Divinyl sulfone (DVS) has been used to activate agarose beads. The DVS activated agarose resulted quite stable in the pH range 5-10 at 25 °C under wet conditions, and can react rapidly with α-amides ...of Cys and His, at pH 5-10, with Lys mainly at pH 10 and with Tyr in a much slower fashion. After blocking with different nucleophiles, the support lost all reactivity, confirming that this protocol could be useful as an enzyme-support reaction end point. Then, chymotrypsin was immobilized on this support at pH 5, 7 and 10. Even though the enzyme was immobilized at all pH values, the immobilization rate decreased with the pH value. The effect of the immobilization on the activity depended on the immobilization pH, at pH 7 the activity decreased (to 50%) more than at pH 10 (by a 25%), while at pH 5 the immobilization has no effect. Then, the effect of blocking with different reagents was analyzed. It was found that blocking with ethylenediamine improved the enzyme activity by 70% and gave the best stability. The stability of all enzyme preparations improved when 24 h incubation was performed at pH 10, but the qualitative stabilization depended on the inactivation conditions. The analysis of the amino acids of the preparation immobilized at pH 10 showed that Lys, Tyr and Cys residues were involved in the immobilization, involving a minimum of 10 residues (glyoxyl agarose gave 4 Lys involved in the immobilization). The new preparation was 4-5 fold more stable than glyoxyl agarose preparation, considered a very stable one, and in some instances was more active than the free enzyme (170% for the enzyme immobilized at pH 10). Thus, DVS activated supports are very promising to permit the multipoint covalent attachment of enzymes, and that way to improve their stability.
DVS supports are very suitable to stabilize enzymes
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multipoint covalent attachment.
In this paper, the combination of Solid Surface-Room Temperature Phosphorescence (SS-RTP) and nanotechnology has led to a new approach in the detection of biogenic amines in complex matrices. This ...novel approach allows, for the first time, the direct determination of the concentration of tryptamine in beers. The novelty of the proposed optical sensor resides in its simplicity, rapidity, absence of complex chromatographic separation, sample clean-up, preconcentration, and derivatization protocols. Therefore, this novel methodology simplifies and reduces considerably the time and cost of the analysis, resolving the two major problems of the determination of tryptamine in beer up to now: low sensitivity and matrix effects.
The proposed sensor is based on a novel white, uncharged, and non-luminescent functional nonwoven nanofibre mat (Tiss®-Link) formed by hydrophilic nanofibres of 300nm of diameter functionalized with a high concentration of active vinyl groups (330µmolg−1). It is used to carry out a kinetically controlled covalent immobilisation of tryptamine via Michael type-reaction. The transduction of the sensor is phosphorescence; the covalently immobilized tryptamine is quantified by SS-RTP, obtaining a detection limit of 6ngmL−1 with short response times (15min). The applicability of the sensor was demonstrated by analysing tryptamine in 10 different varieties of beers, obtaining recovery percentages close to 100%.
This paper describes a novel biosensor which combines the use of nanotechnology (non-woven nanofibre mat) with Solid Surface-Room Temperature Phosphorescence (SS-RTP) measurement for the ...determination of serotonin in human serum. The developed biosensor is simple and can be directly applied in serum; only requires a simple clean-up protocol. Therefore it is the first time that serotonin is analysed directly in serum with a non-enzymatic technique. This new approach is based on the covalent immobilization of serotonin directly from serum on a functional nanofibre material (Tiss®-Link) with a preactivated surface for direct covalent immobilization of primary and secondary amines, and the subsequent measurement of serotonin phosphorescent emission from the solid surface. The phosphorescent detection allows avoiding the interference from any fluorescence emission or scattering light from any molecule present in the serum sample which can be also immobilised on the nanofibre material. The determination of serotonin with this SS-RTP sensor overcomes some limitations, such as large interference from the matrix and high cost and complexity of many of the methods widely used for serotonin analysis.
The potential applicability of the sensor in the clinical diagnosis was demonstrated by analysing serum samples from seven healthy volunteers. The method was validated with an external reference laboratory, obtaining a correlation coefficient of 0.997 which indicates excellent correlation between the two methods.
•We have developed a novel optical sensor for analysing serotonin in complex matrices.•It is free of pre-concentration and derivatization protocols.•It is fast, simple, chip and the most environmental friendly method published so far.•It is the first time that serotonin is analysed directly in serum with a non-enzymatic technique.
A multifunctional material based on co-electrospinning has been developed as a basic material for the development of biosensors with optical oxygen transduction. It is based on coaxial nanofibres: ...inner fibres containing an oxygen sensitive dye and outer fibres containing aldehyde groups to allow the formation of Schiff bases with the amino groups of the enzyme. The resulting material preserves the oxygen sensing properties of the inner optical transducer as well as exhibits a high capacity for immobilizing molecules on its surface.
Uricase has been selected as model enzyme and several parameters (temperature, pH, reaction time, buffer, and enzyme concentration) have been optimised to demonstrate the versatility of this novel multifunctional material in the development of biosensors with optical oxygen transduction for determining uric acid in serum samples. It suggests that the proposed multifunctional material can provide a promising multifunctional platform for biosensing applications.
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•A coaxial multifunctional material for biosensors has been developed.•It allows the enzyme immobilization and optical oxygen transduction.•It shows a detection limit of 4 μM and a linear range of 15–500 μM.•It has been used for detecting uric acid in serum samples.
In this study, we have optimised the sterically directed attachment of biomolecules on the surface of coaxial membranes prepared by co-electrospinning which have been proved to be a material with ...very high performance for the development of biosensors with optical oxygen transduction. Uricase has been used as model enzyme.
Two sterically directed strategies: a) covalent attachment via maleimide, and b) affinity bonding via biotin-streptavidin interaction, have been tested in order to preserve the enzymatic activity of uricase and to improve the analytical figures of merits on the determination of uric acid. The best results were obtained with biotin-streptavidin affinity interaction and using a biotinylation reagent containing a polyethylene glycol chain. The developed biosensor showed high sensitivity towards uric acid with a detection limit of 0.5 µM, a quantification limit of 1.8 µM and linear range from 1.8 to 250 µM. The applicability of the membrane as biosensor with optical oxygen transduction was proved by determining uric acid in serum samples. The obtained results showed a good correlation (0.999) with those obtained by an external reference laboratory.
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•Optimization the sterically directed attachment of biomolecules on multifunctional material.•Uricase has been selected as model enzyme.•It shows a detection limit of 0.5 µM and a linear range of 0.5–250 µM.•The most sensitive optical method for analysing uric acid.•It has been used for detecting uric acid in serum samples.
In this work, a multifunctional material with a core–shell structure containing an inner optical oxygen transducer (PdTFPP) has been successfully used for the immobilization of glucose oxidase on its ...outer surface and the subsequent determination of glucose. The material was fabricated by co-electrospinning and immobilizing the enzyme by physical adsorption. The sensing mechanism is based on glucose oxidase oxidation of glucose that creates a localized decrease in the dissolved oxygen amount and consequently produces a measurable increase in the luminescence intensity of the inner oxygen transducer. The material was applied to detect glucose at room temperature, and exhibited a good luminescent response.
Furthermore, this coaxial material was integrated into a microfluidic chip and its sensitivity to glucose was tested (LOD of 35μM and LOQ of 105μM), obtaining higher sensing properties than using the membrane alone under ambient conditions. This improvement in the sensing response can be explained by considering that the chip limits the oxygen transfer from the ambient air to the coaxial membrane, creating a more controlled environment in where to carry out the measurements. Therefore, the combination of this core–shell material with microfluidic devices could have great potential in the fabrication of oxygen dependent optical biosensors.
Four different types of polymeric particles with different functional groups on their surface have been evaluated to develop biosensors using glucose oxidase as a model enzyme. Direct covalent ...immobilization was achieved on particles functionalized with chloride, epoxy and vinyl groups
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the reactive functional groups on the surface, whereas particles functionalized with carboxylic groups, used as reference materials, were pre-activated with carbodiimide. Immobilization was successfully performed under very mild conditions (20 °C, pH 8.0). In order to determine the advantages and disadvantages of each functional group, both the amounts of immobilized enzymes and their relative activities were fully investigated. In order to demonstrate their applicability on the design on biosensing with oxygen optical transduction, the functionalized particles were co-immobilized in gold chips with oxygen sensing particles (PSMA-PtTFPP) using electrophoretic deposition and characterised for glucose determination in solution.
Functional groups have been evaluated for covalent immobilisation of enzymes in the development of biosensors with oxygen optical transduction.
Divinyl sulfone (DVS) has been used to activate agarose beads. The DVS activated agarose resulted quite stable in the pH range 5-10 at 25 degree C under wet conditions, and can react rapidly with ...alpha -amides of Cys and His, at pH 5-10, with Lys mainly at pH 10 and with Tyr in a much slower fashion. After blocking with different nucleophiles, the support lost all reactivity, confirming that this protocol could be useful as an enzyme-support reaction end point. Then, chymotrypsin was immobilized on this support at pH 5, 7 and 10. Even though the enzyme was immobilized at all pH values, the immobilization rate decreased with the pH value. The effect of the immobilization on the activity depended on the immobilization pH, at pH 7 the activity decreased (to 50%) more than at pH 10 (by a 25%), while at pH 5 the immobilization has no effect. Then, the effect of blocking with different reagents was analyzed. It was found that blocking with ethylenediamine improved the enzyme activity by 70% and gave the best stability. The stability of all enzyme preparations improved when 24 h incubation was performed at pH 10, but the qualitative stabilization depended on the inactivation conditions. The analysis of the amino acids of the preparation immobilized at pH 10 showed that Lys, Tyr and Cys residues were involved in the immobilization, involving a minimum of 10 residues (glyoxyl agarose gave 4 Lys involved in the immobilization). The new preparation was 4-5 fold more stable than glyoxyl agarose preparation, considered a very stable one, and in some instances was more active than the free enzyme (170% for the enzyme immobilized at pH 10). Thus, DVS activated supports are very promising to permit the multipoint covalent attachment of enzymes, and that way to improve their stability.
Painful lesions on the plantar aspect of the first interphalangeal joint (IPJ) of the hallux can be attributed to structures called ossicles, nodules, or sesamoids. The aims of the present study were ...first to verify that ultrasonography (US) is a high-sensitivity tool for diagnosing an interphalangeal ossicle (IO), and second to prove that US-guided-shaving surgery ("milling") is a safe and feasible technique for remodeling the IO. The study is divided into three parts. In the first part, the prevalence of IOs was estimated in 12 cadaver feet using US, anatomical dissection, and fluoroscopy. In the second, a detailed US and morphological description of the IO was obtained. In the third, six cadaver feet were subjected to surgical milling. IO prevalence was 41.6% in gross anatomy, 41.6% in US examination and just 16.6% in fluoroscopy. The ossicles had a mean length of 4 mm (± 2 mm) and a width of 7 mm (± 2 mm). The ossicles could be completely shaved in all specimens without injuring important anatomical structures. Our results indicate that US is a more precise tool for diagnosing an IO than X-ray. Moreover, our US-guided mini-invasive surgical technique appears feasible and safe.
BACKGROUND:The objetive was investigate the long-term impact of low level viremia (LLV) on all-cause mortality, AIDS and non-AIDS events (NAEs), and virological failure in patients receiving ART.
...METHODS:We analyzed ART-naïve adults from the cohort of the Spanish AIDS Research Network (CoRIS) who initiated ART from 2004 to 2015 and achieved plasma viral load (VL) below 50 copies/ml. LLV50-199 was defined as two consecutive VL between 50 and 199 copies/ml, and LLV200-499 as two consecutive VL between 50 and 499 copies/ml with at least one between 200 and 499 copies/ml. Multivariable Cox models were used to estimate the association of LLV with AIDS events/death, NAEs and virological failure.
RESULTS:Of 5986 patients included, 237 (4.0%) experienced LLV50–199 and 168 (2.8%) developed LLV200–499. One hundred seventy-one patients died or developed an AIDS event, 245 had any serious NAE and 280 had virological failure. LLV200-499 was strongly associated with a higher risk of both AIDS events/death adjusted hazard ratio (aHR), 2.89; 95% confidence interval (CI), 1.41 – 5.92 and virological failure (aHR, 3.25; 95% CI, 1.77 – 5.99), while no differences were observed between LLV50-199 and no LLV neither for AIDS events/death (aHR, 1.84; 95% CI, 0.89 – 3.82) nor virological failure (aHR, 1.42; 95% CI, 0.78 – 2.58). LLV was not associated with the occurrence of any serious-NAE.
CONCLUSION:In this cohort, LLV200–499 was strongly associated with AIDS events/death and virological failure, but not with any serious NAE. Therefore, vigorous treatment should be implemented in patients with more than 200 copies/ml.
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