Reynaldo Villalonga

Preparation of core–shell Fe3O4@poly(dopamine) magnetic nanoparticles for biosensor construction

Novel core-shell Fe3O4@poly(dopamine) magnetic nanoparticles were prepared through an in situ self-polymerization method. The hybrid nanomaterial showed an average core diameter of 11 ± 3 nm and a polymer thin film thickness of 1.8 ± 0.2 nm. The core-shell nanoparticles were employed as solid supports for the covalent immobilization of horseradish peroxidase (HRP), and the resulting biofunctionalized magnetic nanoparticles were employed to construct an amperometric biosensor for H2O2. The enzyme biosensor showed a high sensitivity of 442.14 mA M-1 cm-2, a low limit of detection of 182 nM, a wide linear range from 6.0 × 10-7 to 8.0 × 10-4 M and high stability for 1 month.

Adamantane/β-cyclodextrin affinity biosensors based on single-walled carbon nanotubes

One challenging goal for the development of biosensors is the conception of three-dimensional biostructures on electrode surfaces. With the aim to develop 3D architectures based on single-walled carbon nanotubes (SWCNTs) frameworks a novel adamantane-pyrrole monomer was synthesized. After electrochemical polymerization at 0.95V in acetonitrile, the resulting polypyrrole film provided affinity interactions with beta-cyclodextrin. SWCNT coatings were thus functionalized with poly(adamantane-pyrrole) and applied to the anchoring of glucose oxidase (GOX), modified with beta-cyclodextrin. By using this affinity system adamantine-cyclodextrin, beta-cyclodextrin-modified gold nanoparticles were attached onto the functionalized SWCNT deposit as intermediate layer. This allows the immobilization of adamantane-tagged GOX. The responses of these biosensors to glucose were measured by potentiostating the modified electrodes at 0.7V versus saturated calomel electrode (SCE) in order to oxidize the enzymatically generated hydrogen peroxide in the presence of glucose and oxygen. The highest sensitivity and maximum current density were recorded for the configuration based on beta-cyclodextrin-modified gold particles as intermediate layer between adamantine-functionalized SWCNTs and GOX (31.02 mAM(-1)cm(-2) and 350 microAcm(-2), respectively). The similar configuration without SWCNTs exhibits a sensitivity and J(max) of 0.98 mAM(-1)cm(-2) and 75 microAcm(-2), respectively. The resulting supramolecular assemblies were characterized by scanning electron microscopy (SEM). Advantages and disadvantages of the different preparation methods and the performance of each affinity sensor setup are discussed in detail.

Preparation and functional properties of trypsin modified by carboxymethylcellulose

Abstract Trypsin from bovine pancreas was modified by the polyaldehyde derivative of carboxymethylcellulose (CMC) via reductive alkylation with NaBH 4 . The modified enzyme contained 57% carbohydrate by weight, resulting from the modification of 52% of the amino groups of the protein. In comparison with the native protease, the modified trypsin retained 62% and 42% of the esterolytic and proteolytic activity, respectively. The value of K m for CMC–trypsin complex was 2.2 times lower than for the native enzyme. The thermostability and pH stability was improved for trypsin by this modification. The conjugate was also more resistant to the action of the anionic surfactant sodium dodecylsulphate and denaturing agents such as 8 M urea and 6 M guanidinium chloride. This modification also protected the enzyme against autolysis at alkaline pH and improved the stability of the enzyme in the presence of methanol.

Designing Electrochemical Interfaces with Functionalized Magnetic Nanoparticles and Wrapped Carbon Nanotubes as Platforms for the Construction of High-Performance Bienzyme Biosensors

The design of a novel biosensing electrode surface, combining the advantages of magnetic ferrite nanoparticles (MNPs) functionalized with glutaraldehyde (GA) and poly(diallyldimethylammonium chloride) (PDDA)-coated multiwalled carbon nanotubes (MWCNTs) as platforms for the construction of high-performance multienzyme biosensors, is reported in this work. Before the immobilization of enzymes, GA-MNP/PDDA/MWCNT composites were prepared by wrapping of carboxylated MWCNTs with positively charged PDDA and interaction with GA-functionalized MNPs. The nanoconjugates were characterized by scanning electron microscopy (SEM) and electrochemistry. The electrode platform was used to construct a bienzyme biosensor for the determination of cholesterol, which implied coimmobilization of cholesterol oxidase (ChOx) and peroxidase (HRP) and the use of hydroquinone as redox mediator. Optimization of all variables involved in the preparation and analytical performance of the bienzyme electrode was accomplished. At an applied potential of -0.05 V, a linear calibration graph for cholesterol was obtained in the 0.01-0.95 mM concentration range. The detection limit (0.85 μM), the apparent Michaelis-Menten constant (1.57 mM), the stability of the biosensor, and the calculated activation energy can be advantageously compared with the analytical characteristics of other CNT-based cholesterol biosensors reported in the literature. Analysis of human serum spiked with cholesterol at different concentration levels yielded recoveries between 100% and 103%

Stabilization of invertase by modification of sugar chains with chitosan

Chitosan was linked to invertase by covalent conjugation to periodate-activated carbohydrate moieties of the enzyme. The thermostability of modified enzyme was enhanced by about 10 °C. The half-life at 65 °C was increased from 5 min to 5 h. The enzyme stability was enhanced by 20% at pH below 3.0. The half-life of denaturation by 6 M urea was increased by 2 h.

Ultrasensitive detection of adrenocorticotropin hormone (ACTH) using disposable phenylboronic-modified electrochemical immunosensors.

This work reports for the first time an electrochemical immunosensor for the determination of adrenocorticotropin hormone (ACTH). The immunoelectrode design involves the use of amino phenylboronic acid for the oriented immobilization of anti-ACTH antibodies onto screen-printed carbon modified electrode surfaces. A competitive immunoassay between the antigen and the biotinylated hormone for the binding sites of the immobilized antibody was performed. The electroanalytical response was generated by using alkaline phosphatase-labelled streptavidin and 1-naphtyl phosphate as the enzyme substrate. The electrochemical oxidation of the enzyme reaction product, 1-naphtol, measured by differential pulse voltammetry was employed to monitor the affinity reaction. Under the optimized working conditions, an extremely low detection limit of 18 pg/L was obtained. Cross-reactivity was evaluated against other hormones (cortisol, estradiol, testosterone, progesterone, hGH and prolactin) and the obtained results demonstrated an excellent selectivity. The developed immunosensor was applied to a human serum sample containing a certified amount of ACTH with good results.

Functional stabilization of invertase by covalent modification with pectin

Pectin was attached to ethylenediamine-activated carbohydrate moieties of invertase using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide as coupling agent. The modified enzyme retained 57% of the original activity and contained 2.7 mol polymer per mol holoenzyme. Its optimum temperature was increased by 8 °C and its thermostability by 7.3 °C. The half-life at 65 °C was increased from 5 min to 2 days. The enzyme stability was enhanced by 33% at pH 2.0, and also by 27% at pH 12.0. The conjugate retained about 96% of its initial activity after 3 h incubation in 6 M urea.

Glucose-triggered release using enzyme-gated mesoporous silica nanoparticles

A new gated nanodevice design able to control cargo delivery using glucose as a trigger and cyclodextrin-modified glucose oxidase as a capping agent is reported.

Amperometric biosensor for xanthine with supramolecular architecture

Xanthine oxidase modified with 1-adamantanyl residues was supramolecularly immobilized on Au electrodes coated with Au nanoparticles coated with a perthiolated beta-cyclodextrin polymer; the analytical response of the electrode toward xanthine was evaluated.

Thermal stabilization of trypsin by enzymic modification with β-cyclodextrin derivatives

Streptoverticillum sp. transglutaminase was used as catalyst for the attachment of several β‐cyclodextrin derivatives to the glutamine residues in bovine pancreatic trypsin. The modifying agents used were mono‐6‐ethylenediamino‐6‐deoxy‐β‐cyclodextrin, mono‐6‐propylenediamino‐6‐deoxy‐β‐cyclodextrin, mono‐6‐butylenediamino‐6‐deoxy‐β‐cyclodextrin and mono‐6‐hexylenediamino‐6‐deoxy‐β‐cyclodextrin. The transformed trypsin preparations contained about 3 mol of oligosaccharides/mol of protein. The specific esterolytic activity of trypsin was increased by about 4–21% after conjugation. The Km values for cyclodextrin–trypsin complexes represented about 58–87% of that corresponding to the native enzyme. The optimum temperature for esterolytic activity of trypsin was increased by about 5–10 °C after enzymic modification with the cyclodextrin derivatives. The thermostability was increased by 16 °C for the modified trypsin. Thermal inactivation at different temperatures ranging from 45 to 60 °C was markedly increased for the oligosaccharide–trypsin complexes. This modification also protected the enzyme against autolysis at alkaline pH.