Combined confocal and magnetic resonance microscopy

Confocal fluorescence optical microscopy and magnetic resonance microscopy are each used to study live cells in a minimally invasive way. Both techniques provide complementary information. Therefore, by examining cells simultaneously with both methodologies, more detailed information is obtained than is possible with each microscope individually. In this paper two configurations of a combined confocal and magnetic resonance microscope are described. The first configuration is capable of studying large single cells or three-dimensional cell agglomerates, whereas the second configuration is designed for the investigation of monolayers of mammalian cells. In both cases the sample compartment is part of a temperature regulated perfusion system. Images obtained with the combined system are shown forXenopus laevis oocytes, model JB6 tumor spheroids, and a single layer of Chinese hamster ovary cells. Finally, potential applications of the combined microscope are discussed.     

Enzyme entrapped nanoporous scaffolds formed through flow-induced gelation in a microfluidic filter device for sensitive biosensing of organophosphorus compounds

A novel and versatile processing method was developed for the formation of gel scaffolds with in situ AChE-AuNPs immobilization for biosensing of organophosphorus compounds. The biosensor designed by our new approach shows high sensitivity, selectivity and reactivation efficiency. This flow-induced immobilization technique opens up new pathways for designing a simple, fast, biocompatible, and cost-effective process for enhanced sensor performance and on-site monitoring of a variety of toxic organophosphorus compounds.     

Anionic forensic signatures for sample matching of potassium cyanide using high performance ion chromatography and chemometrics

Potassium cyanide was used as a model toxicant to determine the feasibility of using anionic impurities as a forensic signature for matching cyanide salts back to their source. In this study, portions of eight KCN stocks originating from four countries were separately dissolved in water and analyzed by high performance ion chromatography (HPIC) using an anion exchange column and conductivity detection. Sixty KCN aqueous samples were produced from the eight stocks and analyzed for 11 anionic impurities. Hierarchal cluster analysis and principal component analysis were used to demonstrate that KCN samples cluster according to source based on the concentrations of their anionic impurities. The Fisher-ratio method and degree-of-class separation (DCS) were used for feature selection on a training set of KCN samples in order to optimize sample clustering. The optimal subset of anions needed for sample classification was determined to be sulfate, oxalate, phosphate, and an unknown anion named unk5. Using K-nearest neighbors (KNN) and the optimal subset of anions, KCN test samples from different KCN stocks were correctly determined to be manufactured in the United States. In addition, KCN samples from stocks manufactured in Belgium, Germany, and the Czech Republic were all correctly matched back to their original stocks because each stock had a unique anionic impurity profile. The application of the Fisher-ratio method and DCS for feature selection improved the accuracy and confidence of sample classification by KNN.     

Graphene-based immunosensor for electrochemical quantification of phosphorylated p53 (S15)

We reported a graphene-based immunosensor for electrochemical quantification of phosphorylated p53 on serine 15 (phospho-p53¹⁵), a potential biomarker of gamma-radiation exposure. The principle is based on sandwich immunoassay and the resulting immunocomplex is formed among phospho-p53 capture antibody, phospho-p53¹⁵ antigen, biotinylated phospho-p53¹⁵ detection antibody and horseradish peroxidase (HRP)-labeled streptavidin. The introduced HRP results in an electrocatalytic response to reduction of hydrogen peroxide in the presence of thionine. Graphene served as sensor platform not only promotes electron transfer, but also increases the surface area to introduce a large amount of capture antibody, thus increasing the detection sensitivity. The experimental conditions including blocking agent, immunoreaction time and substrate concentration have been optimized. Under the optimum conditions, the increase of response current is proportional to the phospho-p53¹⁵ concentration in the range of 0.2–10 ng mL⁻¹, with the detection limit of 0.1 ng mL⁻¹. The developed immunosensor exhibits acceptable stability and reproducibility and the assay results for phospho-p53¹⁵ are in good correlation with the known values. This easily fabricated immunosensor provides a new promising tool for analysis of phospho-p53¹⁵ and other phosphorylated proteins.     

Probing the intrinsic electronic structure of the cubane [4Fe-4S] cluster: nature's favorite cluster for electron transfer and storage

The cubane [4Fe-4S] is the most common multinuclear metal center in nature for electron transfer and storage. Using electrospray, we produced a series of gaseous doubly charged cubane-type complexes, [Fe4S4L4]2- (L = -SC2H5, -SH, -Cl, -Br, -I) and the Se-analogues [Fe4Se4L4]2- (L = -SC2H5, -Cl), and probed their electronic structures with photoelectron spectroscopy and density functional calculations. The photoelectron spectral features are similar among all the seven species investigated, revealing a weak threshold feature due to the minority spins on the Fe centers and confirming the low-spin two-layer model for the [4Fe-4S](2+) core and its "inverted level scheme". The measured adiabatic detachment energies, which are sensitive to the terminal ligand substitution, provide the intrinsic oxidation potentials of the [Fe4S4L4]2- complexes. The calculations revealed a simple correlation between the electron donor property of the terminal thiolate as well as the bridging sulfide with the variation of the intrinsic redox potentials. Our data provide intrinsic electronic structure information of the [4Fe-4S] cluster and the molecular basis for understanding the protein and solvent effects on the redox properties of the [4Fe-4S] active sites.     

An electrodynamics-based model for ion diffusion in microbial polysaccharides

An electrodynamics-based model was formulated for simulation of ion diffusion in microbial polysaccharides. The fixed charges and electrostatic double layers that may associate with microbial polysaccharides and their effects on ion diffusion were explicitly built into the model. The model extends a common multicomponent ion diffusion formulation that is based on irreversible thermodynamics under a zero ionic charge flux condition, which is only applicable to the regions without fixed charges and electrostatic double layers. An efficient numerical procedure was presented to solve the differential equations in the model. The model well described key features of experimental observations of ion diffusion in negatively charged microbial polysaccharides including accelerated diffusive transport of cations, exclusion of anions, and increased rate of cation transport with increasing negative charge density. The simulated diffusive fluxes of cations and anions were consistent with a cation exchange diffusion concept in negatively charged polysaccharides at the interface of plant roots and soils; and the developed model allows to mathematically study such diffusion phenomena. An illustrative example was also provided to simulate dynamic behavior of ionic current during ion diffusion within a charged bacterial cell wall polysaccharide and the effects of the ionic current on the compression or expansion of the bacterial electrostatic double layer at the interface of the cell wall and bulk solution.     

Thermodynamic model for the solubility of NdPO4(c) in the aqueous Na+-H+-H2PO4–HPO42–OH–Cl–H2O system

The solubility of NdPO₄(c) was studied at 23±2 °C from both the over and undersaturation directions, with pH ranging from 0 to 9, P concentrations ranging from 0.0003 to 1.00M, and equilibration periods ranging from 6 to 57 days. Equilibrium was reached in <6 days. From the H⁺, Nd, and P concentrations in equilibrated solutions, the logarithm of the thermodynamic equilibrium constant for the reaction (NdPO₄(c) Nd³⁺+PO₄ ^3-) was calculated to be -24.65±0.23 and the value of the Pitzer ion-interaction parameter ⁽²⁾for Nd³⁺-H₂PO₄ ^- was determined to be -92.9. Predictions based on these thermodynamic quantities were in excellent agreement with the experimental data.     

Magnetic beads-based bioelectrochemical immunoassay of polycyclic aromatic hydrocarbons

A simple, rapid, and sensitive bioelectrochemical immunoassay method based on magnetic beads (MBs) has been developed to detect polycyclic aromatic hydrocarbons (PAHs). The principle of this bioassay is based on a direct competitive enzyme-linked immunosorbent assay using PAH-antibody-coated MBs and horseradish peroxidase (HRP)-labeled PAH (HRP-PAH). A magnetic process platform was used to mix and shake the samples during the immunoreactions and to separate free and unbound reagents after the liquid-phase competitive immunoreaction among PAH-antibody-coated MBs, PAH analyte, and HRP-PAH. After a complete immunoassay, the HRP tracers attached to MBs were transferred to a substrate solution containing 3,3′,5,5′-tetramethylbenzidine (TMB) and hydrogen peroxide (H₂O₂) for electrochemical detection. The voltammetric characteristics of the substrate were investigated, and the reduction peak current of TMB was used to quantify the concentration of PAH. The different parameters, including the amount of HRP-PAH conjugates, the enzyme catalytic reaction time, and the pH of the supporting electrolyte that governs the analytical performance of the immunoassay have been studied in detail and optimized. The detection limit of 50 pg mL⁻¹ was obtained under optimum experimental conditions. The performance of this bioelectrochemical magnetic immunoassay was successfully evaluated with tap water spiked with PAHs, indicating that this convenient and sensitive technique offers great promise for decentralized environmental applications.     

Determination of organophosphate pesticides at a carbon nanotube/organophosphorus hydrolase electrochemical biosensor

An amperometric biosensor for oganophosphorus (OP) pesticides based on a carbon nanotube (CNT)-modified transducer and an organophosphorus hydrolase (OPH) biocatalyst is described. A bilayer approach with the OPH layer atop of the CNT film was used for preparing the CNT/OPH biosensor. The CNT layer leads to a greatly improved anodic detection of the enzymatically generated p-nitrophenol product, including higher sensitivity and stability. The sensor performance was optimized with respect to the surface modification and operating conditions. Under the optimal conditions the biosensor was used to measure as low as 0.15 μM paraoxon and 0.8 μM methyl parathion with sensitivities of 25 and 6 nA/μM, respectively.