Electrode surface confinement of self-assembled enzyme aggregates using magnetic nanoparticles and its application in bioelectrocatalysis

Self-assembled enzyme aggregates, prepared from magnetic iron oxide nanoparticles, avidin, and a biotinylated redox enzyme, were shown particularly useful for the simple, fast, and efficient construction of highly enzyme-loaded electrodes with the help of a magnet. The approach was illustrated in the case of the bioelectrocatalytic oxidation of NADH by a diaphorase oxidoreductase in the presence of a ferrocene mediator. Two different self-assembling procedures were tested, taking advantage of the spontaneous aggregation of the nanoparticles in the presence of avidin and also of the multivalency binding of biotinylated diaphorase toward avidin. Activities of the bound and unbound diaphorase were systematically controlled allowing determination of the number of active biotinylated diaphorase per nanoparticle incorporated within each magnetic enzyme aggregate. An active enzyme loading capacity of up to 2.35 nmol mg-1 was found for the best nanostructured enzyme assembly, which is 200 times better than for commercialized magnetic micrometer-sized beads coated with streptavidin and saturated with diaphorase. With the help of a permanent magnet, the magnetic enzyme aggregates were finally magnetically collected as a film on the surface of a small screen-printed carbon electrode and the catalytic currents recorded by cyclic voltammetry. From the analysis of the steady-state catalytic current responses and the kinetic rate constants of biotinylated diaphorase, it was possible to determine the enzyme concentration within the magnetic films. Owing to the high enzyme loading in the aggregates of nanoparticles (i.e., 130 microM), the catalytic current responses were definitely higher than the ones measured at an electrode coated with a closed-packed monolayer of diaphorase or at an electrode covered with a film of magnetic micrometer-sized streptavidin beads saturated with diaphorase.     

Electrochemically programmed release of biomolecules and nanoparticles

The controlled release of molecules or nanoparticle conjugates is an important tool for a wide range of applications in science and engineering. Here we demonstrate electrochemically programmed release of biomolecules and nanoparticles immobilized on patterned gold electrodes using the thiol-gold linkage. This technique exploits the reductive desorption of self-assembled monolayers and allows both spatially controlled release and regeneration of small molecules (e.g., drugs), biopolymers (e.g., peptides, proteins, DNA), protein assemblies (e.g., viruses), and nanoparticles (e.g., particle-DNA conjugates). Fluorescence microscopy is used to image the release of avidin and nanoparticles in phosphate-buffered saline and to determine the kinetics of desorption. We also demonstrate that the electrodes can be regenerated using the same conjugation scheme.     

Detection of abasic sites on individual DNA molecules using atomic force microscopy

We have developed an atomic force microscopy-based method for detecting abasic sites (AP sites) on individual DNA molecules. By using uracil and uracil DNA glycosylase, we first prepared a 250-bp DNA template consisting of two AP sites at specific locations. We then detected the AP sites by marking them with biotinylated aldehyde-reactive probes and monomeric avidin. We demonstrate here that (i) the location of monomeric avidin bound on a single DNA molecule was detectable by atomic force microscopy; (ii) the observed location of avidin was in good agreement to the predicted AP sites at a few nanometer resolution; and (iii) by end-labeling the 5'-terminus of one DNA strand, the AP sites were determined without directional ambiguity. The technique described here will provide a sensitive way of locating AP sites and contribute to screen DNA damages from individual molecules.     

Core-shell nanostructured nanoparticle films as chemically sensitive interfaces

This paper reports the results of an investigation of vapor molecule sorption at different types of nanostructured nanoparticle films. Core-shell nanoparticles of two different core sizes, AU2-nm and Au5-nm, and molecular linkers of two different binding properties, 1,9-nonanedithiol and 11-mercaptoundecanoic acid, are utilized as building blocks for constructing chemically sensitive interfaces. The work couples measurements of two different transducers, interdigitated microelectrodes and quartz crystal microbalance, to determine the correlation of the electronic resistance change and the mass loading with vapor sorption. The responses to vapor sorption at these nanostructured interfaces are demonstrated to be dependent on the core size of nanoparticles and the chemical nature of linking molecules. The difference of molecular interactions of vapor molecules at the nanostructured interface is shown to have a significant impact on the response profile and sensitivity. For the tested vapor molecules, while there are small differences for the sorption of nonpolar and hydrophobic vapor molecules, there are striking differences for the sorption of polar and hydrophilic vapor molecules at these nanostructured interfacial materials. The implication of the findings to the delineation of design parameters for constructing core-shell nanoparticle assemblies as chemically sensitive interfacial materials is also discussed.     

Enzyme-modulated cleavage of dsDNA for supramolecular design of biosensors.

Supramolecular docking and immobilization of biotinylated dsDNA onto a self-assembled monolayer of avidin have been measured using impedance spectroscopy and quartz crystal microbalance technique. The formation of the serial assembly was first achieved by linearizing circular plasmid dsDNA using BamH I endonuclease enzyme. This was followed by a bisulfite-catalyzed transamination reaction in order to biotinylate the dsDNA. The reaction is single-strand specific, and it specifically targets unpaired cytosine bases generated during the enzyme cleavage. The biotinylated dsDNA was then used as a ligand at a gold electrode containing avidin. The process was monitored by ac impedance spectroscopy that was used to probe the changes in interfacial electron-transfer resistance upon binding and a microgravimetric quartz crystal microbalance that reflected in situ mass changes on the dsDNA-functionalized substrates. Our results demonstrated that this approach could be employed for the determination of small-molecular-weight organics such as cisplatin, daunomycin, bisphenol A, chlorinated phenols, and ethidium bromide. A detection limit in the magnitude of ca. 10 nM was achieved. This immobilization technique provides a generic approach for dsDNA-based sensor development and for the monitoring of DNA-analyte interactions.     

Assembly of CdS nanoparticles in patterned structures by a novel ion-entrapment process in thermally evaporated fatty acid films

The formation of cadmium sulfide nanoparticle assemblies in a patterned manner on suitable substrates is described. The protocol for realizing such structures comprises the following steps. In the first step, patterned films of a fatty acid are thermally evaporated onto solid supports using suitable masks (e.g., a transmission electron microscope grid). Thereafter, the fatty acid film is immersed in cadmium sulfate solution and Cd2+ ions entrapped in the lipid matrix by electrostatic complexation with the carboxylate ions of the fatty acid molecules. The final step involves reaction of the entrapped Cd2+ ions with Na2S, leading to the in situ generation of cadmium sulfide nanoparticles within the patterned lipid matrix. This approach shows promise for generating patterned nanoparticle assemblies of different chemical compositions.     

Tunable electronic interfaces between bulk semiconductors and ligand-stabilized nanoparticle assemblies

Interfaces between nanoscale and bulk electroactive materials are important for the design of electronic devices using solution-processed nanoparticles. We report that thin films of hexanethiolate-capped gold nanoparticles with a core diameter of 2.1+/-0.4 nm deposited onto n-InP wafers form Schottky contacts whose barrier height can be actively tuned from 0.27+/-0.03 to 1.11+/-0.09 eV by electrochemically adjusting the nanoparticle Fermi level. This result is remarkable because interfacial barriers at conventional metal-semiconductor contacts are largely insensitive to the initial Fermi level of the metal. Furthermore, it highlights two general features of solution-processed nanoparticle assemblies in comparison with traditional bulk electronic materials: (1) the ability of ions to permeate the nanoparticle assembly enables the electrochemical injection of charges and hence active control of the Fermi level, and (2) ligand passivation of nanoparticle surfaces prevents interfacial reactions with the semiconductor that could otherwise lead to strong Fermi-level pinning.     

Application of avidin-biotin technology and adsorptive transfer stripping square-wave voltammetry for detection of DNA hybridization and avidin in transgenic avidin maize

The proteins streptavidin and avidin were electrochemically detected in solution by adsorptive transfer stripping square wave voltammetry (AdTS SWV) at a carbon paste electrode (CPE). AdTS SWV was used to quantify biotinylated oligonucleotides, DNA hybridizations, and avidin in extracts of transgenic avidin maize. The detection limits of denatured and native streptavidin were 6 pM and 120 nM, respectively. The results demonstrated that streptavidin/avidin AdTS SWV is a sensitive and specific method for quantifying DNA and proteins in biological samples such as foods and tissue extracts, including genetically modified crops (avidin maize) and other plants in neighboring fields.     

Development of an "electroptode" immunosensor: indium tin oxide-coated optical fiber tips conjugated with an electropolymerized thin film with conjugated cholera toxin B subunit

We demonstrate that it is possible to create surface-conductive fiber optics, upon which may be electropolymerized a biotinylated polypyrrole thin film, which may then be used to affinity coat the fiber with molecular recognition probes. This fiber-optic electroconductive surface modification is done by the deposition of a thin layer of indium tin oxide. Thereafter, biotin-pyrrole monomers are electropolymerized onto the conductive metal oxide surface and then exposed to avidin. Avidin-biotin interactions were used to modify the fiber optics with biotin-conjugated cholera toxin B subunit molecules, for the construction of an immunosensor to detect cholera antitoxin antibodies. The biosensor was tested for sensitivity, nonspecificity, and overall practicality.     

In vivo detection of gold-imidazole self-assembly complexes: NIR-SERS signal reporters

Here we report in vitro and in vivo detection of self-assembled Au-imidazole by using near-infrared surface-enhanced Raman scattering (NIR-SERS). In vivo, the Au-imidazole structures were administered into tumor-bearing mice and detected noninvasively. The self-assembled Au-imidazole complexes were generated by the adsorption of imidazole molecules onto Au nanoparticles (NP) and were then characterized as aqueous suspensions by using NIR-SERS, angle-dependent light scattering with fractal dimension analysis, and visible extinction spectroscopy. The structure and optical activity was sensitive to imidazole concentration and Au NP size. Specifically, the Au-imidazole assemblies formed at lower imidazole concentrations had the lowest fractal dimension (D(f) = 1.2) and the largest Raman enhancement factors for the dominant NIR-SERS feature, a ring-breathing vibrational mode at 954 cm(-1). Changes in elastic scattering intensity, fractal dimension, and surface plasmon absorption were observed with increasing imidazole concentration. The Raman enhancement factor was also found to range between 10(6) and 10(9) with different primary Au nanoparticle sizes. For the higher enhancement factor systems, NIR-SERS detection of Au-imidazole was performed with data acquisitions time of only 5 s. The largest enhancement was observed for the 954-cm(-1) feature at an imidazole concentration of 1.9 microM when coupled to 54-nm-diameter Au NPs (the largest NP tested). Finally, we show the first demonstration of in vivo, noninvasive, and real-time SERS detection.     

Surface-enhanced Raman scattering inside Au@Ag core/shell nanorods

The design and synthesis of plasmonic nanoparticles with Raman-active molecules embedded inside them are of significant interest for sensing and imaging applications.However,direct synthesis of such nanostructures with controllable shape,size,and plasmonic properties remains extremely challenging.Here we report on the preparation of uniform Au@Ag core/shell nanorods with controllable Ag shells of 1 to 25 nm in thickness.1,4-Aminothiophenol (4-ATP) molecules,used as the Raman reporters,were located between the Au core and the Ag shell.Successful embedding of reporter molecules inside the core/shell nanoparticles was confirmed by the absence of selective oxidation of the amino groups,as measured by Raman spectroscopy.The dependence of Raman intensity on the location of the reporter molecules in the inside and outside of the nanorods was studied.The molecules in the interior showed strong and uniform Raman intensity,at least an order of magnitude higher than that of the molecules on the nanoparticle surface.In contrast to the usual surface-functionalized Raman tags,aggregation and clustering of nanoparticles with embedded molecules decreased the surface-enhanced Raman scattering (SERS) signal.The findings from this study provide the basis for a novel detection technique of low analyte concentration utilizing the high SERS response of molecules inside the core/shell metal nanostructures.As an example,we show robust SERS detection of thiram fungicide as low as 10-9 M in solutions.     

Sensing of H2O2 at low surface density assemblies of Pt nanoparticles in polyelectrolyte

Amperometric detection of H2O2 was studied at random arrays of 2.5 nm polyacrylate-capped Pt nanoparticles (NP) assembled in poly(diallydimethylammonium chloride), PDDA, as a function of NP surface coverage. The arrays were assembled by varying the adsorption time of PDDA-modified electrodes in the nanoparticles solution. Pt NP-on-PDDA assemblies exhibited significant sensitivity and stability facing constant anodic polarization and a low limit of detection at small Pt mass in submonolayer coverage. The current output was measured at approximately 0.5 A M(-1) cm(-2)(geom) over a linear range from 42 nM to 0.16 mM H2O2 at a loading of 0.87 microg(Pt)/cm(2) or an estimated coverage of 0.4 of an assumed monolayer, or higher, and decreased with decreasing NP surface density to 0.2 A M(-1) cm(-2)(geom) at a Pt loading of 190 ng/cm. On the other hand, the intrinsic sensitivity measured relative to the real Pt surface area increased with decreasing coverage and reached a significant limiting value of 0.9 A M(-1) cm(-2) real at approximately 190-380 ng/cm(2). The behavior shows a significant effective turnover rate per Pt site and mass (1 A M(-1)/microg of Pt) in loosely packed assemblies, while overlap of individual diffusion fields (of particles or islands) and inaccessibility of some active sites lowers the sensitivity per nanoparticle in densely packed arrays. The reported trend agrees with the behavior of ultramicroelectrode arrays.     

Multiparameter magnetic relaxation switch assays

Magnetic nanoparticles (NPs) can serve as magnetic relaxation switches (MRSw's), switching from a dispersed to a clustered state, or the reverse, due to the presence of molecular targets, with changes in the spin-spin relaxation time of water (T2). Biotinylated NP probes reacted with an avidin molecular target to form stable NP clusters, which permitted several NMR parameters to be measured as a function of cluster size. Associated with avidin-induced NP cluster formation was an increase in the spin-spin relation rate (1/T2), while the spin-lattice relaxation rate (1/T1)was unaffected. On the basis of the selective effects of NP cluster formation on T2, we developed a T1/T2 interrogation method where NP probe concentration and avidin analyte were unknown and both were determined. A third NMR parameter examined was the replication of T2 measurements, which were used to rapidly determine whether the ratio of avidin to biotinylated NP was optimal or whether additional biotinylated NP was needed. The T1/T2 and T2 replication interrogation methods illustrate how MRSw assays can employ multiple parameters, instead of relying only on T2, to obtain information about the reaction of NPs with molecular targets.     

Development of transferrin functionalized poly(ethylene glycol)/poly(lactic acid) amphiphilic block copolymeric micelles as a potential delivery system targeting brain glioma

The aim of present study is to conceive a biodegradable poly(ethylene glycol)–polylactide (PEG–PLA) copolymer nanoparticle which can be surface biofunctionalized with ligands via biotin–avidin interactions and used as a potential drug delivery carrier targeting to brain glioma in vivo. For this aim, a new method was employed to synthesize biotinylated PEG–PLA copolymers, i.e., esterification of PEG with biotinyl chloride followed by copolymerization of hetero-biotinylated PEG with lactide. PEG–PLA nanoparticles bearing biotin groups on surface were prepared by nanoprecipitation technique and the functional protein transferrin (Tf) were coupled to the nanoparticles by taking advantage of the strong biotin–avidin complex formation. The flow cytometer measurement demonstrated the targeting ability of the nanoparticles to tumor cells in vitro, and the fluorescence microscopy observation of brain sections from C6 glioma tumor-bearing rat model gave the intuitive proof that Tf functionalized PEG–PLA nanoparticles could penetrate into tumor in vivo.     

Importance of nanoparticle size in colorimetric and SERS-based multimodal trace detection of Ni(II) ions with functional gold nanoparticles

Colorimetric detection of analytes using gold nanoparticles along with surface-enhanced Raman spectroscopy (SERS) are areas of intense research activity since they both offer sensing of very low concentrations of target species. Multimodal detection promotes the simultaneous detection of a sample by a combination of different techniques; consequently, surface chemistry design in the development of multimodal nanosensors is important for rapid and sensitive evaluation of the analytes by diverse analytical methods. Herein it is shown that nanoparticle size plays an important role in the design of functional nanoparticles for colorimetric and SERS-based sensing applications, allowing controlled nanoparticle assembly and tunable sensor response. The design and preparation of robust nanoparticle systems and their assembly is reported for trace detection of Ni(II) ions as a model system in an aqueous solution. The combination of covalently attached nitrilotriacetic acid moieties along with the L-carnosine dipeptide on the nanoparticle surface represents a highly sensitive platform for rapid and selective detection of Ni(II) ions. This systematic study demonstrates that significantly lower detection limits can be achieved by finely tuning the assembly of gold nanoparticles of different core sizes. The results clearly demonstrate the feasibility and usefulness of a multimodal approach.     

Versatile apoferritin nanoparticle labels for assay of protein

A versatile bioassay label based on marker-loaded apoferritin nanoparticles (MLANs) has been developed for sensitive protein detection. Dissociation and reconstitution characteristics at different pH as well as the special cavity structure of apoferritin provides a facile route to prepare nanoparticle labels and avoid the complicated and tedious synthesis process of conventional nanoparticle labels. The optical and electrochemical characteristics of the prepared nanoparticle labels are easily controlled by loading different optical or electrochemical markers. A fluorescence marker (fluorescein anion) and a redox marker [hexacyanoferrate(III)] were used as model markers to load into the cavity of apoferritin nanoparticles for microscopic fluorescence immunoassay and electrochemical immunoassay, respectively. Detection limits of 0.06 (0.39 pM) and 0.08 ng mL(-1) (0.52 pM) IgG were obtained with fluorescein MLAN and hexacyanoferrate MLANs, respectively. The new nanoparticle labels hold great promise for multiplex protein detection (in connection with nanoparticles loaded with different markers) and for enhancing the sensitivity of other bioassays.     

Quartz crystal microbalance study of DNA immobilization and hybridization for nucleic Acid sensor development

The immobilization of two 30-mer oligonucleotides, one biotinylated (biotin-DNA) and the other having a mercaptohexyl group at the 5'-phosphate end (BS1-SH), onto modified gold surfaces has been examined using a quartz crystal microbalance (QCM). Both single-layer and multilayer DNA films were prepared. The single-layer films of biotin-DNA were constructed by binding to a precursor layer of avidin, which had been attached to the QCM either covalently using a water-soluble carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) or via electrostatic interaction with poly(allylamine hydrochloride) (PAH). Single-layer films of BS1-SH were also formed on PAH via the electrostatic attraction between the amine groups on PAH and the negatively charged phosphate backbone of DNA. Multilayer films of DNA were fabricated by the successive deposition of avidin and poly(styrenesulfonate) (PSS), up to a total of nine avidin/PSS layers, followed by DNA adsorption. DNA immobilization and hybridization of the immobilized DNAs was monitored in situ from QCM frequency changes. Hybridization was induced by exposure of the DNA-containing films to complementary DNA in solution. Equal frequency changes were observed for the DNA immobilization and hybridization steps for the single-layer films, indicating a DNA probe-to-hybridized DNA target ratio of 1:1. The multilayer DNA films also exhibited DNA hybridization, with a greater quantity of DNA hybridized compared with the single-layer films. The multilayer films provide a novel means for the fabrication of DNA-based thin films with increased capacity for nucleic acid detection.     

Development of binding assays in microfabricated picoliter vials: an assay for biotin

A homogeneous binding assay for the detection of biotin in picoliter vials was developed using the photoprotein aequorin as the label. The binding assay was based on the competition of free biotin with biotinylated aequorin (AEQ-biotin) for avidin. A sequential protocol was used, and modification of the assay to reduce the number of steps was examined. Results showed that detection limits on the order of 10(-14) mol of biotin were possible. Reducing the number of steps provided similar detection limits but only if the amount of avidin used was decreased. These binding assays based on picoliter volumes have potential applications in a variety of fields, including microanalysis and single-cell analysis, where the amount of sample is limited. In addition, these assays are suitable for the high-throughput screening of biopharmaceuticals.     

Comparison of DNA detection methods using nanoparticles and silver enhancement

DNA microarrays are an emerging technology for the parallel detection of DNA molecules. Fluorescent molecules are the current standard for a DNA array's optical readout but they possess some drawbacks including the stability of the dyes and the cost of the scanners. Therefore alternative labelling strategies are of considerable interests. One such strategy is the use of nanoparticles which offers several advantages in terms of stability and versatility of the detection mode. The authors present a review on the different ways DNA can be detected, mainly onto a solid support, using nanoparticle labels.     

A general method for patterning gradients of biomolecules on surfaces using microfluidic networks

This report outlines a general method for the fabrication of immobilized gradients of biomolecules on surfaces. This method utilizes a microfluidic network that generates a gradient of avidin in solution and immobilizes this protein on the surface of glass or poly(dimethylsiloxane) by physical adsorption. The immobilized gradient of avidin is then translated into gradients of biotinylated ligands (e.g., small molecules, oligomers of DNA, polysaccharides) using the specific interaction between biotin and avidin. This method can also generate immobilized gradients of certain proteins and artificial polymers by a direct transfer of gradients from solution onto the surface. The major advantage of this method is that almost any type of molecule can, in principle, be immobilized in a well-defined surface gradient of arbitrary shape with dimensions of a few micrometers to a few centimeters. It is possible to tailor the precise shapes of gradients on surfaces from gradients in solution, either kinetically or competitively. Kinetic methods rely on controlling the time that the surface is exposed to the gradient in solution: when a single protein adsorbs from solution, the amount that adsorbs depends both on its concentration in solution and on the time allowed for adsorption. Competitive methods rely on exposure of the surface to a complementary gradient of two proteins in solution (In these experiments, the sum of the concentrations of the proteins in solution is independent of positions although the concentration of each, individually, depends on the position. In this procedure, the relative amount of each protein, at saturation on the surface, depends only on its concentration.).