Comparative determination of some phytohormones in wild-type and genetically modified plants by gas chromatography-mass spectrometry and high-performance liquid chromatography-tandem mass spectrometry.

The analytical performances of two optimized analytical methodologies used for the determination of auxins, cytokinins, and abscisic acid in plant samples were critically compared. Phytohormones were extracted from Nicotiana glauca samples using a modified Bieleski solvent and determined both by gas chromatography-mass spectrometry (GC-MS), after derivatization with N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA), and by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) on the Bieleski extract without any further treatment. HPLC-MS/MS gave better results in terms of higher coefficients of determination of the calibration curves, higher and more reproducible recoveries, lower limits of detection, faster sample preparation, and higher sample throughput. Thus, two sets of N. glauca and N. langsdorffii samples, both wild-type and genetically modified by inserting the glucocorticoid receptor (GR) gene encoding for the rat glucocorticoid receptor, were first characterized by reverse transcriptase-polymerase chain reaction (RT-PCR) analysis and then analyzed by HPLC-MS/MS. Significant differences in the phytohormone content between the two sample sets were found and are very important in terms of understanding the mechanisms and effects on growth processes and the development of transgenic plants.

A DNA-based piezoelectric biosensor: strategies for coupling nucleic acids to piezoelectric devices.

A DNA-based piezoelectric biosensor has been here studied in terms of probe immobilisation and DNA sample pre-treatment. The biosensor is specific for the detection of the mecA gene of methicillin-resistant Staphylococcus aureus (MRSA). Methicillin-resistant S. aureus is responsible of several infections in humans, like pneumonia, meningitis and endocarditic. MRSA is also a major cause of hospital-acquired infections worldwide. The antibiotics resistance is conferred by the gene mecA, codifying for an anomalous protein. Two different immobilisation procedures of the probe specific for mecA gene are reported: immobilisation via streptavidin-biotin interaction and direct immobilisation of thiolated probes. After the study with synthetic oligonucleotides, the system has been applied to the analysis of bacterial DNA from MRSA, amplified by polymerase chain reaction. These samples were pre-treated with two different denaturation procedures and the performances of the sensor in the two cases were compared. The two immobilisation methods and denaturation protocols were here used to study the influences of these parameters on the performances of the sensor, applied here to the detection of the mecA gene. Better results in terms of sensitivity and reproducibility were obtained when using the biotinylated probe and the PCR-amplified samples treated by a denaturation procedures involving the use of high temperature and blocking oligonucleotides.