Loading Behavior of {Chitosan/Hyaluronic Acid}<i>_n</i> Layer-by-Layer Assembly Films toward Myoglobin: An Electrochemical Study

When {CS/HA}n layer-by-layer films assembled by oppositely charged chitosan (CS) and hyaluronic acid (HA) were immersed in myoglobin (Mb) solution at pH 5.0, Mb was gradually loaded into the {CS/HA}n films, designated as {CS/HA}n−Mb. The cyclic voltammetric (CV) peak pair of Mb FeIII/FeII redox couple for {CS/HA}n−Mb films on pyrolytic graphite (PG) electrodes was used to investigate the loading behavior of {CS/HA}n films toward Mb. The various influencing factors, such as the number of bilayers (n), the pH of Mb loading solution, and the ionic strength of solution, were investigated by different electrochemical methods and other techniques. The results showed that the main driving force for the bulk loading of Mb was most probably the electrostatic interaction between oppositely charged Mb in solution and HA in the films, while other interactions such as hydrogen bonding and hydrophobic interaction may also play an important role. Other polyelectrolyte multilayer (PEM) films with different components were compared with {CS/HA}n films in permeability and Mb loading, and electroactive probes with different size and surface charge were compared in their incorporation into PEM films. The results suggest that due to the unique structure of CS and HA, {CS/HA}n films with relatively low charge density are packed more loosely and more easily swelled by water, and have better permeability, which may lead to the higher loading amount and shorter loading time for Mb. The protein-loaded PEM films provide a new route to immobilize redox proteins on electrodes and realize the direct electrochemistry of the proteins

Triply Responsive Films in Bioelectrocatalysis with a Binary Architecture: Combined Layer-by-Layer Assembly and Hydrogel Polymerization

In this work, triply responsive films with a specific binary architecture combining layer-by-layer assembly (LbL) and hydrogel polymerization were successfully prepared. First, concanavalin A (Con A) and dextran (Dex) were assembled into {Con A/Dex}5 LbL layers on electrode surface by the lectin-sugar biospecific interaction between them. The poly(N,N-diethylacrylamide) (PDEA) hydrogels with entrapped horseradish peroxidase (HRP) were then synthesized by polymerization on the surface of LbL inner layers, forming {Con A/Dex}5–(PDEA-HRP) films. The films demonstrated reversible pH-, thermo-, and salt-responsive on–off behavior toward electroactive probe Fe(CN)63– in its cyclic voltammetric responses. This multiple stimuli-responsive films could be further used to realize triply switchable electrochemical reduction of H2O2 catalyzed by HRP immobilized in the films and mediated by Fe(CN)63– in solution. The responsive mechanism of the films was explored and discussed. The pH-sensitive property of the system was attributed to the electrostatic interaction between the {Con A/Dex}5 inner layers and the probe at different pH, and the thermo- and salt-responsive behaviors should be ascribed to the structure change of PDEA hydrogels for the PDEA-HRP outermost layers under different conditions. The concept of binary architecture was also used to fabricate {Con A/Dex}5–(PDEA-GOD) films on electrodes, where GOD = glucose oxidase, which was applied to realize the triply switchable bioelectrocatalysis of glucose by GOD in the films with ferrocenedicarboxylic acid as the mediator in solution. This film system with the unique binary architecture may establish a foundation for fabricating a novel type of multicontrollable biosensors based on bioelectrocatalysis with immobilized enzymes

Interaction between Myoglobin and Hyaluronic Acid in Their Layer-by-Layer Assembly: Quartz Crystal Microbalance and Cyclic Voltammetry Studies

Myoglobin (Mb), with different net surface charges at different pH in buffers and negatively charged hyaluronic acid (HA) at pH 5.0 in solutions were alternately adsorbed onto various solid surfaces and successfully assembled into {Mb/HA}n layer-by-layer films. The Mb in {Mb/HA}n films showed a quasi-reversible cyclic voltammetry (CV) response for its heme FeIII/FeII redox couple. Quartz crystal microbalance (QCM) and CV were used to confirm the film growth and characterize the films. The interaction between Mb and HA and the influencing factors for Mb adsorption on HA surface, such as pH, Mb concentration, and ionic strength, were investigated in detail. The assembly driving force for {Mb/HA}n films, especially for the films assembled with like-charged Mb and HA, was found to be of electrostatic origin, while the secondary interaction such as hydrophobic interaction also plays an important role in some circumstances. Although the growth of {Mb(pH 7.0)/HA}n and {Mb(pH 9.0)/HA}n films was linear with the adsorption step, the exponential growth of {Mb(pH 5.0)/HA}n films was observed, especially when the films became thick. This exponential increase of mass and thickness with deposition step for {Mb(pH 5.0)/HA}n films was most probably attributed to the diffusion mechanism in which some HA molecules could diffuse in to and out of the whole films during the film assembly. Atomic force microscopy (AFM) results supported this speculation. UV−vis and IR spectroscopies of {Mb/HA}n films, combined with the comparative CV experiments of {Mb/HA}n and {catalase/HA}n films, suggest that Mb in the {Mb/HA}n multilayer films retains its near-native structure

Increment of Density of Au Nanoparticles Deposited in Situ within Layer-by-Layer Films and Its Enhancement on the Electrochemistry of Ferrocenecarboxylic Acid and Bioelectrocatalysis

Poly(allylamine hydrochloride) (PAH) and SiO2 nanoparticles were assembled into {PAH/SiO2}n layer-by-layer (LbL) films on the surface of pyrolytic graphite (PG) electrodes. The films were then placed in HAuCl4 solutions to load Au(III) ions into the films, followed by chemical reduction of Au(III) into Au(0) nanoparticles (AuNPs) by immersion of the films in NaBH4 solutions. This procedure of in situ deposition of AuNPs could be repeated for several cycles to enhance the density of AuNPs in the films. The presence of AuNPs in the films with high density improved the electrochemical responses of ferrocenecarboxylic acid (Fc(COOH)) greatly and then enhanced the electrocatalytic oxidation of glucose by glucose oxidase (GOD) with Fc(COOH) as a mediator. Various techniques, such as quartz crystal microbalance (QCM), scanning electron microscopy (SEM), energy dispersive X-ray (EDX) analysis, and different electrochemical approaches, were used to characterize the films. The mechanism of enhancement of AuNPs in the films on Fc(COOH) electrochemistry was explored and discussed in detail

pH-Controllable Bioelectrocatalysis Based on “On−Off” Switching Redox Property of Electroactive Probes for Spin-Assembled Layer-by-Layer Films Containing Branched Poly(ethyleneimine)

Weak polybase branched poly(ethyleneimine) (BPEI) and strong polyacid poly(styrenesulfonate) (PSS) were assembled into BPEI/{PSS/BPEI}n layer-by-layer (LBL) films on electrodes by electrostatic interaction between them with spin-coating approach. The cyclic voltammetric (CV) response of ferrocenedicarboxylic acid (Fc(COOH)2) at BPEI/{PSS/BPEI}n film electrodes was very sensitive to the pH of the testing solutions. At pH 4.0, the probe showed a well-defined CV peak pair with relatively large peak currents for the films, while, at pH 7.0, the CV response was significantly depressed. By switching the film electrodes in buffers between pH 4.0 and 7.0, the CV peak currents changed periodically between a relatively high value at the “on” state and a very low value at the “off” state, indicating that the pH-sensitive “on−off” switching function of the films toward the probe is reversible. A series of comparative experiments indicates that the electrostatic interaction between the films and the probe plays a predominant role in deciding the pH-sensitive behavior of the films. This pH-dependent property of the films could be used to control or modulate the bioelectrocatalysis of glucose by glucose oxidase (GOD) with Fc(COOH)2 as the mediator by changing the surrounding pH. This “smart” bioelectrocatalytic film system may establish a foundation for fabricating novel pH-controllable electrochemical biosensors

Dual-Switchable Bioelectrocatalysis Synergistically Controlled by pH and Perchlorate Concentration Based on Poly(4-vinylpyridine) Films

Poly(4-vinylpyridine) (P4VP) films were electropolymerized on a pyrolytic graphite (PG) electrode surface. The cyclic voltammetric (CV) response of ferrocenedicarboxylic acid (Fc(COOH)2) at P4VP film electrodes was very sensitive to the pH and perchlorate (ClO4−) concentration in testing solutions. Fc(COOH)2 was at the “on” state with a relatively large CV oxidation peak current for the films at pH 4.0 but showed the “off” state with significantly suppressed CV response at pH 7.0. The reversible ClO4− concentration-sensitive on−off property of P4VP films toward Fc(COOH)2 at pH 4.0 was also observed. In particular, the influence of pH and ClO4− concentration on the on−off behavior of the system is not independent or separate but synergetic or cooperative, and the electrostatic interaction between the films and the probe plays a predominant role in deciding the pH- and/or ClO4− concentration-dependent behavior for the system. The dual-responsive property of the P4VP films toward Fc(COOH)2 could also be used to control the bioelectrocatalysis of glucose by glucose oxidase. This synergetic-triggered bioelectrocatalysis on the basis of the intelligent interface system may establish a foundation for fabricating novel multiple factor-controllable biosensors based on enzymatic electrocatalysis

pH-Switchable Bioelectrocatalysis of Hydrogen Peroxide on Layer-by-Layer Films Assembled by Concanavalin A and Horseradish Peroxidase with Electroactive Mediator in Solution

The lectin protein concanavalin A (Con A) and the glycoenzyme horseradish peroxidase (HRP) were assembled into {Con A/HRP}n layer-by-layer films on electrodes mainly by biospecific affinity between them. The cyclic voltammetric (CV) response of ferricyanide (Fe(CN)63−) at {Con A/HRP}n film electrodes was very sensitive to the environmental pH. The peak currents of Fe(CN)63− were quite large at pH 4.0 but greatly suppressed at pH 9.0, demonstrating reversible pH-sensitive “on−off” behavior. This property could be used to realize pH-controlled electrochemical reduction of H2O2 catalyzed by HRP immobilized in {Con A/HRP}n films and mediated by Fe(CN)63− in solution. The modulation of the solution pH was also realized by in situ biochemical reactions with various enzymes in solution and was used to tune the pH-switchable bioelectrocatalysis. The possible mechanism of the pH-responsive on−off behavior of the films toward the probe was explored, and the electrostatic interaction between the films and the probe is believed to play a key role in deciding the pH-sensitive behavior of the films. This “smart” interface may be used to establish a foundation for fabricating novel pH-controllable electrochemical biosensors based on bioelectrocatalysis with immobilized enzymes

“On−Off” Switchable Bioelectrocatalysis Synergistically Controlled by Temperature and Sodium Sulfate Concentration Based on Poly(<i>N</i>-isopropylacrylamide) Films

In this work, poly(N-isopropylacrylamide) (PNIPAm) films were synthesized on a Au electrode surface through the electrochemically induced free-radical polymerization method. The “coil-to-globule” phase transition of PNIPAm films was sensitive to both environmental temperature and sodium sulfate (Na2SO4) concentration in solution and was detected by cyclic voltammetry (CV) of ferrocenecarboxylic acid (Fc(COOH)) probe. For example, in solutions containing no Na2SO4 at 25 °C, the probe demonstrated a well-defined CV peak pair with large peak currents, showing the “on” state; at 35 °C, the CV response was significantly suppressed, showing the “off” state. By switching the film electrodes in solution between 25 and 35 °C, the CV peak currents cycled between the on and off states, demonstrating the reversible thermosensitive switching function of the films. Similarly, the reversible Na2SO4−concentration-sensitive “on−off” property of PNIPAm films toward Fc(COOH) was also observed. In particular, the influence of temperature and Na2SO4 concentration on the on−off behavior of the films was not independent or separate, but synergetic or cooperative. The dual-responsive property of the films could also be used to switch the on−off bioelectrocatalysis. That is, the electrochemical oxidation of glucose catalyzed by glucose oxidase (GOx) and mediated by Fc(COOH) in solution could be controlled or modulated by changing the surrounding temperature, Na2SO4 concentration, or both. This dual- and synergetic-triggered bioelectrocatalysis based on the “smart” PNIPAm interface system may establish a foundation for fabricating novel multiple factor-controllable biosensors

Electrostatic Adsorption of Heme Proteins Alternated with Polyamidoamine Dendrimers for Layer-by-layer Assembly of Electroactive Films

A novel thin film of heme proteins, including hemoglobin (Hb), myoglobin (Mb), and catalase (Cat), was successfully assembled layer by layer with polyamidoamine (PAMAM) dendrimers on different solid surfaces. At pH 7.0, protonated PAMAM possesses positive surface charges, whereas the proteins have net negative surface charges at pH above their isoelectric points. Thus, layer-by-layer {PAMAM/protein}n films were assembled with alternate adsorption of oppositely charged PAMAM and proteins from their aqueous solutions mainly by electrostatic interaction. The assembly process was monitored by quartz crystal microbalance (QCM), UV−vis spectroscopy, and cyclic voltammetry (CV). The growth of the protein multilayer films was regular and linear, whereas the electroactivity of the films was only extended to a few bilayers. CVs of {PAMAM/protein}n films showed a pair of well-defined and nearly reversible peaks characteristic of the protein heme Fe(III)/Fe(II) redox couples. Although {PAMAM/Hb}n and {PAMAM/Mb}n films showed very similar properties, {PAMAM/Cat}n films displayed different and unique characters. The substrates with biological or environmental significance, such as oxygen, hydrogen peroxide, trichloroacetic acid, and nitrite, were catalytically reduced at {PAMAM/protein}n film electrodes, showing the potential applicability of the films as new types of biosensors or bioreactors based on direct electrochemistry of the proteins. Both the electrochemical and electrocatalytic activity of {PAMAM/protein}n films can be tailored precisely by controlling the number of bilayers or the film thickness

pH-Controllable On−Off Bioelectrocatalysis of Bienzyme Layer-by-Layer Films Assembled by Concanavalin A and Glucoenzymes with an Electroactive Mediator

Mediated electrochemical biosensors consisting of two enzymes have attracted increasing interest because of their wider applicability. In this work, concanavalin A (Con A) and two glycoenzymes, horseradish peroxidase (HRP) and glucose oxidase (GOD), were assembled into {Con A/HRP/Con A/GOD}n layer-by-layer films on an electrode surface mainly by lectin−sugar biospecific interaction between Con A and glycoenzymes. The cyclic voltammetry (CV) response of Fe(CN)63− at the bienzyme film electrodes was very sensitive to the environmental pH: at pH 4.0, the CV peak currents were quite large and the films were at the “on” state; at pH 8.0, however, the electrochemical response was significantly suppressed and the films were at the “off” state. By switching the film electrodes in solution between pH 4.0 and pH 8.0, the CV peak currents cycled between the on and off states, demonstrating the reversible pH-sensitive on−off switching. The pH-responsive property of the films toward the probe could be used to switch the on−off bioelectrocatalysis of glucose. That is, the electrochemical oxidation of glucose catalyzed by GOD and HRP in the films mediated by Fe(CN)63− in solution could be controlled by changing the surrounding pH, allowing the reversible transition of bioelectrocatalysis between the on and off states. CV, electrochemical impedance spectroscopy, amperometry, and quartz crystal microbalance studies were used to characterize the {Con A/HRP/Con A/GOD}n films. The mechanism of pH-sensitive switchable behavior of the films was further explored by comparative experiments and should be attributed to the different electrostatic interactions between the films and the probes at different pH values. This pH-switchable bioelectrocatalysis based on the smart bienzyme interface may pave the way for designing novel controllable biosensors