Application of Antibody-Powered Triplex-DNA Nanomachine to Electrochemiluminescence Biosensor for the Detection of Anti-Digoxigenin with Improved Sensitivity Versus Cycling Strand Displacement Reaction.

The accurate and rapid quantitative detection of antibodies had a significant influence in controlling and preventing disease or toxin outbreaks. In this work, we first introduce the antibody-powered triplex-DNA nanomachine to release cargo DNA as a substitute target for sensitive electrochemiluminescence (ECL) detection of anti-digoxigenin based on a novel ternary ECL system. It is worth noting that the cargo DNA as a substitute target of antibody can further participate in an enzyme-assisted cycling strand displacement reaction to achieve ECL signal amplification and improve the sensitivity of antibody detection. Additionally, porous palladium nanospheres with a considerable catalytic activity were first applied as a coreaction accelerator to efficiently enhance the intensity of the ECL system of rubrene microblocks as luminophore and dissolved O2 as an endogenous coreactant. With the resultant ternary ECL system as a biosensing platform, a significantly enhanced initial signal was achieved in advance. Then, the ferrocene-labeled quenching probes were employed to reduce initial signal and obtain the low-background signal. Eventually, the cargo DNA made the quenching probes release and recover the signal in the presence of anti-digoxigenin. Thereupon, the wide linear range (0.01-200 nM) and low limit of detection (6.7 pM) were obtained, and this method not only reduces conjugation steps but also provides a sensitive and novel ECL analysis platform for the trace detection of other antibodies and antigen.

A novel phytochemical, digoxigenin-3-O-rutin in the amelioration of isoproterenol-induced myocardial infarction in rat: a comparison with digoxin.

INTRODUCTION: The commonly used cardiac glycoside, digoxin (DIG), has a narrow therapeutic window. Although some investigations were made to counteract its toxic effects, no alternate phytochemical is available till date that is more potent and safer than DIG. AIMS: Our main aim was to isolate a novel cardenolide from the seeds of Trigonella foenum graceium and to evaluate its relative potential in comparison to that of DIG. EXPERIMENTAL DESIGN: In one experiment effects of the isolated compound at 2.5, 5.0, and 10 mg/kg (p.o.) were evaluated in isoproterenol (ISO)-induced cardiovascular problems in rats. As the test drug (TDR) reversed most of the ISO-induced changes, it was subjected to the phytochemical analyses and was identified as digoxigenin-3-O-rutin. In another experiment effects of DIG and rutin (Rtn) were compared with those of TDR or DIG alone. The hydroxyl radical scavenging activity was also measured by electron spin resonance (EPR). RESULTS: digoxigenin-3-O-rutin at 10 mg/kg markedly reduced the ISO-induced increase in cardiac lipid peroxidation and in the levels of serum creatinine phosphokinase-MB, glutamate oxaloacetate transaminase, glutamate pyruvate transaminase, lactate dehydrogenase, and creatinine. It also reversed the ISO-induced changes in the cardiac histomorphology. Interestingly TDR appeared to be more effective than DIG alone or DIG and Rtn in combination. CONCLUSION: The newly isolated digoxigenin-3-O-rutin appears to be more potent and safe than digoxin. Its higher efficacy could be due to its structural specificity and might have been mediated through its better free radical scavenging action.

Immunoaffinity screening with capillary electrochromatography.

Highly efficient capillary electrochromatographic separations of cardiac glycosides and other steroids are presented. Employing butyl-derivatized silica particles as stationary phase resulted in a nearly three times faster electroosmotic flow (EOF) compared to capillary electrochromatography (CEC) with octadecyl silica particles. On-column focusing with a preconcentration factor of 180 was performed and separation efficiencies of up to 240,000 plates per meter were obtained. Using label-free standard UV absorbance, detection limits of 10-80 nM were reached for all steroids tested. For screening of cardiac glycosides, e.g., digoxin and digitoxin in mixtures of steroids, CEC was combined with immunoaffinity extraction using immobilized polyclonal anti-digoxigenin antibodies and F(ab) fragments. Simply adding small amounts of antibody carrying particles to the samples and comparing chromatograms before and after antibody addition allowed screening for high affinity antigens in mixtures with moderate numbers of compounds. Under conditions of competing antigens, affinity fingerprints of immobilized anti-digoxigenin and anti-digitoxin antibodies were obtained, reflecting the cross-reactivity of eleven steroids. The method provides high selectivity due to the combination of bioaffinity interaction with highly efficient CEC separation and UV detection at several wavelengths in parallel. This selectivity was exploited for the detection of four cardiac glycosides in submicromolar concentrations in an untreated urine sample.

Efficient synthesis of 3-aminodigoxigenin and 3-aminodigitoxigenin probes.

Oxidation of digoxigenin and digitoxigenin to the 3-ketones followed by reductive amination produced a mixture of amine epimers. The inability to separate the epimeric mixtures of chemiluminescent digoxigenin probes derived by conjugation to the acridinium label prompted us to develop an HPLC method to separate the amines. Labeling of the pure amines resulted in good yields of the isomerically pure probes.