Site-Directed Immobilization of Proteins Through Electrochemical Deprotection on Electroactive Self-Assembled Monolayers (original) (raw)
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Journal of Electroanalytical Chemistry, 2008
Different immobilization procedures for horseradish peroxidase (HRP) were investigated using, as support matrices, self-assembled monolayers (SAM) formed on gold electrodes. The influences of these immobilization processes in the biosensor performance were also evaluated. For this, monolayers were prepared from thiols with different structures, carbon chain sizes and terminal groups. It was shown that the thiol carbon chain size influences especially in monolayer coverage and, consequently, in the biomolecule immobilization efficiency. From the studies carried out for SAM characterization on the electrode surface it was verified that thiols with shorter chains (n < 9) tend to form monolayers with a considerable amount of defects on the gold surface that lead to a lower coverage. However, thiols with a longer carbon chain present a higher coverage degree, which are not suited as substrate to the development of electrochemical biosensors, because they passivate the transduction interface, making difficult the electron transfer and, consequently, reducing electrode sensitivity. In relation to the enzyme immobilization on gold electrodes, it was verified, using different techniques, that monolayers that possess-NH 2 terminal groups provided the best results, probably due to the use of glutaraldeyde as ligand during the immobilization process. Analyzing biosensor performances for hydrogen peroxide, it was verified that SAM formed by cysteamine is the most adequate for HRP immobilization, because it provides better efficiency for enzyme immobilization associated to high sensitivity for H 2 O 2 .
Analytical Sciences, 2001
The fabrication of enzyme electrodes using self-assembled monolayers (SAMs) has attracted considerable interest because of the spatial control over the enzyme immobilization. A model system of glucose oxidase covalently bound to a gold electrode modified with a SAM of 3-mercaptopropionic acid was investigated with regard to the effect of fabrication variables such as the surface topography of the underlying gold electrode, the conditions during covalent attachment of the enzyme and the buffer used. The resultant monolayer enzyme electrodes have excellent sensitivity and dynamic range which can easily be adjusted by controlling the amount of enzyme immobilized. The major drawback of such electrodes is the response which is limited by the kinetics of the enzyme rather than mass transport of substrates. Approaches to bringing such enzyme electrodes into the mass transport limiting regime by exploiting direct electron transfer between the enzyme and the electrode are outlined.
Journal of Nanobiotechnology, 2011
Background: The interest in introducing ecologically-clean, and efficient enzymes into modern industry has been growing steadily. However, difficulties associated with controlling their orientation, and maintaining their selectivity and reactivity is still a significant obstacle. We have developed precise immobilization of biomolecules, while retaining their native functionality, and report a new, fast, easy, and reliable procedure of protein immobilization, with the use of Adenylate kinase as a model system. Methods: Self-assembled monolayers of hexane-1,6-dithiol were formed on gold surfaces. The monolayers were characterized by contact-angle measurements, Elman-reagent reaction, QCM, and XPS. A specifically designed, mutated Adenylate kinase, where cysteine was inserted at the 75 residue, and the cysteine at residue 77 was replaced by serine, was used for attachment to the SAM surface via spontaneously formed disulfide (S-S) bonds. QCM, and XPS were used for characterization of the immobilized protein layer. Curve fitting in XPS measurements used a Gaussian-Lorentzian function. Results and Discussion: Water contact angle (65-70°), as well as all characterization techniques used, confirmed the formation of self-assembled monolayer with surface SH groups. X-ray photoelectron spectroscopy showed clearly the two types of sulfur atom, one attached to the gold (triolate) and the other (SH/S-S) at the ω-position for the hexane-1,6-dithiol SAMs. The formation of a protein monolayer was confirmed using XPS, and QCM, where the QCM-determined amount of protein on the surface was in agreement with a model that considered the surface area of a single protein molecule. Enzymatic activity tests of the immobilized protein confirmed that there is no change in enzymatic functionality, and reveal activity~100 times that expected for the same amount of protein in solution. Conclusions: To the best of our knowledge, immobilization of a protein by the method presented here, with the resulting high enzymatic activity, has never been reported. There are many potential applications for selective localization of active proteins at patterned surfaces, for example, bioMEMS (MEMS-Micro-Electro-Mechanical Systems. Due to the success of the method, presented here, it was decided to continue a research project of a biosensor by transferring it to a high aspect ratio platform-nanotubes.
Journal of The American Chemical Society, 2002
The development of bioelectronic enzyme applications requires the immobilization of active proteins onto solid or colloidal substrates such as gold. Coverage of the gold surface with alkanethiol selfassembled monolayers (SAMs) reduces nonspecific adsorption of proteins and also allows the incorporation onto the surface of ligands with affinity for complementary binding sites on native proteins. We present in this work a strategy for the covalent immobilization of glycosylated proteins previously adsorbed through weak, reversible interactions, on tailored SAMs. Boronic acids, which form cyclic esters with saccharides, are incorporated into SAMs to weakly adsorb the glycoprotein onto the electrode surface through their carbohydrate moiety. To prevent protein release from the electrode surface, we combine the affinity motif of boronates with the reactivity of epoxy groups to covalently link the protein to heterofunctional boronateepoxy SAMs. The principle underlying our strategy is the increased immobilization rate achieved by the weak interaction-induced proximity effect between slow reacting oxyrane groups in the SAM and nucleophilic residues from adsorbed proteins, which allows the formation of very stable covalent bonds. This approach is exemplified by the use of phenylboronates-oxyrane mixed monolayers as a reactive support and redoxenzyme horseradish peroxidase as glycoprotein for the preparation of peroxidase electrodes. Quartz crystal microbalance, atomic force microscopy, and electrochemical measurements are used to characterize these enzymatic electrodes. These epoxy-boronate functional monolayers are versatile, stable interfaces, ready to incorporate glycoproteins by incubation under mild conditions.
Biosensors and …, 1997
Functionalized monolayer electrodes provide the grounds for bioelectronic and optobioelectronic devices. Reconstitution of apo-glucose oxidase, apo-GOx, onto a pyrroloquinoline quinone-FAD diad, assembled as a monolayer on a Au-electrode, yields an aligned bioelectrocatalytically active enzyme on the electrode surface. The resulting reconstituted enzyme electrode exhibits superior electrical contact with the electrode surface and acts as an amperometric glucose sensing electrode. The enzyme electrode operates under oxygen and is unaffected by interfering substrates such as ascorbic acid.
Journal of Electroanalytical Chemistry, 2020
A study of biosensors formed with the enzyme horseradish peroxidase immobilized onto gold electrodes with cyclic voltammetry is presented here. This enzyme was chemically linked to self-assembled monolayers of 3-mercaptopropionic, 8-mercaptooctanoic and 11-mercaptoundecanoic acids. Gold electrodes were used as holders. The goal of this work was the determination of the optimum concentration of the enzyme contained in the deposition solution that was used to modify the carboxylic ending of the monolayers just to form the biosensors. This concentration was established at 5, 7 and 10 μmol L −1 for the 11-mercaptoundecanoic, the 8-mercaptooctanoic and the 3-mercaptopropanoic acids respectively. UV-visible spectroscopy experiments allowed this fact. Furthermore, the good quality of the formation of the thiols monolayers was confirmed by using potassium ferricyanide as an electrochemical probe and reductive desorption voltammograms. Additionally, the substrate guaiacol was employed successfully to validate the developed biosensors. To conclude, comparisons between the three monolayers suggested that the shortest monolayer was the less compact, although the one with the best sensibility.
Potential control of horseradish peroxidase immobilization on gold electrode
Science in China Series B, 2004
A new approach based on potential control was firstly used for the immobilization of horseradish peroxidase (HRP) as the model protein. The self-assembly monolayer (SAM) was prepared with 2-aminoethanethiol (AET) on the gold electrode. The charge on HRP was adjusted by means of the acidity of the phosphate buffer solution (PBS) for dissolving the HRP. The influence of electric potential on HRP immobilization was investigated by means of colorimetric immunoassay of enzyme-substrate interaction (CIESI) using an automatic plate reader. The HRP modified electrodes were characterized with X-ray photoelectron spectroscopy (XPS) as well as atomic force microscope (AFM) on template-stripped gold surface. The potential for maximum immobilization of HRP was near the zero charge potential. The result indicates that controlled potential can affect the course of HRP immobilization without the loss of enzymic activity. It is of great significance for the control of biomolecular self-assembly and the intrinsic electric device.
Supramolecular multilayer structures of wired redox enzyme electrodes
Physical Chemistry Chemical Physics, 2005
Supramolecular multilayer structures comprised of glucose oxidase (GOx), and Os complex derivatised poly(allylamine) (PAH-Os) have been built by alternate layer-by-layer (LBL) electrostatic adsorption in a selfassembly process. The resulting modified electrodes with integrated mediator were tested as reagentless glucose biosensors. The enzyme kinetic parameters and the surface concentration of ''wired'' enzyme G E have been obtained by analysis of the catalytic current dependence on glucose concentrations for the ping-pong mechanism of glucose oxidation. An average osmium volume concentration was estimated by integration of the redox charge in the absence of glucose and the ellipsometric thickness. The total enzyme surface concentration was measured with a quartz crystal microbalance (QCM) during each adsoption step and the fraction of ''wired'' enzyme and the bimolecular rate constant for FADH 2 oxidation by the redox polymer for the different multilayers. The catalytic current increases with the number of LBL layers because the increase in the enzyme loading while the efficiency of enzyme FADH 2 oxidation by the Os redox polymer, except for the first dipping cycle remains almost constant at about 2 Â 10 4 M À1 s À1 .
2007 29th Annual International Conference of the IEEE Engineering in Medicine and Biology Society, 2007
In this paper, a new approach to improve long term stability of electro-enzymatic glucose sensors is presented. Self-assembled monolayers (SAMs) are employed for surface treatment of the gold electrodes to improve adhesion. Three types of functional alkylthiols, namely 11-amino-1undecanethiol hydrochloride, 1-hexadecanethiol (1hexadecanthiol) and 1,9-nonanedithiol, and bovine serum albumin (BSA) are investigated in our study. Alkylhiols are used for surface treatment of gold electrodes and BSA is mixed in enzyme (glucose oxidase) solution to realize the proposed sensors. Furthermore, gold is investigated as an electrode material. Both reference and active electrodes are fabricated using only gold. The current response of the modified sensors showed long term stability. However, the unmodified sensors showed very short term stability because glucose oxidase does not adhere well to the electrodes. The proposed modified sensors also show lower drift than the unmodified sensors without surface treatment and BSA.