Enhanced Performance of Reagent-Less Carbon Nanodots Based Enzyme Electrochemical Biosensors

This work reports on the advantages of using carbon nanodots (CNDs) in the development of reagent-less oxidoreductase-based biosensors. Biosensor responses are based on the detection of H2O2, generated in the enzymatic reaction, at 0.4 V. A simple and fast method, consisting of direct adsorption of the bioconjugate, formed by mixing lactate oxidase, glucose oxidase, or uricase with CNDs, is employed to develop the nanostructured biosensors. Peripherical amide groups enriched CNDs are prepared from ethyleneglycol bis-(2-aminoethyl ether)-N,N,N′,N′-tetraacetic acid and tris(hydroxymethyl)aminomethane, and used as precursors. The bioconjugate formed between lactate oxidase and CNDs was chosen as a case study to determine the analytical parameters of the resulting L-lactate biosensor. A linear concentration range of 3.0 to 500 µM, a sensitivity of 4.98 × 10−3 µA·µM−1, and a detection limit of 0.9 µM were obtained for the L-lactate biosensing platform. The reproducibility of the biosensor was found to be 8.6%. The biosensor was applied to the L-lactate quantification in a commercial human serum sample. The standard addition method was employed. L-lactate concentration in the serum extract of 0.9 ± 0.3 mM (n = 3) was calculated. The result agrees well with the one obtained in 0.9 ± 0.2 mM, using a commercial spectrophotometric enzymatic kit.

Improved radioimmunoassay of melatonin in serum.

Melatonin was extracted from serum by using Baker reversed-phase C-18 columns. More than 99% of the applied melatonin was retained by the columns, and more than 97% was eluted from the columns in 300 microL of methanol. We then determined melatonin in the serum extract by a modification of a standard radioimmunoassay, using filtration instead of centrifugation to collect the [3H]melatonin-antibody complex precipitated by saturated ammonium sulfate. These modifications allow more rapid, accurate, and reproducible determination of melatonin than do previously published procedures.

Unsaturated fatty acids as endogenous inhibitors of tamoxifen binding to anti-oestrogen-binding sites

It is known that triphenylethylene anti-oestrogens such as tamoxifen bind to specific high-affinity anti-oestrogen-binding sites, which are distinct from oestrogen receptors. These binding sites are widely distributed in human and animal tissues, but their function and endogenous ligands are unknown. By using [3H]tamoxifen and a rat liver microsomal fraction, a radio-ligand-binding assay was developed in an attempt to identify endogenous ligands for the anti-oestrogen-binding sites in the rat. An ether extract of rat serum inhibited [3H]tamoxifen binding to rat liver binding sites in a dose-dependent manner. Identification of the active serum constituents that inhibited [3H]tamoxifen binding was achieved by g.l.c.-mass spectrometry after preliminary purification of a rat serum extract by silica-gel t.l.c. Three unsaturated fatty acids (oleic, linoleic and arachidonic) accounted for about 50% of the total inhibiting activity of the serum extract. The concentrations of these fatty acids required to inhibit [3H]tamoxifen binding were in the range of 10-100 microM, comparable with those found in the rat circulation under physiological conditions. Saturated fatty acids present in rat serum (palmitic and stearic) did not inhibit [3H]tamoxifen binding. A survey of other fatty acids revealed that, in general, unsaturated fatty acids were far more potent than saturated fatty acids in inhibiting [3H]tamoxifen binding. These studies demonstrate that unsaturated fatty acids are quantitatively the most important circulating inhibitors of [3H]tamoxifen binding to the anti-oestrogen-binding sites. The biological significance of their interaction with these sites, however, remains to be clarified.

Improved determination of serum theophylline by gas chromatography with use of a nitrogen-phosphorus detector.

We describe a method for the 7-min determination of theophylline in less than 100 muL of serum. The procedure requires no centrifugation or solvent evaporation after extraction. Butylation is done on the gas-chromatographic column by injecting the serum extract followed by a butylating mixture which contains 1-iodobutane as the alkylating agent. The method is precise, accurate, and free of interference. Results correlate well with those by ultraviolet spectrophotometry.

FURTHER STUDIES WITH TOXIC SERUM EXTRACTS OF HEMOLYTIC STREPTOCOCCI

1. The same Streptococcus hemolyticus organisms may be subjected to extraction six times in 2 days with untreated inactivated serum with no loss in potency of the later extracts when the organisms are kept frozen solid during the night between the extractions. 2. The serum extract toxins of hemolytic streptococci can be preserved without deterioration for at least 6 months if kept frozen solid. 3. No toxins stronger than those containing 10 units per cc. for mice have been prepared. Reasons for thinking that this is due to the saturation of the serum with the toxin at this point are given. 4. Half saturation with (NH4)2SO4 precipitates out practically all of the hemotoxin in a preparation. 5. Serum extracts were made from strains of hemolytic streptococci other than the Gay strain and attempts were made to correlate the virulence and toxin production from each strain. No such correlation could be established. 6. The principal pathologic finding in mice inoculated with the streptococcus serum extract toxin is a marked degeneration of the tubular epithelium of the kidney.