Mixed Self-Assembled Monolayers of Mercaptoundecanoic Acid and Thiolactic Acid for the Construction of an Enzymatic Biosensor for Hydroquinone Determination
Authors: Pessoa Christiana Andrade , Santos Cleverson Siqueira , Mossanha Rosana 
Published: February 2020

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.