Applications Of Nanoporous Metals In Biocatalysis And Biosensing

In this paper, dealloying AuAg, CuAl, and AgAl alloys is used for the preparation of various nanoporous metals. These nanoporous metal materials with outstanding physicochemical properties are characterized and used for biocatalyst enzyme immobilization. We studied the basic enzymatic properties and direct electron transfer of the immobilized enzymes and the electrocatalytic activities of nanoporous metals in detail. We also tried to fabricate electrochemical biosensors/biofuel cells based on the combination of the electrocatalytic activities of nanoporous metals and the biocatalytic activities of enzymes. In addition, the optical (SERS) properties of nanoporous silver have also been explored for its potential application in sensing.1. Immobilization of laccase on nanoporous gold:comparative studies on the immobilization strategies and the particle size effectsNPG prepared simply by dealloying Ag from Au/Ag alloy, was used in the present study as a carrier for laccase immobilization. Three immobilization strategies, i.e., physical adsorption, electrostatic attraction, and covalent coupling, were used to immobilize laccase on NPG. A detailed comparison among the three strategies was made in light of the loading, the specific activity, and the leakage of laccase. The present results indicated that the physical adsorption strategy was the best one for laccase immobilization on NPG. This was because of the potential covalent linkage between the nanoscale gold surface and the amino groups of the residue amino acids of laccase. The effects of the particle size of NPG on laccase loading and enzyme kinetics were also investigated. When the particle size of NPG got smaller, more laccase could access the inner pore and be immobilized. The kinetic study showed that the crushed NPG not only enhanced mass transfer of the substrate and its oxidation product but also favored the exposure of the active sites of the immobilized laccase to the substrate, i.e., the crushing facilitated the enhancement of the catalytic efficiency of laccase.2. Adsorption of laccase on the surface of nanoporous gold and the direct electron transfer between themNPG with different pore sizes was obtained by simple dealloying and thermal annealing methods. The morphology of the NPG was characterized by scanning electron microscopy and nitrogen adsorption technique. Laccase was immobilized on the surface of the NPG by physical adsorption. Detailed studies were made on the effect of the pore size on laccase immobilization. NPG with pore size of 40-50 nm was demonstrated to be a suitable support for laccase immobilization. Compared with free enzyme, the optimum pH of immobilized laccase did not change; the optimum temperature, however, rose from 40 to 60℃. Both thermal and storage stabilities of laccase improved markedly via the immobilization. Laccase immobilized on NPG (100 nm in thickness) was used for enzyme electrode construction. Direct electrochemistry of laccase on NPG supported by glassy carbon electrode (NPG/GC) was achieved with high efficiency due to the outstanding physicochemical characteristics of the NPG. The laccase-loaded NPG/GC electrode also exhibited a strong electrocatalytic activity toward O2 reduction. When stored at 4℃for 1 month, the electrode showed no obvious changes in its response. All results presented in the paper indicated that NPG was an excellent carrier for laccase immobilization and would have potential applications in biofuel cell and/or biosensor areas.3. Immobilization of lignin peroxidase on nanoporous gold:Enzymatic properties and in situ release of H2O2 by co-immobilized glucose oxidaseImmobilization of enzymes on porous inorganic materials is very important for biocatalysis and biotransformation. In this chapter, NPG was used as a support for lignin peroxidase (LiP) immobilization. NPG with a pore size of 40-50 nm was prepared by dealloying Au/Ag alloy (50:50 wt%) for 17 h. By incubation with LiP aqueous solution, LiP was successfully immobilized on NPG. The optimal temperature of the immobilized LiP was ca.40,10℃higher than that of free LiP. After 2 h incubation at 45℃,55% of the initial activity of the immobilized LiP was still retained while the free LiP was completely deactivated. In addition, a high and sustainable LiP activity was achieved via in situ release of H2O2 by a co-immobilized glucose oxidase. The present co-immobilization system was demonstrated to be very effective for LiP-mediated dye decolourization.4. Enzyme-modified nanoporous gold-based electrochemical biosensorsOn the basis of the unique physical and chemical properties of nanoporous gold, which was obtained simply by dealloying Ag from Au/Ag alloy, an attempt was made in the present study to develop NPG-based electrochemical biosensors. The NPG-modified glassy carbon electrode (NPG/GCE) exhibited high-electrocatalytic activity toward the oxidation of nicotinamide adenine dinucleotide (NADH) and hydrogen peroxide (H2O2), which resulted in a remarkable decrease in the overpotential of NADH and H2O2 electro-oxidation when compared with the gold sheet electrode. The high density of edge-plane-like defective sites and large specific surface area of NPG should be responsible for the electrocatalytic behavior. Such electrocatalytic behavior of the NPG/GCE permitted effective low-potential amperometric biosensing of ethanol or glucose via the incorporation of alcohol dehydrogenase (ADH) or glucose oxidase (GOx) within the three-dimensional matrix of NPG The ADH-and GOx-modified NPG-based biosensors showed good analytical performance for biosensing ethanol and glucose due to the clean, reproducible and uniformly distributed microstructure of NPG. The stabilization effect of NPG on the incorporated enzymes also made the constructed biosensors very stable. After 1 month storage at 4℃, the ADH-and GOx-based biosensors lost only 5.0% and 4.2% of the original current response. All these indicated that NPG was a promising electrode material for biosensors construction.5. A novel nanoporous gold modified electrode for the selective determination of dopamine in the presence of ascorbic acidThe electrochemical detection of DA at conventional solid electrodes was interfered by the coexisted AA. To circumvent this problem, many modified electrodes were tried. In this chapter, an attempt was made to use NPG as modifying materials. The NPGs with different pore sizes were prepared simply by dealloying Ag from Au/Ag alloy with concentrated nitric acid. The glassy carbon electrode (GCE) based modified electrode was fabricated by simply affixing the NPG film on the surface of a GCE. The electrochemical behaviors of AA and DA at the modified electrode were studied. The results indicated that the NPG/GCE exhibited substantial enhancement in electrochemical sensitivity for DA and AA due to its large surface area. Results also showed that the oxidation of AA at the electrode was a diffusion-controlled process, but for DA it was an adsorption-controlled process. This result, together with the different anodic peak potentials of the two species, made it possible the selective determination of DA in the presence of AA. Due to the interaction of the amino group of DA with the surface of nanoscale gold, DA could be accumulated on the NPG/GCE, while AA could not. This permitted the coexistence of large amount of AA. When differential pulse voltammetry (DPV) was used, a sub-micro level of DA could be detected in the millimolar level of AA with a detection limit of 17 nM at a signal-to-noise ratio of 3. In a word, the modified electrode showed good sensitivity, selectivity and reproducibility.6. Immobilization of horseradish peroxidase on nanoporous copper and its potential applicationsBecause gold is a noble metal, its high price may inhibit the large-scale usage of NPG. Thus, in this section we tried nanoporous copper (NPC) as a support for enzyme immobilization. NPC with a pore size of 100-200 nm was prepared by simply dealloying A160Cu40 alloy in a 5 wt.% HCl solution. The NPC was characterized by scanning electron microscopy and nitrogen adsorption techniques. Horseradish peroxidase (HRP) was immobilized on NPC by adsorption. Compared with free enzyme, the thermal stability of the immobilized enzyme was greatly improved due to the multiple attachments between the enzyme molecule and the NPC surface. After 2 h incubation at 50℃, the immobilized HRP retained ca.90% of the initial activity while only ca.10% initial activity remained for the free enzyme. The interaction between HRP and the porous surface also made the Km and Kcat values of the immobilized enzyme increase (from 0.43 to 0.80 mM) and decrease (from 8.1×103 to 2.2 X 103 min-1), respectively. Based on the good electric conductivity and electrocatalytic activity of the NPC electrode, an electrochemical biosensor for O-phenylenediamine (OPD) was made. The calibration curve of the biosensor was linear from 0.5μM to 14.5μM OPD with a sensitivity of 0.37μAμM-1. The stability and reproducibility of the biosensor were also demonstrated to be good. When positioned at-0.45 V for 200 s, its current response toward 10μM OPD remained ca. 80% of its initial value. For five HRP-loaded NPC electrodes, the relative standard deviation (RSD) of the current response toward 10μM OPD was ca.4.5%. All these results indicated that NPC was a good support for the HRP immobilization and its low price would facilitate its large-scale application.7. Dealloying Ag-Al Alloy to Prepare Nanoporous Silver as Substrate for Surface-Enhanced Raman Scattering:Effects of Structural Evolution and Surface ModificationIn previous sections, we studied the electrochemical behavior of nanoporous metals and their applications in electrochemical sensing. These novel nanoporous metals also have unique optical properties, such as surface plasmon absorption. However, relative studies are still in its early stage. In this section, we studied the SERS properties of nanoporous Ag using R6G as the probe.It is known that sensitive detection of molecules using SERS technique depends on the nanostructured metallic substrate, and many efforts have been devoted to the preparation of SERS substrates with high sensitivity, stability and reproducibility. In this article, we reported on the fabrication of stable monolithic nanoporous silver (NPS) by chemical dealloying of Ag-Al precursor alloys with the emphasis being put on the effect of the structural evolution on SERS signals. It was found that the dealloying conditions had great influence on the morphology (the ligament/pore size) and the crystallization status, which determined the SERS signal of rhodamine 6G on the NPS. NPS with a small pore, low residual Al and perfect crystallization gave high SERS signal. A high enhancement factor of 7.5×105 was observed on a bare NPS obtained by dealloying Ag3oAl70 in 2.5 wt.% HCl at room temperature followed by 15 min aging at ca.85℃. After coated with Ag nanoparticles on the NPS surface, the enhancement factor increased to 1.6×108 due to the strong near-field coupling between the ligaments and nanoparticles.