Biological investigation using a shear horizontal surface acoustic wave sensor: small "click generated" DNA hybridization detection.

We have used a 104 MHz lithium tantalate (LiTaO(3)) surface acoustic wave (SAW) sensor to investigate DNA probes grafting and their further hybridization with natural and click generated (Cg-DNA) oligonucleotides. Natural DNA targets of different strand lengths, tosyl-di(tri, tetra) thymidine and azido-di(tri, tetra) thymidine oligonucleotides were tested. In our case, and besides the follow-up of a 34mer DNA hybridization, we detected complementarity between natural DNA probes and azido-tetra-thymidine for the first time, whereas previous hybridization studies reported a minimal of 10-mer oligonucleotides recognition length. We also demonstrated that contrarily to natural DNA, the synthesized oligonucleotides present stable bonds with complementary DNA strands. Frequency responses of both grafting and hybridization have shown the same shape: an exponential decay with different time constants, (187±1)s and (68±19) s for grafting and hybridization respectively. We have also shown that recognition between DNA strands and tetranucleotide analogues is comparable to natural 34mer DNA bases and presents the same time constant within uncertainties.

On the use of electrospray ionization and desorption electrospray ionization mass spectrometry for bulk and surface polymer analysis.

Electrospray ionization (ESI) and desorption electrospray ionization (DESI) mass spectrometry were for the first time compared to investigate degradation products resulting from radiolysis of polyurethane (PUR) irradiated using accelerated electrons. First, DESI optimization was performed to obtain the best results in terms of sensitivity for surface polymer analysis. Then, a comparison was performed between DESI-MS and ESI-MS characterization of the degradation products of irradiated PUR with different doses up to 10 MGy. Similar results were obtained despite a lower sensitivity of DESI. Finally, DESI results obtained by direct surface analysis of irradiated PUR films as a function of depth were compared to bulk analysis results obtained by ESI. This comparison shows that degradation products of PUR are very comparable at the surface or within the polymer, confirming the radio-oxidation homogeneity.

Molecularly imprinted conducting polymer based electrochemical sensor for detection of atrazine.

An original electrochemical sensor based on molecularly imprinted conducting polymer (MICP) is developed, which enables the recognition of a small pesticide target molecule, atrazine. The conjugated MICP, poly(3,4-ethylenedioxythiophene-co-thiophene-acetic acid), has been electrochemically synthesized onto a platinum electrode following two steps: (i) polymerization of comonomers in the presence of atrazine, already associated to the acetic acid substituent through hydrogen bonding, and (ii) removal of atrazine from the resulting polymer, which leaves the acetic acid substituents open for association with atrazine. The obtained sensing MICP is highly specific towards newly added atrazine and the recognition can be quantitatively analyzed by the variation of the cyclic voltammogram of MICP. The developed sensor shows remarkable properties: selectivity towards triazinic family, large range of detection (10(-9) mol L(-1) to 1.5 x 10(-2) mol L(-1) in atrazine) and low detection threshold (10(-7) mol L(-1)).

Electroacoustic miniaturized DNA-biosensor.

A micrometer-sized electroacoustic DNA-biosensor was developed. The device included a thin semi-crystalline polyethylene terephthalate (PET) dielectric layer with two Ag microband electrodes on one side and a DNA thiol-labeled monolayer adsorbed on a gold surface on the other. A resonance wave was observed at 29 MHz with a network analyzer, upon AC voltage application between the two Ag electrodes, corresponding to electromechanical coupling induced by molecular dipoles of the PET polymer chain in the dielectric layer. It was found that the device size and geometry were well adapted to detect DNA hybridization, by measuring the capacity of the resonance response evolution: hybridization induced polarization of the dielectric material that affected the electromechanical coupling established in the dielectric layer. The 0.2 mm(2) sensor sensitive area allows detection in small volumes and still has higher detection levels for bioanalytical applications, the non-contact configuration adopted avoids electric faradic reactions that may damage biosensor sensitive layers, and finally, PET is a costless raw material, easy to process and well adapted for large scale production. The well-balanced technological and economic advantages of this kind of device make it a good candidate for biochip integration.

5Alpha-androstane-3beta,7alpha,17beta-triol and 5alpha-androstane-3beta,7beta,17beta-triol as substrates for the human 11beta-hydroxysteroid dehydrogenase type 1.

Several studies have shown that the native 7alpha-hydroxy-dehydroepiandrosterone (7alpha-hydroxy-DHEA) is a substrate for the human 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) which converts the 7alpha- into the 7beta-epimer through an oxido-reduction process. Research on the 11beta-HSD1 has investigated its function and structure through using native glucocorticoid substrates and known inhibitors. Other steroid substrates are also of interest. Among testosterone metabolites, 5alpha-androstane-3beta,17beta-diol (Adiol) is a substrate for the cytochrome P450 7B1 which produces 5alpha-androstane-3beta,7alpha,17beta-triol (7alpha-Adiol). This steroid may be a substrate for the 11beta-HSD1. We used recombinant yeast-expressed 11beta-HSD1 with NADP(H)-regenerating systems for examining the products obtained after incubation with 7alpha-Adiol, 7beta-Adiol or 7-oxo-Adiol. Oxidative conditions for the 11beta-HSD1 provided no trace of 7-oxo-Adiol but the inter-conversion of 7alpha- and 7beta-hydroxy-Adiol with V(max)/K(M) (pmol min(-1) microg(-1)/microM) values of 2 and 0.5, respectively. This state was maintained under reductive conditions. The use of a 7-oxo-Adiol substrate under reductive conditions led to the production of both 7alpha- and 7beta-hydroxy-Adiol with V(max)/K(M) values of 3.43 and 0.22, respectively. These findings support the hypothesis that the oxido-reductase and epimerase activities of 11beta-HSD1 depend on the positioning of the steroid substrates within the active site and may provide insight into its fine structure and mechanism of action.

7alpha- and 7beta-hydroxy-epiandrosterone as substrates and inhibitors for the human 11beta-hydroxysteroid dehydrogenase type 1.

The human 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) catalyzes both the NADP(H)-dependent oxido-reduction of cortisol and cortisone and the inter-conversion of 7alpha- and 7beta-hydroxy-dehydroepiandrosterone (DHEA) through a 7-oxo-DHEA intermediate. As shown with human liver and intestine fractions, 7alpha-hydroxy-epiandrosterone (7alpha-hydroxy-EpiA) and 7beta-hydroxy-EpiA were readily inter-converted with no evidence for a 7-oxo-EpiA intermediate. Whether this inter-conversion resulted from action of the 11beta-HSD1 or from an unknown epimerase is unresolved. Furthermore, whether these steroids could inhibit the cortisol-cortisone oxido-reduction remains a question. The recombinant human 11beta-HSD1 was used to test these questions. NADP(+) supplementation only provided the production of 7beta-hydroxy-EpiA out of 7alpha-hydroxy-EpiA with a V(max)/K(M) ratio at 0.1. With NADPH supplementation, both 7alpha-hydroxy-EpiA and 7beta-hydroxy-EpiA were formed in low amounts from 7beta-hydroxy-EpiA and 7alpha-hydroxy-EpiA, respectively. These inter-conversions occurred without a trace of the putative 7-oxo-EpiA intermediate. In contrast, the 7-oxo-EpiA substrate was efficiently reduced into 7alpha-hydroxy-EpiA and 7beta-hydroxy-EpiA, with V(max)/K(M) ratios of 23.6 and 5.8, respectively. Competitive and mixed type inhibitions of the 11beta-HSD1-mediated cortisol oxidation were exerted by 7alpha-hydroxy-EpiA and 7beta-hydroxy-EpiA, respectively. The 11beta-HSD1-mediated cortisone reduction was inhibited in a competitive manner by 7-oxo-EpiA. These findings suggest that the active site of the human 11beta-HSD1 may carry out directly the epimeric transformation of 7-hydroxylated EpiA substrates. The low amounts of these steroids in human do not support a physiological importance for modulation of the glucocorticoid status in tissues.