On-line surface plasmon resonance biosensing of vascular endothelial growth factor signaling in intact-human hepatoma cell lines.

Surface plasmon resonance (SPR) monitoring of biorecognition events at intracellular levels is a valuable tool for studying the angiogenic response of carcinoma living cells during tumor growth and proliferation. We report here a comparative study of two different strategies to detect human hepatoma cell interactions between transmembrane vascular endothelial growth factor receptor (VEGFR2) and vascular endothelial growth factor (VEGF). To monitor VEGFR2 activation after VEGF stimulation, intact hepatocellular carcinoma HepG2 or Huh7 cells (2 × 10(5) cells per mL) were directly immobilized on the sensor chip. Distinguishable SPR sensorgrams were obtained for each cell line depending on the time required for VEGFR2 activation. SPR signals for VEGF-VEGFR2 binding were inhibited by the VEGFR inhibitor, CBO-P11. The SPR response after VEGF stimulation/inhibition was in good agreement with the results observed by immunoblotting analysis. In a second approach we used intact cell lines as analytes. SPR analysis was done by injecting HepG2 and HuH7 cell suspensions (2-4 × 10(4) cells per mL) onto a sensor surface previously immobilized with VEGF via a thiol self-assembled monolayer (SAM). Specificity and reproducibility were evaluated reusing the same chip surface over more than 60 complete regeneration cycles. Comparison between both methods yielded differences in terms of reliability, making the latter strategy more effective for the analysis of real samples. The investigation of VEGF signaling in intact human hepatoma living cells by SPR monitoring comprises a novel and promising design for the study of tumor angiogenesis via downregulation of VEGF and VEGFR2 pathways. Further investigation on VEGFR activation and vascular function could contribute to establish a robust and meaningful tool for early cancer diagnostics.

Direct surface plasmon resonance immunosensing of pyraclostrobin residues in untreated fruit juices.

A surface plasmon resonance (SPR) immunoassay for on-line detection of the strobilurin fungicide pyraclostrobin in untreated fruit juices is presented. The analysis of pyraclostrobin residues is accomplished in apple, grape, and cranberry samples by monitoring the recognition events occurring separately in a two-channel home-made SPR biosensor. Covalent coupling of the analyte derivative results in a reversible method, enabling more than 80 measurements on the same sensor surface. Optimization of the immunoassay conditions provides limits of detection as low as 0.16 µg L(-1). The selectivity and reproducibility of the analysis is ensured by studying both non-specific interactions with unrelated compounds and inter-assay coefficients of variation. Excellent recovery ranging from 98 to 103% was achieved by a simple 1:5 dilution of fruit juice with assay buffer before the analysis. The lack of previous cleaning and homogenization procedures reduces the analysis time of a single food sample to only 25 min, including the regeneration cycle.

Proteolytic activity, mycotoxins and andrastin A in Penicillium roqueforti strains isolated from Cabrales, Valdeón and Bejes-Tresviso local varieties of blue-veined cheeses.

High quality local varieties of blue-veined cheese are made in the villages of the valleys of Cabrales, Valdeón and Bejes-Tresviso in North Spain. Penicillium roqueforti strains have been isolated from each of those blue cheeses and compared with the collection strain P. roqueforti CECT 2905 (ATCC 10110) and a strain 'Valdeón-industrial' used for large scale production of Valdeón cheese. Using molecular genetics techniques and 5.8S and 18S rRNAs and the D1-D2 regions of 28S rRNA all strains were identified as authentic P. roqueforti. These strains from local varieties of blue cheese could be distinguished from the Valdeón-industrial strain and the control strain CECT 2905 by the mitochondrial DNA restriction pattern. The industrial strain showed high levels of aspartylprotease AspA, whereas the culture collection strain showed barely detectable levels of this enzyme, as shown by proteolysis tests and by immunodetection with anti-AspA antibodies. The lipolytic activity was similar in the strains isolated from the three types of local blue cheeses. The strains isolated from the local varieties of the blue cheese produced moderate levels of PR toxin, whereas the Valdeón-industrial strains showed a higher content of this mycotoxin. All strains (except the control strain CECT 2905) showed similar levels of roquefortine C. The antitumoral compound andrastin A was produced by all strains at different levels. P. roqueforti CECT 2905 showed high ability to synthesize this compound. Andrastin A was present in all industrial and local varieties of blue cheese. The content of andrastin A was similar to that of other well-known blue cheeses from France and Denmark.

On-line determination of 3,5,6-trichloro-2-pyridinol in human urine samples by surface plasmon resonance immunosensing.

An immunochemical method for the analysis of 3,5,6-trichloro-2-pyridinol (TCP), a major urinary metabolite of chlorpyrifos, is developed using a surface plasmon resonance (SPR)-based biosensor. The stability of the assay was assessed by covalently linking the analyte derivative to a thin, gold-modified sensor surface. For optimization of analyte derivative immobilization, sensor chips were activated via alkanethiol monolayers with terminal amine or carboxyl groups. Binding inhibition tests were performed in untreated urine samples and compared to those obtained in distilled water and PBS was used as control. In all cases, similar detection limits, at the micrograms per litre level (0.1-0.24 microg L(-1)), were attained for TCP assays independently of the dilution buffer. Reproducibility of measurements was studied throughout more than 130 regeneration cycles, which allowed the repeated use of the same immunosensor surface without significant variation of the SPR signal. All measurements were developed in real-time in only 10 min, using a SPR portable system. The device could be applied as a valuable analytical method to both environmental screening and clinic diagnostics.

Optical immunosensor for fast and sensitive detection of DDT and related compounds in river water samples.

A surface plasmon resonance (SPR) based immunosensor has been developed for the monitoring of environmentally persistent pollutants like DDT, its metabolites and analogues in real water samples. A reusable immunosurface is provided via the covalent attachment of the analyte derivative to a self-assembled alkanethiol monolayer formed onto the SPR gold-thin layer. The regeneration of the sensor surface allowed the performance of 270 assay cycles within an analysis time of 20 min for each assay cycle. Immunoassays based on a binding inhibition format were performed by using two monoclonal antibodies (MAbs) with different selectivity. Low limits of detection (LODs), in the sub-nanogram per litre range, were attained for DDT-selective (15 ng L-1) and DDT group-selective immunoassays (31 ng L-1). Both assays were carried out in spiked river water samples without significant effect of the matrix. SPR measurements were validated using gas-chromatography-mass spectrometry. The comparison between methods was in good agreement showing an excellent correlation coefficient (r2=0.995). The SPR analysis of DDT proved to be three times more sensitive than colorimetric ELISAs without the need of labelling and a much lower time of response. Our SPR biosensor portable platform (beta-SPR) is already commercialised by the company SENSIA, S.L. (Spain).

Multi-analyte SPR immunoassays for environmental biosensing of pesticides.

Multi-analyte detection of environmentally relevant pesticides is performed by using a two-channelled surface plasmon resonance (SPR) biosensor. The special design of the SPR instrument allows the determination of several analytes (DDT, chlorpyrifos and carbaryl) via different immobilization formats. First, simultaneous pesticide monitoring is possible by flowing chlorpyrifos, carbaryl or DDT samples separately over each channel of the SPR system, wherein their corresponding recognition element was previously immobilized. The second approach is based on the multiple and combined immobilization of several analyte recognition elements on the sensing surface of one individual flow cell. In this format, the analysis time for all three pesticides varied from 40 to 60 min depending on the number of regeneration cycles. In most cases, similar detection limits were attained for the target analyte irrespective of the assay format, with sensitivity values at the nanogram per litre level (18-50 ng L(-1)). The assay reproducibility was proved through the repeated use of the same sensor surface for over more than 200 assay cycles, whereas the absence of biosensor response to non-related analytes showed the specificity and reliability of the analysis. The SPR instrument, including optics, electronics and microfluidics, is already commercialised by the company SENSIA, SL.

Determination of carbaryl in natural water samples by a surface plasmon resonance flow-through immunosensor.

The analysis of carbaryl in natural water samples was accomplished using a portable immunosensor based on surface plasmon resonance (SPR) technology. The assay was based on a binding inhibition immunoassay format with the analyte derivative covalently immobilized on the sensor surface. An alkanethiol self-assembled monolayer (SAM) was formed onto the gold-coated sensor surface to allow the reusability of the same sensing surface during 220 regeneration cycles. Reproducibility was evaluated by performing three independent assays in triplicate on 3 different days. The batch-assay variability was also calculated using three different gold-coated sensor surfaces. The intra- and inter-day relative standard deviation were 8.6 and 15.3%, respectively, whilst a variation of 7.4% in assay sensitivity was obtained by employing different sensor chips. The lowest detection limit, calculated as the concentration providing a 10% decrease of the blank signal, was of 1.38 microg L(-1). Matrix effects were also evaluated in different water types, showing I50 values (carbaryl concentrations that produced a 50% decrease of the blank signal) within the range of carbaryl standard curves in distilled water (2.78-3.55 microg L(-1)). The carbaryl immunoassay performance was validated with respect to conventional high-performance liquid chromatography-mass spectrometry (HPLC-MS). The correlation between methods was in good agreement (r2 = 0.998, 0.999 and 0.999) for the three types of natural water samples tested. A complete assay cycle, including regeneration, is accomplished in 20 min. All measurements were carried out with the SPR sensor system (beta-SPR) commercialised by the company SENSIA, SL (Spain). The small size and low-time of response of the beta-SPR platform would allow its utilization in real contaminated locations.

Determination of environmental organic pollutants with a portable optical immunosensor.

A portable surface plasmon resonance (SPR) optical biosensor device is described as a direct immunosensing system to determine organic pollutants in natural water samples. Monitoring of organochlorine (DDT), organophosphorus (chlorpyrifos) and carbamate (carbaryl) compounds within the concentration levels stipulated by the European legislation, can be accomplished using this immunosensor. The lowest limit of detection (LOD) was obtained for DDT, at 20 ng L(-1), whilst 50 ng L(-1) and 0.9 microg L(-1), were achieved for chlorpyrifos and carbaryl, respectively. Matrix effects were evaluated for the carbaryl immunoassay in different water types with detection limits within the range of carbaryl standard curves in distilled water (0.9-1.4 microg L(-1)). The covalent immobilization of the analyte derivative through an alkanethiol self-assembled monolayer (SAM) allowed the reusability of the sensor surface during more than 250 regeneration cycles. The quality of the regeneration was proved over a 1-month period of continuous working. The analysis time for a complete assay cycle, including regeneration, comprises 24 min. Our portable SPR-sensor system is already a market product, commercialized by the company SENSIA, SL. The size and electronic configuration of the device allow its portability and utilization on real contaminated locations.

[The rifampicin test in the diagnosis of Gilbert syndrome]. / La prueba de la rifampicina en el diagnóstico del síndrome de Gilbert.

OBJECTIVE: To show the effectiveness and feasibility of the Rifampicin test in diagnosing Gilbert's syndrome. DESIGN: A prospective, descriptive study. SITE. General Medical Service of the Navarra Hospital. PATIENTS: 17 patients with bilirubin levels on two or more occasions and 6 healthy patients without these data. Both in- and out-patients. MAIN MEASUREMENTS AND RESULTS: The Rifampicin test. Bilirubin levels were determined when fasting and one, two, three and four hours after taking 900 mg of Rifampicin (Rimactan). After the test, those patients who had Gilbert's disease presented bilirubin levels significantly higher than the healthy patients, who remained within normal levels. CONCLUSIONS: In the group studied, the Rifampicin test demonstrated its effectiveness in the diagnosis of suspected Gilbert's disease. It would therefore be possible to use this test in the Primary Care field.