Label-free technologies for quantitative multiparameter biological analysis

In the postgenomic era, information is king and information-rich technologies are critically important drivers in both fundamental biology and medicine. It is now known that single-parameter measurements provide only limited detail and that quantitation of multiple biomolecular signatures can more fully illuminate complex biological function. Label-free technologies have recently attracted significant interest for sensitive and quantitative multiparameter analysis of biological systems. There are several different classes of label-free sensors that are currently being developed both in academia and in industry. In this critical review, we highlight, compare, and contrast some of the more promising approaches. We describe the fundamental principles of these different methods and discuss advantages and disadvantages that might potentially help one in selecting the appropriate technology for a given bioanalytical application.

Interaction between blood plasma proteins and magnetite nanoparticles

Spin label EPR spectroscopy and dynamic and Rayleigh light scattering are employed to study the interaction between magnetite nanoparticles with a diameter of 17 nm and plasma proteins (fibrinogen and albumin). Protein molecules are shown to be adsorbed on nanoparticle surface with the formation of multilayer shells. When a buffer solution (pH 8.5) contains 0.01 vol % nanoparticles, 90–100 fibrinogen molecules are adsorbed per one particle and the thickness of an adsorbed layer is 30–40 nm. For albumin, the layer thickness is 10–15 nm. In a constant magnetic field, large linear microsized aggregates oriented parallel to field lines are formed in dispersions of nanoparticles covered with adsorbed protein molecules. The study of fibrin gel formation resulting from the action of thrombin enzyme on fibrinogen suggests that, in the presence of nanoparticles, the rate of gelation decreases by a factor of approximately two, while the ratio between the average mass and average length of fibrin polymer fibers rises.     

CE-ICP-MS for studying interactions between metals and biomolecules

Metal-biomolecule interactions comprise an important research area in metallomics, and are significant for biology, medicine, pharmacy, nutrition, metabolism, and environmental science. Hybrid techniques are preferred for studying interactions between metals and biomolecules. Of all the separation techniques, capillary electrophoresis (CE) exhibits high resolution, minimal sample and reagent consumption, and rapid and efficient separations with minor disturbance of the existing equilibrium between the metal species and their biomolecular complexes. Inductively coupled plasma mass spectrometry (ICP-MS) presents high sensitivity to most of elements and offers multi-element detection.This article provides an overview of CE-ICP-MS for the study of metal-biomolecule interactions. We discuss applications of CE-ICP-MS to the study of interactions between metals or metalloids and natural ligands, such as humic substances or fulvic acids, and the interchange of metal complexes with metal species in metalloproteins.     

Water-induced interactions between carbon nanoparticles

Molecular dynamics simulations were carried out in order to study the hydration of C60 fullerenes, carbon nanotubes, and graphene sheets in aqueous solution and the nature of water-induced interactions between these carbon nanoparticles. The hydration of these nonpolar carbon nanoparticles does not exhibit classical hydrophobic character due to the high density of surface atoms (carbon) resulting in strong water-surface dispersion interactions. Water was found to wet the nanoparticle surfaces independent of nanoparticle surface curvature, with the decrease in the extent of water-water hydrogen bonding with decreasing surface curvature being offset by stronger water-surface interactions. While all carbon nanoparticles investigated are anticipated to aggregate in water due to strong direct nanoparticle-nanoparticle interactions, the water-induced interactions between nanoparticles were found to be repulsive and, in contrast to the wetting behavior, were observed to exhibit strong dependence on surface curvature. The strength of the water-induced interaction between carbon nanoparticles was found to correlate well with the number of hydration water molecules displaced upon particle aggregation, which, relative to the amount of direct nanoparticle-nanoparticle contact engendered upon aggregation, decreases with decreasing surface curvature.     

Ion-mediated interactions between charged and neutral nanoparticles

Monte-Carlo simulations are used to study the ion-mediated effective interaction between weakly charged and highly charged nanoparticles in an implicit solvent. Three models of nanoparticles are successively studied, from crude charged hard spheres to dipolar and non-spherical nanoparticles. The analysis of the effective potential revealed that in an electrolyte solution, even a neutral nanoparticle feels an important repulsive force in the presence of a charged nanoparticle, with a typical range similar to the Debye length. When the two nanoparticles carry charges of opposite sign, we have shown that this repulsion can reverse the effect of the direct attractive electrostatic potential at short distances. This also yields the change of sign of the effective potential as a function of the relative orientations of two anisotropic nanoparticles. Moreover, we found that the 3-body terms of the effective potentials can overcome the 2-body terms, which is not observed in the case of symmetrically charged nanoparticles.     

Label-free detection of adenosine based on fluorescence resonance energy transfer between fluorescent silica nanoparticles and unmodified gold nanoparticles

A sensitive and convenient strategy was developed for label-free assay of adenosine. The strategy adapted the fluorescence resonance energy transfer property between Rhodamine B doped fluorescent silica nanoparticles (SiNPs) and gold nanoparticles (AuNPs) to generate signal. The different affinities of AuNPs toward the unfolded and folded aptamers were employed for the signal transfer in the system. In the presence of adenosine, the split aptamer fragments react with adenosine to form a structured complex. The folded aptamer cannot be adsorbed on the surface of AuNPs, which induces the aggregation of AuNPs under high ionic concentration conditions, and the aggregation of AuNPs leads to the decrease of the quenching ability. Therefore, the fluorescence intensity of Rhodamine B doped fluorescent SiNPs increased along with the concentration of adenosine. Because of the highly specific recognition ability of the aptamer toward adenosine and the strong quenching ability of AuNPs, the proposed strategy demonstrated good selectivity and high sensitivity for the detection of adenosine. Under the optimum conditions in the experiments, a linear range from 98 nM to 100 μM was obtained with a detection limit of 45 nM. As this strategy is convenient, practical and sensitive, it will provide a promising potential for label-free aptamer-based protein detection.     

Limitation of immunoaffinity column for the removal of abundant proteins from plasma in quantitative plasma proteomics

In plasma proteomics, before a proteome analysis, it is essential to prepare protein samples without high‐abundance proteins, including albumin, via specific preparation techniques, such as immunoaffinity capture. However, our preliminary experiments suggested that functional changes with use alter the ability of the immunoaffinity column. Thus, in this study, to evaluate the changes of the removal ability of abundant proteins from plasma by the immunoaffinity column, plasma proteome analysis was performed for the long‐term test for the reproducibility of the affinity column using the fluorogenic derivatization–liquid chromatography–tandem mass spectrometry method combined with an IgY column. The specific adsorption for albumin decreased with an increase in the number of the column usage before its expiration date. Moreover, it was demonstrated that hydrophobic high molecular weight compounds in plasma adsorbed onto the column materials surface contributed to the functional changes from specific immunoaffinity adsorption into hydrophobic interaction. These results suggested that, in quantitative plasma proteomics studies, it is important to keep in mind the risk of not only the nonselective loss but also the changes in the adsorption ability of the immunoafinity column. Copyright © 2008 John Wiley & Sons, Ltd.     

Isolation and quantitative analysis of road dust nanoparticles

Nanoparticles are capable of preconcentrating various elements, including toxic ones; they have high mobility in the environment and can easily penetrate into a human body. The study of the chemical composition and properties of road dust nanoparticles is an urgent task of analytical chemistry, which needs to be addressed in the monitoring of the anthropogenic load on the environment and the assessment of the potential danger of pollution to human health. In the present paper, we propose a new approach for the isolation, characterization, and quantitative elemental analysis of road dust nanoparticles. Conditions are selected for the separation of nanoparticles from Moscow dust samples by field-flow fractionation in a rotating coiled column; the resulting fractions are characterized by independent methods (using static light scattering and electron microscopy); the method for calculating the concentration of elements in the nanoparticle fraction according to inductively coupled plasma atomic emission spectrometry and mass spectrometry is improved; elements in a water-soluble form are isolated and determined; and the role of soluble organic matter in the binding of trace elements is discussed. It is shown that the total concentration of most elements in the samples of Moscow dust is comparable to the average values for urban soils. Abnormally high concentrations of several elements (Cu, Zn, Ag, Cd, Sn, Sb, Hg, Pb, Tl, and Bi) are revealed in the fraction of nanoparticles; the enrichment factor with respect to the total concentration ranges from 10 to 450. The source of contamination of road dust nanoparticles with copper, zinc, antimony, and cadmium is highly probable wearing-off of brake pads and car tires. The developed procedure of separation, characterization, and analysis of nanoparticles can be used for other polydisperse environmental samples (for example, volcanic ash).     

Interfacial interactions between proteins and mammalian lipases

The effects of proteins, both endogenous and exogenous, on the activity of lipases against water soluble and water insoluble substrates have been reviewed. The enzymes considered are pancreatic and gastric lipases, and lipoprotein, bile-salt-stimulated human milk and pancreatic carboxyl ester lipases. A brief account is given of the function of each enzyme and of the physical properties of the interacting proteins, which include albumins, lysozymes, globulins and immunoglobulins, myoglobin, transferrins, alpha-lactalbumin and melittin. With few exceptions (for example, the effect of colipase on pancreatic lipase), the interaction of proteins with lipases which act at the lipid-water interface of water insoluble substrates results in deactivation of enzymic activity. It seems that the amphiphilic nature of proteins allows them to aggregate at interfaces, thereby altering the nature of the interface and decreasing accessibility of the substrate to the enzyme. This discussion gives consideration to association of the proteins with the enzyme or the interface and to whether the interactions with specific binding sites or interfacial inactivation are responsible for the observations. However, the effect of proteins on lipases acting against water soluble substrates varies from protein to protein. Activation of enzyme-activity occurs if the interacting proteins are able to act as acyl transfer agents and thus introduce another catalytic hydrolysis pathway into the reaction mechanism. Inhibition may be caused by specific interactions between the protein and the enzyme or the substrate.     

Label-free detection of protein-protein interactions at the GaAs/water interface through surface infrared spectroscopy: discrimination between specific and nonspecific interactions by using secondary structure analysis

Here, we propose a label-free detection of protein-protein interactions that enables simultaneous qualitative analysis of target proteins by using Fourier transform infrared (FTIR) absorption spectroscopy in multiple internal reflection geometry (MIR-FTIR). Using this method, the target proteins were detected based on the peak height of the amide I and amide II bands, while discrimination of specific and nonspecific signals is made based on the secondary structure of the analytes, which is determined through second-derivative analysis of the amide I band. As a model system, an antigen peptide was immobilized on the surface of GaAs, which was transparent to mid-infrared light, and the interaction with its antibody was examined in aqueous media. We demonstrated that the binding of the antibody to the antigen immobilized on a GaAs surface selectively gave rise to beta-sheet amide I vibrations (1639 and 1690 cm-1), while no structurally related signals were induced by nonspecifically adsorbed proteins. The peak height of the beta-peak (1639 cm-1) in the amide I band linearly increased with the antiserum concentration as well as that of the amide II band. The detection limit (S/N = 3) was a 1:36 000 dilution for the amide I signal. In addition, through the use of surface-sensitive MIR-FTIR, the present sensor selectively detected the antigen-antibody interactions at the surfaces without being affected by the presence of bulk species, enabling rapid and wash-free detection. Our method provides not only rapid label-free detection of protein-protein interactions but a more accurate discrimination between specific and nonspecific interactions through the use of the secondary structure of the target proteins as a measure for the specific signals.     

Particle-particle interactions between layered double hydroxide nanoparticles

Changes in chemical properties of nanoscale particles include quantum size effect, changes in the cell parameters and lattice symmetry, and surface and interface effects. In the case of layered double hydroxides (LDHs), surface and interface effects dominate for nanoparticles of MgAl LDHs. Using TEM micrographs of nanoparticle-sized LDHs, we have found that the increased number of surface atoms relative to the internal atoms increases the surface-to-surface interparticle attractions. As a result, nanosize LDH particles are able to form continuous oriented films that adhere well to a polar substrate.     

Thiol-specific and nonspecific interactions between DNA and gold nanoparticles

The contribution of nonspecific interactions to the overall interactions of thiol-ssDNA and dsDNA macromolecules with gold nanoparticles was investigated. A systematic investigation utilizing dynamic light scattering and cryogenic transmission electron microscopy has been performed to directly measure and visualize the changes in particle size and appearance during functionalization of gold nanoparticles with thiol-ssDNA and nonthiolated dsDNA. The results show that both thiol-ssDNA and dsDNA do stabilize gold nanoparticle dispersions, but possible nonspecific interactions between the hydrophobic DNA bases and the gold surface promote interparticle interactions and cause aggregation within rather a short period of time. We also discuss the adsorption mechanisms of dsDNA and thiol-ssDNA to gold particles.     

A study of the interactions between carboplatin and blood plasma proteins using size exclusion chromatography coupled to inductively coupled plasma mass spectrometry

To study the carboplatin–protein interaction, a sensitive method using size exclusion chromatography coupled to inductively coupled plasma mass spectrometry (SEC–ICP–MS) was developed. The complexes formed between plasma proteins and carboplatin were monitored and identified with this method. Composite blood plasma samples from patients who were undergoing chemotherapy were analyzed, and carboplatin was found to bind plasma proteins. In addition, blank plasma samples were spiked with carboplatin and were analyzed as a time course study, and the results confirmed that carboplatin formed complexes with plasma proteins, primarily albumin and γ-globulin. To further substantiate the study, these two proteins were incubated with carboplatin. The binding between carboplatin and these proteins was then characterized qualitatively and quantitatively. In addition to a one-to-one binding of Pt to protein, protein aggregation was observed. The kinetics of the binding process of carboplatin to albumin and γ-globulin was also studied. The initial reaction rate constant of carboplatin binding to albumin was determined to be 0.74 M⁻¹ min⁻¹, while that for γ-globulin was 1.01 M⁻¹ min⁻¹, which are both lower than the rate constant of the cisplatin–albumin reaction previously reported.     

Size exclusion chromatography coupled to electrospray ionization mass spectrometry for analysis and quantitative characterization of arsenic interactions with peptides and proteins

Arsenic‐binding proteins are of toxicological importance since enzymatic activities can be blocked by arsenic interactions. In the present work, a novel methodology based on size exclusion chromatography coupled to electrospray ionization mass spectrometry (SEC‐ESI‐MS) was developed with special emphasis to preserve the intact proteins and their arsenic bindings. The eluent composition of 25 mM Tris/HCl, pH 7.5, with the addition of 100‐mM NaCl optimized for SEC with UV detection provided the highest SEC separation efficiency, but was not compatible with the ESI‐MS because of the non‐volatility of the buffer substance and of the salt additive. In order to find the best compromise between chromatographic separation and ionization of the arsenic‐binding proteins, buffer type and concentration, pH value, portion of organic solvent in the SEC eluent as well as the flow rate were varied. In the optimized procedure five different arsenic‐binding peptides and proteins (glutathione, oxytocin, aprotinin, α‐lactalbumin, thioredoxin) covering a molar mass range of 0.3–14 kDa could be analyzed using 75% 10‐mM ammonium formate, pH 5.0/25% acetonitrile (v : v) as eluent and a turbo ion spray source operated at 300 °C and 5.5 kV. A complete differentiation of all peptides and proteins involved in the arsenic‐binding studies as well as of their arsenic‐bound forms has become feasible by means of the extracted ion chromatograms (XIC) of the mass spectrometric detection. The new method offered the possibility to estimate equilibrium constants for the reaction of phenylarsine oxide with different thiol‐containing biomolecules by means of the XIC peak areas of reactants and products. Limits of detection in the range of 2–10 µM were obtained by SEC‐ESI‐MS for the individual proteins. Copyright © 2009 John Wiley & Sons, Ltd.     

Computer simulation studies on the interactions between nanoparticles and cell membrane

In recent times,nanoparticles(NPs)have received intense attention not only due to their potential applications as a candidate for drug delivery,but also because of their undesirable effects on human health.Although extensive experimental studies have been carried out in literature in order to understand the interaction between NPs and a plasma membrane,much less is known about the molecular details of the interaction mechanisms and pathways.As complimentary tools,coarse grained molecular dynamics(CGMD)and dissipative particle dynamics(DPD)simulations have been extensively used on the interaction mechanism and evolution pathway.In the present review we summarize computer simulation studies on the NP-membrane interaction,which developed over the last few years,and particularly evaluate the results from the DPD technique.Those studies undoubtedly deepen our understanding of the NP-membrane interaction mechanisms and provide a design guideline for new NPs.     

Electrostatic interactions between amphoteric latex particles and proteins

The electrostatic interactions between amphoteric polymethyl methacrylate latex particles and proteins with different pI values were investigated. These latex particles possess a net positive charge at low pH, but they become negatively charged at high pH. The nature and degree of interactions between these polymer particles and proteins are primarily controlled by the electrostatic characteristics of the particles and proteins under the experimental conditions. The self-promoting adsorption process from the charge neutralization of latex particles by the proteins, which have the opposite net charge to that of the particles, leads to a rapid reduction in the zeta potential of the particles (in other words colloidal stability), and so strong flocculation occurs. On the other hand, the electrostatic repulsion forces between similarly charged latex particles and the proteins retard the adsorption of protein molecules onto the surfaces of the particles. Therefore, latex particles exhibit excellent colloidal stability over a wide range of protein concentrations. A transition from net negative charge to net positive charge, and vice versa (charge reversal), was observed when the particle surface charge density was not high enough to be predominant in the protein adsorption process.     

A quantitative analysis of the through-space and the through-bond interactions between lone-pairs and σ-frames: S-triazine and S-tetrazine

The CNDO/2 and INDO calculations were performed on s-triazine and s-tetrazine. The s-triazine has three lone-pairs in a molecule, and these can be combined into three combinations, A, n_s and n_A. Among the three, n_s and n_A are degenerated when the whole interaction conserves its molecular point symmetry, D_3h. The s-tetrazine has four lone-pairs, which can be transformed into four combinations, SS,SA, AS and AA. The energies of these orbitals show interesting behavior. The results were subjected to an analysis from the standpoint of the through-space and through-bond interactions using the localized molecular orbitals. As a result of these analyses, the interactions were expressed by several interaction terms.     

Applied environmental metrology for studying health-pollution interactions

Nuclear and isotopic techniques are valuable tools in assessing the levels of environmental pollution by toxic elements and for studying how these contaminants affect human health. More than 90 counterparts from 55 countries around the world have participated in projects on these topics, supported by the International Atomic Energy Agency (IAEA), during last ten years. With the support of the IAEA, for example, an appropriate metrology for compositional characterization of size fractionated airborne particulate matter was developed, verified and implemented in more than 40 countries. This paper reviews the development and application of environmental metrology tools involving nuclear analytical and isotopic techniques, as seen from the particular perspective of IAEA programmes.     

Carbohydrate chips for studying high-throughput carbohydrate-protein interactions

Carbohydrate-protein interactions play important biological roles in living organisms. For the most part, biophysical and biochemical methods have been used for studying these biomolecular interactions. Less attention has been given to the development of high-throughput methods to elucidate recognition events between carbohydrates and proteins. In the current effort to develop a novel high-throughput tool for monitoring carbohydrate-protein interactions, we prepared carbohydrate microarrays by immobilizing maleimide-linked carbohydrates on thiol-derivatized glass slides and carried out lectin binding experiments by using these microarrays. The results showed that carbohydrates with different structural features selectively bound to the corresponding lectins with relative binding affinities that correlated with those obtained from solution-based assays. In addition, binding affinities of lectins to carbohydrates were also quantitatively analyzed by determining IC(50) values of soluble carbohydrates with the carbohydrate microarrays. To fabricate carbohydrate chips that contained more diverse carbohydrate probes, solution-phase parallel and enzymatic glycosylations were performed. Three model disaccharides were in parallel synthesized in solution-phase and used as carbohydrate probes for the fabrication of carbohydrate chips. Three enzymatic glycosylations on glass slides were consecutively performed to generate carbohydrate microarrays that contained the complex oligosaccharide, sialyl Le(x). Overall, these works demonstrated that carbohydrate chips could be efficiently prepared by covalent immobilization of maleimide-linked carbohydrates on the thiol-coated glass slides and applied for the high-throughput analyses of carbohydrate-protein interactions.