High-Throughput Analysis of Bacterial Toxic Lipopolysaccharide in Water by Dual-Wavelength Monitoring Using a Ratiometric Fluorescent Chemosensor.

Lipopolysaccharide (LPS) is a bacterial toxin that causes fever in humans. Our small-molecule chemosensor named Zn-dpa-C2OPy shows rapid ratiometric fluorescence response to LPS in water with a detection limit of 11 pM, which is lower than that of our previously reported sensor. Spectroscopic measurements (fluorescence, absorbance, 1H NMR, and fluorescence lifetime), dynamic light scattering measurements, and transmission electron microscopy observations revealed that the fluorescence response was induced by the changes in the aggregation state via multi-point recognition of LPS through hydrophobic and electrostatic interactions, in addition to the coordination between the zinc(II)-dipicolylamine moiety of the chemosensor and the phosphate group of LPS. The proposed Zn-dpa-C2OPy chemosensor was applied to an original flow injection analysis (FIA) system with a self-developed dual-wavelength fluorophotometer, and a high throughput of 36 samples per hour was achieved. These results demonstrate the feasibility of this unique methodology combining a ratiometric fluorescent chemosensor and FIA for continuous online monitoring of LPS in water.

Simple and Rapid Endotoxin Recognition Using a Dipicolylamine-Modified Fluorescent Probe with Picomolar-Order Sensitivity.

Endotoxin is a lipopolysaccharide (LPS) that is found in the outer membrane of the cell wall of Gram-negative bacteria. Due to its high toxicity, the allowable endotoxin limit for water for injection is set at a very low value. Conventional methods for endotoxin detection are time-consuming and expensive and have low reproducibility. A previous study has shown that dipicolylamine (dpa)-modified pyrene-based probes exhibit fluorescence enhancement in response to LPS; however, the application of such probes to the sensing of LPS is not discussed. Against this backdrop, we have developed a simple and rapid endotoxin detection method using a dpa-modified pyrenyl probe having a zinc(II) center (Zn-dpa-C4Py). When LPS was added into Zn-dpa-C4Py solution, excimer emission of the pyrene moiety emerged at 470 nm. This probe can detect picomolar concentrations of LPS (limit of detection = 41 pM). The high sensitivity of the probe is ascribed to the electrostatic and hydrophobic interactions between the probe and LPS, which result in the dimer formation of the pyrene moieties. We also found that Zn-dpa-C4Py has the highest selectivity for LPS compared with other phosphate derivatives, which is probably caused by the co-aggregation of the probe with LPS. We propose that Zn-dpa-C4Py is a promising chemical sensor for the detection of endotoxin in medical and pharmaceutical applications.

Track-etched membrane-based dual-electrode coulometric detector for microbore/capillary high-performance liquid chromatography.

The electrochemical flow cell containing track-etched microporous membrane electrodes was applied to a dual-electrode coulometric detector for microbore/capillary HPLC with a small injection volume and low eluent flow rate. The proposed flow cell with a 0.1-mm diameter inlet channel gave a detection volume of 0.08 nL per electrode, which was determined by the eluent flow through the electrode. For the dual-electrode detector, the calculated volume was 0.24 nL. The efficiency of electrooxidation of l-ascorbic acid increased as the flow rate decreased and was close to 100% when the flow rate was below 50 µL min-1, which is a common flow rate in microbore or capillary liquid chromatography. Catecholamines, such as noradrenaline, adrenaline, and dopamine, were detected by total conversion with two-electron oxidation in the potential range from 0.8 to 1.0 V vs. Ag/AgCl after separation with a microbore column. These peaks were accompanied by corresponding cathodic peaks derived from quasi-stable electrooxidation products of the catecholamines. The detection limits of noradrenaline, adrenaline, and dopamine were 0.1, 0.1, and 0.2 µM, respectively. The RSD values for five replicate measurements of 5.0 µM of these compounds were 0.9%, 0.7%, and 1.5%, respectively. Coulometric detection was also demonstrated by determination of catecholamines in pharmaceuticals.

Determination of ultra-trace mercury(II) by flow-injection/anodic stripping voltammetry using a track-etched microporous membrane electrode.

A new flow-injection/anodic stripping voltammetry has been demonstrated to assess ultra-trace mercury(II) using track-etched microporous membrane electrodes. The electrodes were prepared by the sputtering of gold or platinum onto both sides of a membrane filter with a smooth flat surface and with cylindrical pores having uniform diameter. The deposition of mercury from a mercury(II) solution was performed while the sample solution flowed through the membrane electrodes. After the deposition step, an anodic stripping voltammogram was obtained by sweeping the potential from 0 to +0.8 V vs. Ag/AgCl. In this case, the sample solution flowed through the pores of the 10-µm-thick membrane filters. Efficient electrolysis occurred during passage of the sample solution through the electrode, of which the pore size was 0.4 µm. In this study, the voltammetry described above was demonstrated using an FIA system. The continuous-flow mode showed a detection limit of 0.04 µg L(-1) when the experimental conditions of the flow rate and the deposition time were set at 0.5 mL min(-1) and 180 s. In the sample-injection mode equipped with a 1-mL sample loop, a linear relation was found for 0.5-4.0 µg L(-1) of a mercury(II) standard solution (r = 0.995). The detection limit was 0.05 µg L(-1). This method was applied to the ultra-trace determination of mercury(II) in river-water samples.

A dual-electrode flow sensor fabricated using track-etched microporous membranes.

A new dual-electrode flow sensor has been fabricated by piling the microporous membrane electrodes which have 7-10µm thickness. The electrode was prepared by sputtering of platinum onto both sides of the membrane filter which contain a smooth flat surface as well as cylindrical pores with uniform diameters. The electrolysis is performed when the sample solution flows through the membrane electrode, and a generated analyte on the first working electrode is instantaneously transported to the surface of second working electrode which is located at the downstream of the first one. In this case, the sample solution surely flows through the pores of the membrane filters. As the result, highly efficient electrolysis was achieved at each electrode, and the collection efficiency values as high as 100% were obtained in the wide range of flow rate. Good responses to the injections of sample solutions were also confirmed in the FIA system.

Adsorption of divalent transition metal ions with a chelating agent on octadecyl silica gel.

This investigation looked at the extraction ability of divalent transition metal ions onto an octadecyl silica gel (C18g) with a 4,4-trifluoro-1-(2-thienyl)-1,3-butadione (TTA) chelating agent. A method of retaining TTA onto C18g (TTA-C18g) was developed in order to adsorb the metal ions. The difference in the half-adsorption and half-extraction pH values between transition metals Ni2+-Co2+ was found to be 0.7 in this system. This is better than previously published results of 0.3 for the conventional extraction method using TTA in nitrobenzene. More than 96% of the metal ions in aqueous solution could be adsorbed onto TTA-C18g. Our system, which has no organic phase, can achieve a better removal or separation of transition metal ions than the conventional solvent-extraction methods using TTA in toluene or nitrobenzene.