A sensitive photothermometric biosensor based on redox reaction-controlled nanoprobe conversion from Prussian blue to Prussian white.

As a new low-cost photothermal nanoprobe, Prussian blue nanoparticles (PB NPs) have been demonstrated to have more potential in photothermometric-based point-of-care testing (POCT) application. However, most of the existing PB NP-based photothermometric sensors were constructed mainly relying on in situ generation of PB NPs or their combination with antigens and antibodies, therefore usually suffering from the inherent defects like complicated preparation and cumbersome surface process as well as high-cost modification. To break this limitation of PB NP-based photothermometric POCT, we proposed an ingenious redox reaction-controlled nanoprobe conversion strategy and successfully applied to photothermometric detection of ascorbate oxidase (AAO). In this design, the heat of PB NP photothermal system under 808-nm laser irradiation dramatically decreased with the addition of AA, due to a unique AA-induced Prussian blue to Prussian white (PB-to-PW) conversion. Upon AAO addition, the heat of reaction system increased because of the enzymatic catalytic reaction between AAO and AA, which led to a significant reduction of AA and resultantly inhibited PB-to-PW conversion. Such target-mediated nanoprobe conversion resulted in an obvious temperature change that could be easily detected by a common thermometer and exhibited good linear ranges from 0.25 to 14 mU/mL with a detection limit as low as 0.21 mU/mL for POCT analysis of AAO. This facile, convenient, and portable photothermometric sensing platform provides an innovative route for the design of PB NP nanoprobe-based photothermometric detection methods. A sensitive photothermometric AAO sensor based on a redox reaction-controlled nanoprobe conversion strategy from Prussian blue to Prussian white.

Manganese(II)-doped zinc/germanium oxide nanoparticles as a viable fluorescent probe for visual and time-resolved fluorometric determination of ascorbic acid and its oxidase.

A method is described for the determination of ascorbic acid (AA) in complex biological fluids. It based on maganese(II)-doped zinc/germanium oxide nanoparticles (Mn@ZnGe NPs) with appealing time-resolved phosphorescence (TRP). TRP can provide a background-free reporter signal in analytical methods. The absorption of AA overlaps the excitation band of Mn@ZnGe NPs at 254 nm. This reduces the intensity of fluorescence via an inner filter effect (IFE) with increasing concentration of AA. Typical experimental conditions include an emission peak at 536 nm, a delay time of 50 µs and a counting time of 2 ms. This method can detect AA in a range of 5-500 µM with a 0.13 µM limit of detection. If AA is oxidized by the enzyme AA oxidase (AAOx), dehydroascorbic acid will be formed which doesn't absorb at 254 nm. Hence, the IFE cannot occur and fluorescence is not reduced. The strategy can be used to quantify AAOx in the activity range of 1-4 U·mL-1. By using a handheld UV lamp and a smart phone with a color-scanning feature, the feasibility for visual detection and real-time/onsite quantitative scanometric monitoring of AA and AAOx is demonstrated. Graphical abstract Schematic presentation of a fluorometric method for determination of ascorbic acid (AA) and ascorbic oxidase and a scanometric visual assay. It based on the use of maganese(II)-doped zinc/germanium oxide nanoparticles (Mn@ZnGe NPs) with appealing time-resolved phosphorescence (TRP) and the inner-filter effect (IFE) between AA and Mn@ZnGe NPs.

Redox properties of transitional milk from mothers of preterm infants.

AIM: There is a discrepancy between the amount of transitional milk produced by mothers of preterm infants and the low capacity of premature infants to consume it. This milk can be used in milk banks, but previous studies found that there are large variations in the level of host-defence proteins in individual samples of milk from mothers of premature infants, which implies that large individual variations in antioxidative defence composition are also possible. METHODS: Milk samples were collected from 20 healthy mothers of preterm infants. We determined the values for non-enzymatic antioxidative capacity parameters (oxygen radical absorbance capacity (ORAC)), static oxidation-reduction potential (ORP), activities of antioxidant defence enzymes and the amount of vitamin C in whole milk, skim and whey fractions of transitional milk. RESULTS: The main low-molecular-weight antioxidant in transitional milk is vitamin C and most of it is contained in whey. ORAC is higher in whole transitional milk than in skim milk and whey, and ORP is lower in whole transitional milk than that in skim milk and whey. Antioxidative enzyme activities are similar in all individual samples of transitional milk from mothers of preterm infants. CONCLUSIONS: Our results indicate that transitional milk of mothers of preterm infants shows slow individual variations in antioxidative defence composition; therefore, it can be used in human milk banks.

Real-time detection of L-ascorbic acid and hydrogen peroxide in crude food samples employing a reversed sequential differential measuring technique of the SIRE-technology based biosensor.

Detection of the common electrochemical interferents, ascorbic acid and hydrogen peroxide, using a SIRE (Sensors based on Injection of the Recognition Element) technology based biosensor in reverse mode operation is reported. The differential measuring principle employed in the SIRE biosensor during operation in reverse mode is such that the sample is measured first in the presence of enzyme (yielding matrix signal only), and then measured again in the absence of enzyme (yielding signal from matrix+analyte). Subtraction of the signal obtained in the presence of enzyme from the signal obtained in the absence of enzyme gives a specific signal for the analyte only and correlates directly to its concentration in solution. The linear range for the determination of ascorbic acid and hydrogen peroxide was 0-3 mM and 0-2 mM, respectively, with an enzyme concentration of 25 U ascorbate oxidase/ml and 1000 U catalase/ml. The reproducibility was 5% for ascorbic acid (R.S.D. n=15) and 10% for hydrogen peroxide (R.S.D. n=18). The cost per measurement was 0.28 USD for ascorbic acid analysis and 0.0008 USD for hydrogen peroxide analysis. The degradation of ascorbic acid in cereal was followed in real-time, as was the stabilization of low pH on the degradation process.

Continuous spectrophotometric assay for ascorbate oxidase based on a novel chromophoric substrate, 2-aminoascorbic acid.

Recently, we reported the development of a sensitive continuous spectrophotometric assay for the ascorbate-dependent mammalian enzyme dopamine beta-monooxygenase based on the novel chromophoric electron donor 2-aminoascorbic acid [K. Wimalasena and D.S. Wimalasena (1991) Anal. Biochem. 197, 353-361]. We now report that ascorbate oxidase (EC 1.10.3.3, L-ascorbate:O2 oxidoreductase) also catalyzes the oxidation of 2-aminoascorbic acid to chromophoric 2,2'-nitrilodi-2(2')-deoxy-L-ascorbic acid (red pigment). The reaction is kinetically well behaved, displaying the expected stoichiometry for an oxidase-catalyzed reaction with respect to oxygen and the oxidation product (red pigment), demonstrating that 2-aminoascorbic acid is a well-behaved alternative substrate for the enzyme. Ascorbate oxidase is a very efficient enzyme toward its natural substrate, ascorbic acid. Although 2-amino-ascorbic acid is a significantly weak substrate for the enzyme in comparison to ascorbic acid, as indicated by the apparent initial rate kinetic parameters, the high extinction coefficient of the red pigment under our assay conditions suggests that this novel reactivity of the enzyme could be used to design a sensitive, convenient, and continuous spectrophotometric assay for ascorbate oxidase. While this assay is more convenient than the existing oxygen monitor assay, its adaptability to measure the activity of the enzyme in the immobilized form may be helpful in the development of technologies for the automated detection of ascorbic acid in biological fluids for industrial or clinical applications. In addition, this novel reactivity of the enzyme may be used to examine the substrate specificity and the mechanism of action of the enzyme.

An electrophoretic procedure to detect three key enzymes of the ascorbate system.

A method for detection of ascorbic acid oxidase, ascorbic acid peroxidase and dehydroascorbic acid reductase is reported. This method allows the qualitative determination of the presence of these enzymes, also in conditions where the commonly used spectrophotometric assays are unreliable.

The blue oxidases, ascorbate oxidase, laccase and ceruloplasmin. Modelling and structural relationships.

On the basis of the spatial structure of ascorbate oxidase [Messerschmidt, A., Rossi, A., Ladenstein, R., Huber, R., Bolognesi, M., Gatti, G., Marchesini, A., Petruzzelli, R. & Finazzi-Agro, A. (1989) J. Mol. Biol. 206, 513-529], an alignment of the amino acid sequence of the related blue oxidases, laccase and ceruloplasmin is proposed. This strongly suggests a three-domain structure for laccase closely related to ascorbate oxidase and a six-domain structure of ceruloplasmin. These domains demonstrate homology with the small blue copper proteins. The relationships suggest that laccase, like ascorbate oxidase, has a mononuclear blue copper in domain 3 and a trinuclear copper between domain 1 and 3 and ceruloplasmin has mononuclear copper ions in domains 2, 4 and 6 and a trinuclear copper between domains 1 and 6.

The reaction of nitric oxide with copper proteins and the photodissociation of copper-NO complexes.

The reactivity with nitric oxide was investigated for a number of type-1, type-2 and type-3 copper proteins azurin from Pseudomonas aeruginosa (type-1 copper); bovine superoxide dismutase, diamine oxidase from pig kidney and galactose oxidase from Dactylium dendroides (type-2 copper); haemocyanin from Helix pomatia (type-3 copper); the blue oxidases ceruloplasmin from pig serum, and ascorbate oxidase from Cucurbita pepo medullosa. Type-1 copper formed complexes with NO in the oxidised state, which complexes were only fully formed at low temperatures and could be photodissociated at 77K. Complex formation led to the disappearance of the EPR signal of type-1 copper and of the optical absorbance band in the 600 nm region. In azurin, photodissociation caused the reappearance of the original 625 nm absorbance band, but in the blue oxidases, a new band with lower intensity was found at 595 nm instead of the original absorbance band at 610 nm. In all cases, the EPR signal of type-1 copper did not return. These results are best explained by the formation of a photolabile type-1 Cu1+-NO+ complex. They also indicate that in the complex formed, the type-1 copper structure is probably not disrupted, and that after illumination, the nitric oxide molecule is still in the near vicinity of the copper atom. Type-2 copper did not react at all with nitric oxide, and type-3 copper formed complexes with nitric oxide in both the oxidised and the reduced state, but photodissociation of these complexes could not be demonstrated.