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An important factor affecting the performance of electrochemical genosensors is the immobilization strategy used to anchor the capture probes (CP) on the electrode surface. One of the most common immobilization systems involves the formation of self‐assembled monolayers of thiolated probes on gold surfaces, mainly due to its simplicity and effectiveness. Recent studies have shown that in two‐component thiolated DNA and mercaptohexanol (MCH) monolayers, there is incomplete backfilling that leads to defective surface blockage, producing unspecific background signal. In order to solve these problems, ternary monolayers have been proposed, in which besides MCH as primary diluent, a second diluent is added to enhance the hybridization efficiency and anti‐fouling properties of the surface. Herein, we report a thorough evaluation of different surface chemistries using two different sensors, one for the detection of a peanut allergen encoding sequence and the other one targeting a gluten‐encoding sequence. We assessed the relationship between CP length and the chemical characteristics of the diluents. Factors such as concentration, nature of the functional groups and chain length of the second diluent were evaluated. We found that length of diluents affects primarily the hybridization efficiency, while the type of functional groups, e.g. hydroxyl, carboxyl or a second thiol group, influences the degree of surface coverage. Results diverged between the two sensors evaluated.

A new ultra HPLC (UHPLC) method using both MS and fluorescence detection (FD) was developed for the determination of five fluoroquinolones in wastewaters. Systematic method development approach was compared with a conventional one. During the systematic approach, a possibility of automatic switching among four independent analytical columns of different chemistries has been used. Acidic as well as basic pH using ACN and methanol as organic modifiers was tested. The best separation of fluoroquinolones was obtained on phenyl analytical column at pH 10.5, which is a completely novel approach for separation of fluoroquinolones. Further, a new SPE procedure was developed for the sample preparation using basic pH as well. The sensitivity and selectivity of FD and MS detection were compared. FD at basic pH 10.5 demonstrated lower sensitivity than at acidic pH, which is conventionally performed. At basic pH, UHPLC-MS/MS was found about two orders of magnitude more sensitive than FD. Both methods were validated and subsequently UHPLC-FD method was used for the evaluation of stability of fluoroquinolones. UHPLC-MS/MS method was used for the analysis of wastewater samples. Norfloxacin and ciprofloxacin were detected in samples of influent and effluent from wastewater treatment plant. Ofloxacin was detected only in influent from wastewater treatment plant.

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Biomarkers, 25-hydroxyvitamin D3 and 25-hydroxyvitamin D2 , are important indicators of the vitamin D general status and are monitored in several pathophysiological disorders, such as osteoporosis, diabetes, heart disease, etc. A novel ultra-HPLC with MS/MS methodology for the analysis of 25-hydroxyvitamin D derivatives coupled with a very simple and highly rapid sample preparation step was developed. Analytical parameters obtained showed linearity (R(2) ) above 0.999 for both vitamins with accuracies between 95.8 and 102%. The LODs were as low as 0.22 and 0.67 nmol/L for 25-hydroxyvitamin D3 and 25-hydroxyvitamin D2 , respectively. Intra-assay precision (%RSD) was lower than 4.5%, and inter-assay precision (%RSD) was lower than 6.5%. The feasibility of the developed methodology to be applied in clinical routine analysis has been proved by its application in blood samples from non-agenarian patients, patients with familial hypercholesterolemia and patients suffering from age-related macular degeneration.