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At least 865 records · Page 48Linked to original sources
PubMed2001

Mediatorless biosensor for H(2)O(2) based on recombinant forms of horseradish peroxidase directly adsorbed on polycrystalline gold.

Four forms of horseradish peroxidase (HRP) have been used to prepare peroxidase-modified gold electrodes for mediatorless detection of peroxide: native HRP, wild type recombinant HRP, and two recombinant forms containing six-His tag at the C-terminus and at the N-terminus, respectively. The adsorption of the enzyme molecules on gold was studied by direct mass measurements with electrochemical quartz crystal microbalance. All the forms of HRP formed a monolayer coverage of the enzyme on the gold surface. However, only gold electrodes with adsorbed recombinant HRP forms exhibited high and stable current response to H(2)O(2) due to its bioelectrocatalytic reduction based on direct electron transfer between gold and HRP. The sensitivity of the gold electrodes modified with recombinant HRPs was in the range of 1.4-1.5 A M(-1) cm(-2) at -50 mV versus Agmid R:AgCl. The response to H(2)O(2) in the concentration range 0.1-40 microM was not dependent on the presence of a mediator (i.e. catechol) giving strong evidence that the electrode currents are diffusion limited. Lower detection limit for H(2)O(2) detection was 10 nM at the electrodes modified with recombinant HRPs.

Adsorption↗
PubMed2000

Biosensors based on membrane-bound enzymes immobilized in a 5-(octyldithio)-2-nitrobenzoic acid layer on gold electrodes.

Gold electrodes were modified through chemisorption of 5-(octyldithio)-2-nitrobenzoic acid (ODTNB). ODTNB includes a long chain in a short-length thio acid, providing a heterogeneous-like alkanethiol layer after adsorption on gold electrodes. Membrane-bound enzymes, in particular D-fructose dehydrogenase (FDH), D-gluconate dehydrogenase (GADH), and L-lactic dehydrogenase (cytochrome b2) (Cyb2), were immobilized onto ODTNB-modified gold electrodes simply by adsorption. The short-length thio acid may provide electrostatic interactions with enzyme surface charges, while the alkanethiolate enables hydrophobic interaction with the largely lipophilic, membrane-bound enzymes. The immobilization of FDH, GADH, and Cyb2 onto ODTNB-modified gold surfaces has been studied with the quartz crystal microbalance (QCM). Spectrophotometric and electrochemical assays indicate that the immobilized enzyme retains its enzymatic activity after immobilization onto the ODTNB-modified gold surface. The amount of immobilized (and active) enzyme was estimated from QCM to be of the order of 2.5 x 10(-12)-5.3 x 10(-12) mol x cm(-2). A fructose biosensor was developed, making use of a gold surface modified with ODTNB and fructose dehydrogenase, employing hydroxymethylferrocene as a mediator in solution. Calibration curves exhibited a linear relation between the biosensor response and the substrate concentration up to 0.7 mM. Statistical analysis gave an excellent linear correlation (r = 0.9993) and a sensitivity of 6.1 mM(-1) fructose. The biosensor shows a significant stable catalytic current for at least 25 days.

Biosensing Techniques↗
PubMed2000

A fluorescence-based method for determining the surface coverage and hybridization efficiency of thiol-capped oligonucleotides bound to gold thin films and nanoparticles.

Using a fluorescence-based method, we have determined the number of thiol-derivatized single-stranded oligonucleotides bound to gold nanoparticles and their extent of hybridization with complementary oligonucleotides in solution. Oligonucleotide surface coverages of hexanethiol 12-mer oligonucleotides on gold nanoparticles (34 +/- 1 pmol/cm2) were significantly higher than on planar gold thin films (18 +/- 3 pmol/cm2), while the percentage of hybridizable strands on the gold nanoparticles (1.3 +/- 0.3 pmol/cm2, 4%) was lower than for gold thin films (6 +/- 2 pmol/cm2, 33%). A gradual increase in electrolyte concentration over the course of oligonucleotide deposition significantly increases surface coverage and consequently particle stability. In addition, oligonucleotide spacer sequences improve the hybridization efficiency of oligonucleotide-modified nanoparticles from approximately 4 to 44%. The surface coverage of recognition strands can be tailored using coadsorbed diluent oligonucleotides. This provides a means of indirectly controlling the average number of hybridized strands per nanoparticle. The work presented here has important implications with regard to understanding interactions between modified oligonucleotides and metal nanoparticles, as well as optimizing the sensitivity of gold nanoparticle-based oligonucleotide detection methods.

Gold↗
PubMed2005

Gold nanoparticle-modified etched capillaries for open-tubular capillary electrochromatography.

The use of gold nanoparticles in conjunction with etched capillary-based open-tubular capillary electrochromatography (OTCEC) to improve the efficiency of separation and the selectivity between selected solutes is described. The fused-silica capillaries (50-microm i.d.) were etched with ammonium hydrogen difluoride, followed by prederivatization of the new surface with (3-mercaptopropyl)trimethoxysilane (MPTMS) for the immobilization of dodecanethiol gold nanoparticles, for OTCEC. The electrochromatography of a "reversed-phase" test mixture and of selected polycylic aromatic hydrocarbons was investigated, and efficient separations and high theoretical plate numbers per meter were obtained. The electroosmotic flow characteristics of the etched gold nanoparticle capillary, unetched gold nanoparticle capillary, bare capillary, and etched bare capillary were studied by varying the percentage of organic modifier in buffer, buffer pH, and separation voltage. Optical microscopy and scanning electron microscopy were used to examine the process of etching and modification and the surface features of the etched gold nanoparticle capillary. The results confirm that dodecanethiol gold nanoparticles bonded on the etched inner wall of the fused-silica capillary can provide sufficient solute-bonded phase interactions to obtain OTCEC separations with reproducible retention, as well as characteristic reversed-phase behavior, even with the inner diameter of the capillary of 50 microm.

Capillary Electrochromatography↗
PubMed2004

Realization and characterization of porous gold for increased protein coverage on acoustic sensors.

Immunosensors show great potential for the direct detection of biological molecules. The sensitivity of these affinity-based biosensors is dictated by the amount of receptor molecules immobilized on the sensor surface. An enlargement of the sensor area would allow for an increase of the binding capacity, hence a larger amount of immobilized receptor molecules. To this end, we use electrochemically deposited "gold black" as a porous sensor surface for the immobilization of proteins. In this paper, we have analyzed the different parameters that define the electrochemical growth of porous gold, starting from flat gold surfaces, using different characterization techniques. Applied potentials of -0.5 V versus a reference electrode were found to constitute the most adequate conditions to grow porous gold surfaces. Using cyclic voltammetry, a 16 times increase of the surface area was observed under these electrochemical deposition conditions. In addition, we have assessed the immobilization degree of alkanethiols and of proteins on these different porous surfaces. The optimized deposition conditions for realizing porous gold substrates lead to a 11.4-fold increase of thiol adsorption and a 3.3-fold increase of protein adsorption, using the quartz crystal microbalance (QCM-D) as a biological transducer system. Hence, it follows that the high specific area of the porous gold can amplify the final sensitivity of the original flat surface device.

Adsorption↗
PubMed2005

SPR sensor chip for detection of small molecules using molecularly imprinted polymer with embedded gold nanoparticles.

Molecularly imprinted polymer gel with embedded gold nanoparticle was prepared on a gold substrate of a chip for a surface plasmon resonance (SPR) sensor for fabricating an SPR sensor sensitive to a low molecular weight analyte. The sensing is based on swelling of the imprinted polymer gel that is triggered by an analyte binding event within the polymer gel. The swelling causes greater distance between the gold nanoparticles and substrate, shifting a dip of an SPR curve to a higher SPR angle. The polymer synthesis was conducted by radical polymerization of a mixture of acrylic acid, N-isopropylacrylamide, N,N'-methylenebisacrylamide, and gold nanoparticles in the presence of dopamine as model template species on a sensor chip coated with allyl mercaptan. The modified sensor chip showed an increasing SPR angle in response to dopamine concentration, which agrees with the expected sensing mechanism. Furthermore, the gold nanoparticles were shown to be effective for enhancing the signal intensity (the change of SPR angle) by comparison with a sensor chip immobilizing no gold nanoparticles. The analyte binding process and the consequent swelling appeared to be reversible, allowing one the repeated use of the presented sensor chip.

Gold↗
PubMed2006

Surface coverage and structure of mixed DNA/alkylthiol monolayers on gold: characterization by XPS, NEXAFS, and fluorescence intensity measurements.

Self-assembly of thiol-terminated single-stranded DNA (HS-ssDNA) on gold has served as an important model system for DNA immobilization at surfaces. Here, we report a detailed study of the surface composition and structure of mixed self-assembled DNA monolayers containing a short alkylthiol surface diluent [11-mercapto-1-undecanol (MCU)] on gold supports. These mixed DNA monolayers were studied with X-ray photoelectron spectroscopy (XPS), near-edge X-ray absorption fine structure spectroscopy (NEXAFS), and fluorescence intensity measurements. XPS results on sequentially adsorbed DNA/MCU monolayers on gold indicated that adsorbed MCU molecules first incorporate into the HS-ssDNA monolayer and, upon longer MCU exposures, displace adsorbed HS-ssDNA molecules from the surface. Thus, HS-ssDNA surface coverage steadily decreased with MCU exposure time. Polarization-dependent NEXAFS and fluorescence results both show changes in signals consistent with changes in DNA orientation after only 30 min of MCU exposure. NEXAFS polarization dependence (followed by monitoring the N 1s --> pi* transition) of the mixed DNA monolayers indicated that the DNA nucleotide base ring structures are oriented more parallel to the gold surface compared to DNA bases in pure HS-ssDNA monolayers. This indicates that HS-ssDNA oligomers reorient toward a more-upright position upon MCU incorporation. Fluorescence intensity results using end-labeled DNA probes on gold show little observable fluorescence on pure HS-ssDNA monolayers, likely due to substrate quenching effects between the fluorophore and the gold. MCU diluent incorporation into HS-ssDNA monolayers initially increases DNA fluorescence signal by densifying the chemisorbed monolayer, prompting an upright orientation of the DNA, and moving the terminal fluorophore away from the substrate. Immobilized DNA probe density and DNA target hybridization in these mixed DNA monolayers, as well as effects of MCU diluent on DNA hybridization in complex milieu (i.e., serum) were characterized by surface plasmon resonance (SPR) and (32)P-radiometric assays and reported in a related study (Gong, P.; Lee, C.-Y.; Gamble, L. J.; Castner, D. G.; Grainger, D. W. Anal. Chem. 2006, 78, 3326-3334.).

Alkanes↗
PubMed2006

Fabrication of carbohydrate microarrays on gold surfaces: direct attachment of nonderivatized oligosaccharides to hydrazide-modified self-assembled monolayers.

This paper describes a new and simple microarray platform for presenting multiple nonderivatized oligosaccharides to protein targets, with utility for mapping carbohydrate-protein recognition events. The approach is based on the creation of a hydrazide-derivatized, self-assembled monolayer on a gold surface in a single or two-step procedure, for efficient and selectively oriented anchoring of oligosaccharide probes via their reducing ends, with detection using fluorescence detection of bound proteins. The biggest hurdles in employing gold-based substrate for fluorescence-based microarray detection include fluorescence quenching and nonspecific surface adsorption of proteins. We found that the quenching effect could be minimized by introducing a omega-thiolated fatty acid (C16) self-assembled monolayer between the gold surface and hydrazide groups, followed by detection involving three successive binding protein layers covering the gold surface. In addition, an effective blocking scheme involving poly(ethylene glycol) aldehyde and bovine serum albumin was employed to reduce nonspecific protein adsorption to the chip surface. As proof of principle, we demonstrate here that sulfated oligosaccharide probes from heparin can be effectively and covalently attached without prior derivatization onto the hydrazide-modified, self-assembled monolayer on gold-coated slide surfaces in a microarray format. This platform is used to assess binding of specific heparin-binding protein targets at very high sensitivity, and we also demonstrate that the approach can be extended to nonsulfated sugars. Direct attachment of nonderivatized sugar probes on the chip is advantageous since it avoids the need for laborious prederivatization and cleanup steps. This versatile fluorescence microarray platform provides a facile approach for interrogating multiple carbohydrate-protein interactions in a high-throughput manner and has potential as a common gold surface platform for other diverse interrogations by MALDI-MS, surface plasmon resonance, and quartz crystal microbalances.

Gold↗
PubMed2000

Oriented immobilization of antibodies onto the gold surfaces via their native thiol groups.

The general approach for site-oriented immobilization of antibodies onto gold supports is reported. The immobilization is carried out using the native sulfide groups of immunoglobulin (IgG). To liberate the thiol groups, the intact IgG was split into two half-IgG fragments without destruction of the binding site of the antibody. The immobilization of half-IgG fragments on the gold surface was carried out by simple adsorption. The antigen binding capacity of the half-IgG modified gold supports is similar to that of the gold surfaces with the traditionally linked antibodies and is much higher than for nonspecifically adsorbed intact IgGs. The immobilized antibodies, according to the proposed approach, maintain high antigen binding constants. The immobilization procedure provides orientation of IgG fragments in terms of the similar distance between the binding site of the antibody and the surface of the gold support, which does not cause the distribution of the apparent affinity constants. The high operational stability of half-IgG modified gold electrodes makes them applicable for analytical applications.

Electrochemistry↗
PubMedDate not supplied

Immobilization of Candida bombicola cells on free-standing organic-gold nanoparticle membranes and their use as enzyme sources in biotransformations.

Preparation of chemically functionalized biocompatible surfaces is of current interest, with application in the immobilization of various bioactive species such as DNA, enzymes, whole cells, etc. We report herein the one-step synthesis of a self-supporting gold nanoparticle membrane, its surface modification, and application in the immobilization of Candida bombicola (yeast) cells. The gold nanoparticle membrane is prepared by the spontaneous reduction of aqueous chloroaurate ions by a diamine at a liquid-liquid interface. The gold nanoparticles in the polymeric membrane may be capped with octadecylamine (ODA) molecules, thereby rendering the nanoparticle membrane hydrophobic. Exposure of the hydrophobized organic-gold nanoparticle membrane to C. bombicola yeast cells results in their binding to the membrane, possibly through nonspecific interactions such as hydrophobic interactions between the yeast cell walls and the ODA molecules. The enzyme cytochrome P450 present in the yeast cells immobilized on the organic-gold nanoparticle membrane was then used in the transformation of the arachidonic acid (AA) to sophorolipids followed by acid hydrolysis to form 20-hydroxyeicosatetraneoic acid (20-HETE). The organic-gold nanoparticle membrane-C. bombicola bioconjugate could be easily separated from the reaction medium and reused a number of times.

Arachidonic Acid↗
PubMed2006

Chiral N-isobutyryl-cysteine protected gold nanoparticles: preparation, size selection, and optical activity in the UV-vis and infrared.

We have prepared gold nanoparticles covered with N-isobutyryl-l-cysteine and N-isobutyryl-d-cysteine, respectively. These particles with a mean particle size smaller than 2 nm are highly soluble in water and are amenable to chiroptical techniques such as vibrational circular dichroism (VCD) and circular dichroism (CD) spectroscopy. Density functional theory shows that the VCD spectra are sensitive toward the conformation of the adsorbed thiol. Based on the comparison between the experimental VCD spectrum and the calculated VCD spectra for different conformers, a preferential conformation of the thiol adsorbed on the gold particles can be proposed. In this conformation the carboxylate group interacts with the gold particle in addition to the sulfur. The particles could furthermore be separated according to their charge and size into well-defined compounds. The optical absorption spectra revealed a well-quantized electronic structure and a systematic red-shift of the absorption onset with increasing gold core size, which was manifested in a color change with particle size. Some compounds showed basically identical absorption spectra as analogous gold particles protected with l-glutathione. This shows that these particles have identical core sizes (10-12, 15 and 18 gold atoms, respectively) and indicates that the number and arrangement of the adsorbed thiol are the same, independent of the two thiols, which have largely different sizes. Some separated compounds show strong optical activity with opposite sign when covered with the d- and l-enantiomer, respectively, of N-isobutyryl-cysteine. The origin of the optical activity in the metal-based transitions is discussed. The observations are consistent with a mechanism based on a chiral footprint on the metal core imparted by the adsorbed thiol.

Cysteine↗
PubMed2006

Development of multianalyte flow-through and lateral-flow assays using gold particles and horseradish peroxidase as tracers for the rapid determination of carbaryl and endosulfan in agricultural products.

Membrane-based competitive immunoassays using gold particles and horseradish peroxidase (HRP) as tracers in flow-through and lateral-flow formats for multianalysis of carbaryl and endosulfan were developed. For gold-based immunoassay, membrane strips were coated with goat anti-rabbit IgG (control line) and carbaryl hapten-ovalbumin (OVA) and endosulfan hapten-OVA (test lines). The visual detection limits for carbaryl and endosulfan were 100 and 10 microg/L in gold-based assays, respectively. For immunoassay using HRP as tracer, anti-carbaryl and anti-endosulfan antibodies were separately coated on the membrane as test lines, and the visual detection limits were 10 microg/L for carbaryl and 1 microg/L for endosulfan. The developed assays used gold particles and HRP as labels, respectively; 10 times enhancement in the visual detection limit using HRP label was obtained in the study. Matrix interference was eliminated by appropriate dilution of sample extracts with buffer. For the validation of the multianalyte assay, the samples were screened by multianalyte gold-based assay and confirmed by HPLC for carbaryl determination and by GC for endosulfan determination. The results of multianalyte gold-based flow-through assay for the determination of carbaryl and endosulfan were in good agreement with the results of instrumental analysis (HPLC with ultraviolet detection and GC with electron capture detection). The developed multianalyte immunoassays for which the results were interpreted visually can be used as convenient qualitative tools for on-site rapid screening of carbaryl and endosulfan simultaneously in agricultural products.

Avena↗
PubMed2005

Mechanism of gold metal ion reduction, nanoparticle growth and size control in aqueous amphiphilic block copolymer solutions at ambient conditions.

Spontaneous formation and efficient stabilization of gold nanoparticles with an average diameter of 7 approximately 20 nm from hydrogen tetrachloroaureate(III) hydrate (HAuCl4.3H2O) were achieved in air-saturated aqueous poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (PEO-PPO-PEO) block copolymer solutions at ambient temperature in the absence of any other reducing agent. The particle formation mechanism is considered here on the basis of the block copolymer concentration dependence of absorption spectra, the time dependence (kinetics) of AuCl4- reduction, and the block copolymer concentration dependence of particle size. The effects of block copolymer characteristics such as molecular weight (MW), PEO block length, PPO block length, and critical micelle concentration (cmc) are explored by examining several PEO-PPO-PEO block copolymers. Our observations suggest that the formation of gold nanoparticles from AuCl4- comprises three main steps: (1) reduction of metal ions by block copolymer in solution, (2) absorption of block copolymer on gold clusters and reduction of metal ions on the surface of these gold clusters, and (3) growth of metal particles stabilized by block copolymers. While both PEO and PPO blocks contribute to the AuCl4- reduction (step 1), the PEO contribution appears to be dominant. In step 2, the adsorption of block copolymers on the surface of gold clusters takes place because of the amphiphilic character of the block copolymer (hydrophobicity of PPO). The much higher efficiency of particle formation attained in the PEO-PPO-PEO block copolymer systems as compared to PEO homopolymer systems can be attributed to the adsorption and growth processes (steps 2 and 3) facilitated by the block copolymers. The size of the gold nanoparticles produced is dictated by the above mechanism; the size increases with increasing reaction activity induced by the block copolymer overall molecular weight and is limited by adsorption due to the amphiphilic character of the block copolymers.

Gold↗
PubMed2005

Structure of tert-butyl carbamate-terminated thiol chemisorbed to gold.

Monolayers of tert-butyl carbamate-terminated thiol were formed by adsorption of the molecules onto polycrystalline gold substrate. The adsorbates were studied using techniques as X-ray photoelectron spectroscopy (XPS), near-edge X-ray absorption fine structure spectroscopy (NEXAFS), and infrared reflection-absorption spectroscopy (IRAS). The results provide the electronic structure, composition, characteristic fingerprint, and orientation of the molecular adsorbate. XPS verified that the thiolate group is chemically bonded to the gold surface and that a complete chemisorption of the molecule occurs. Elemental depth profiling by varying the excitation energy in XPS supports the angle-dependent XPS results. Both techniques showed that the tert-butyl group is oriented away from the gold surface. A nearly parallel orientation of the carbonyl group relative to the gold surface is deduced from the IRAS results. The main molecular axis is estimated to have an average tilt angle of about 38 degrees relative to the gold surface normal on the basis of the NEXAFS results. Cyclic voltammetry indicates a less blocking capability of the adsorbates. Overall, the molecules are oriented in an upright manner with indications of presence of pinholes and/or defects possibly due to steric hindrance of the bulky tert-butyl group. This molecular system is envisioned to be of use for surface-based organic synthesis on gold substrates.

Absorption↗
PubMed2006

New preparation method of gold nanoparticles on SiO2.

It is shown that adsorption of the [Au(en)(2)](3+) cationic complex can be successfully employed for the deposition of gold nanoparticles (1.5 to 3 nm) onto SiO(2) with high metal loading, good dispersion, and small Au particle size. When the solution pH increases (from 3.8 to 10.5), the Au loading in the Au/SiO(2) samples increases proportionally (from 0.2 to 5.5 wt %), and the average gold particle size also increases (from 1.5 to 2.4 nm). These effects are explained by the increase in the amount of negatively charged sites present on the SiO(2) surface, namely, when the solution pH increases, a higher number of [Au(en)(2)](3+) species can be adsorbed. Extending the adsorption time from 2 to 16 h gives rise to an increase in the gold loading from 3.3 to 4.0 wt % and in the average particle size from 1.8 to 2.9 nm. Different morphologies of gold nanoparticles are present as a function of the particle size. Particles with a size of 3-5 nm show defective structure, some of them having a multiple twinning particle (MTP) structure. At the same time, nanoparticles with an average size of ca. 2 nm exhibit defect-free structure with well-distinguishable {111} family planes. TEM and HAADF observations revealed that Au particles do not agglomerate on the SiO(2) support: gold is present on the surface of SiO(2) only as small particles. Density functional theory calculations were employed to study the mechanisms of [Au(en)(2)](3+) adsorption, where neutral and negatively charged silica surfaces were simulated by neutral cluster Si(4)O(10)H(4) and negatively charged cluster Si(4)O(10)H(3), respectively. The calculation results are totally consistent with the suggestion that the deposition of gold takes place according to a cationic adsorption mechanism.

Adsorption↗
PubMed2005

Gold nanoparticle localization at the core surface by using thermosensitive core-shell particles as a template.

We report novel thermosensitive hybrid core-shell particles via in situ gold nanoparticle formation using thermosensitive core-shell particles as a template. This method for the in situ synthesis of gold nanoparticles with microgel interiors offers the advantage of eliminating or significantly reducing particle aggregation. In addition, by using thermosensitive microgel structures in which the shell has thermosensitive and gel properties in water--whereas the core itself is a water-insoluble polymer--we were able to synthesize the gold nanoparticles only at the surface of the core, which had reactive sites to bind metal ions. After the gold nanoparticles were synthesized, electroless gold plating was carried out to control the thickness of the gold nanoshells. The dispersions of the obtained hybrid particles were characterized by dynamic light scattering and UV-vis absorption spectroscopy, and the dried particles were also observed by electron microscopy. Adaptation of the technique shown here will create a number of applications as optical, electronic, and biomedical functional materials.

Gold↗
PubMed2006

Facile synthesis of highly biocompatible poly(2-(methacryloyloxy)ethyl phosphorylcholine)-coated gold nanoparticles in aqueous solution.

Diblock copolymers comprising a highly biocompatible poly(2-(methacryloyloxy)ethyl phosphorylcholine) (PMPC) block and a poly(2-(dimethylamino)ethyl methacrylate) (PDMA) block were evaluated for the synthesis of sterically stabilized gold nanoparticles in aqueous solution. The PDMA block becomes partially protonated on addition of HAuCl4, and the remaining nonprotonated tertiary amine groups reduce the AuCl4- counterion to zerovalent gold in situ. This approach results in the adsorption of the PDMA block onto the gold nanoparticle surface while the PMPC chains serve as a stabilizing block, producing highly biocompatible gold sols in aqueous solution at ambient temperature without any external reducing agent. The size and shape of gold nanoparticles could be readily controlled by tuning synthesis parameters such as the block composition and the relative and absolute concentrations of the PMPC-PDMA diblock copolymer and HAuCl4. These highly biocompatible gold sols have potential biomedical applications.

Biocompatible Materials↗
PubMed2001

P-chip and P-chip bienzyme electrodes based on recombinant forms of horseradish peroxidase immobilized on gold electrodes.

Adsorption and bioelectrocatalytic activity of native horseradish peroxidase (HRP) and its recombinant forms on polycrystalline gold electrodes were studied. Recombinant forms of HRP were produced by a genetic engineering approach using an E. coli expression system. According to direct mass measurements with a quartz crystal microbalance, all the forms of HRP formed monolayer coverage of the enzyme on the gold surface. However, only gold electrodes modified with the recombinant HRP forms (non-glycosylated) exhibited high and stable current response to H2O2 due to its bioelectrocatalytic reduction based on direct electron transfer (ET) between gold and the active site of the enzyme. Introduction of a six-His tag either at the C-terminus or at the N-terminus of the enzyme molecule additionally increased the strength of the enzyme binding with the gold surface and the efficiency of direct ET. Immobilization of recombinant forms of HRP containing histidine functional groups on the surface of the gold electrode was used both for the development of a P-chip, a biosensor for hydrogen peroxide determination based on direct ET, and for the development of a bienzyme biosensor electrode for the determination of L-lysine based on co-immobilized recombinant forms of HRP and L-lysine-alpha-oxidase.

Adsorption↗