Amplifying the electrical hybridization signals of DNA array by multilayer assembly of Au nanoparticle probes

This paper describes a versatile method for amplifying the signals of Au-nanoparticle-based DNA hybridization detecting systems. The Au nanoparticles usually serve as labels to enhance DNA hybridization signal. We further assembled several layers of nanoparticles to selectively increase the number of labelled nanoparticles. Through silver enhancement, the multilayer nanoparticles may produce significantly higher amounts of metal silver on the their surfaces than the monolayer nanoparticles did. This finally accounts for the greatly enhanced DNA hybridization signal. Particularly, the amplification of electrical detection system was demonstrated here. Electrical measuring results indicated that the current values were enhanced by approximately 3 orders of magnitude, and the single nucleotide mismatch discrimination ratio was enlarged to approximately 10(9):1.     

Polymer-coated ferromagnetic colloids from well-defined macromolecular surfactants and assembly into nanoparticle chains

A novel synthetic route to polymer-coated ferromagnetic colloids of metallic cobalt has been developed. Well-defined end-functional polystyrenes were synthesized using controlled radical polymerization and used as surfactants in the thermolysis of dicobaltoctacarbonyl to afford uniform ferromagnetic nanoparticles. The presence of the polymer shell enabled prolonged colloidal stability of dispersions in a wide range of organic solvents and formed glassy encapsulating coatings around ferromagnetic cores in the solid state. These polymer-coated colloids assembled into robust, micron-sized nanoparticle chains when cast onto supporting surfaces due to dipolar associations of magnetic cores. Hierarchical assemblies were also prepared by blending polystyrene-coated cobalt colloids with larger silica beads.     

Activity of Subtilisin Carlsberg in macromolecular crowding

Enzymatic activity of a proteolytic enzyme Subtilisin Carlsberg (SC) in anionic sodium dodecyl sulfate (SDS) micellar medium has been explored and found to be retarded compared to that in bulk buffer. Circular dichroism (CD) study reveals that SDS, which is a potential protein denaturant, has an insignificant denaturation effect on SC. The structural integrity of the protein offers an opportunity to study the functionality of the enzyme SC in a macromolecular crowding of micelles. Dynamic light scattering (DLS) data indicates no sandwich-like micelle-SC complex formation ruling out the possibility of interaction of the enzyme with the hydrophobic core of the micelle. However, steady state and time resolved emission studies on specific and nonspecific fluorescent probes indicate the proximity effect at the surface of the enzyme due to macromolecular crowding of the micelles. The agreement of retarded enzymatic activity in the micellar crowd with a theoretical model ascribed to the facts that substrates are compartmentalized in the micelles and enzyme interacts with the micelle through stern layer.     

Residue-level interrogation of macromolecular crowding effects on protein stability

Theory predicts that macromolecular crowding affects protein behavior, but experimental confirmation is scant. Herein, we report the first residue-level interrogation of the effects of macromolecular crowding on protein stability. We observe up to a 100-fold increase in the stability, as measured by the equilibrium constant for folding, for the globular protein chymotrypsin inhibitor 2 (CI2) in concentrations of the cosolute poly(vinylpyrrolidone) (PVP) that mimic the protein concentration in cells. We show that the increased stability is caused by the polymeric nature of PVP and that the degree of stabilization depends on both the location of the individual residue in the protein structure and the PVP concentration. Our data reinforce the assertion that macromolecular crowding stabilizes the protein by destabilizing its unfolded states.     

Kinetics of bioconjugate nanoparticle label binding in a sandwich-type immunoassay

Nanoparticle labels have enhanced the performance of diagnostic, screening, and other measurement applications and hold further promise for more sensitive, precise, and cost-effective assay technologies. Nevertheless, a clear view of the biomolecular interactions on the molecular level is missing. Controlling the ratio of molecular recognition over undesired nonspecific adhesion is the key to improve biosensing with nanoparticles. To improve this ratio with an aim to disallow nonspecific binding, a more detailed perspective into the kinetic differences between the cases is needed. We present the application of two novel methods to determine complex binding kinetics of bioconjugate nanoparticles, interferometry, and force spectroscopy. Force spectroscopy is an atomic force microscopy technique and optical interferometry is a direct method to monitor reaction kinetics in second-hour timescale, both having steadily increasing importance in nanomedicine. The combination is perfectly suited for this purpose, due to the high sensitivity to detect binding events and the ability to investigate biological samples under physiological conditions. We have attached a single biofunctionalized nanoparticle to the outer tip apex and studied the binding behavior of the nanoparticle in a sandwich-type immunoassay using dynamic force spectroscopy in millisecond timescale. Utilization of the two novel methods allowed characterization of binding kinetics in a time range spanning from 50 ms to 4 h. These experiments allowed detection and demonstration of differences between specific and nonspecific binding. Most importantly, nonspecific binding of a nanoparticle was reduced at contact times below 100 ms with the solid-phase surface.

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Figure A single biofunctionalized nanoparticle was attached to the outer tip apex and the binding behavior of the nanoparticle in a sandwich-type immunoassay, A) without analyte, B) with analyte and C) saturating analyte concentration, was recorded using dynamic force spectroscopy in millisecond timescale. The setting allowed measurement of the association speed of nonspecific binding.

     

The influence of macromolecular crowding on HIV-1 protease internal dynamics

High macromolecular concentrations, or crowded conditions, have been shown to affect a wide variety of molecular processes, including diffusion, association and dissociation, and protein folding and stability. Here, we model the effect of macromolecular crowding on the internal dynamics of a protein, HIV-1 protease, using Brownian dynamics simulations. HIV-1 protease possesses a pair of flaps which are postulated to open in the early stages of its catalytic mechanism. Compared to low concentrations, close-packed concentrations of repulsive crowding agents are found to significantly reduce the fraction of time that the protease flaps are open. Macromolecular crowding is likely to have a major effect on in vivo enzyme activity, and may play an important regulatory role in the viral life cycle.     

Simultaneous identification of point mutations via DNA ligase-mediated gold nanoparticle assembly

Multiplex single nucleotide polymorphisms analysis has found a great demand in human genetics and pharmacogenetics. The present study reports a novel approach for a genotyping assay that could achieve simultaneous identification of multiple point mutations via a ligase-mediated gold nanoparticle assembly. Based on the allelic specificity of DNA ligase, gold nanoparticles modified by oligonucleotide probes perfectly matched to the DNA targets were assembled into a thermally-stable aggregate, while a single-base mismatch would result in the dissociation of the gold nanoparticle assembly at high temperature. Then, DNA targets and their point mutations could be differentiated using a multi-step temperature elevation analysis monitored by ultraviolet-visible measurements. This approach offered a direct colorimetric discrimination of multiple point mutations without stringent temperature control. The proposed approach is demonstrated using a model system for the identification of single-base mutations in codon 17 and position -28 of the beta-thalassemia gene. The results reveal that the wild and the mutant types could be simultaneously determined successfully. Owing to its ease of operation and high specificity, it was expected that the proposed procedure might hold great promise in both research-oriented and clinical genomic assays.     

Surface-enhanced Raman scattering of p-aminothiophenol on a Au(core)/Cu(shell) nanoparticle assembly.

Surface-enhanced Raman scattering (SERS) of p-aminothiophenol (PATP) molecules adsorbed onto assemblies of Au(core)/Cu(shell) nanoparticles is reported. We compare it with the SERS spectrum of PATP adsorbed onto gold nanoparticles: both the absolute and relative scattered intensities of various bands in the two spectra are very different. The difference in relative intensity can be ascribed to chemical effects; the chemical enhancement ratio of the two substrates is approximately 3-5. A theoretical analysis based on a charge-transfer model is carried out, which yields a consistent result and shows that the difference in chemical enhancement is mainly due to the state densities and Fermi levels of the substrates. The difference in absolute intensity originates from electromagnetic (EM) enhancement. EM enhancement of Au(core)/Cu(shell) nanoparticles is unlike that of single-component gold or copper SERS-active substrates. The core/shell particle size for optimal enhancement is about 20 nm in the case of a 632.8 nm incident laser (the size ratio of the core and shell layers is about 0.6).     

Misfolding of amyloidogenic proteins at membrane surfaces: the impact of macromolecular crowding

The presence of inert macromolecular crowding agents mimics the situation in vivo where amyloidogenic proteins are released into an aqueous, congested intracellular environment. By using the amphiphatic Alzheimer Abeta-protein as the model system, the presence of a three-dimensional macromolecular crowding environment enhanced significantly its misfolding behavior if charged membrane surfaces as two-dimensional aggregation templates were present.     

The role of macromolecular crowding in single-entity electrochemistry: Friend or foe?

The recent development of nanoscale probes has enabled the study of single molecules and single cells with unprecedented resolution and the expansion of the field of single-entity electrochemistry. There is a growing evidence suggesting that highly crowded intracellular environment facilitate nanoelectrochemical measurements in cells by improving the signal-to-noise ratio. In this opinion piece, we discuss the concept of macromolecular crowding and its implications in single-entity electrochemistry.     

Effect of molecular crowding on the response of an electrochemical DNA sensor

E-DNA sensors, the electrochemical equivalent of molecular beacons, appear to be a promising means of detecting oligonucleotides. E-DNA sensors are comprised of a redox-modified (here, methylene blue or ferrocene) DNA stem-loop covalently attached to an interrogating electrode. Because E-DNA signaling arises due to binding-induced changes in the conformation of the stem-loop probe, it is likely sensitive to the nature of the molecular packing on the electrode surface. Here we detail the effects of probe density, target length, and other aspects of molecular crowding on the signaling properties, specificity, and response time of a model E-DNA sensor. We find that the highest signal suppression is obtained at the highest probe densities investigated, and that greater suppression is observed with longer and bulkier targets. In contrast, sensor equilibration time slows monotonically with increasing probe density, and the specificity of hybridization is not significantly affected. In addition to providing insight into the optimization of electrochemical DNA sensors, these results suggest that E-DNA signaling arises due to hybridization-linked changes in the rate, and thus efficiency, with which the redox moiety collides with the electrode and transfers electrons.     

Effect of precursor-layer surface charge on the layer-by-layer assembly of polyelectrolyte/nanoparticle multilayers

In this Article, we investigate the effect of a precursor layer, which is composed of four bilayers of polyethyleneimine (PEI) and poly(sodium styrene sulfonate) (PSS), on the subsequent LBL assembly of hybrid films composed of indium tin oxide (ITO) nanoparticles and PSS. A precursor polyelectrolyte layer is usually deposited to minimize interference by the substrate. It is shown here that the "effective" surface charge of the precursor layer can significantly affect the subsequent assembly behavior of [ITO/PSS](9.5) hybrid thin films. Depending on the surface charge of the precursor layer, the subsequent LbL assembly of [ITO/PSS](9.5) hybrid films can exhibit either one or two regimes. When two growth regimes are present, the first one consists of a "recovery regime", and the second is the expected "linear growth regime." The length of the "recovery regime" is dependent on how much positive charge the precursor layer possesses and how fast this surface charge can be compensated. This work reveals for the first time that changes in the surface charge of the precursor layer can have a significant effect on the subsequent LBL assembly process. The surface charge of the precursor layer was investigated using ζ-potential measurements on model silica microspheres. These experiments showed that the surface charge of the precursor layer, [PEI/PSS](4), is dependent on the pH of the solution in which it is immersed, and that it can reverse from a negatively charged surface to a positively charged one, at sufficiently low pH due to the protonation of PEI, despite having the negatively charged PSS layer as the outermost layer.     

纳米粒子组装体系的研究进展

制备纳米粒子组装体系是构筑纳米结构的重要方法之一，本文综述了纳米粒子组装体系的制备方法及其性质和应用研究。     

Magnetic nanoparticle assembly on surfaces using click chemistry

Controlled assembly of ferromagnetic nanoparticles on surfaces is of crucial importance for a range of spintronic and data storage applications. Here, we present a novel method for assembling monolayers of ferromagnetic FePt nanoparticles on silicon oxide substrates using "click chemistry". Reaction of alkyne-functionalized FePt nanoparticles with azide-terminated self-assembled monolayers (SAMs), on silicon oxide, leads to the irreversible attachment of magnetic nanoparticles to the surface via triazole linkers. Based on this covalent interaction, well-packed monolayers of FePt nanoparticles were prepared and nanoparticle patterns are generated on surfaces via microcontact printing (μCP).     

Toward realistic modeling of dynamic processes in cell signaling: quantification of macromolecular crowding effects

One of the major factors distinguishing molecular processes in vivo from biochemical experiments in vitro is the effect of the environment produced by macromolecular crowding in the cell. To achieve a realistic modeling of processes in the living cell based on biochemical data, it becomes necessary, therefore, to consider such effects. We describe a protocol based on Brownian dynamics simulation to characterize and quantify the effect of various forms of crowding on diffusion and bimolecular association in a simple model of interacting hard spheres. We show that by combining the elastic collision method for hard spheres and the mean field approach for hydrodynamic interaction (HI), our simulations capture the correct dynamics of a monodisperse system. The contributions from excluded volume effect and HI to the crowding effect are thus quantified. The dependence of the results on size distribution of each component in the system is illustrated, and the approach is applied as well to the crowding effect on electrostatic-driven association in both neutral and charged environments; values for effective diffusion constants and association rates are obtained for the specific conditions. The results from our simulation approach can be used to improve the modeling of cell signaling processes without additional computational burdens.     

Suppression of linear side products by macromolecular crowding in nonribosomal enterobactin biosynthesis

Nonribosomal enterobactin synthetase of Escherichia coli was found to prematurely release a large amount of linear precursors in an in vitro reconstitution. However, these side products are suppressed to negligible levels by polymeric cosolvents that create macromolecular crowding, a prominent feature of the intracellular environment. These findings show that macromolecular crowding is essential to normal functioning of the nonribosomal peptide synthetase and suggest that it may be crucial to biotechnological utilization of similar enzyme systems.