Urease
has been covalently immobilized on a 3-D networking silica
gel (SG) using dimethyldichlorosilane (DMDCS) as second generation
silane coupling reagent and m-nitroaniline as linker
component in a robust methodology and subsequently characterized as
[{Si(OSi)4(H2O)0.05}205.2]
n=4{OSi(CH3)2–NH–C6H4–NN-urease}·282.5H2O (molecular mass 263 445 g or 263.4 kDa). Selective coupling
of tyrosine residue with an identifiable m-nitroaniline
modified SG unit prevents enzyme–enzyme cross-linking leading
to enhancement of enzymatic activity. The material worked at room
temperature and its activity (luminescent and ammonia releasing efficiency)
was enhanced by 3-fold (for both synthetic and real sample) compared
to native enzyme values at neutral pH. Up to 30 days and 30 cycles,
this 3-fold activity remains as such but reduces gradually to native
enzyme level after 60 days and 60 cycles of reuse.
A strategy for rapid in situ elimination of interfering substances that are present in extracts of food samples during assay is described in this article. The novel feature of this method is that the sample purification is carried out as a part of the assay, and a separate sample cleanup step is not required. The assay procedure involves the sequential addition of standard or sample, cleaning solutions, and aflatoxin B1-horseradish peroxidase conjugate (AFB1-HRP) over antibody-spotted zones of a membrane, and 3,3'-diaminobenzidine was used as the substrate for visualization. We have determined that trifluoroacetic acid and propionic acids at concentrations of 100 mM are highly effective for cleaning groundnut, wheat, corn, and poultry feed samples and that NaHCO3 (100 mM) is successful in cleaning processed soybean. In all cases, subsequent washing was performed with phosphate-buffered saline solution to facilitate the removal of traces of adhering interfering substances. A batch of 12 samples can be analyzed within 8 min either by visual comparison of the color intensity (inversely related to the analyte concentration) of a sample spot with those of reference standards or, more precisely, by densitometry. The method was tested for the analysis of AFB1 in groundnut, wheat, corn, processed soybean, chili, and poultry feed. The detection limit obtained was 5 microg/kg, except for chili, where it was 10 microg/kg. The average recoveries from different noninfected food samples spiked with AFB1 at concentrations of 5 to 100 microg/kg were between 99 and 105%. The values obtained for infected corn and groundnut samples correlated well with the estimates obtained by high-pressure liquid chromatography. The absence of a sample extraction step reduces the cost and labor involved in the assay. The method may be potentially applicable to the assay of other mycotoxins and environmental pollutants.
By
the use of a new silane coupling reagent, dimethyldichlorosilane
(DMDCS), effective and instantaneous immobilization of 8-hydroxyquinoline
(HQ) on an inorganic carrier (silica gel, SG) has been carried out
for the facile synthesis of an extractor material (composition: {Si(OSi)
p=4(H2O)
x=0.16}
n=11[−Si(CH3)2–NH–C6H4–NN–HQ]
z=4·25H2O; molar mass: 4010.3
g/mol). The material (thermal stability: ≤100 °C; chemical
stability: ≤8 M HNO3) possesses a high Brunauer–Emmett–Teller
surface area (BET-SAFe(III): 1170 m2·g–1), an appreciable preconcentration factor (PFFe(III): 145.1), and high breakthrough capacity (BTCFe(III): column exchange capacity, 269 μmol·g–l; Langmuir Q
0, 278.6 μmol·g–1) for Fe(III). Along with these discernible analytical
qualities, a high level of reusability (<800 cycles @ 95% recovery)
reflects the material warranty. Fe(III), present as [Fe(OH)(H2O)5]2+ at the recommended pH (1.90 ±
0.15), binds at the highest occupied molecular orbital (HOMO) of the
sorbent (η = 7.69 eV) through hard–soft binding with
an appreciable binding energy (−14.2 eV). The breakthrough
capacity (BTC: 269–278.6 μmol·g–1) was found to be the product of the amount of extractor HOMO (280
μmol·g–1) and the degree of polymerization
of the adsorbed metal ion, x (i.e., BTC = [amount
of HOMOextractor (μmol·g–1)] × x for monomeric (x =
1) and polymeric (x > 1) species). The findings
reveal
substantial improvement of Weetall–Hill immobilization of chelating
ligands on inorganic carriers.
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