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This paper presents a simple and effective flow-through electrochemical biosensor, consisting of Trametes versicolor laccase (Lac)-based mini-reactor and a tubular detector of silver solid amalgam (TD-AgSA), capable of rapid and selective detection of phenolic compounds. Amperometric detection relies on the reduction of the quinone molecule (formed during the enzymatic reaction in a mini-reactor) on TD-AgSA at -50 mV vs SCE. Since different enzyme immobilization techniques may contribute to differing biosensor performances, four covalent strategies for Lac attachment were compared: (i) through glutaraldehyde to supports -NH

The novel and highly sensitive methods for individual determination of two food azo dyes Amaranth (AM, E 123) and Allura Red AC (AR, E 129) in the commercial beverages were successfully developed on two types of silver solid amalgam electrodes, namely mercury meniscus modified (m-AgSAE) and liquid mercury free polished (p-AgSAE) amalgam electrodes, using differential pulse adsorptive stripping voltammetry (DP-AdSV) and direct current adsorptive stripping voltammetry (DC-AdSV) for the first time. In addition, the reduction processes of AM and AR on amalgam electrodes were compared with the processes on hanging mercury drop electrode. The influence of pH, accumulation potential, and accumulation time on the signal enhancement of AM and AR were investigated. The number of electrons and the number of protons participating in the rate-determining step of the reduction process for azo dyes were calculated. Due to the significantly increasing reduction peak currents of AM and AR on m-AgSAE and p-AgSAE caused by their adsorption at the electrode surface, the amalgam electrodes exhibit the wide linear ranges and good sensitivity to the determination of AM and AR. For example, the limits of detection were found to be 2.1 × 10−9 mol L−1 for AM and 3.4 × 10−9 mol L−1 for AR on m-AgSAE using DC-AdSV. Moreover, the amalgam electrodes showed good repeatability (RSD lower than 5.0% for 5 × 10−8 mol L−1 of tested azo dyes) and stability and it was confirmed that these electrodes are useful tools to azo dye monitoring in a food safety control field.

Silver amalgam screen-printed electrodes (AgA-SPEs) were designed, prepared and tested for the first time as perspective representatives among SPEs that allow measuring at high negative potentials. For this purpose, the precise electrochemical (coulometric) preparation procedure for mercury deposition at the silver working electrode surface of the commercial silver-SPE was developed and a universal method of preparing amalgam electrodes from different metal-SPEs was described. An optimal electrolyzer construction for the mercury deposition, electrolyte composition, electrolysis potential, and a way to activate the electrode surface for its uniform coverage by mercury were proposed. The effect of the mercury content in amalgam on the potential of hydrogen evolution (PHE) at AgA-SPEs was investigated. The maximum PHE value (−1979 ± 4 mV; RSD = 0.17 %; N = 5) was found on AgA-SPE with 50 % (w/w) of Hg content in 0.1 mol L−1 NaOH, which is more negative than in the case of the original silver-SPE for 387 mV (−1592 ± 12 mV; RSD = 0.69 %; N = 5). Determination of the iodine content in iodinated table salt was used for the AgA-SPEs practical testing. Measurement parameters were optimized for three electrochemical cell arrangements: i) the electrodes area of AgA-SPE was covered by 50 μL of the sample solution and differential pulse voltammetric (DPV) measurements were carried out; ii) 3-electrode batch arrangement with only working electrode from AgA-SPE connected with an analyzer for DPV-measurements; iii) amperometric measurements in a flow system. The results of iodine analysis by all three methods were consistent with the manufacturer declared values.

This is the first report of a novel strategy for uric acid (UA) determination via newly developed highly stable flow biosensors based on different enzymatic mini-reactors in combination with a four-electron reduction of the consumed oxygen. The design of the biosensors provides a spatial separation of the detection (working electrode) and biorecognition (uricase-based mini-reactor) part. To prepare the most efficient enzymatic mini-reactor, a comparative study focused on three different techniques of the covalent uricase immobilization at two kinds of mesoporous silica powders (SBA−15 and MCM−41) and the physical enzyme adsorption at four types of carbonaceous powders (glassy carbon, graphite, Starbon® 300, and Starbon® 800) has been performed. All investigations were carried out for two kinds of uricase: from Candida sp. (UOx(c)) and from Bacillus fastidiosus (UOx(b)) to estimate the influence of uricase source. The flow-through cell with tubular detector of silver solid amalgam was used for amperometric monitoring of four-electron oxygen reduction at the highly negative potential (−1100 mV vs. SCE). It was found that the developed UA biosensors responses are strongly dependent on the enzyme immobilization strategy. UOx(c) bounded to glassy carbon by physical adsorption has provided good results in terms of sensitivity, stability, and repeatability for UA determination. The biosensor with the mini-reactor based on the covalent immobilization of UOx(b) at glutaraldehyde activated NH2 functionalized MCM–41 has shown extremely high stability (more than 1 year) and reusability (at least 600 measurements) The biosensor’s practical applicability has been tested by successful determination of UA in human urine.

A stable and simple amperometric biosensor based on the separated bienzyme (acetylcholinesterase (AChE) – choline oxidase (ChOx)) part has been successfully developed for the determination of acetylcholine (ACh) using flow injection analysis for the first time. The bienzyme part consists of two consecutively connected enzymatic mini-reactors filled by mesoporous silica powders (SBA − 15) covalently covered by AChE and ChOx, respectively. The oxygen consumption as the result of two sequential enzymatic reactions was monitored amperometrically. The wall-jet cell with working silver solid amalgam electrode covered by mercury film was used for four-electron oxygen reduction at the highly negative potential. The experimental parameters including the amounts of immobilized enzymes, pH of the carrier solution, the detection potential, flow rate, and injection volume of ACh were optimized. The detection limit was found to be 4.1 μmol L−1. The interfering effect of 1.0 mmol L−1 glucose, 0.1 mmol L−1 uric acid, and 0.1 mmol L−1 ascorbic acid to 0.1 mmol L−1 ACh was 0.6%, 2.2%, and 2.8%, respectively. The developed amperometric ACh biosensor showed 59.8% of its initial response after >400 measurements over 100 days.