Electroactive layer-by-layer films of heme protein-coated polystyrene latex beads with poly(styrene sulfonate)

In this work, a novel two-step construction strategy for protein layer-by-layer assembly films was proposed. In the first step, positively charged hemoglobin (Hb) or myoglobin (Mb) at pH 5.0 was adsorbed on the negatively charged surface of 500 nm diameter-sized polystyrene (PS) latex beads, forming core-shell structured PS-protein particles. In the next step, the PS-protein particles were further assembled layer by layer with oppositely charged poly(styrene sulfonate)(PSS) on various solid surfaces under suitable conditions. Cyclic voltammetry (CV), quartz crystal microbalance (QCM), and UV-vis spectroscopy were used to monitor the growth of {(PS-protein)/PSS}(n) films. The stable {(PS-protein)/PSS}(n) films modified on pyrolytic graphite (PG) electrodes demonstrated good electroactivity in protein-free buffer, which was originated from protein heme Fe(III)/Fe(II) redox couples, and the electroactivity extended to six (PS-protein)/PSS bilayers. UV-vis spectroscopy showed that Hb and Mb in the films retained their near-native structure in the medium pH range. {(PS-protein)/PSS}(n) films catalyzed electrochemical reduction of oxygen, hydrogen peroxide, trichloroacetic acid (TCA) and nitrite with a significant lowering of overpotential, and displayed better catalytic activity than corresponding cast PS-protein films.     

Direct electron transfer and electrocatalysis of hemoglobin in layer-by-layer films assembled with Al-MSU-S particles

In this paper, layer-by-layer (LBL) {MSU/Hb}(n)/PDDA films assembled by alternate adsorption of positively charged hemoglobin (Hb) and negatively charged mesoporous molecular sieves of Al-MSU-S onto a glassy carbon electrode (GCE) were reported. Al-MSU-S was synthesized by the precursor of zeolite Y and ionic liquids 1-hexadecane-3-methylimidazolium bromide (CMIMB) as a template in basic medium. It exhibited larger pore diameter, pore volume and surface area. Direct electrochemical and electrocatalytic properties of Hb in these layer-by-layer films were investigated. A pair of well-defined nearly reversible cyclic voltammetric peaks was observed and the formal potential of the heme Fe(III)/Fe(II) redox couple was found to be -0.295V (vs. SCE). The influences of layer's number and the pH of the external solution to the electron transfer behavior of Hb in {MSU/Hb}(n)/PDDA films were also estimated by cyclic voltammetry and a set of optimized conditions for film fabrication was inferred. The hemoglobin in{MSU/Hb}(n)/PDDA films displayed a good electrocatalytic activity to the reduction of hydrogen peroxide, which had linear current responses from 1.0 x 10(-6) to 1.86 x 10(-4)mol/L with the detection limit of 5.0 x 10(-7)mol/L (S/N=3). The apparent Michaeli-Menten constant (K(m)(app)) was 0.368 mmol/L. Thus, this methodology shows potential application of the preparation of third-generation biosensors.     

Comparative bioelectrochemical study of core-shell nanocluster films with ordinary layer-by-layer films containing heme proteins and CaCO3 nanoparticles

Negatively charged heme protein hemoglobin (Hb) or myoglobin (Mb) at pH 9.0 and positively charged poly(diallyldimethylammonium) (PDDA) were alternately adsorbed on the surface of CaCO(3) nanoparticles, forming core-shell CaCO(3)-[PDDA/(protein/PDDA)(m)] ([protein-m]) nanoclusters. Oppositely charged [protein-m] and poly(styrenesulfonate) (PSS) were then assembled layer by layer on various solid substrates, forming {[protein-m]/PSS}(n) films. In the meantime, ordinary layer-by-layer films of heme proteins with CaCO(3) nanoparticles ({protein/CaCO(3)}(n)) were also grown on solid surfaces. Transmission electron microscopy (TEM), ultraviolet-visible (UV-vis) spectroscopy, quartz crystal microbalance (QCM), and cyclic voltammetry (CV) were used to characterize the nanoclusters and monitor the growth of the two types of films. Both kinds of protein films assembled on pyrolytic graphite (PG) electrodes exhibited well-defined, nearly reversible CV reduction-oxidation peaks, characteristic of heme Fe(III)/Fe(II) redox couples, and were used to catalyze the electrochemical reduction of hydrogen peroxide. The {[protein-m]/PSS}(n) films demonstrate distinct advantages over the {protein/CaCO(3)}(n) films due to their larger fraction of electroactive proteins, higher catalytic efficiency, and better thermostability. The penetration experiments of the electroactive probe into these films indicate that the {[protein-m]/PSS}(n) nanocluster films possess more pores or channels than the simple {protein/CaCO(3)}(n) films, which may be beneficial to counterion transport in the charge-hopping mechanism and helpful for the diffusion of catalysis substrates into the films. In addition, the electrochemical and biocatalytic activity of protein nanocluster films can be tailored by controlling the number of bilayers assembled on the nanoparticle cores (m) as well as the film thickness or the number of nanocluster layers on the electrodes (n).     

Electroactive core-shell nanocluster films of heme proteins, polyelectrolytes, and silica nanoparticles

Novel protein core-shell nanocluster films were assembled layer by layer on solid surfaces. In the first step, positively charged heme protein hemoglobin (Hb) or myoglobin (Mb) and negatively charged poly(styrenesulfonate) (PSS) were alternately adsorbed on the surface of SiO2 nanoparticles, forming core-shell SiO2-(protein/PSS)m nanoclusters. In the second step, the SiO2-(protein/PSS)m nanoclusters and polycationic poly(ethylenimine) (PEI) were assembled layer by layer on various solid substrates, forming [[SiO2-(protein/PSS)m]/PEI]n films. Various techniques were used to characterize the nanoclusters and monitor the film growth. [[SiO2-(protein/PSS)m]/PEI]n films at pyrolytic graphite (PG) electrodes exhibited well-defined, chemically reversible cyclic voltammetric reduction-oxidation peaks characteristic of the heme Fe(III)/Fe(II) redox couples. The proteins in the films retained near native conformations in the medium pH range, and the films catalyzed electrochemical reduction of oxygen and hydrogen peroxide. Advantages of the nanocluster films over the simple [SiO2/protein]n layer-by-layer films include a larger fraction of electroactive protein and higher specific biocatalytic activity. Using this approach, biocatalytic activity can be tailored and controlled by varying the number of bilayers deposited on the nanoparticle cores and the number of nanocluster layers on electrodes.     

Layer-by-layer films of hemoglobin or myoglobin assembled with zeolite particles: electrochemistry and electrocatalysis

Positively charged hemoglobin (Hb) or myoglobin (Mb) at pH 5.0 in solutions and negatively charged zeolite particles in dispersions were alternately adsorbed onto solid surfaces forming [zeolite/protein](n) layer-by-layer films, which was confirmed by quartz crystal microbalance (QCM) and cyclic voltammetry (CV). The protein films assembled on pyrolytic graphite (PG) electrodes exhibited a pair of well-defined, nearly reversible CV peaks at about -0.35 V vs. SCE at pH 7.0, characteristic of the heme Fe(III)/Fe(II) redox couples. Hydrogen peroxide (H(2)O(2)) and nitrite (NO(2)(-)) in solution were catalytically reduced at [zeolite/protein](7) film modified electrodes, and could be quantitatively determined by CV and amperometry. The shape and position of infrared amide I and II bands of Hb or Mb in [zeolite/protein](7) films suggest that the proteins retain their near-native structure in the films. The penetration experiments of Fe(CN)(6)(3-) as the electroactive probe into these films and scanning electron microscopy (SEM) results indicate that the films possess a great amount of pores or channels. The porous structure of ]zeolite/protein](n) films is beneficial to counterion transport, which is crucial for protein electrochemistry in films controlled by the charge-hopping mechanism, and is also helpful for the diffusion of catalysis substrates into the films. The proteins with negatively charged net surface charges at pH 9.0 were also successfully assembled with like-charged zeolite particles into layer-by-layer films, although the adsorption amount was less than that assembled at pH 5.0. The possible reasons for this were discussed, and the driving forces were explored.     

Construction of hyaluronan-silver nanoparticle–hemoglobin multilayer composite film and investigations on its electrocatalytic properties

In this work, hyaluronan-silver nanoparticles (HSNPs) were prepared by UV-initiated photoreduction, and protein hemoglobin (Hb) was then alternately assembled with the prepared negatively charged HSNPs into layer-by-layer (LBL) films on solid surface. The electrochemical behavior and electrocatalytic activities toward oxygen and hydrogen peroxide of the resulting films were studied. It was found that the HSNPs greatly enhanced the electron transfer reactivity of Hb as a bridge. The assembly films showed a pair of nearly reversible redox peaks with a formal potential of −0.32 V (vs. Ag/AgCl) for the heme Fe(III)/Fe(II) redox couple. The immobilized Hb in the films maintained its biological activity, showing a surface-controlled process with a heterogeneous electron transfer rate constant (k _s) of 1.0 s⁻¹ and displaying the same features of a peroxidase in the electrocatalytic reduction of oxygen and hydrogen peroxide. This work provides a novel model to fabricate LBL films with protein, polysaccharide and nanoparticles, which may establish a foundation for fabricating new type of biosensors based on the direct electron transfer of redox proteins immobilized in nanocomposite multilayer films with underlying electrodes.     

Fabrication of free-standing multilayer films by using pH-responsive microgels as sacrificial layers

A facile way to prepare free-standing polyelectrolyte multilayer films of poly(sodium 4-styrenesulfonate)(PSS)/poly(diallyldimethylammonium)(PDDA) was developed by applying a new pH-dependent sacrificial system based on cross-linked poly(N,N-dimethylaminoethyl methacrylate) (PDMAEMA) microgels. The tertiary amine groups of PDMAEMA microgels can be protonated in acidic environment, and the protonated microgels were deposited by layer-by-layer (LbL) technique with PSS. PSS/PDDA multilayer films were constructed on the top of the PSS/microgels sacrificial layers. The LbL assembly process was investigated by UV–vis spectroscopy. Further study shows that the free-standing PSS/PDDA multilayer films can be obtained within 3 min by treating the as-prepared films in alkali aqueous solution with a pH of 12.0. The pH-triggered exfoliation of PSS/PDDA multilayer films provides a simple and facile way to prepare LbL assembled free-standing multilayer films.     

Oligosilsesquioxanes as versatile building blocks for the preparation of self-assembled thin films

A self-assembly approach to the preparation of nanocomposite siliceous thin films by using oligosilsesquioxanes as building blocks is presented. Poly(styrene-4-sulfonate), PSS, and octa(3-aminopropyl)silsesquioxane, NSi8, were layer-by-layer (LbL) assembled onto planar substrates and polystyrene (PS) particles, thus forming composite multilayers. We have clarified the binding properties of NSi8 to PSS by examining the pH influence on film buildup by microelectrophoresis (zeta-potential) and quartz crystal microgravimetry (QCM). The regular growth of PSS/NSi8 multilayers on planar supports was confirmed by surface plasmon resonance (SPR) spectroscopy and QCM. By applying the LbL coating procedure to spherical templates, we prepared compact, microporous hollow silica spheres by calcining PS spheres coated with (poly(allylamine hydrochloride) (PAH)/PSS)(2)/(NSi8/PSS)(n) (n varying from 3 to 12), at 750 degrees C, because of sintering of the octameric clusters (NSi8). Hollow spheres derived from coatings with n = 3 drastically altered in size (relative to the template core), depending on the size of the PS particles used. The novelty of this method for the nanofabrication of siliceous films stems from the use of well-defined and discrete building blocks, such as NSi8, leading to homogeneous organic-silica composite films as well as individual siliceous particles of variable size and shape.     

Assembly of layer-by-layer films of electroactive hemoglobin and surfactant didodecyldimethylammonium bromide

When a solid substrate with negative surface charges was placed in an aqueous didodecyldimethylammonium bromide (DDAB) vesicle dispersion, the cationic surfactant DDAB with two hydrocarbon chains could be assembled into the biomembrane-like tail-to-tail double-layer structure on the solid surface with the positively charged head groups toward outside, making the surface charge reverse from negative to positive. After the solid substrate with DDAB was immersed in a hemoglobin (Hb) solution at pH 9.0, the negatively charged Hb was adsorbed on the surface of DDAB layer by electrostatic attraction, forming a DDAB/Hb film. By repeating this adsorption cycle, the {DDAB/Hb}(n) layer-by-layer films were assembled on solid surfaces, which was confirmed by UV-vis spectroscopy, quartz crystal microbalance (QCM), and cyclic voltammetry (CV). The stable {DDAB/Hb}(n) films assembled on pyrolytic graphite (PG) electrodes showed two pairs of nearly reversible redox peaks at about -0.22 and -1.14 V vs SCE in pH 7.0 buffers, characteristic of the Hb heme Fe(III)/Fe(II) and Fe(II)/Fe(I) redox couples, respectively. The direct electrochemistry of Hb in the films could be used to electrocatalyze reduction of various substrates. UV-vis and IR spectroscopic results and comparison experiments with {DDAB/hemin}(n) films indicate that Hb in the {DDAB/Hb}(n) films essentially retains its native structure. Atomic force microscopy (AFM) was used to characterize the morphology of the films with different outermost layers.     

Redox switching of polyoxometalate-methylene blue-based layer-by-layer films

Iron-substituted crown-type polyoxometalate (POM) [P(8)W(48)O(184)Fe(16)(OH)(28)(H(2)O)(4)](20-) has been successfully immobilized onto glassy carbon electrode surfaces by means of the layer-by-layer (LBL) technique employing the cationic redox active dye, methylene blue (MB). The constructed multilayers exhibit pH-dependent redox activity for both the anionic POM and the cationic dye moieties, which is in good agreement with their solution behavior. The films have been characterized by alternating current impedance, atomic force microscopy, and X-ray photoelectron spectroscopy, whereby the nature of the outer layer within the assemblies was found to have an effect upon the film's behavior. Preliminary investigations show that the POM dye-based films show electrocatalytic ability toward the reduction of hydrogen peroxide, however, only when there is an outer anionic POM layer.     

Driving forces for layer-by-layer self-assembly of films of SiO2 nanoparticles and heme proteins

Heme protein hemoglobin (Hb) or myoglobin (Mb) and silica nanoparticles in a variety of charge states were assembled layer-by-layer into films on solid surfaces to investigate the driving forces for film assembly. Cyclic voltammetry (CV), quartz crystal microbalance (QCM), X-ray photoelectron spectroscopy (XPS), and UV-vis and reflectance absorption infrared (RAIR) spectroscopy were used to characterize the different [SiO2/protein]n films. Even when the proteins and silica were both negatively charged, stable layer-by-layer [SiO2/protein]n films were successfully fabricated, although amounts of protein were smaller than when nanoparticles and proteins had opposite charges. Results suggest the importance of localized Coulombic attractions between the negative nanoparticle surface and positively charged amino acid residues on the Mb or Hb surfaces in the assembly and for the stability of [SiO2/protein]n films.     

Electrochemistry of ITO electrode modified by multilayer ultrathin films based on crown-shaped polyoxomolybdate

The electrochemical multilayer films of crown-shaped polyoxomolybdate Na21{[Na5(H2O)14] intersection[Mo(V)(20)Mo(VI)(26)O134(OH)10(mu-CH3COO)4]}.CH3COONa.90H2O (Mo46) and polyelectrolytes by layer-by-layer assembly were investigated. The stable multilayer films were assembled by alternate adsorption of negatively charged POM and positively charged polyelectrolytes is from their aqueous dispersions. UV-vis spectroscopy and cyclic voltammetry were used to monitor the regular growth of the multilayer films. The multilayer films-modified ITO electrode was used for the detection of electrocatalytic activity toward the reduction of nitrite, bromate, and hydrogen peroxide. The proposed novel immobilized method exhibited good stability, reproducibility and high sensitivity for the determination of electrocatalytic, which is important for practical application.     

辣根过氧化物酶活性膜结构及生物电催化性能

通过分子沉积法研究了在聚对苯二甲酸乙二醇酯（PET）表面及金电极表面组装辣根过氧化物酶（HRP）/聚对苯乙烯磺酸钠（PSS）多层生物活性膜，用原子力显微镜(AFM)研究了组装膜的表面形貌，并研究了组装膜的形貌、粗糙度和活性关系.应用循环伏安法（CV）研究了组装HRP膜后电极对H2O2的电化学催化还原作用.实验发现，采用亚甲基蓝（MB）溶液为介质，在H2O2浓度为0.2～5.0 mmol•L－1时，其响应电流对H2O2浓度变化基本呈线性.     

Direct Electrochemistry of Hemoglobin in Layer‐by‐Layer Films Assembled with DNA and Room Temperature Ionic Liquid

Based on electrostatic interaction and electrodeposition, poly‐anionic deoxyribonucleic acid (DNA), room temperature ionic liquid 1‐butyl‐3‐methyl‐imidazolium tetrafluoroborate (BMIMBF₄), hemoglobin (Hb) and Poly(diallyldimethylammonium chloride) (PDDA) were successfully assembled into Hb/IL/DNA/PDDA layer‐by‐layer complex films on the surface of ITO electrode. FTIR spectroscopy, electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) were used to characterize the composite film. The obtained results demonstrated that the Hb molecule in the film kept its native structure and showed its good electrochemical behavior. A pair of well‐defined redox peaks of Hb with the formal potentials (E°′) of ?0.180 V (vs. SCE) was appeared in phosphate buffer solution (PBS, pH 7.0). The Hb/IL/DNA/PDDA/ITO modified electrode also showed an excellent electrocatalytic behavior to the reduction of hydrogen peroxide (H₂O₂). Therefore, the IL/DNA/PDDA complex film as a novel matrix open up a possibility for further study on the direct electrochemistry of other proteins and the fabrication of the third‐generation electrochemical biosensors.     

Preparation and characterization of polyoxometalate/protein ultrathin films grown on electrode surfaces using layer-by-layer assembly

We report a new electrostatic layer-by-layer assembly method for the controlled deposition of electrocatalytically active enzymes onto electrode surfaces using polyoxometalate as the counteranion. Cytochrome c (cyt c), a redox active protein, and P(2)W(18)O(62)(6-), a Dawson-type polyoxometalate, were deposited onto glassy carbon electrodes by two procedures: static dipping and electrochemical cycling. Cyclic voltammetry and UV-vis spectroscopy reveal that approximately 1.5 x 10(-10) mol/cm(2) of P(2)W(18)O(62)(6-) and 2.2 x 10(-11) mol/cm(2) of cytochrome c are deposited per cycle, which correspond to approximately one monolayer of each molecule. The thicknesses of the resulting films measured by atomic force microscopy also indicate that the films are formed in a layer-by-layer fashion. Experimental factors that affect electron-transfer rate in these films, such as scan rate and film thickness, were systematically analyzed. The use of {P(2)W(18)O(62)(6-)/cyt c}n films to catalyze hydrogen peroxide reduction was demonstrated.     

聚电解质PDDA/PSS层层自组装膜的渗透汽化性能

采用聚电解质层层自组装(LbL)技术, 在不同盐浓度下制备了聚(二烯丙基二甲基氯化铵)/聚苯乙烯磺酸钠(PDDA/PSS) 多层自组装膜, 并用于渗透汽化性能的研究. 重点考察了组装溶液中NaCl的浓度、组装层数及操作温度对自组装膜的异丙醇脱水性能的影响. 同时, 用扫描电镜观测了不同条件下制备膜的表面形貌. 结果表明, 在高NaCl含量的聚电解质溶液中只需组装几个双层的LbL膜, 即能获得较高的分离因子和较大的通量, 并解释了该LbL膜呈现反“trade-off”现象的原因.     

High-flux nanofiltration membranes prepared by adsorption of multilayer polyelectrolyte membranes on polymeric supports

Layer-by-layer deposition of anionic and cationic polyelectrolytes readily converts polymeric ultrafiltration membranes into materials capable of nanofiltration. ATR-FTIR spectra confirm that layer-by-layer deposition occurs on the ultrafiltration substrates, and adsorption of as few as 2.5 bilayers of poly(styrenesulfonate) (PSS)/protonated poly(allylamine) (PAH) or 3.5 bilayers of PSS/poly(diallyldimethylammonium chloride) (PDADMAC) reduces the molecular weight cutoff of polyethersulfone ultrafiltration supports from 50 kDa to <500 Da. Deposition of multilayer polyelectrolyte films on 300 and 500 kDa membranes also decreases molecular weight cutoffs, but solute rejections are significantly lower when using these supports, suggesting that the polyelectrolyte films do not completely cover large (0.2-0.4 microm in diameter) pores. On the 50 kDa substrates, PSS/PDADMAC films containing 3.5 bilayers exhibit a 95% rejection of SO(4)(2-) and a chloride/sulfate selectivity of 27, whereas 4.5-bilayer PSS/PAH coatings show a glucose/raffinose selectivity of 100. Pure water flux for [PSS/PAH](3)PSS-coated membranes at 4.8 bar is 1.6 m(3)/(m(2)day), which is more than 2-fold higher than that through a commercial 500 Da membrane.     

Direct Electrochemistry of Hemoglobin in Layer-by-layer {PDDA/Hb}_n Films Assembled on Pyrolytic Graphite Electrodes

The principle of alternate adsorption can be used to design and control specific molecular architectures1. Protein-polyion layer-by-layer assembly opens a possibility of organizing proteins with specific molecular architectural plan1 and studying redox proteins with electrochemical methods2. Recently, we reported electrochemistry of layer-by-layer {PSS/Mb}n films grown on PG electrodes1. In this work, layer-by-layer {PDDA/Hb}n films were fabricated on PG electrodes. Electrochemistry and …     

Electrochemical behavior of hemoglobin in neutral surfactants with different poly(ethylene oxide) unit lengths adsorbed on an electrode

The direct electron transfer and adsorption behavior of hemoglobin(Hb) in a series of surfactants with different poly(ethylene oxide)(PEO) unit lengths on a glassy carbon electrode have been studied.With a surfactant of appropriate PEO unit length,the surfactant film-modified electrode exhibited a more stable adsorption state with a larger surface coverage of Hb and a more positive formal potential,which can be attributed to the effect of hydrogen bonding between proteins and surfactants.The electrochemical behavior of surfactants with different PEO unit lengths is discussed in detail.Moreover,UV-visible spectroscopy demonstrated that the structure of Hb was not destroyed in the surfactant films.The electrocatalytic activity of hydrogen peroxide on three neutral surfactant-modified electrodes has also been investigated.     

以聚电解质多层膜为纳米反应器制备Au@Pt核壳纳米粒子

以聚二烯丙基二甲基氯化铵和聚苯乙烯磺酸钠为构筑单元,通过静电层层自组装制备了多层膜,利用薄膜中存在的抗衡阴离子,选择AuCl-4和PtCl2-6作为Au和Pt的前驱体,通过连续的阴离子交换/还原,原位制备了Au-Pt双金属纳米粒子。 紫外-可见分光光度法、透射电子显微镜和能量色散X射线能谱数据表明,在聚电解质多层薄膜中成功地制备了具有核壳结构的Au@Pt双金属纳米粒子。 这种纳米粒子在电化学催化、燃料电池方面具有潜在的应用价值。