Edward P. C. Lai

Determination of theophylline in serum by molecularly imprinted solid-phase extraction with pulsed elution.

The technique of molecular imprinting is used to produce an extensively cross-linked poly(methacrylic acid-co-ethylene dimethacrylate) material that contains theophylline as a print molecule. After Soxhlet extraction of the theophylline, binding sites are formed in the polymer with complementary size, shape, and positioning of chemical functionalities. The molecularly imprinted polymers (MIP) high theophylline selectivity, chemical stability, and physically robust nature make it an ideal stationary-phase material in columns for HPLC separation of theophylline from other structurally related drug compounds. Mobile-phase tests confirm that a retention mechanism typical of normal-phase chromatography governs the separation, and selectivity of the MIP column can be controlled by a combination of the mobile phase and the sample solvent. Under optimal conditions, the MIP column functions like a solid-phase sorbent for theophylline extraction. Rapid elution of the bound theophylline can be accomplished in a pulsed format through injection of 20 μL of a solvent that has the proper polarity and protic nature to disrupt the electrostatic interactions and hydrogen bonding between theophylline and binding sites. A concentration detection limit of 120 ng/mL is obtained using direct UV absorption detection at 270 nm, which corresponds to a mass detection limit of 2.4 ng. This new technique, molecularly imprinted solid-phase extraction with pulsed elution (MISPE-PE), permits on-line preconcentration of theophylline from a large volume of dilute sample solution. Using a sample volume of 300 μL, a 40 ng/mL standard solution produces a detectable peak signal. Application of MISPE-PE in serum analysis further demonstrates the high capability of the MIP column to selectively isolate theophylline from other matrix components for fast, accurate determination.

Capillary electrophoresis: the state-of-the-art in metal speciation studies

An overview of the state-of-the-art of capillary electrophoresis (CE) for metal speciation analysis is presented. Various CE separation approaches and detection modes applied are discussed. A comprehensive description of reported methods to date in CE speciation studies of individual metals in different oxidation states, metal complexes with inorganic and organic ligands, oxoanions and organometallic compounds is demonstrated. The potential of CE for characterisation of metal interactions with large molecules like humic substances and proteins is also described. Several examples are presented to demonstrate CEs ability to solve real-world speciation problems. Further, the current strengths and limitations of CE with regard to speciation studies are discussed. The performance of CE is also critically evaluated in relationship to conventional chromatographic techniques used in metal speciation determination.

Determination of nicotine in tobacco by molecularly imprinted solid phase extraction with differential pulsed elution

A molecularly imprinted solid phase extraction-differential pulsed elution (MISPE-DPE) method has been developed for the determination of nicotine in tobacco. Nicotine and a trace alkaloid myosmine were able to bind to the molecularly imprinted polymer (MIP) packed in a micro-column when acetonitrile was used as the mobile phase. However, over 95% of the bound myosmine could be desorbed and washed away by a 20 µl pulse of methanol, compared to only 43% of the bound nicotine. The remaining quantity of bound nicotine was desorbed by a 20 µl pulse of 1% trifluoroacetic acid in water, for direct UV detection at 254 nm. This MISPE-DPE method provided inherent selectivity for nicotine that allowed both a shorter analysis time (3 min) and a lower analysis cost than liquid chromatographic methods. A detection limit of 1.8 µg ml–1 and a linear dynamic range up to 1000 µg ml–1 were obtained. Preconcentration of 845 µl of a 10 ng ml–1 nicotine standard solution produced a detectable peak signal. These analytical figures of merit are superior to those reported previously for several nicotine–MIP-based methods.

Capillary electrophoresis for the characterization of quantum dots after non-selective or selective bioconjugation with antibodies for immunoassay

Capillary electrophoresis coupled with laser-induced fluorescence was used for the characterization of quantum dots and their conjugates to biological molecules. The CE-LIF was laboratory-built and capable of injection (hydrodynamic and electrokinetic) from sample volumes as low as 4 μL via the use of a modified micro-fluidic chip platform. Commercially available quantum dots were bioconjugated to proteins and immunoglobulins through the use of established techniques (non-selective and selective). Non-selective techniques involved the use of EDCHCl/sulfo-NHS for the conjugation of BSA and myoglobin to carboxylic acid-functionalized quantum dots. Selective techniques involved 1) the use of heterobifunctional crosslinker, sulfo-SMCC, for the conjugation of partially reduced IgG to amine-functionalized quantum dots, and 2) the conjugation of periodate-oxidized IgGs to hydrazide-functionalized quantum dots. The migration times of these conjugates were determined in comparison to their non-conjugated QD relatives based upon their charge-to-size ratio values. The performance of capillary electrophoresis in characterizing immunoconjugates of quantum dot-labeled IgGs was also evaluated. Together, both QDs and CE-LIF can be applied as a sensitive technique for the detection of biological molecules. This work will contribute to the advancements in applying nanotechnology for molecular diagnosis in medical field.

Molecularly imprinted polymers for ochratoxin a extraction and analysis.

Molecularly imprinted polymers (MIPs) are considered as polymeric materials that mimic the functionality of antibodies. MIPs have been utilized for a wide variety of applications in chromatography, solid phase extraction, immunoassays, and sensor recognition. In this article, recent advances of MIPs for the extraction and analysis of ochratoxins are discussed. Selection of functional monomers to bind ochratoxin A (OTA) with high affinities, optimization of extraction procedures, and limitations of MIPs are compared from different reports. The most relevant examples in the literature are described to clearly show how useful these materials are. Strategies on MIP preparation and schemes of analytical methods are also reviewed in order to suggest the next step that would make better use of MIPs in the field of ochratoxin research. The review ends by outlining the remaining issues and impediments.

Rapid determination of theophylline in serum by selective extraction using a heated molecularly imprinted polymer micro-column with differential pulsed elution

Molecular imprinting of theophylline in poly(methacrylic acid ethylene dimethacrylate) form binding sites with complementary size, shape and chemical functionalities to theophylline. This molecularly imprinted polymer (MIP) can be packed into a micro-column for selective solid phase extraction (SPE) of theophylline from 20 microl of sample solution. Its chemical inertness and thermal stability allow the use of various organic solvents and elevated column temperatures for effective binding of theophylline. Non-specific adsorption of interfering drugs on the MIP surface is eliminated by an intermediate wash with 20 microl of acetonitrile, prior to quantitative desorption of the bound theophylline by 20 microl of methanol for in-line UV spectrophotometric determination. In this differential pulsed elution (DPE) technique, both the column temperature and solvent flow rate can be optimized to enhance selectivity. Application of this micro-analytical method, molecularly imprinted solid phase extraction DPE (MISPE-DPE), is demonstrated for accurate determination of theophylline in human blood serum. The method is validated over a linear range from 2 microg/ml to at least 20 microg/ml.

Molecularly imprinted solid phase extraction for rapid screening of metformin

Abstract A molecularly imprinted solid phase extraction (MISPE) method has been developed for the rapid screening of metformin. Newly synthesized molecularly imprinted polymer (MIP) particles were slurry-packed into a micro-column for selective solid phase extraction (SPE) of metformin. With CH 3 CN flowing at 0.5 ml/min, a total binding capacity of 1600 ng metformin was determined for the 20 mg of MIP particles. A broad range of MISPE conditions was evaluated with respect to sample solvent, pH, and buffer compositions. A 95±2% binding could be achieved from one 20-μl injection of sample solution in acetonitrile plus phosphate buffer, up to 1200 ng of metformin. However, the micro-column interacted indiscriminately with phenformin, a structural analogue, to attain 49±2% binding. Separation of phenformin from metformin was ultimately achieved, using differential pulsed elution (DPE) with 1 M trifluoromethacrylic acid in acetonitrile. Final pulsed elution (FPE) using 3% trifluoroacetic acid in methanol was good for the quantitative elution of metformin. The MISPE–DPE–FPE method, with UV detection at 240 nm, afforded a detection limit of 0.8 μg/ml (or 16 ng) for metformin. Each MISPE–DPE–FPE analysis required less than 5 min to complete.

Separation of carbonyl 2,4-dinitrophenylhydrazones by capillary electrochromatography with diode array detection.

The applicability of capillary electrochromatography (CEC) with photodiode array detection for the analysis of carbonyl hydrazones is presented. The CEC separation of thirteen hydrazones was optimized by a systematic variation of conditions using a commercially available CE system and a 3-micron porous C18-bonded silica capillary column. The separation profile obtained under optimal isocratic conditions (60% actonitrile-4% tetrahydrofuran-5 mM Tris, pH 8) is similar to those reported for gradient HPLC, with significant improvements in efficiency (to 150,000-250,000 theoretical plates/m) and analysis time (by a factor of 4). The retention time reproducibility is better than 0.2% (RSD) from run to run and 1% from day to day. The limits of detection for individual carbonyl hydrazones range between 0.1 and 0.5 microgram/ml. Applications to ambient air and automobile exhaust are shown.

Time‐of‐flight mass spectrometry of bioorganic molecules by laser ablation of silver thin film substrates and particles

A laser desorption/ionization (LDI) technique, which uses laser ablation of a thin silver film substrate under vacuum conditions to desorb and ionize bioorganic molecules, was developed for molecular mass and structural reactivity analysis in time-of-flight mass spectrometry (TOF-MS). After a sample overlayer is deposited by solvent evaporation on a thin silver film substrate, it is subjected to 355 or 532 nm Nd:YAG laser light by back-irradiation. Photoablation of the silver film substrate occurs with sufficient laser fluence, producing Agn+ (n=1–9) cluster cations which can react with the desorbed bioorganic molecules in the gas phase to form M+ or [M+H]+ and [M+Ag]+ ions for TOF-MS analysis. This LDI technique has been successfully applied to dithizone, benzo[e]pyrene, 1,4,8,11-tetraazocyclotetradecane, dicyclohexyl-18-crown-6, [5]-helicene dendrimer, gramicidin S, substance P and melittin. One advantage of this method over conventional LDI techniques is that the sample does not need to have appreciable spectral absorption at the laser wavelength. The use of silver in thin-film substrates affords analyte-dependent efficiencies that may serve for the direct and accurate mass analysis of specific groups of bioorganic molecules in sample mixtures. In a new sample preparation method, gramicidin S is added to a Tollen’s reagent mixture for direct impregnation on to silver particles during their formation and growth in the colloidal solution. These silver particles provide a silver matrix for the analyte molecules, which can enhance the LDI efficiency to produce greater [M+H]+ and [M+Ag]+ signals.

Antimicrobial effect of polydopamine coating on Escherichia coli

Discovery of new antimicrobials is highly desired due to the emergence of microorganisms that have multi-drug resistant capability. The purpose of this study was to investigate the antimicrobial effect of dopamine (DA) on Escherichia coli. DAs inhibitory activity was tested at different initial E. coli cell concentrations. Significant 3.5 and 4.2 log inhibitions were observed for 1 × 108 and 1 × 107 cells per mL, when compared to controls, corresponding to 99.97 and 99.99% inhibitions. Microscopic techniques (optical, fluorescence, and scanning electron) and Fourier transform infrared spectroscopy confirmed a polydopamine coating on the bacterial cells. Capillary electrophoresis with ultra-violet detection indicated a significant change in the cell structure caused by a low dose (100 mg L−1) of DA within 200 min of incubation.