohydrate-specific interactions: a comparison with conventional analytical techniques. A critical review.

Carbohydrates (glycans) and their conjugates with proteins and lipids contribute significantly to many biological processes. That makes these compounds important targets to be detected, monitored and identified. The identification of the carbohydrate content in their conjugates with proteins and lipids (glycoforms) is often a challenging task. Most of the conventional instrumental analytical techniques are time-consuming and require tedious sample pretreatment and utilising various labeling agents. Surface plasmon resonance (SPR) has been intensively developed during last two decades and has received the increasing attention for different applications, from the real-time monitoring of affinity bindings to biosensors. SPR does not require any labels and is capable of direct measurement of biospecific interaction occurring on the sensing surface. This review provides a critical comparison of modern analytical instrumental techniques with SPR in terms of their analytical capabilities to detect carbohydrates, their conjugates with proteins and lipids and to study the carbohydrate-specific bindings. A few selected examples of the SPR approaches developed during 2004-2011 for the biosensing of glycoforms and for glycan-protein affinity studies are comprehensively discussed.

Surface plasmon resonance for real-time study of lectin-carbohydrate interactions for the differentiation and identification of glycoproteins.

A study of specific interactions between lectins and glycoproteins has been carried out using surface plasmon resonance (SPR) in a flow-injection mode. Lectins were covalently immobilised on the surfaces of the microfluidic sensor chip via amine coupling and serum glycoproteins were injected into the flow channels. Specific lectin-glycoprotein interactions caused the shift of refractive index proportional to the mass concentration accumulated on the channel surface. Lectins showed different affinity to the tested glycoproteins and each glycoprotein displayed its own lectin-binding pattern. It is possible to distinguish and identify even glycoproteins with similar sugar structures by simple and quick screening. The working conditions of the assay were optimised. The lectin-based SPR made it possible to carry out the label-free detection of glycoproteins within a broad concentration range with a good linearity. Regeneration conditions for the surface of the sensor chip were found and optimised. Combination of 10mM HCl and 10mM glycine-HCl (pH 2.5) removes the bound glycoproteins from the lectin surface without damaging it. The kinetic and affinity parameters of lectin-glycoprotein binding were evaluated. The proposed method was tested on human glycosylated serum. Combination of the lectin panel with SPR is suitable both for specific screening and for sensitive assay of serum glycoproteins.

Lectin typing of Campylobacter jejuni using a novel quartz crystal microbalance technique.

Seven Campylobacter jejuni strains were characterised by a lectin typing assay. The typing system was based on a quartz crystal microbalance technique (QCM) with four commercially available lectins (wheat germ agglutinin, Maackia amurensis lectin, Lens culinaris agglutinin, and Concanavalin A), which were chosen for their differing carbohydrate specificities. Initially, the gold surfaces of the quartz crystals were modified with 11-mercaptoundecanoic acid followed by lectin immobilisation using a conventional amine-coupling technique. Bacterial cells were applied for lectin typing without preliminary treatment, and resonant frequency and dissipation responses were recorded. The adhesion of microorganisms on lectin surfaces was confirmed by atomic force microscopy. Scanning was performed in the tapping mode and the presence of bacteria on lectin-coated surfaces was successfully demonstrated. A significant difference in the dissipation response was observed for different C. jejuni strains which made it possible to use this parameter for discriminating between bacterial strains. In summary, the QCM technique proved a powerful tool for the recognition and discrimination of C. jejuni strains. The approach may also prove applicable to strain discrimination of other bacterial species, particularly pathogens.

A study of glycoprotein-lectin interactions using quartz crystal microbalance.

A study of biospecific interactions between lectins and glycoproteins using a quartz crystal microbalance biosensor with dissipation monitoring (QCM-D) was reported. Four lectins were covalently immobilised on the thiol-modified gold electrode of the QCM chips in order to obtain sensing surfaces. The frequency shift served as analytical signal and the dissipation shift provided additional information about the viscoelastic properties of the glycoprotein-lectin complex formed on the surface of the QCM chip. The working conditions of the assay were optimised. The interaction between different lectins and glycoproteins was characterised by specific frequency shifts and each glycoprotein displayed its own unique lectin-binding pattern. This lectin pattern can serve as a finger print for the discrimination between various glycoproteins. The biosensor enabled quantitative determination of glycoproteins in the concentration range of 50 microg mL(-1) to 1 mg mL(-1) with good linearity and R.S.D. of less than 6.0%. An additional advantage of the proposed biosensor was the possibility to re-use the same lectin surfaces during a long period of time (2 month) without changes in analytical response. This was experimentally achieved by the application of a proper regeneration solution (10 mM glycine-HCl, pH 2.5). The lectin-based quartz crystal microbalance technique is suitable both for rapid screening and for quantitative assay of serum glycoproteins.