A disposable electrochemical immunosensor for prolactin involving affinity reaction on streptavidin-functionalized magnetic particles

A novel electrochemical immunosensor was developed for the determination of the hormone prolactin. The design involved the use of screen-printed carbon electrodes and streptavidin-functionalized magnetic particles. Biotinylated anti-prolactin antibodies were immobilized onto the functionalized magnetic particles and a sandwich-type immunoassay involving prolactin and anti-prolactin antibody labelled with alkaline phosphatase was employed. The resulting bio-conjugate was trapped on the surface of the screen-printed electrode with a small magnet and prolactin quantification was accomplished by differential pulse voltammetry of 1-naphtol formed in the enzyme reaction using 1-naphtyl phosphate as alkaline phosphatase substrate. All variables involved in the preparation of the immunosensor and in the electrochemical detection step were optimized. The calibration plot for prolactin exhibited a linear range between 10 and 2000 ng mL(-1) with a slope value of 7.0 nA mL ng(-1). The limit of detection was 3.74 ng mL(-1). Furthermore, the modified magnetic beads-antiprolactin conjugates showed an excellent stability. The immunosensor exhibited also a high selectivity with respect to other hormones. The analytical usefulness of the immnunosensor was demonstrated by analyzing human sera spiked with prolactin at three different concentration levels.     

Comments on the linear relation between reaction rate and affinity

In the case of catalytic dehydrogenation of cyclohexane, the linear relation between reaction rate and affinity holds at an appreciable distance away from equilibrium on both sides. In fact, in order to explain this observation, it is necessary to invoke a high value of three for the stoichiometric number of the rate determining step. This in turn can be explained by a reasonable mechanism for the reaction.     

Kinetic analysis of interaction between lipopolysaccharide and biomolecules

Lipopolysaccharide (LPS) is a major component of the outer membrane of all gram-negative bacteria. It interacts with some biomolecules and triggers a toxic reaction. In this paper, we studied the interaction between LPS from Salmonella Minnesota and some biomolecules using a surface plasmon resonance (SPR) biosensor. Biomolecules were immobilized on a CM5 sensor chip using the amino coupling method and LPS was injected over the immobilized surfaces. The affinity constant K_A of LPS with serum albumin, hemoglobin, chitosan and lysozyme was 2.36 × 10⁷, 2.03 × 10⁸, 7.58 × 10⁶, 2.82 × 10⁴ L·mol⁻¹, respectively. However, LPS could not interact with ferritin. __________ Translated from Chinese Journal of Analytical Chemistry, 2007, 35(5): 677–680 [译自: 分析化学]     

Separation of biomolecules using electrophoresis and nanostructures

A large number of nanostructures have the potential to be used together with electrophoresis as separation media or separation additive in capillary electrophoresis, micellar electrokinetic chromatography, capillary electrochromatography, and other analytical techniques. Among those structures are nanotubes, nanocavities, nanowires, nanoposts, nanocones, nanospheres, molecular imprints, nanoparachutes (conical monodendrons), and general nanoparticles with random structures. This review is focused only on publications describing experimental works using molecular imprints, nanoposts, and nanospheres that are fabricated and applied for the purpose of separation media in electrophoresis-driven separations. The review follows an approximate chronological order in each section. As shown, the most popular are those resulting from molecular imprinting technologies. These biomimetic receptors are used in a great variety of fields, which includes electrophoresis, micellar electrokinetic chromatography, capillary electrochromatography, and other fields not reviewed in this work. A few examples of these other fields are, e.g., liquid chromatography, membranes, extractor or preconcentration techniques, immunosorbent assays, and sensing devices. The second topic scanned in the present work is the nanostructures that are used as obstacles to replace gels or polymers solutions in electrophoresis. Finally, the nascent field of nanospheres of gold and other materials as separation media is also reviewed.     

THE weighted histogram analysis method for free-energy calculations on biomolecules. I. The method

The Weighted Histogram Analysis Method (WHAM), an extension of Ferrenberg and Swendsen's Multiple Histogram Technique, has been applied for the first time on complex biomolecular Hamiltonians. The method is presented here as an extension of the Umbrella Sampling method for free-energy and Potential of Mean Force calculations. This algorithm possesses the following advantages over methods that are currently employed: (1) It provides a built-in estimate of sampling errors thereby yielding objective estimates of the optimal location and length of additional simulations needed to achieve a desired level of precision; (2) it yields the “best” value of free energies by taking into account all the simulations so as to minimize the statistical errors; (3) in addition to optimizing the links between simulations, it also allows multiple overlaps of probability distributions for obtaining better estimates of the free-energy differences. By recasting the Ferrenberg–Swendsen Multiple Histogram equations in a form suitable for molecular mechanics type Hamiltonians, we have demonstrated the feasibility and robustness of this method by applying it to a test problem of the generation of the Potential of Mean Force profile of the pseudorotation phase angle of the sugar ring in deoxyadenosine. © 1992 by John Wiley & Sons, Inc.     

A new method for preparation of magnetic polymer particles

Synthesis of magnetic polymer particles (MPP) was carried out through two steps. Firstly, the iron oxide particles carrying vinyl groups were synthesized, and in the second step, styrene (ST), divinyl benzene (DVB), and 2-hydroxyethyl methacrylate (HEMA) were polymerized on the surface of modified iron oxide through an emulsifier-free emulsion polymerization technique. The final particles were characterized by a Fourier transform infrared spectroscopy (FTIR), Fourier transform infrared attenuated total reflectance spectroscopy (FTIR-ATR), X-ray diffractometer (XRD), thermal gravimetry analysis (TGA), Fritsch particle sizer, scanning electron microscope (SEM), and vibrating sample magnetometer (VSM). Experimental analysis confirmed that all the iron oxide particles were embedded in a polymer phase and the final particles have more than 67 % iron oxide content. According to magnetometry data, the shape of the hysteresis loops evidences the ferromagnetic character of the particles.     

Adsorption behaviors of emulsifiers and biomolecules on temperaturesensitive polymer particles

Adsorption and desorption behaviours of emulsifiers and biomolecules on the two kinds of temperature-sensitive composite polymer particles were compared. One (I) was produced by seeded emulsion copolymerization of dimethylaminoethyl methacrylate and ethylene glycol dimethacrylate with 0.17 m-sized polystyrene seed particles. The other (II) was produced by seeded emulsion copolymerization of N-isopropylacrylamide (NIPAM) and N,N-methylenebisacrylamide with 0.36 m-sized styrene-NIPAM copolymer particles. The amount of adsorption at temperatures above each lower critical solution temperature (LCST) was found to be much higher for I than II. In both cases, at temperatures below the LCST, almost all lactalbumin hydrolysate molecules adsorbed above the LCST were desorbed but the desorptions of adsorbed egg albumin and lysozyme molecules were not so high. The adsorption and desorption were reversible for both particles and the efficiency was better for I than II. From these results, it is concluded that the adsorption/desorption of protein onto the temperature-sensitive polymer particles is controllable by changing the temperature below and above the LCST of the shell layer and the sensitivity is based on the surface property of the composite polymer particles.Part CXLIX of the series Studies on Suspensions and Emulsions     

Raman spectroscopy for the analysis of biomolecules

Spurred on by technological advances, Raman spectroscopy is becoming an important tool in the analysis of biological materials. It can provide unique information on composition and molecular structure. For biological chromophores resonance Raman effectively transforms absorption spectroscopy into a high information technique.     

Investigation of the reaction between triallyl-boron and methyl borate by the method of proton magnetic resonance

Conclusions Triallylboron enters into an equilibrium exchange reaction with methyl borate, forming esters of allyl- and diallylboric acids; the ratio of the reaction products depends upon the ratio of the starting materials.Translated from Izvestiya Akademii Nauk SSSR, Seriya Khimicheskaya, No. 6, pp. 1117–1118, June, 1966.     

A new method for reversible immobilization of thiol biomolecules based on solid-phase bound thiolsulfonate groups

A new method for the reversible immobilization of thiol bimolecules, e.g., thiolpeptides and thiolproteins, to beaded agarose and other solid phases is reported. The method consists of an activation and a coupling step. The activation is based on oxidation of disulfides (or thiol groups via disulfides) present in a solid phase by hydrogen peroxide at moderately acidic pH. This oxidation leads to disulfide oxides (thiolsulfinate groups of which the majority are further oxidized to thiolsulfonate). The thiolsulfonate groups react easily with thiol compounds, which become immobilized via disulfide bonds. The pH range for thiol coupling is wide (pH 5-8), but for most thiols the reaction seems to proceed faster at pH>7. The stability of the reactive group to hydrolysis, especially at neutral and weakly acidic pH, is very high. The activated gel, therefore, can be stored as a suspension at pH 5 for extended periods. The method has been used to reversibly immobilize glutathione, β-galactosidase, alcohol dehydrogenase, urease, and papain, all with exposed thiol groups as well as thiolated bovine serum albumin and sweet-potato β-amylase. Depending on the thiol content of starting thiol-agarose, thiol-sulfonate-agarose derivatives with different binding capacities can be obtained. Thus, up to 5.0 mg (16 μmol) glutathione and 15 mg thiol-protein/mL gel derivative have been immobilized.     

Separation of mistletoe lectins based on the degree of glycosylation using boronate affinity chromatography

A mixture of two mistletoe lectins (MLs) has been separated according to the degree of glycosylation using boronate affinity chromatography. The mistletoe lectins, mistletoe lectin I (MLI) and mistletoe lectin III (MLIII) with degrees of glycosylation of 6.1 and 3.8%, respectively, were used in the investigation. MLI exhibited a higher retention time than MLIII due to its higher degree of glycosylation. Separation was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The developed method may lead to new applications for the boronate affinity technique, as well as provide an alternative separation method for MLs.     

Nanoparticle-based mass spectrometry for the analysis of biomolecules

Nanoparticles (NPs) are useful as matrixes for the analyses of several types of biomolecules (including aminothiols, peptides, and proteins) and for mass spectrometric imaging through surface-assisted laser desorption/ionization mass spectrometry (SALDI-MS), mainly because of their large surface area, strong absorption in the ultraviolet-near-infrared region, and ready functionalization. Metallic NPs, metal oxide NPs, and semiconductor quantum dots, unmodified or functionalized with recognition ligands, have a strong affinity toward analytes; therefore, they allow the enrichment of biomolecules, leading to improved sensitivity with minimal matrix interference in their mass spectra. SALDI-MS using NPs overcomes the two major problems commonly encountered in matrix-assisted laser desorption/ionization mass spectrometry: the presence of "sweet spots" and the high background signals in the low-mass region. In this tutorial review, we discuss the roles played by the nature, size, and concentration of the NPs, the buffer composition, and the laser energy in determining the sensitivity and mass ranges for the analytes. We describe internal standard SALDI-MS methods that allow the concentrations of analytes to be determined with low variation (relative standard deviations: <10%) and we highlight how the simplicity, sensitivity, and reproducibility of SALDI-MS approaches using various NPs allow the analyses of proteins and small analytes and the imaging of cells.     

Protein ligand docking based on empirical method for binding affinity estimation

An empirical protein-ligand binding affinity estimation method, SCORE, was incorporated into a popular docking program, DOCK4. The combined program, ScoreDock, was used to reconstruct the 200 protein-ligand complex structures and found to give good results for the complexes with high binding affinities. A quality assessment method for docking results from ScoreDock was developed based on the whole test set and tested by additionally selected complexes. The method significantly improves the docking accuracy and was shown to be reliable in docking quality assessment. As a docking tool in structural based drug design, ScoreDock can screen out final hits directly based on the predicted negative logarithms of dissociation equilibrium constants of protein-ligand complexes, and can explicitly deal with structure water molecules, as well as metal atoms.     

Interpolyelectrolyte reaction between the particles of oppositely charged microgels

Processes that take place in oppositely charged microgel particles were studied. It was shown that water-swollen microgel particles about 100 μm in size were capable of interacting in dilute and concentrated dispersions to produce continuous dispersed systems. This interaction is accompanied by the release of water and low-molecular-mass counterions of network polyelectrolytes. Their liberation is due to the interpolyelectrolyte reaction between peripheral regions of unlike charged microgel particles in the particle contact zone.     

Separation parameters in the reaction between volatile halides and hydrogen

Equations for calculating the separation coefficient and factor in reactions of volatile halides with hydrogen from the equilibrium constants of the corresponding reactions and initial reactant concentrations were obtained. The concept of the limiting impurity concentration was introduced. The values specified were calculated for the reduction of volatile chlorides with hydrogen.     

Mass probe-assisted ionization method for total analysis of biomolecules with electrospray ionization-mass spectrometry

In this paper, we review the mass probes used for the derivation of a variety of biomolecules efficiently detected by the electrospray ionization-mass spectrometry and mass probe-assisted ionization method for total analysis and determination by consecutive detection with a single instrument. We describe mass probes for a variety of molecules including proteins, nucleobases, metallic cations, and other small molecules.     

Flow-through particles for the high-performance liquid chromatographic separation of biomolecules: perfusion chromatography

This paper reports a new technique for reducing resistance to stagnant mobile phase mass transfer without sacrificing high adsorbent capacity or necessitating extremely high pressure operation. The technique involves the flow of liquid through a porous chromatographic particle, and has thus been termed "perfusion chromatography". This is accomplished with 6000-8000 A pores which transect the particle. Data from electron microscopy, column efficiency, frontal analysis and theoretical modelling all suggest that mobile phase will flow through these large pores. In this manner, solutes enter the interior of the particles through a combination of convective and diffusional transport, with convection dominating for Peclet numbers greater than one. The implications of flow through particles on bandspreading, resolution and dynamic loading capacity are examined. It is shown that the rate of solute transport is strongly coupled to mobile phase velocity such that bandspreading, resolution of proteins and dynamic loading capacity are unaffected by increases in mobile phase velocity up to several thousand centimeters per hour. The surface area of this very large-pore diameter material is enhanced by using a network of smaller, 500-1500 A interconnecting pores between the throughpores. Scanning electron micrographs show that the pore network is continuous and that no point in the matrix is more than 5000-10,000 A from a through-pore. As a consequence, diffusional path lengths are minimized and the large porous particles take on the transport characteristics of much smaller particles but with a fraction of the pressure drop. Capacity and resolution studies show that these materials bind and separate an amount of protein equivalent to that of conventional high-performance liquid chromatography as well as low performance agarose-based media at greater than 10-100 times higher mobile phase velocity with no loss in resolution.     

Separation and characterisation of products of the reaction between propionamide and formaldehyde

Summary Propionamide-formaldehyde reaction products have been characterised using micellar electrokinetic chromatography (MEKC), HPLC-electrospray-MS (HPLC-ES-MS) and time-of-flight-MS (TOF-MS). HPLC-MS-MS was used to distinguish between isomeric species. The MEKC separation of all reaction products was obtained with good resolution and efficiency. Comparison of water-micelle distribution constants (P _MEKC) for propionamide-formaldehyde with those for caprolactam-formaldehyde reaction products suggests that the concentration of sodium dodecyl sulfate appropriate for use in MEKC is inversely related toP _MEKC. Quantification of all species containing one or two amide units was achieved using standard calibration, mass balance and the assumption of identical absorption coefficients for the same functional groups in monomers and dimers.     

Chemical composition and reaction analysis of single aerosol particles

In situ measurements of gas-liquid surface reactions of single aerosol microdroplets are presented. By means of optical levitation in combination with elastic (Mie) and inelastic (Raman) light scattering it is possible to get information on the chemistry of e.g. acid/base reactions as well as the physical behavior of single microparticles.     

Chemical composition and reaction analysis of single aerosol particles

In situ measurements of gas-liquid surface reactions of single aerosol microdroplets are presented. By means of optical levitation in combination with elastic (Mie) and inelastic (Raman) light scattering it is possible to get information on the chemistry of e.g. acid/base reactions as well as the physical behavior of single microparticles.