Current Analytical Chemistry - Volume 12, Issue 1, 2016

This review introduces the concept of enzyme biosensors. As the major feature of an enzyme is its selectivity and specificity, we briefly highlighted the principles of enzyme production and enzyme classification. Moreover, enzyme immobilization methods, including physical adsorption and entrapment techniques with special attention on cross-linking and covalent binding are discussed followed by different electrochemical methods used in the biosensors fabrication. Furthermore, a comprehensive discussion about horseradish peroxidase (HRP) as a model enzyme is presented with investigation of some modified enzyme electrodes such as screen-printed and carbon paste electrodes. Some approaches to enhance biosensors performances and their applications are also highlighted.

Mercury is one of the most toxic elements that may be found in natural water bodies and can negatively affect living organisms, human health as well as drinking water quality. In addition, water is the main transportation and transformation pathway of mercury into the environment and living organisms. Therefore, specific strategies have been developed to reduce mercury releases and to remediate mercury contaminated areas. Mercury analysis in water is of great importance, in order to monitor water quality and associated environmental stresses, thus a considerable number of new publications appear every year. Many of these publications deal with the determination of mercury’s chemical form; others with the development of new analytical techniques, while there are also studies that focus on areas of particular environmental interest. Classic analytical chemistry methods for mercury analysis and speciation in water, namely CVAAS and ICP-MS, have achieved sub-ppt detection limits; while even though, atomic and nuclear analytical methods are promising, especially XRF which has achieved sub-ppb detection limits, they are scarcely used. Other promising analytical methods include CVAFS, FIA-AFS, CVAES, Raman probe, Raman scattering and anodic stripping voltammetry. This paper is a review of the state of the art of mercury analysis and speciation in water (including seawater) with emphasis given on the last 5 years. It highlights the novelties of the referenced papers; the applied analytical techniques, mercury speciation, preconcentration procedures, selectivity, achieved detection limits, the type of the analyzed waters; systems for on-site analysis and mercury analysis in real water samples.

An overview on the progress in the organophosphorus hydrolase based electrochemical sensors design and application for direct organophosphorus pesticides quantification is presented in this work. The key analytical performances of the enzyme and of the bacterial potentiometric and amperometric organophosphorus hydrolase based sensors, as well as their advantages and deficiencies are commented in detail.

This paper presents a new disposable electrochemical biosensor for the analysis of Haptoglobin (Hp) which shows many important functionalities such as antioxidant capabilities, antiinflammatory activities and regulation of the immune system. Aminopropyltriethoxysilane (APTES) was self-assembled onto an indium tin oxide (ITO) substrate to fabricate a disposable biosensor. Anti- Hp antibodies were immobilized onto the modified ITO surface covalently. The optimization of the construction parameters and the analytical performance of the biosensor were examined and compared. The methods, cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), and scanning electron microscopy (EIS) were operated for detailed characterization of the different surfaces of the biosensor. Kramers-Kronig Transform was also applied to raw EIS data for checking the verification of obtained experimental EIS data. Moreover, a useful method, single frequency impedance (SFI), was successfully operated to characterize the binding of Hp to the electrode surface modified with anti-Hp. At last, the presented biosensor was applied to the analysis of artificial serum samples, which were spiked with Hp.

In this work, an enzymatic biofuel cell (BFC) was developed by combining nanomaterial modified and plain glassy carbon paste electrodes (GCPE). Because of the composite nature of GCPE, very practical and economic bio-anode and bio-cathode electrodes were formed. Bio-anode electrode was obtained by modification of composite GCPE with glucose oxidase (GOx) and gold nanoparticle (Au np). The effects of various nanomaterials like, aluminum titanate, manganese (IV) oxide nanoparticle and Fe3O4-Au bimetallic nanostructure on GCPE performance were also searched. pbenzoquinone mediator was used for the electron transfer between enzyme redox center and bio-anode electrode where glucose analyte was used as substrate. Optimization of experimental parameters (e.g. substrate concentration, mediator concentration, enzyme amount, temperature and pH) of bio-anode were carried out by linear sweep voltammetry. Laccase (Lac) modified plain GCPE was used as bio-cathode electrode. Then Au np modified GCPE and Lac modified GCPE were combined in a single cell and membraneless biofuel cell was obtained. The power density of single cell BFC was found as 6.7 μW cm-2 at a cell potential of 92 mV and current density of this system was obtained as 154.8 μA cm-2 in phosphate buffer solution.

Hydrophilic interaction liquid chromatography (HILIC) has been used to investigate the retention characteristics of quercetin and its glycosides typically present in plant samples. Two stationary phases – bare silica and with zwitterionic sulfoalkylbetaine groups on silica surface (ZIC) were used. The effect of acetonitrile content and the presence of salt in the mobile phase were studied. The analyte retention factors were observed to be inversely proportional to the water content in the mobile phase on ZIC column, which is characteristic of typical hydrophilic partitioning mechanism. Using the bare silica column, U-shaped curves were obtained, which shows more or less apparent dual retention mechanism. The potential of HILIC separation was demonstrated for the analysis of bioactive compounds in the aqueous extracts of popular medicinal herbs St John`s wort (Hypericum perforatum) and lemon balm (Melissa officials L).

The use of immobilized enzymes in proteomics studies has increased over the last decade not only due to their ease of use in fluidic systems but also because autoproteolysis is suppressed allowing use of high ratios of enzyme-to-substrate for rapid digestion. Chymotrypsin immobilization by crosslinking with excess aqueous glutaraldehyde (GA) has been used to produce a gel-like agglomerated pellet of high enzyme loading capable of digesting protein. The simple procedure involved dropwise addition of 80 μL GA (2.5% in water) to 80 μL chymotrypsin in pH 6.4 phosphate buffer, reaction for 2 h at room temperature without stirring, washing with buffer and NaCl, then deactivation of any free aldehyde groups by reaction with 200 mM glycine for 3 h. The enzyme was ready for use after washing with buffer and water. The efficiency of the immobilized enzyme for protein digestion was followed by capillary electrophoretic (CE) peptide mapping with absorbance detection at 200 nm. A study comparing preparation methods showed that making a small immobilized enzyme pellet leads to better digestions (i.e., peptide maps) compared to using a five-fold larger pellet or a part of the latter. When three different buffers for the chymotrypsin crosslinking reaction were compared, sodium phosphate at pH 6.4 showed better batch-to-batch reproducibility and activity of the immobilized enzyme: overall peak area RSD was 27% versus 39% and 43% for the other pHs (with 11% of this variability due to the CE method) and up to 3 times more peptide peaks were detected. The immobilized chymotrypsin pellet could be used for two consecutive digestions of denatured BSA and was able to partially digest native BSA.

Melamine was quantified in liquid milk without performing the usual liquid-liquid or solidphase extraction steps. Following a simple clean up procedure (extraction by acetonitrile/water mixture, centrifugation and filtration by 0.45 μm membrane), melamine was accurately quantified in fullfat liquid milk down to 0.057 mg kg-1 using second order multivariate calibration. Second order data was created from absorbance-pH measurements (batch-experiment method) taking the advantage of acid/base properties of melamine. The study was carried out over the spectral domain 200-250 nm and pH 2.0-8.0. Although the number of pH points was lower than the spectral points (4 vs. 51), the data was satisfactorily analyzed by second order multivariate calibration methods. Quantification of melamine in spiked milk samples (0.5-2.0 mg kg-1) is carried out using unfolded partial least squares-residual bilinearization U-PLS/RBL, parallel factor analysis PARAFAC and multivariate curve resolution-alternating least squares MCR-ALS. U-PLS/RBL outperformed other methods with mean recoveries and relative errors of prediction of 100.1 (2.3), 99.3 (6.2), and 93.8 (16.4) for U-PLS/RBL, MCR-ALS, and PARAFAC, respectively. The reason for success of U-PLS/RBL is attributed to its effectiveness for resolving melamine-signal (the basic form) from the signal of milk-extract. PARAFAC and MCR-ALS showed a modest performance and this is attributed to the rank-deficiency in the collected matrices (for test samples), linear dependency (in pH-dimension), and intense spectral-overlapping in the spectral mode. The performance of the proposed method is critically compared with other published methods.