This paper describes the development of a label-free electrochemical immunosensor for the qualitative detection of American trypanosomiasis in serum samples. The immunosensor was constructed using self assembled monolayer technique with gold substrate modified with monolayer of 3-mercaptopropionic acid. The T. cruzi antigens were immobilized after activation carboxylic groups in monolayer with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide. The parameters that can interfere the impedimetric response of the immunosensor response such as, concentration and incubation time in the solution of T. cruzi antigens, concentration of bovine serum albumin and incubation time in the solution of T. cruzi antibodies were optimized in the presence of the probe molecule hexacyanoferrate(II)/(III), using cyclic voltammetry and electrochemical impedance spectroscopy. The interaction between antigen–antibody was monitored using electrochemical impedance spectroscopy and the results were expressed in terms of the increase of the charge transfer resistance across the interface. The immunosensor showed selective and sensitive in the qualitative detection of T. cruzi antibodies in serum samples infected with toxoplasma antibodies. Therefore, the proposed methodology is a promising alternative for the development of a immunosensor for serological qualitative diagnosis of Chagas disease.
A horseradish peroxidase (HRP) biosensor was constructed using binary self-assembled monolayers (SAM) of 11-mercaptoundecanoic acid (MUA) and thiolactic acid (TLA) on gold surface. The advantages of using mixed SAM for the enzyme immobilization is that the long carbon chain molecules act as a support for the enzyme while the short chain molecules favor the electron transfer process. In order to obtain this modified surface, the gold electrode was incubated in a solution containing different proportions of MUA and TLA and the best concentration ratio of these molecules was 0.5 and 1.0 mmol L −1 , respectively. The preparation steps and the biosensor response were monitored by electrochemical techniques. The biosensor proposed was applied to determine hydroquinone in a 0.10 mol L −1 phosphate buffer solution containing H 2 O 2 0.3 mmol L −1 . The Au-SAM mix -HRP electrode, in the presence of hydrogen peroxide, catalyzes the oxidation of hydroquinone to the corresponding quinone, which is electrochemically reduced back to hydroquinone at −0.08 V vs Ag/AgCl. The analytical curve was linear for hydroquinone concentrations from 5.0 to 30 μmol L −1 and the detection limit was 1.26 μmol L −1 . The lifetime of this biosensor was 15 days. The modified electrode displayed good reproducibility, sensitivity and stability for the determination of hydroquinone.The modification of electrode surfaces using the self-assembled monolayers (SAM) technique is more convenient, because they form spontaneously, easy to handle mechanically and relatively stable in electrolyte solutions. 1 The main interest in developing mixed selfassembled monolayers (SAM mix ) is related to the development of attractive methods to promote different arrangements on the electrode surface, which enables the control of specific reaction sites. In this way, SAM mix can be configured as an appropriate platform for the immobilization of biomolecules 2 which have been frequently applied in electroanalysis for the development of biosensors, 3,4 since well-organized and compact monolayers present advantages like selectivity, sensitivity and reduced overpotentials in electrocatalytic reactions. The SAM mix can be obtained by combining the properties of alkanethiols with different carbon chain lengths or different functional group. 5 Several methods for the preparation of mixed SAMs are reported in the literature, being the most commonly used the coadsorption of different thiols, 6,7 and also by electrochemical substitution modification. 8,9 The SAM mix obtained through co-adsorption of different functional groups was described by Ngunjiri et al, 10 formed by molecules octadecanethiol and 11-mercaptoundecanoic acid for immobilization of proteins. Another example, Ji et al. 11 developed the SAM mix formed by thioctic acid (T-COOH) and thioctic acid amide (T-NH 2 ) which was used to immobilize tyrosinase to construct an electrochemical biosensor for phenolic compounds. Studies with this biosensor showed that mixed SAMs improved protein adsorption and they were more co...
Self-assembled monolayers (SAMs) have attracted much interest due to their potential applications in biosensors, biomolecular electronics and nanotechnology. Due to its homogeneity, ease of preparation and ability to vary both the length of the chains and terminal functional groups, which provide a good versatility for immobilizing biocatalytically active compounds. Mixed SAM (designated as SAMmix), that combines the properties of alkanethiols with different carbon chain lengths or different functional groups, is one of the most attractive methods for promoting different arrangements on the electrode surface. SAMmix of long and short chains are reported to show better electron transfer rates than unicomponent SAMs due to the flexibility of redox species distribution at the SAM interface. Therefore, in this work, a horseradish peroxidase (HRP) biosensor was constructed using methods of physical adsorption and covalent binding for the enzyme immobilization on binary SAMs of 11-mercaptoundecanoic acid (MUA) and thiolactic acid (TLA) on gold surface for biosensor construction. The characterization of the biosensors was evaluated by voltammetry and electrochemical impedance spectroscopy. The immobilization method by covalent binding provided greater stability when compared to the adsorption method. For the preparation of the mixed self-assembled monolayers (SAMmix), the gold electrode was incubated in a solution containing different proportions of MUA and TLA and the best concentration ratio of these molecules was 0.5 and 1.0 mmol L-1, respectively. The results obtained by cyclic voltammetry and electrochemical impedance spectroscopy showed that the electrode surface dramatically changes when varying the concentration of these molecules. The rate of electron transfer was substantially affected because TLA enables an increase in the conductivity due to the formation of SAMmix "islands", whereas MUA although partially block the surface, it gives stability to the monolayer for the enzyme immobilization. The SAMmix obtained using 1 mmol L-1 of MUA had the lowest sensitivity due to the high concentration of the long-chain molecule. The HRP enzyme was efficiently immobilized on the SAMmix, designated as Au-SAMmix-HRP. The HRP enzyme was immobilized by the method of covalent attachment, in which the reaction occurs between the -COOH end groups of the SAM ligands with EDC (N-(3-9 dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride) and NHS (N-hydroxysuccinimide ester), which favors a more stable immobilization of the enzyme. This biosensor was applied for the determination of hydroquinone (HQ). It was observed that the presence of the enzyme on the monolayer increases the peak current compared to SAMmix, however best results were obtained for determination of HQ when a fixed concentration of H2O2 was added in solution. Therefore, the effect of the H2O2 concentration on the biosensor response was investigated. It was observed that as the concentration of H2O2 was increased, there was a linear increase in the biosensor current response from of 30.0 to 300.0 µmmol L-1 and 0.1 to 1.0 mmol L-1, with sensitivities of 10.35 μA/mol L-1 and and 2.38 μA/mol L-1, respectively. Lower sensitivity was observed at high concentrations of the substrate, probably due to the saturation of the active site of the enzyme, reducing its sensitivity. Michaelis–Menten kinetics, KM app of 0.4 mmol L-1 was obtained, indicating that the electrode architecture employed presents advantages for the fabrication of enzymatic biosensor. The resulting Au-SAMmix-HRP exhibited an excellent electrocatalytic activity toward the hydroquinone, which presents a wide linear range from 3.0 to 30.0 mmol L with a linear equation, I= 2209.69 [HQ] - 0.00534 with a correlation coefficient of 0.985. The standard deviation for the calibration curve was estimated at SB = 0.00101. This data was used to calculate the detection limit and quantification limit of the biosensor which were DL = 1.37 µmol L-1 and QL = 4.57 µmol L-1. The repeatability of the biosensor Au-SAMmix-HRP was performed on a series of 10 measurements (n = 10) in a solution containing HQ concentration of 0.3 mmol L-1 ([H2O2]= 0.3 mmol L-1) in PBS buffer by means of cyclic voltammetry, with 0.99% RSD. The biosensor stability was evaluated after 100 consecutive cycles by cyclic voltammetry in these same conditions and only a slight decay of the cathodic current peak was observed, at about 14 %. The long-term stability of the biosensor was also investigated and the biosensor was stable for at least 6 days without change in the response. After this period, the signal decreased 10% over a period of 15 days. Therefore, the biosensor Au-SAMmix-HRP displayed good reproducibility, sensitivity and stability for the determination of hydroquinone.
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