New nanostructured electrochemical biosensors based on three-dimensional (3-mercaptopropyl)-trimethoxysilane network.

The design and characterization of a new nanostructured organic-inorganic hybrid material and its application to L-lactic acid determination are described. This material is based on the integration of the enzyme lactate oxidase (LOx) and gold nanoparticles (AuNPs) into a sol-gel 3D polymeric network derived from (3-mercaptopropyl)-trimethoxysilane (MPTS) previously formed onto a gold surface. MPTS presents the advantage of forming a 3D polymeric network containing a large number of thiol tail groups distributed throughout its structure that enable both its anchoring onto gold surfaces and the AuNPs incorporation. Moreover, this matrix provides a biocompatible environment that preserves the catalytic activity of LOx after its immobilization and allows the incorporation of a high amount of enzyme, which is expected to improve the sensitivity of the final biosensing device. Characterization of the designed biosensing platform was performed using quartz crystal microbalance (QCM), scanning electron microscopy (SEM) and atomic force microscopy (AFM) techniques. From the conjunction of these techniques, information about (i) the kinetic of LOx adsorption process in real time, (ii) the amount of LOx incorporated into the network, and (iii) the morphological characteristics at the nanometre level of the designed biosensing material was obtained. This information is very useful on the development of successful biosensing devices. Finally, the response of the biosensor to L-lactic acid was evaluated. The biosensor responds linearly to L-lactic acid in the range of 50 µM to 0.25 mM, with a sensitivity of 3.4 µA mM(-1) and a detection limit of 4.0 µM.

Carbon nanotubes/pentacyaneferrate-modified chitosan nanocomposites platforms for reagentless glucose biosensing.

The design, characterization and applicability of a nanostructured biosensor platform are described. The biosensor is developed through the immobilization of three components: a polymeric chitosan network previously modified with a redox mediator (denoted as PCF-Pyr-Ch), an enzyme (glucose oxidase, chosen as a model) and carbon nanotubes onto a solid glassy carbon electrode (C). In order to assess the influence of the nanomaterial in the performance of the resulting analytical device, a second biosensor, free of carbon nanotubes, is developed. The characterization of both biosensing platforms was performed in aqueous phosphate buffer solutions using atomic force microscopy technique. In the presence of glucose, both systems exhibit a clear electrocatalytic activity, and glucose could be amperometrically determined at +0.35 V versus Ag/AgCl. The performance of both biosensors was evaluated in terms of sensitivity, detection limit and linear response range. Finally, the enhancement of the analytical response induced by the presence of carbon nanotubes was evaluated.

Disposable sensors for rapid screening of mutated genes.

A screening method for rapid detection of gene mutations directly in polymerase chain reaction (PCR) of genomic DNA is described. The method involves the development of a disposable screen-printed gold electrode modified with a thiolated capture probe directly obtained from denaturated PCR genomic DNA, which recognizes (by hybridization) its fully complementary sequence (wild type), giving a signal, whereas no signal is obtained for single-mismatched target (mutant). The detection of the hybridization event is achieved by changes in the metal redox center electroactivity of the complex [Ru(NH(3))(5) L](2+), where L is [3-(2-phenanthren-9-yl-vinyl)-pyridine], at -0.200 V. This complex binds to double-stranded DNA in a very selective form. The method allows discrimination between the wild type and the mutant of gene MRP3 directly in large PCR amplicons extracted from blood cells, without the need to use either synthetic probes or labeled targets. The mutation involves the presence of a single-nucleotide polymorphism (SNP) at base 54 of a 145-base-pair sequence from exon 21 of gene MRP3. Since the presence of this SNP might lead to a variety of hereditary liver disorders, its identification in a rapid and easy form may provide novel therapeutic targets for the future. The screening method proposed has excellent signal reproducibility, with a relative standard deviation of 10%. In addition, with the method developed as little as 6.6 ng/muL PCR product can be detected.

One-step covalent microcontact printing approach to produce patterns of lactate oxidase.

A comparative study of three different strategies to pattern lactate oxidase (LOx) onto bare gold substrates by microcontact printing (muCP) is presented. The quality of the resulting patterns in terms of homogeneity, compactness and stability has been evaluated by atomic force microscopy in both air and aqueous conditions. The following approaches have been tested: (i) LOx was directly stamped to a bare gold surface; (ii) LOx was previously covalently bonded to a thiolated molecule, dithiodipropionic acid di(N-succinimidyl ester) (DTSP), and this conjugate (LOx/DTSP) was transferred from an elastomeric stamp to a bare gold substrate; (iii) formation of a LOx/DTSP micropattern on a bare gold surface (as described in approach ii) was followed by exposure to a solution containing hexadecylmercaptane (HDM). In all cases, the catalytic activity of the final LOx patterns has been assessed by electrochemical measurements. From comparison of the three strategies, it can be concluded that the third one gives rise to LOx patterns that present a high stability and compactness, also offering the advantage of reducing the number of microcontact printing steps to one.

Dual-stage DNA sensing: recognition and detection.

Selective polynucleotide recognition and detection based on a dual-stage method are described. The method involves the development of a recognition surface based on gold nanoparticles modified with a thiolated capture probe able to hybridize with its complementary sequence (target). After hybridization, this sensing surface is removed from the solution and electrodeposited on an electrode surface. The detection of the hybridization event is achieved using the complex [Ru(NH(3))(5)L](2+), were L is [3-(2-phenanthren-9-yl-vinyl)-pyridine], as electrochemical indicator. This complex binds to double strand DNA more efficiently than to single stranded DNA. The advantage of this dual-stage DNA sensing method is the high selectivity derived from the separation of the hybridization event (occurring on one surface) from the detection step (on a different surface), enabling the analysis of long target DNAs, which is usually the case in real DNA sequence analysis. In addition, this approach not only quantifies pmol of a complementary target sequence but also is sensitive to the presence of a single mismatch and its position in the sequence.

Architectures based on the use of gold nanoparticles and ruthenium complexes as a new route to improve genosensor sensitivity.

The preparation of DNA-sensing architectures based on gold nanoparticles (Au-NPs) in conjunction with an "in situ" prepared ruthenium complex as a new route to improve the analytical properties of genosensors is described. In the development of these architectures several strategies to obtain Au-NPs modified gold electrodes (Au-NP/Au) have been essayed, in particular covalent binding and electrochemical deposition from a solution containing Au-NPs previously synthesized. UV-vis absorption measurements in conjunction with transmission electron microscope (TEM) images reveal that the synthesized Au-NPs are stable for at least 4 weeks and have a narrow size distribution. Atomic force microscopy (AFM) was employed to characterize the morphology and to estimate the Au-NPs surface coverage of the modified gold electrodes obtained following the different modification strategies. In order to assess the utility of these architectures as DNA-sensing devices, a thiolated capture probe sequence from Helicobacter pylori was immobilized onto the as-prepared surface. This sequence was chosen as a case of study within the framework of developing approaches of wide applicability. The hybridization event is detected using a water-soluble pentaamin ruthenium [3-(2-phenanthren-9-yl-vinyl)-pyridine] complex (Ru(NH(3))(5)L) prepared "in situ". This complex, due to its intercalative character, is able to bind to double stranded DNA more efficiently than to single stranded DNA. In addition, the metal provides with a redox center that can be used as an electrochemical indicator. On the basis of this strategy, complementary target sequences of H. pylori have been detected over the range of 40-800 pmol with a detection limit of 25+/-2 pmol.

Diamond nanoparticles as a way to improve electron transfer in sol-gel L-lactate biosensing platforms.

In the present work, we have included for the first time diamond nanoparticles (DNPs) in a sol-gel matrix derived from (3-mercaptopropyl)-trimethoxysilane (MPTS) in order to improve electron transfer in a lactate oxidase (LOx) based electrochemical biosensing platform. Firstly, an exhaustive AFM study, including topographical, surface potential (KFM) and capacitance gradient (CG) measurements, of each step involved in the biosensing platform development was performed. The platform is based on gold electrodes (Au) modified with the sol-gel matrix (Au/MPTS) in which diamond nanoparticles (Au/MPTS/DNPs) and lactate oxidase (Au/MPTS/DNPs/LOx) have been included. For the sake of comparison, we have also characterized a gold electrode directly modified with DNPs (Au/DNPs). Secondly, the electrochemical behavior of a redox mediator (hydroxymethyl-ferrocene, HMF) was evaluated at the platforms mentioned above. The response of Au/MPTS/DNPs/LOx towards lactate was obtained. A linear concentration range from 0.053 mM to 1.6 mM, a sensitivity of 2.6 µA mM(-1) and a detection limit of 16 µM were obtained. These analytical properties are comparable to other biosensors, presenting also as advantages that DNPs are inexpensive, environment-friendly and easy-handled nanomaterials. Finally, the developed biosensor was applied for lactate determination in wine samples.

Electrocatalytic processes promoted by diamond nanoparticles in enzymatic biosensing devices.

We have developed a biosensing platform for lactate determination based on gold electrodes modified with diamond nanoparticles of 4nm of nominal diameter, employing the enzyme lactate oxidase and (hydroxymethyl)ferrocene (HMF) as redox mediator in solution. This system displays a response towards lactate that is completely different to those typically observed for lactate biosensors based on other nanomaterials, such as graphene, carbon nanotubes, gold nanoparticles or even diamond nanoparticles of greater size. We have observed by cyclic voltammetry that, under certain experimental conditions, an irreversible wave (E(0)=+0.15V) appears concomitantly with the typical Fe(II)/Fe(III) peaks (E(0)=+0.30V) of HMF. In this case, the biosensor response to lactate shows simultaneous electrocatalytic peaks at +0.15V and +0.30V, indicating the concurrence of different feedback mechanisms. The achievement of a biosensor response to lactate at +0.15V is very convenient in order to avoid potential interferences. The developed biosensor presents a linear concentration range from 0.02mM to 1.2mM, a sensitivity of 6.1µAmM(-1), a detection limit of 5.3µM and excellent stability. These analytical properties compare well with those obtained for other lactate-based biosensors that also include nanomaterials and employ HMF as redox mediator.

PTEN tumour suppressor is linked to the cell cycle control through the retinoblastoma protein.

The tumour suppressor PTEN, also named MMAC1 or TEP1, is associated with a number of malignancies in human populations. This protein has a dual protein phosphatase activity, being also capable to dephosphorylate phosphatidylinositol 3,4,5 triphosphate. We have studied the mechanism of growth suppression attributable to PTEN. We observed that PTEN overexpression inhibits cell growth in a variety of normal and transformed, human and murine cells. Bromodeoxyuridine (BrdU) incorporation and TUNEL labelling experiments in transiently transfected cells demonstrate that this inhibition is due to a cell cycle arrest rather than induction of apoptosis. Given that PTEN is unable to cause cell growth arrest in retinoblastoma (Rb)-deficient cell lines, we have explored the possible requirement for pRb in the PTEN-induced inhibition of cell proliferation. We found that the co-expression of SV40 antigen, but not a mutant form (which binds exclusively to p53), and cyclin D1/cdk4 are able to overcome the PTEN-mediated growth suppression. In addition, the reintroduction of a functional pRb, but not its relatives p107 or p130, in Rb-deficient cells restores the sensitivity to PTEN-induced arrest. Finally, the hyperphosphorylation of transfected pRb is inhibited by PTEN co-expression and restored by PI-3K co-expression. Accordingly, PTEN gene is mostly expressed, in parallel to Akt, in mid-late G1 phase during cell cycle progression prior to pRb hyperphosphorylation. Finally, we have studied the signal transduction pathways modulated by PTEN expression. We found that PTEN-induced growth arrest can be rescued by the co-expression of active PI-3K and downstream effectors such as Akt or PDK1, and also certain small GTPases such as Rac1 and Cdc42, but not by active Ha-ras, raf or RhoA. Collectively, our data link the tumour suppressor activities of PTEN to the machinery controlling cell cycle through the modulation of signalling molecules whose final target is the functional inactivation of the retinoblastoma gene product.

Diamond nanoparticles based biosensors for efficient glucose and lactate determination.

In this work, we report the modification of a gold electrode with undoped diamond nanoparticles (DNPs) and its applicability to the fabrication of electrochemical biosensing platforms. DNPs were immobilized onto a gold electrode by direct adsorption and the electrochemical behavior of the resulting DNPs/Au platform was studied. Four well-defined peaks were observed corresponding to the DNPs oxidation/reduction at the underlying gold electrode, which demonstrate that, although undoped DNPs have an insulating character, they show electrochemical activity as a consequence of the presence of different functionalities with unsaturated bonding on their surface. In order to develop a DNPs-based biosensing platform, we have selected glucose oxidase (GOx), as a model enzyme. We have performed an exhaustive study of the different steps involved in the biosensing platform preparation (DNPs/Au and GOx/DNPs/Au systems) by atomic force microscopy (AFM), field emission scanning electron microscopy (FE-SEM) and cyclic voltammetry (CV). The glucose biosensor shows a good electrocatalytic response in the presence of (hydroxymethyl)ferrocene as redox mediator. Once the suitability of the prototype system to determine glucose was verified, in a second step, we prepared a similar biosensor, but employing the enzyme lactate oxidase (LOx/DNPs/Au). As far as we know, this is the first electrochemical biosensor for lactate determination that includes DNPs as nanomaterial. A linear concentration range from 0.05 mM to 0.7 mM, a sensitivity of 4.0 µA mM(-1) and a detection limit of 15 µM were obtained.

Cyclosporine increases renal parathyroid hormone-related protein expression in vivo in the rat.

BACKGROUND: Clinical use of cyclosporine (CsA) is limited by its known nephrotoxicity. Parathyroid hormone (PTH)-related protein (PTHrP) increases after acute renal ischemia and stimulates proliferation of renal cells in culture. Herein, we have examined whether the renal expression of PTHrP and its PTH/PTHrP receptor is affected by chronic CsA nephrotoxicity. METHODS: Rats were randomly assigned to receive daily intramuscular injections of either CsA (25 mg/kg) or the same volume of the vehicle olive oil (control) for 3 weeks. At this time interval, under ether anesthesia, rat blood and kidneys were obtained for analytical determinations, and total RNA isolation or immunohistochemistry, respectively. RESULTS: Serum urea was 11+/-2 and 6+/-1 mmol/L (P < 0.01) in CsA-treated and control rats, respectively. We found that PTH/PTHrP receptor mRNA was unchanged, but PTHrP mRNA, and also transforming growth factor-beta1 mRNA expression as positive control, was about twofold increased in the kidney of CsA-treated rats. This was accompanied by increased PTHrP immunostaining in renal cortical tubules, associated with tubule vacuolation. CONCLUSION: This study demonstrates an up-regulation of PTHrP, associated with chronic CsA-induced nephrotoxicity. Our findings support a role for PTHrP in the CsA-injured kidney.

No association between particular DRD3 and DAT gene polymorphisms and manic-depressive illness in a Spanish sample.

We have carried out an association study of a polymorphism in the 3'UTR of the dopamine transporter gene (DAT) and a polymorphism in the coding region of the D3 receptor gene (DRD3) in Spanish patients with manic depression and in controls. No significant differences in allelic and genotypic frequencies of either of these polymorphisms was found in patients compared with controls.

Parathyroid hormone-related protein increases DNA synthesis in proximal tubule cells by cyclic AMP- and protein kinase C-dependent pathways.

The present study was performed to characterize the possible involvement of cAMP synthesis and protein kinase C (PKC) activation in the DNA synthesis-stimulating effect of parathyroid hormone-related protein (PTHrP) in proximal tubule cells. We found that DNA synthesis was stimulated by 10 microM 8BrcAMP, and 1 microM Sp-cDBIMPS, two cAMP analogs, and also by 1 microM phorbol 12-myristate 13-acetate (PMA) and 100 microM 1,2-dioctanoyl-sn-glycerol, two PKC activators, and 10 nM [Cys23] human (h)PTHrP (24-35) amide in rabbit proximal tubule cells (PTC). Both Sp-cDBIMPS and PMA, at 1 microM, also increased DNA synthesis in SV40-immortalized mouse proximal tubule cells MCT. Human PTHrP (7-34) amide [PTHrP (7-34)] dose dependently stimulated DNA synthesis in a similar manner as [34Tyr]PTHrP (1-34) amide [PTHrP (1-34)], in PTC. PMA pre-treatment for 20 h, which downregulates PKC, completely blocked the effect induced by PTHrP (7-34), but not that of PTHrP (1-34), in the latter cells. In contrast, the same PMA pre-treatment abolished the DNA synthesis stimulation by PTHrP (1-34) and PTHrP (7-34) in MCT cells, which appear to have PTH receptors mainly coupled to phospholipase C and not adenylate cyclase. Our results indicate that the stimulatory effect of PTHrP on DNA synthesis in proximal tubule cells is mediated by a cAMP- and PKC-dependent mechanism.

[Diffuse thoracic lymphangiomatosis: diagnosis and treatment]. / Linfangiomatosis torácica difusa: diagnóstico y tratamiento.

Histologically, lymphangiomatosis is a rare type of benign neoplasm caused by abnormal development and proliferation of the lymphatic system. Thoracic lymphangiomatosis can present in a localized (lymphangioma) or diffuse form (lymphangiomatosis). In most cases the disease progresses to serious morbidity or even death. The treatment of choice for localized disease is usually surgery or, less frequently, local injection of sclerosing agents (streptococcus antigen OK-432). However, in diffuse forms there is a gelatinous infiltrate without defined limits. In these cases the main treatment option is radiotherapy. We report 2 cases of diffuse thoracic lymphangiomatosis with pulmonary infiltrate. In both cases radiotherapy in appropriate doses successfully eliminated pulmonary infiltrates, pleural effusion, dyspnea, and general discomfort. Surgery was needed to resolve complications of the disease and for diagnosis.

Laccase biosensors based on different enzyme immobilization strategies for phenolic compounds determination.

Different enzyme immobilization approaches of Trametes versicolor laccase (TvL) onto gold surfaces and their influence on the performance of the final bioanalytical platforms are described. The laccase immobilization methods include: (i) direct adsorption onto gold electrodes (TvL/Au), (ii) covalent attachment to a gold surface modified with a bifunctional reagent, 3,3'-Dithiodipropionic acid di (N-succinimidyl ester) (DTSP), and (iii) integration of the enzyme into a sol-gel 3D polymeric network derived from (3-mercaptopropyl)-trimethoxysilane (MPTS) previously formed onto a gold surface (TvL/MPTS/Au). The characterization and applicability of these biosensors are described. Characterization is performed in aqueous acetate buffer solutions using atomic force microscopy (AFM), providing valuable information concerning morphological data at the nanoscale level. The response of the three biosensing platforms developed, TvL/Au, TvL/DTSP/Au and TvL/MPTS/Au, is evaluated in the presence of hydroquinone (HQ), used as a phenolic enzymatic substrate. All systems exhibit a clear electrocatalytic activity and HQ can be amperometrically determined at -0.10 V versus Ag/AgCl. However, the performance of biosensors - evaluated in terms of sensitivity, detection limit, linear response range, reproducibility and stability - depends clearly on the enzyme immobilization strategy, which allows establishing its influence on the enzyme catalytic activity.

Gold nanoparticles-induced enhancement of the analytical response of an electrochemical biosensor based on an organic-inorganic hybrid composite material.

The design and characterization of a new organic-inorganic hybrid composite material for glucose electrochemical sensing are described. This material is based on the entrapment of both gold nanoparticles (AuNPs) and glucose oxidase, which was chosen as a model, into a sol-gel matrix. The addition of spectroscopic grade graphite to this system, which confers conductivity, leads to the development of a material particularly attractive for electrochemical biosensor fabrication. The characterization of the hybrid composite material was performed using atomic force microscopy and scanning electron microscopy techniques. This composite material was applied to the determination of glucose in presence of hydroxymethylferrocene as a redox mediator. The system exhibits a clear electrocatalytic activity towards glucose, allowing its determination at 250 mV vs Ag/AgCl. The performance of the resulting enzyme biosensor was evaluated in terms of sensitivity, detection limit, linear response range, stability and accuracy. Finally, the enhancement of the analytical response of the resulting biosensor induced by the presence of gold nanoparticles was evaluated by comparison with a similar organic-inorganic hybrid composite material without AuNPs.

Bioanalytical device based on cholesterol oxidase-bonded SAM-modified electrodes.

A rapid, simple and reproducible two-step method for constructing cholesterol biosensors by covalently bonding cholesterol oxidase (ChOx) to a 3,3'-dithiodipropionic acid di(N-succinimidyl ester) (DTSP)-modified gold electrode is described. Exhaustive characterizations of both the immobilization process and the morphological properties of the resulting ChOx monolayer were performed via a quartz crystal microbalance (QCM) and atomic force microscopy (AFM) operated under liquid conditions, respectively. In addition, scanning electrochemical microscopy (SECM) measurements were performed in order to check that the immobilized enzyme retains its catalytic activity. The replacement of the natural electron acceptor (O(2)) in the enzymatic reaction with an artificial mediator, hydroxymethylferrocene (HMF), was also studied. Finally, cholesterol was amperometrically determined by measuring the hydrogen peroxide produced during the enzymatic reaction at +0.5 V. The optimized cholesterol biosensor exhibited a sensitivity of 54 nA mM(-1) and a detection limit of 22 microM.

Microscopic and voltammetric characterization of bioanalytical platforms based on lactate oxidase.

A microscopic and voltammetric characterization of lactate oxidase- (LOx-) based bioanalytical platforms for biosensor applications is presented. In this context, emphasis is placed on amperometric biosensors based on LOx that have been immobilized by direct absorption on carbon surfaces, in particular, glassy carbon (GC) and highly ordered pyrolytic graphite (HOPG). The immobilized LOx layers have been characterized using atomic force microscopy (AFM) under liquid conditions and cyclic voltammetry. In addition, spatially resolved mapping of enzymatic activity has been carried out using scanning electrochemical microscopy (SECM). In the presence of lactate with hydroxymethylferrocene (HMF) as a redox mediator in solution, biosensors obtained by direct adsorption of LOx onto GC electrodes exhibited a clear electrocatalytic activity, and lactate could be determined amperometrically at 300 mV versus SSCE. The proposed biosensor also exhibits good operating performance in terms of linearity, detection limit, and lifetime.

Comprehensive study of bioanalytical platforms: xanthine oxidase.

A comprehensive study of a general bioanalytical platform for biosensor applications is presented using xanthine oxidase (XnOx) as a case study within the framework of developing approaches of broad applicability. In this context, emphasis is placed on amperometric biosensors based on XnOx, which has been immobilized by covalent binding to gold electrodes modified with dithiobis-N-succinimidyl propionate. The immobilized XnOx layers have been characterized using atomic force microscopy under liquid conditions and quartz crystal microbalance techniques. In addition, spatially resolved mapping of enzymatic activity has been carried out using scanning electrochemical microscopy. Redox dyes of phenothiazine derivatives, specifically, thionine and methylene blue, have been found to work well as electron acceptors for reduced XnOx. The kinetic parameters and equilibrium constants of the mediated enzymatic oxidation of xanthine in the presence of the above-mentioned redox dyes have been calculated. The response of the enzymatic electrode to varying xanthine concentrations has been obtained in the presence of thionine or methylene blue as redox mediator in solution. Under these conditions, xanthine could be determined amperometrically at +0.2 V versus SSCE.

Electrocatalytic oxidation of nitric oxide at indium hexacyanoferrate film-modified electrodes.

The electrocatalytic oxidation of nitric oxide (NO) by indium (III) hexacyanoferrate (III) films has been studied. These films are electrodeposited onto glassy carbon electrodes through potential cycling in acidic solutions containing a potassium electrolyte, indium (III), and potassium hexacyanoferrate. The resulting modified electrodes exhibit a reversible redox response ascribed to the oxidation/reduction of iron atoms presents in the electrodeposited film. The films have a potent and persistent electrocatalytic activity towards NO oxidation at neutral pH. The electrocatalytic oxidation of NO takes place at potentials around +0.75 V which represents a moderate diminution in the overpotential. In addition, interferences due to the presence of nitrate and nitrite have been significantly reduced. According to these results, the described modified electrodes have been used as sensors for the determination of NO generated by decomposition of a typical NO-donor, such as S-nitroso- N -acetyl- d, l -penicillamine (SNAP).