Foodborne Pathogen Screening Using Magneto-fluorescent Nanosensor: Rapid Detection of E. Coli O157:H7.

Enterohemorrhagic Escherichia coli O157:H7 has been linked to both waterborne and foodborne illnesses, and remains a threat despite the food- and water-screening methods used currently. While conventional bacterial detection methods, such as polymerase chain reaction (PCR) and enzyme-linked immunosorbent assays (ELISA) can specifically detect pathogenic contaminants, they require extensive sample preparation and lengthy waiting periods. In addition, these practices demand sophisticated laboratory instruments and settings, and must be executed by trained professionals. Herein, a protocol is proposed for a simpler diagnostic technique that features the unique combination of magnetic and fluorescent parameters in a nanoparticle-based platform. The proposed multiparametric magneto-fluorescent nanosensors (MFnS) can detect E. coli O157:H7 contamination with as little as 1 colony-forming unit present in solution within less than 1 h. Furthermore, the ability of MFnS to remain highly functional in complex media such as milk and lake water has been verified. Additional specificity assays were also used to demonstrate the ability of MFnS to only detect the specific target bacteria, even in the presence of similar bacterial species. The pairing of magnetic and fluorescent modalities allows for the detection and quantification of pathogen contamination in a wide range of concentrations, exhibiting its high performance in both early- and late-stage contamination detection. The effectiveness, affordability, and portability of the MFnS make them an ideal candidate for point-of-care screening for bacterial contaminants in a wide range of settings, from aquatic reservoirs to commercially packaged foods.

Multiparametric Magneto-fluorescent Nanosensors for the Ultrasensitive Detection of Escherichia coli O157:H7.

Enterohemorrhagic Escherichia coli O157:H7 presents a serious threat to human health and sanitation and is a leading cause in many food- and waterborne ailments. While conventional bacterial detection methods such as PCR, fluorescent immunoassays and ELISA exhibit high sensitivity and specificity, they are relatively laborious and require sophisticated instruments. In addition, these methods often demand extensive sample preparation and have lengthy readout times. We propose a simpler and more sensitive diagnostic technique featuring multiparametric magneto-fluorescent nanosensors (MFnS). Through a combination of magnetic relaxation and fluorescence measurements, our nanosensors are able to detect bacterial contamination with concentrations as little as 1 colony-forming unit (CFU). The magnetic relaxation property of our MFnS allow for sensitive screening at low target CFU, which is complemented by fluorescence measurements of higher CFU samples. Together, these qualities allow for the detection and quantification of broad-spectrum contaminations in samples ranging from aquatic reservoirs to commercially produced food.

Inhibition of choline uptake by N-cyclohexylcholine, a high affinity ligand for the choline transporter at the blood-brain barrier.

The blood-brain barrier (BBB) choline transporter (CHT) may have utility as a drug delivery vector for drugs that act in the central nervous system. Previous studies suggested the importance of hydrophobic moieties on the cationic nitrogen of choline for improved affinity for this transporter. In a pilot study, we therefore designed five novel N-cycloalkyl derivatives of choline, one of which showed promising inhibition properties. This choline analogue had a cyclohexyl (UMBB-5) moiety substituting one of the methyl groups attached to the cationic nitrogen in choline. In situ experimental data were obtained from in situ rat brain perfusion studies. The binding affinity for the BBB-choline transporter found for UMBB-5 was K(i)=1.9 microM. Comparative molecular field analysis (CoMFA) suggested that the cyclohexyl moiety orientates towards a steric favourable area. Taken together, the results of these in situ and in silico studies provide further evidence or restrictions that occur with binding to this brain drug delivery vector.

Cation transport specificity at the blood-brain barrier.

UNLABELLED: The molecular identification, expression and cloning of membrane-bound organic cation transporters are being completed in isolated in vitro membranes. In vivo studies, where cation specificity overlaps, need to complement this work. METHOD: Cross-inhibition of [3H]choline and [3H]thiamine brain uptake by in situ rat brain perfusion. RESULTS: [3H]Choline brain uptake was not inhibited by thiamine at physiologic concentrations (100 nM). However, choline ranging from 100 nM to 250 microM inhibited [3H]thiamine brain uptake, though not below levels observed at thiamine concentrations of 100 nM. CONCLUSION: (1) The molecular family of the blood-brain barrier (BBB) choline transporter may be elucidated in vitro by its interaction with physiologic thiamine levels, and (2) two cationic transporters at the BBB may be responsible for thiamine brain uptake.

Molecular modeling studies on the active binding site of the blood-brain barrier choline transporter.

The blood-brain barrier choline transporter may have utility as a drug delivery vector to the central nervous system. Surprisingly, this transporter has as yet not been cloned and expressed. We therefore initiated a 3D-QSAR study to develop predictive models for compound binding and identify structural features important for binding to this transporter. In vivo experimental data were obtained from in situ rat brain perfusion studies. Comparative molecular field analysis (CoMFA) and comparative molecular similarity index analysis (CoMSIA) methods were used to build the models. The best cross-validated CoMFA q(2) was found to be 0.47 and the non-cross-validated r(2) was 0.95. CoMSIA hydrophobic cross-validated q(2) was 0.37 and the non-cross-validated r(2) was 0.85. These models rendered a useful approximation for binding requirements in the BBB-choline transporter and, until such time as the cloned transporter becomes available, may have significant utility in developing a predictive model for the rational design of drugs targeted to the brain.