Novel luminescent affiprobes for molecular detection of Staphylococcus aureus using flow cytometry.

AIMS: Diagnosis of Staphylococcus aureus is important in various diseases from hospital-acquired infections to foodborne diseases. This work reports two new luminescent affiprobes for specific detection of S. aureus. METHODS AND RESULTS: To develop advanced luminescent affiprobes, enhanced green fluorescent protein (EGFP) was flanked by single and double repeats of ZpA963 affibody using molecular biology studies. The recombinant proteins including fluorescent monomeric affibody (fA1 ) and fluorescent dimeric affibody (fA2 ) were expressed in the bacterial expression system, purified and used to identify the S. aureus. Fluorescence microscope and flow cytometry results demonstrated that the treated samples with fA1 and fA2 had relatively high fluorescent mean intensities in comparison to the untreated S. aureus cells. Moreover, it was revealed that 'fA2 ' affiprobe had lower dissociation constant value (about 25-fold) and was more effective for detection of S. aureus than the 'fA1 ' affiprobe. In addition, the binding of the affiprobes for some other pathogenic bacteria i.e. Escherichia coli, Bacillus cereus, Enterococcus faecalis and Staphylococcus saprophyticus was examined. Expectedly, no cross-reaction was observed for binding the constructed affiprobes to these bacteria, eliminating possibilities for false positive results. CONCLUSIONS: The results show that 'fA1 ' affiprobe and 'fA2 ' affiprobe are two new efficient luminescent affiprobes for detecting S. aureus. SIGNIFICANCE AND IMPACT OF THE STUDY: We developed a new approach for detection of Staphylococcus aureus in a simple one-step process and in low concentrations of probes. In the best of our knowledge, this is the first study to direct detection of bacterial cells by affiprobes and may be used to develop new diagnostic kits.

Glycated albumin precipitation using aptamer conjugated magnetic nanoparticles.

To develop a strategy for the elimination of prefibrillar amyloid aggregates, a three-step non-modified DNA aptamer conjugation on silica-coated magnetic nanoparticles was carried out to achieve aptamer conjugated on MNP (Ap-SiMNP). Prefibrillar amyloid aggregates are generated under a diabetic condition which are prominently participated in developing diabetic complications. The binding properties of candidate DNA aptamer against serum albumin prefibrillar amyloid aggregates (AA20) were verified using electrophoretic mobility shift assay (EMSA) and surface plasmon resonance spectroscopy (SPR) analysis. The chloro-functionalized silica-coated MNPs were synthesized then a nano-targeting structure as aptamer conjugated on MNP (Ap-SiMNP) was constructed. Finally, Ap-SiMNP was verified for specific binding efficiency and AA20 removal using an external magnetic field. The candidate aptamer showed a high binding capacity at EMSA and SPR analysis (KD = 3.4 × 10─9 M) and successfully used to construct Ap-SiMNP. Here, we show a proof of concept for an efficient bio-scavenger as Ap-SiMNP to provide a promising opportunity to consider as a possible strategy to overcome some diabetic complications through specific binding/removal of toxic AA20 species.