Nanostructured biosensors built by layer-by-layer electrostatic assembly of enzyme-coated single-walled carbon nanotubes and redox polymers

In this study, we describe the construction of glucose biosensors based on an electrostatic layer-by-layer (LBL) technique. Gold electrodes were initially functionalized with negatively charged 11-mercaptoundecanoic acid followed by alternate immersion in solutions of a positively charged redox polymer, poly[(vinylpyridine)Os(bipyridyl)2Cl(2+/3+)], and a negatively charged enzyme, glucose oxidase (GOX), or a GOX solution containing single-walled carbon nanotubes (SWNTs). The LBL assembly of the multilayer films were characterized by UV-vis spectroscopy, ellipsometry, and cyclic voltammetry, while characterization of the single-walled nanotubes was performed with transmission electron microscopy, Raman spectroscopy, thermogravimetric analysis, and X-ray photoelectron spectroscopy. When the GOX solution contained single-walled carbon nanotubes (GOX-SWNTs), the oxidation peak currents during cyclic voltammetry increased 1.4-4.0 times, as compared to films without SWNTs. Similarly the glucose electro-oxidation current also increased (6-17 times) when SWNTs were present. By varying the number of multilayers, the sensitivity of the sensors could be controlled.     

Alternate assemblies of platinum nanoparticles and metalloporphyrins as tunable electrocatalysts for dioxygen reduction

Through electrostatic layer-by-layer (LBL) assembly, negatively charged citrate-stabilized platinum nanoparticles (PtNPs) and positively charged [tetrakis(N-methylpyridyl)porphyrinato] cobalt were alternately deposited on a 4-aminobenzoic acid-modified glassy carbon electrode and also on indium tin oxide substrates, directly forming the three-dimensional nanostructured materials. Thus-prepared multilayer films were characterized by UV--visible spectroscopy, surface plasmon resonance (SPR) spectroscopy, atomic force microscopy (AFM), and cyclic voltammetry. Regular growth of the multilayer films is monitored by UV--visible spectroscopy and SPR spectroscopy. AFM provides the morphology of the multilayer films. The PtNPs containing multilayer films exhibit high electrocatalytic activity for the reduction of dioxygen with high stability. Rotating disk electrode voltammetry and rotating ring-disk electrode voltammetry demonstrate that the PtNP-containing multilayer films can catalyze an almost four-electron reduction of O(2) to H(2)O in an air-saturated 0.5 M H(2)SO(4) solution. Furthermore, the electrocatalytic activity of the films could be further tailored by simply choosing different cycles in the LBL process or more specifically the amount of the assembly components in the films. The high electrocatalytic activity and good stability for dioxygen reduction make the PtNP-containing multilayer films potential candidates for the efficient cathode material in fuel cells.     

Amperometric hydrogen peroxide biosensor based on a glassy carbon electrode modified with polythionine and gold nanoparticles

We report on a novel hydrogen peroxide biosensor that was fabricated by the layer-by-layer deposition method. Thionine was first deposited on a glassy carbon electrode by two-step electropolymerization to form a positively charged surface. The negatively charged gold nanoparticles and positively charged horseradish peroxidase were then immobilized onto the electrode via electrostatic adsorption. The sequential deposition process was characterized using electrochemical impedance spectroscopy by monitoring the impedance change of the electrode surface during the construction process. The electrochemical behaviour of the modified electrode and its response to hydrogen peroxide were studied by cyclic voltammetry. The effects of the experimental variables on the amperometric determination of H₂O₂ such as solution pH and applied potential were investigated for optimum analytical performance. Under the optimized conditions, the biosensor exhibited linear response to H₂O₂ in the concentration ranges from 0.20 to 1.6?mM and 1.6 to 4.0?mM, with a detection limit of 0.067?mM (at an S/N of 3). In addition, the stability and reproducibility of this biosensor was also evaluated and gave satisfactory results.

Ideal nonideality in adsorption of 2-aminoethanethiol and 2-mercaptoethane sulfonic acid to form electrostatically stabilized binary self-assembled monolayers on Au(111)

In formation of binary self-assembled monolayers (SAMs) composed of 2-aminoethanethiol (AET) and 2-mercaptoethane sulfonic acid (MES) by adsorption from an ethanol solution on Au(111), the adsorption shows nearly ideal nonideality in that the surface ratio of MES to AET in the SAM is unity and does not depend on the mixing ratio of MES to AET in the bathing ethanol solution used for preparing SAMs, chi(soln)MES, over the wide range of chi(soln)MES between 0.01 and 0.95. X-ray photoelectron spectroscopy confirms that at least 80% of AET molecules adsorbed are protonated in this range of chi(soln)MES, indicating that the electrostatic interaction between positively charged AET and negatively charged MES is responsible to the observed nonideality. Correspondingly, there appears only one cathodic peak in a linear-sweep voltammogram of the reductive desorption of the SAM, having a narrow full width at half-maximum of about 20 mV. This suggests the presence of strong lateral attractive interaction between the adsorbed thiolates.     

Formation of metal coating on deoxyribonucleic acid molecule

The Monte Carlo method is employed to study the conditions for nanoconductor formation on a DNA molecule in aqueous solution via electrostatic interactions between negatively charged groups of the matrix molecule and positively charged functionalized gold nanoparticles. A model is developed that explicitly takes into account low-molecular-mass counterions resulting from the dissociation of DNA molecule and surface functional groups of nanoparticles. It is shown that the explicit regard to the counterions is of great importance for investigating conditions of nanoaggregate formation. The model parameters are estimated at which continuous metal coatings are formed via self-assembly.     

Alternate assemblies of polyelectrolyte functionalized carbon nanotubes and platinum nanoparticles as tunable electrocatalysts for dioxygen reduction

A novel method based on electrostatic layer-by-layer self-assembly (LBL) technique for alternate assemblies of polyelectrolyte functionalized multi-walled carbon nanotubes (MWNTs) and platinum nanoparticles (PtNPs) is proposed. The shortened MWNTs can be functionalized with positively charged poly(diallyldimethylammonium chloride) (PDDA) based on electrostatic interaction. Through electrostatic layer-by-layer assembly, the positively charged PDDA functionalized MWNTs (PDWNTs) and negatively charged citrate-stabilized PtNPs were alternately assembled on a 3-mercaptopropanesulfonic sodium (MPS) modified gold electrode and also on other negatively charged surface, e.g. quartz slide and indium–tin-oxide (ITO) plate, directly forming the three-dimensional (3D) nanostructured materials. This is a very general and powerful technique for the assembling three-dimensional nanostructured materials containing carbon nanotubes (CNTs) and nanoparticles. Thus prepared multilayer films were characterized by ultraviolet–visible–near-infrared spectroscopy (UV–vis–NIR), scanning electron microscopy (SEM) and cyclic voltammetry (CV). Regular growth of the mutilayer films is monitored by UV–vis–NIR. SEM provides the morphology of the multilayer films. The PtNPs containing multilayer films exhibit high electrocatalytic activity for the reduction of dioxygen. Furthermore, the electrocatalytic activity of the films could be further tailored by simply choosing different cycles in the LBL process. This assembling method for polyelectrolyte functionalized carbon nanotubes and nanoparticles introduces new opportunities for the incorporation of various functionalities into nanotube devices, which, in turn, opens up the possibility of building more complex multicomponent nanostructures.     

Electrochemical studies of derivatized thiol self-assembled monolayers on gold electrode in the presence of surfactants.

Electrochemical impendence spectroscopy (EIS), cyclic voltammetry (CV) and differential pulse voltammetry (DPV) were performed to investigate the barrier properties and electron transfer of derivatized thiol self-assembled monolayers (SAMs) on gold in the presence of surfactants. The thiol derivatives used included 2-mercaptoethanesulfonic acid (MES), 2-mercaptoacetic acid (MAA), and N-acetyl-L-cysteine (NAC). A simple equivalent circuit was derived to fit the impedance spectra very well. The negative redox probe [Fe(CN)6](3-/4-) was selected to indicate the electron-transfer efficiency on the interface of the studied electrodes. It was found that by changing the surface structure of SAMs, different surfactants could regulate the barrier properties and electron-transfer efficiency in different ways. A positively charged surfactant lowered the electrostatic repulsion between the negative redox probe and negatively charged surface groups of a monolayer, while enhancing the reversibility of electron transfer by virtue of increasing the redox probe concentration within the electric double-layer region. A neutral surfactant showed no significant effect, while a negative surfactant hindered the access and reaction of redox probe by electrostatic repulsion of same-sign charges.     

Composite Assembly of Silver Nanoparticles with Avidin and Biotinylated AChE on Gold for the Pesticidal Electrochemical Sensing

An amperometric pesticide biosensor has been devised by the composite assembly of silver nanoparticles with avidin and biotinylated acetylcholinesterase (AChE) on gold electrodes modified with a biotin‐terminated self assembly monolayer (SAM). This composite assembly strategy takes use of the biospecific recognition avidin with the biotin from the SAM‐terminals and biotinylated AChE, as well as the electrostatic interaction between silver nanoparticles with negatively charged citrate shell and avidin with encounter charge at pH 7.2. The construction process of the composite interface on gold was monitored by surface plasmon resonance (SPR), and its structure was characterized by attenuated total reflection Fourier‐transform infrared spectra, atomic force microscopy and UV‐vis spectra. The composite interface shows excellent electron transfer ability, as characterized by cyclic voltammetry and electrochemical impedance spectroscopy. Under the optimum conditions a quantitative measurement of organophosphate pesticide dimethoate was achieved with the linear range of 0.05 μM to10 μM and the detection limit 0.01 μM, taken as the concentration equivalent to a 10% decrease in signal. Silver nanoparticles conjugated biotin‐avidin system represents a simple and functional approach to the integration of electrode sensing interface with improved biocompatibility and electron transfer ability, which may provide an analytical access to a large group of enzymes for bioelectrochemical application.     

Synthesis and capping of water-dispersed gold nanoparticles by an amino acid: bioconjugation and binding studies

We report a novel strategy for the synthesis of aqueous stable, carboxylated gold nanoparticles (GNPs) by using glutamic acid as the reducing agent. The ratio of chloroaurate ions, AuCl(-)(4) to glutamic acid was optimized in the reaction medium to obtain monodispersed GNPs. Glutamic acid reduced gold nanoparticles were characterized by UV-visible, FTIR, dynamic light scattering and transmission electron microscopy, which demonstrated high stability in aqueous solution over a period of time indicating stabilization via surface-bound amino acid. Functionalized nanoparticles were conjugated with protein molecules through electrostatic attraction between the surface-terminated negatively charged carboxylate groups (COO(-)) of glutamic acid and the positively charged amino groups (NH(+)(3)) of the protein. The conjugation efficiency of the GNP:protein conjugates was confirmed qualitatively and quantitatively through gel electrophoresis and critical flocculation concentration analysis. The interaction between functionalized GNPs with protein molecules was investigated using fluorescence spectroscopy showing the fluorescence quenching of the tryptophan residues of protein molecules after conjugation. Circular dichroism (CD) studies of the conjugates confirmed that the protein undergoes a more flexible conformational state on the boundary surface of GNPs after conjugation. There was substantial conformational transition from alpha-helix to beta-sheet structure after conjugation of protein to GNPs.     

Protein and nanoparticle adsorption on orthogonal, charge-density-versus-net-charge surface-chemical gradients

An orthogonal, charge-density-versus-net-charge, surface-chemical gradient, composed of ternary mixed self-assembled monolayers, has been prepared from three hydrophilic components: positively chargeable amine-terminated, negatively chargeable carboxylic-acid-terminated, and hydroxyl-terminated alkanethiols, with the latter bearing a slight negative charge in electrolytes. The chemical composition and its distribution have been monitored by X-ray photoelectron spectroscopy. The adsorption behavior of negatively charged SiO(2) nanoparticles and positively charged amine-modified SiO(2) nanoparticles has been studied. Additionally, negatively charged proteins (bovine serum albumin and fibrinogen) and positively charged proteins (lysozyme) were adsorbed on the gradients. Negatively charged nanoparticles and proteins adsorb mainly in the positively charged region and vice versa, illustrating that the adsorption behavior is mainly influenced by electrostatic interactions, and showing the potential of the gradient for sorting applications. Despite literature reports to the contrary, no area was found that was completely resistant to protein adsorption.     

Immobilization of gold nanorods onto acid-terminated self-assembled monolayers via electrostatic interactions

We report the immobilization of gold nanorods onto self-assembled monolayers (SAMs) of 16-mercaptohexadecanoic acid (16-MHA). The simple two step protocol involves formation of a SAM of 16-MHA molecules onto gold-coated glass slides and subsequent immersion of these slides into the gold nanorod solution. The nanorods, formed by a seed-mediated, surfactant-assisted synthesis protocol, are stabilized in solution due to surface modification by the surfactant cetyltrimethylammonium bromide (CTAB). Attractive electrostatic interactions between the carboxylic acid group on the SAM and the positively charged CTAB molecules are likely responsible for the nanorod immobilization. UV-vis spectroscopy has been used to follow the kinetics of the nanorod immobilization. The nature of interaction between the gold nanorods and the 16-MHA SAM has been probed by Fourier transform infrared spectroscopy (FTIR). The surface morphology of the immobilized rods is studied by scanning electron microscopy (SEM) and atomic force microscopy (AFM) measurements. SEM was also used to determine the density of the immobilized nanorods as a function of the pH of immobilization. Control over the surface coverage of the immobilized gold nanorods has been demonstrated by simple pH variation. Such well-dispersed immobilized gold nanorods with control over the surface coverage could be interesting substrates for applications such as surface-enhanced Raman spectroscopy (SERS).     

Alpha-helix-inducing dimerization of synthetic polypeptide scaffolds on gold

Designed, synthetic polypeptides that assemble into four-helix bundles upon dimerization in solution were studied with respect to folding on planar gold surfaces. A model system with controllable dimerization properties was employed, consisting of negatively and positively charged peptides. Circular dichroism spectroscopy and surface plasmon resonance based measurements showed that at neutral pH, the peptides were able to form heterodimers in solution, but unfavorable electrostatic interactions prevented the formation of homodimers. The dimerization propensity was found to be both pH- and buffer-dependent. A series of infrared absorption-reflection spectroscopy experiments of the polypeptides attached to planar gold surfaces revealed that if the negatively charged peptide was immobilized from a loading solution where it was folded, its structure was retained on the surface provided it had a cysteine residue available for anchoring to gold. If it was immobilized as random coil, it remained unstructured on the surface but was able to fold through heterodimerization if subsequently exposed to a positively charged polypeptide. When the positively charged peptide was immobilized as random coil, heterodimerization could not be induced, probably because of high-affinity interactions between the charged primary amine groups and the gold surface. These observations are intended to pave the way for future engineering of functional surfaces based on polypeptide scaffolds where folding is known to be crucial for function.     

In situ observation of gamma-Fe2O3 nanoparticle adsorption under different monolayers at the air/water interface

We studied the adsorption of gamma-Fe 2O 3 nanoparticles from an aqueous solution under different charged Langmuir monolayers (stearic acid, stearyl alcohol, and stearyl amine). The aqueous subphase was composed of a colloidal suspension of gamma-Fe 2O 3 nanoparticles. The average hydrodynamic diameter of the nanoparticles measured by dynamic light scattering measurements was 16 nm. The observed zeta potential of +40 mV (at pH 4) results in a long-term stability of the colloidal dispersion. The behavior of the different monolayer/nanoparticle composites were studied with surface pressure/area (pi/ A) isotherms. The adsorption of the nanoparticles under the different monolayers induced an expansion of the monolayers. These phenomena depended on the charge of the monolayers. After the Langmuir/Blodgett transfer on glass substrates, the nanoparticle/monolayer composite films were studied by means of UV-vis spectroscopy. The spectra pointed to increasing adsorption of the nanoparticles with increasing electronegativity of the monolayers. On the basis of these results, we studied the in situ adsorption of nanoparticles under the different monolayers by X-ray reflectivity measurements. Electron density profiles of the liquid/gas interfaces were obtained from the X-ray reflectivity data. The results gave clear evidence for the presence of electrostatic interaction between the differently charged monolayers and the positively charged nanoparticles. While the adsorption process was favored by the negatively charged stearic acid monolayer, the positively charged layer of stearyl amine prevented the formation of ultrathin nanoparticle layers.     

Silver nanocomposite layer-by-layer films based on assembled polyelectrolyte/dendrimer

Silver nanocomposite multilayer films were prepared through the in situ method. Multilayer thin films, prepared through the sequential electrostatic deposition of a positively charged third-generation poly(amidoamine) dendrimer (PAMAM) and negatively charged poly(styrenesulfonate) (PSS) and poly(acrylic acid) (PAA), were utilized as nanoreactors for the formation of silver nanoparticles. The silver ions were preorganized in layer-by-layer (LBL) films composed of PAMAM dendrimers and subsequently reduced with hydrogen to prepare the silver nanoparticles. The UV-vis spectrum and profilometer were used to characterize the regular growth of bilayers. UV-vis absorption from plasmon resonance at 435 nm and TEM images indicated the formation of the silver nanoparticles in the multilayer films. The silver nanocomposite LBL films were also constructed on the indium tin oxide-glass and investigated using cyclic voltammetry. The silver nanoparticles in the multilayer films have a stronger negative redox potential. The silver nanocomposite LBL films may have a potential application in the catalysis of reduction of 4-nitrophenol with sodium borohydride.     

Effects of mutational (Lys to Ala) surface charge changes on the redox properties of electrode-immobilized cytochrome c

Untrimethylated yeast iso-1-cytochrome c (cytc) and its single and multiple Lys to Ala variants at the surface lysines 72, 73, and 79 were adsorbed on carboxyalkanethiol self-assembled monolayers (SAMs) on gold, and the thermodynamics and kinetics of the heterogeneous protein-electrode electron-transfer (ET) reaction were determined by voltammetry. The reaction thermodynamics were also measured for the same species freely diffusing in solution. The selected lysine residues surround the heme group and contribute to the positively charged domain of cytc involved in the binding to redox partners and to carboxyl-terminated SAM-coated surfaces. The E degrees' (standard reduction potential) values for the proteins immobilized on SAMs made of 11-mercapto-1-undecanoic acid and 11-mercapto-1-undecanol on gold were found to be lower than those for the corresponding diffusing species owing to the stabilization of the ferric state by the negatively charged SAM. For the immobilized proteins, Lys to Ala substitution(s) do not affect the surface coverage, but induce significant changes in the E degrees' values, which do not simply follow the Coulomb law. The results suggest that the species-dependent orientation of the protein (and thereby of the heme group) toward the negatively charged SAM influences the electrostatic interaction and the resulting E degree' change. Moreover, these charge suppressions moderately affect the kinetics of the heterogeneous ET acting on the reorganization energy and the donor-acceptor distance. The kinetic data suggest that none of the studied lysines belong to the interfacial ET pathway.     

The role of electrostatic interactions on the rejection of organic solutes in aqueous solutions with nanofiltration

The effects of electrostatic interactions on the rejection of organic solutes with nanofiltration membranes were investigated. For two different membranes, the rejection of selected organic acids, positively and negatively charged pharmaceuticals and neutral pharmaceuticals was investigated at different feed water chemistries (different ionic strengths and pH conditions, with and without the presence of NOM and divalent cations). It was concluded that for negatively charged membranes, electrostatic repulsion leads to an increase of the rejection of negatively charged solutes and electrostatic attraction leads to a decrease of the rejection of positively charged solutes, compared to neutral solutes. Neutral and positively charged solutes engage in hydrophobic interactions with negatively charged membranes, whereas negatively charged solutes do not engage in hydrophobic interactions since they cannot approach the membrane surface. This provides proof for the theory of an increased concentration of positively charged organic solutes and a decreased concentration of negatively charged organic solutes at the membrane surface compared to the bulk fluid. This concept may be denoted as “charge concentration polarisation”. The concept was further used as a modelling tool to predict the effects of electrostatic interactions on the rejection of trace organic solutes.     

Imaging Local Proton Fluxes through a Polycarbonate Membrane by Using Scanning Electrochemical Microscopy and Functionalized Alkanethiols

A new application of scanning electrochemical microscopy (SECM) to probe the transport of protons through membranes is described. Herein, a probe ultramicroelectrode (UME) is modified with a self‐assembled monolayer (SAM) of 11‐mercaptoundecanoic acid to qualitatively image areas within different pH regions above a track‐etched membrane. The current response of the modified electrode in the presence of potassium hexacyanoferrate as electroactive component is different in acidic and alkaline solutions. Depending on the pH value of the solution, the SAM‐covered electrode exposes either a neutral or a negatively charged insulating monolayer at pH 3 or 7, respectively, which leads to an increase/decrease in the faradaic current due to electrostatic interactions between the neutral/charged surface and the charged redox mediator. Therefore, local pH changes in the close vicinity of a membrane‐like substrate lead to different current responses recorded at the tip electrode when scanning above the surface.     

pH响应的介孔二氧化硅纳米颗粒的制备及可控释放

选择带负电荷且溶解度和分子结构对pH值非常敏感的聚丙烯酸作为封堵分子, 采用静电吸附的修饰方法, 制备了pH响应的MCM-41型介孔二氧化硅纳米颗粒. 利用高倍透射电子显微镜(TEM)、 X射线衍射(XRD)、 傅里叶变换红外光谱(FTIR)及比表面积分析等手段表征了介孔二氧化硅纳米颗粒的物理化学性质. 以联钌吡啶染料分子作为模式客体分子, 研究了pH调控下的模式客体分子在介孔二氧化硅纳米颗粒中的包裹及释放行为. 结果表明, 该介孔二氧化硅纳米颗粒对pH具有很好的响应性; 在近中性条件下, 带正电的二氧化硅纳米颗粒通过静电吸附作用吸附带负电的聚丙烯酸, 导致介孔封堵, 使包载的染料分子几乎无释放; 客体分子的释放率随着pH值的降低而升高, 当pH≤5时, 染料分子显著释放, pH=1时客体分子的释放率高达98％, 可以实现对包载客体分子的控制释放. 该pH响应的介孔二氧化硅纳米颗粒载体具有制备简便、 价格低廉和包载量大等优点, 有望应用于药物的控制释放.     

Effect of Electrostatic Interaction on Electrodeposition of Nickel Hexacyanoferrate with Functional MWCNTs and Their Application for the Determination of Persulfate and Tannic Acid

A nanocomposite film of NiHCF electrodeposited onto AFCNT was modified on GCE by the electrostatic interaction of multiple scan cyclic voltammetry. Amino groups have been introduced with a positive charge onto the surface of MWCNTs. The presence of NiHCF‐AFCNT/GCE was characterized by using FE‐SEM, EDS, EIS, and XRD studies. The nanocomposite film of NiHCF‐AFCNT was further utilized to determine persulfate and tannic acid, respectively. The nanocomposite film modified electrode displays linear response from 0.1 to 19 mM and 10 to 170 µM for persulfate and tannic acid with a correlation coefficient of 0.9921 and 0.9869. The detection limits were found to be 0.1 mM and 1 µM, respectively. As results, the electrostatic interaction between positively charged metal ions, amino functionalized CNT and negatively charged hexacyanoferrate can be a new subject for many interesting applications.     

Electrochemical surface plasmon resonance investigation of dodecyl sulfate adsorption to electroactive self-assembled monolayers via ion-pairing interactions

The redox-induced assembly of amphiphilic molecules and macromolecules at electrode surfaces is a potentially attractive means of electrochemically modulating the organization of materials and nanostructures on solid substrates via ion-pairing interactions or charge-transfer complexation. In this regard, we have investigated the potential-induced adsorption and aggregation of dodecyl sulfate, a common anionic surfactant, at a ferrocenylundecanethiolate (FcC11SAu) self-assembled monolayer (SAM)/aqueous solution interface by electrochemical surface plasmon resonance (ESPR) spectroscopy. The surfactant anions adsorb onto the electroactive SAM by specific ion-pairing interactions with the oxidized ferricinium species. The ferricinium charge density (QFc+) obtained by cyclic voltammetry and surface coverage measured by SPR indicate that the dodecyl sulfate forms an interdigitated monolayer, where half of the surfactant molecules have their sulfate headgroups paired to the surface and half have their headgroups exposed to the aqueous solution. The surface coverage of dodecyl sulfate was found to depend on both the ferricinium surface concentration and the surfactant aggregation state in solution. A maximum coverage of dodecyl sulfate on the ferricinium surface is obtained below the critical micelle concentration (cmc), in contrast to dodecyl sulfate adsorption to SAM surfaces of static positive charge. This marked difference in adsorption behavior is attributed to the dynamic generation of ferricinium by potential cycling and the specific nature of the ion-pairing interactions versus pure electrostatic ones. The results presented point to a new way of organizing molecules via electrical stimulus.