Investigations on the generation of oil-in-water (O/W) nanoemulsions through the combination of ultrasound and microchannel.

O/W nanoemulsions are isotropic colloidal systems constituted of oil droplets dispersed in continuous aqueous media and stabilised by surfactant molecules. Nanoemulsions hold applications in more widespread technological domains, more crucially in the pharmaceutical industry. Innovative nanoemulsion-based drug delivery system has been suggested as a powerful alternative strategy through the useful means of encapsulating, protecting, and delivering the poorly water-soluble bioactive components. Consequently, there is a need to generate an emulsion with small and consistent droplets. Diverse studies acknowledged that ultrasonic cavitation is a feasible and energy-efficient method in making pharmaceutical-grade nanoemulsions. This method offers more notable improvements in terms of stability with a lower Ostwald ripening rate. Meanwhile, a microstructured reactor, for instance, microchannel, has further been realised as an innovative technology that facilitates combinatorial approaches with the acceleration of reaction, analysis, and measurement. The recent breakthrough that has been achieved is the controlled generation of fine and monodispersed multiple emulsions through microstructured reactors. The small inner dimensions of microchannel display properties such as short diffusion paths and high specific interfacial areas, which increase the mass and heat transfer rates. Hence, the combination of ultrasonic cavitation with microstructures (microchannel) provides process intensification of creating a smaller monodispersed nanoemulsion system. This investigation is vital as it will then facilitate the creation of new nanoemulsion based drug delivery system continuously. Following this, the fabrication of microchannel and setup of its combination with ultrasound was conducted in the generation of O/W nanoemulsion, as well as optimisation to analyse the effect of varied operating parameters on the mean droplet diameter and dispersity of the nanoemulsion generated, besides monitoring the stability of the nanoemulsion. Scanning transmission electron microscopy (STEM) images were also carried out for the droplet size measurements. In short, the outcomes of this study are encouraging, which necessitates further investigations to be carried out to advance a better understanding of coupling microchannel with ultrasound to produce pharmaceutical-grade nanoemulsions.

Linking otolith microchemistry and surface water contamination from natural gas mining.

Unconventional natural gas drilling and the use of hydraulic fracturing technology have expanded rapidly in North America. This expansion has raised concerns of surface water contamination by way of spills and leaks, which may be sporadic, small, and therefore difficult to detect. Here we explore the use of otolith microchemistry as a tool for monitoring surface water contamination from generated waters (GW) of unconventional natural gas drilling. We exposed Brook Trout in the laboratory to three volumetric concentrations of surrogate generated water (SGW) representing GW on day five of drilling. Transects across otolith cross-sections were analyzed for a suite of elements by LA-ICP-MS. Brook Trout exposed to a 0.01-1.0% concentration of SGW for 2, 15, and 30 days showed a significant (p < 0.05) relationship of increasing Sr and Ba concentrations in all but one treatment. Analyses indicate lesser concentrations than used in this experiment could be detectable in surface waters and provide support for the use of this technique in natural habitats. To our knowledge, this is the first demonstration of how trace elements in fish otoliths may be used to monitor for surface water contamination from GW.

Real-Time Microfluidic Blood-Counting System for PET and SPECT Preclinical Pharmacokinetic Studies.

UNLABELLED: Small-animal nuclear imaging modalities have become essential tools in the development process of new drugs, diagnostic procedures, and therapies. Quantification of metabolic or physiologic parameters is based on pharmacokinetic modeling of radiotracer biodistribution, which requires the blood input function in addition to tissue images. Such measurements are challenging in small animals because of their small blood volume. In this work, we propose a microfluidic counting system to monitor rodent blood radioactivity in real time, with high efficiency and small detection volume (∼1 µL). METHODS: A microfluidic channel is built directly above unpackaged p-i-n photodiodes to detect ß-particles with maximum efficiency. The device is embedded in a compact system comprising dedicated electronics, shielding, and pumping unit controlled by custom firmware to enable measurements next to small-animal scanners. Data corrections required to use the input function in pharmacokinetic models were established using calibrated solutions of the most common PET and SPECT radiotracers. Sensitivity, dead time, propagation delay, dispersion, background sensitivity, and the effect of sample temperature were characterized. The system was tested for pharmacokinetic studies in mice by quantifying myocardial perfusion and oxygen consumption with (11)C-acetate (PET) and by measuring the arterial input function using (99m)TcO4 (-) (SPECT). RESULTS: Sensitivity for PET isotopes reached 20%-47%, a 2- to 10-fold improvement relative to conventional catheter-based geometries. Furthermore, the system detected (99m)Tc-based SPECT tracers with an efficiency of 4%, an outcome not possible through a catheter. Correction for dead time was found to be unnecessary for small-animal experiments, whereas propagation delay and dispersion within the microfluidic channel were accurately corrected. Background activity and sample temperature were shown to have no influence on measurements. Finally, the system was successfully used in animal studies. CONCLUSION: A fully operational microfluidic blood-counting system for preclinical pharmacokinetic studies was developed. Microfluidics enabled reliable and high-efficiency measurement of the blood concentration of most common PET and SPECT radiotracers with high temporal resolution in small blood volume.

Ultrasensitive sensing platform for platelet-derived growth factor BB detection based on layered molybdenum selenide-graphene composites and Exonuclease III assisted signal amplification.

A highly sensitive and ultrasensitive electrochemical aptasensor for platelet-derived growth factor BB (PDGF-BB) detection is fabricated based on layered molybdenum selenide-graphene (MoSe2-Gr) composites and Exonuclease III (Exo III)-aided signal amplification. MoSe2-Gr is prepared by a simple hydrothermal method and used as a promising sensing platform. Exo III has a specifical exo-deoxyribonuclease activity for duplex DNAs in the direction from 3' to 5' terminus, however its activity is limited on the duplex DNAs with more than 4 mismatched terminal bases at 3' ends. Herein, aptamer and complementary DNA (cDNA) sequences are designed with four thymine bases on 3' ends. In the presence of target protein, the aptamer associates with it and facilitates the formation of duplex DNA between cDNA and signal DNA. The duplex DNA then is digested by Exo III and releases cDNA, which hybridizes with signal DNA to perform a new cleavage process. Nevertheless, in the absence of target protein, the aptamer hybridizes with cDNA will inhibit the Exo III-assisted nucleotides cleavage. The signal DNA then hybridizes with capture DNA on the electrode. Subsequently, horse radish peroxidase is fixed on electrode by avidin-biotin reaction and then catalyzes hydrogen peroxide and hydroquinone to produce electrochemical response. Therefore, a bridge can be established between the concentration of target protein and the degree of the attenuation of the obtained signal, providing a quantitative measure of target protein with a broad detection range of 0.0001-1 nM and a detection limit of 20 fM.

Strategy to rapidly discriminate trace isomeric lignan compounds from Gymnotheca chinensis by probe electrospray ionization tandem mass spectrometry.

Probe electrospray ionization (PESI) is a recently developed ionization technique based on electrospray ionization (ESI) that generates electrospray from the tip of a solid needle. High tolerance to salts, requirements of a trace amount of sample and direct ambient sampling- are major advantages of PESI compared with conventional ESI. In this report, three pairs of isomeric lignans bearing tetra-hydrofuran with variable conformations from Gymnotheca chinensis were investigated by probe electrospray tandem mass spectrometry (PESI-MS/MS) in the positive ion mode. The diagnostic characteristics of these compounds were obtained and the isomers could be successfully distinguished by comparison with their breakdown curves, even though the isomers differed only in the conformation of some groups of the isomer pairs. This report provides a rapid and reliable method for the identification of trace amounts of isomeric lignans by PESI-MS/MS. Furthermore, application of PESI and breakdown curves should have value in mass spectrometry studies of isomeric natural products compounds.

Ultrasensitive electrochemical immunoassay for CEA through host-guest interaction of ß-cyclodextrin functionalized graphene and Cu@Ag core-shell nanoparticles with adamantine-modified antibody.

A novel non-enzymatic immunoassay was designed for ultrasensitive electrochemical detection of carcino-embryonic antigen (CEA) using ß-cyclodextrin functionalized Cu@Ag (Cu@Ag-CD) core-shell nanoparticles as labels and ß-cyclodextrin functionalized graphene nanosheet (CD-GN) as sensor platform. CD-GN has excellent conductivity which promoted the electric transmission between base solution and electrode surface and enhanced sensitivity of immunosensor. In addition, owing to supramolecular recognition of CD-GN for the guest molecule, quite a few synthesized adamantine-modified primary antibodies (ADA-Ab1) were immobilized on the CD-GN by supramolecular host-guest interaction between CD and ADA. Cu@Ag-CD as a signal tag could be captured by ADA-modified secondary antibody (ADA-Ab2) through a host-guest interaction, leading to a large loading of Cu@Ag nanoparticles with high electrical conductivity and catalytic activity. The fabricated immunosensor exhibits excellent analytical performance for the measurement of CEA with wide range linear (0.0001-20 ng/mL), low detection limit (20 fg/mL), good sensitivity, reproducibility and stability, which provide an enormous application prospect in clinical diagnostics.

PIXE as a complement to ICP-OES trace metal analysis in Sudanese medicinal plants.

This paper compares trace element concentrations (Ca, K, Sr, Fe, Mn, Zn, Ni, Cu, Co and Cr) in 27 Sudanese medical plants determined in parallel by PIXE and ICP-OES to get information on which technique is preferable at different matrices and element concentrations. PIXE correlates well to ICP-OES for Sr, Mn, Ca, K, Zn and Fe determinations. ICP-OES seems to be the superior technique over PIXE when measuring low concentrated elements (chromium, cobalt, nickel and copper) in the medicinal plants.

Structure-selective hot-spot Raman enhancement for direct identification and detection of trace penicilloic acid allergen in penicillin.

Trace penicilloic acid allergen frequently leads to various fatal immune responses to many patients, but it is still a challenge to directly discriminate and detect its residue in penicillin by a chemosensing way. Here, we report that silver-coated gold nanoparticles (Au@Ag NPs) exhibit a structure-selective hot-spot Raman enhancement capability for direct identification and detection of trace penicilloic acid in penicillin. It has been demonstrated that penicilloic acid can very easily link Au@Ag NPs together by its two carboxyl groups, locating itself spontaneously at the interparticle of Au@Ag NPs to form strong Raman hot-spot. At the critical concentration inducing the nanoparticle aggregation, Raman-enhanced effect of penicilloic acid is ~60,000 folds higher than that of penicillin. In particular, the selective Raman enhancement to the two carboxyl groups makes the peak of carboxyl group at C6 of penicilloic acid appear as a new Raman signal due to the opening of ß-lactam ring of penicillin. The surface-enhanced Raman scattering (SERS) nanoparticle sensor reaches a sensitive limit lower than the prescribed 1.0‰ penicilloic acid residue in penicillin. The novel strategy to examine allergen is more rapid, convenient and inexpensive than the conventional separation-based assay methods.

Novel antioxidative peptides from the protein hydrolysate of oysters (Crassostrea talienwhanensis).

The antioxidative activity of hydrolysate peptides from oysters (Crassostrea talienwhanensis) was investigated. After hydrolysis with subtilisin, the yields of the peptides that were soluble in trichloroacetic acid (TCA-soluble) and the antioxidant activities of the resulting hydrolysate were determined using an orthogonal design and a hydroxyl radical scavenging reaction. The hydrolysate was fractionated using Sephadex G-15 gel filtration chromatography, and the two resulting bioactive peptides were subsequently purified by RP-HPLC with a Kromasil C18 (ODS) column. The amino acid sequences were analyzed by nano-ESI-MS/MS. The critical reaction temperature, pH, hydrolysis time and enzyme-to-substrate (E/S) ratio were determined for the optimum hydrolysis with subtilisin, and the E/S ratio was found to be the most critical reaction condition. The amino acid sequences of the peptides (518 and 440 Da) were proline-valine-methionine-glycine-aspartic acid (PVMGA) and glutamine-histidine-glycine-valine (QHGV), respectively. These two novel peptides exhibited high antioxidative actions based on their hydroxyl and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activities.

Rapid and direct low micromolar NMR method for the simultaneous detection of hydrogen peroxide and phenolics in plant extracts.

A rapid and direct low micromolar ¹H NMR method for the simultaneous identification and quantification of hydrogen peroxide and phenolic compounds in plant extracts was developed. The method is based on the highly deshielded ¹H NMR signal of H2O2 at ∼10.30 ppm in DMSO-d6 and the combined use of picric acid and low temperature, near the freezing point of the solution, in order to achieve the minimum proton exchange rate. Line widths of H2O2 below 3.8 Hz were obtained for several Greek oregano extracts which resulted in a detection limit of 0.7 µmol L⁻¹. Application of an array of NMR experiments, including 2D ¹H-¹³C HMBC, spiking of the samples with H2O2, and variable temperature experiments, resulted in the unequivocal assignment of H2O2 precluding any confusion with interferences from intrinsic phenolics in the extract.

Microdosing studies using accelerated mass spectrometry as exploratory investigational new drug trials.

Innovative attempts have been made to overcome nonproductivity and high expenditure in the clinical stages of new drug development. Microdosing studies using subpharmacological doses provide early insight into the body's disposition toward candidate compounds, and are innovative exploratory trials that can promote productivity in drug development. Highly sensitive analytical technology is crucial in microdosing studies that employ qualitative and quantitative assays of target materials in humans. Accelerator mass spectrometry (AMS) has facilitated the adoption of a human microdosing study in the early phase of clinical drug development. Results derived from AMS microdosing studies using labeled compounds can provide various types of information for candidate selection, including pharmacokinetic characteristics and metabolic profiles of candidate compounds. The applicability of microdosing studies is currently expanding into absolute bioavailability and mass balance studies. Although it remains uncertain whether microdosing adequately predicts the pharmacokinetics of therapeutic doses, further development of microdosing studies using AMS may benefit the field of new drug development and could pose a new challenge to researchers. The use of advanced technology in candidate selection will contribute to improved productivity and competitiveness in pharmaceutical research and development. The introduction of microdosing studies using AMS in Korea will present a newly applicable method for innovative clinical trials and contribute to development potential in global competition.

Development of a certified reference material (NMIJ CRM 7505-a) for the determination of trace elements in tea leaves.

A certified reference material (CRM) for trace elements in tea leaves has been developed in National Metrology Institute of Japan (NMIJ). The CRM was provided as a dry powder (<90 µm) after frozen pulverization of washed and dried fresh tea leaves from a tea plant farm in Shizuoka Prefecture, Japan. Characterization of the property value for each element was carried out exclusively by NMIJ with at least two independent analytical methods, including inductively coupled plasma mass spectrometry (ICP-MS), high-resolution (HR-) ICP-MS, isotope-dilution (ID-) ICP-MS, inductively coupled plasma optical emission spectrometry (ICP-OES), graphite-furnace atomic-absorption spectrometry (GF-AAS) and flame atomic-absorption spectrometry (FAAS). Property values were provided for 19 elements (Ca, K, Mg, P, Al, B, Ba, Cd, Cu, Fe, Li, Mn, Na, Ni, Pb, Rb, Sr, Zn and Co) and informative values for 18 elements (Ti, V, Cr, Y, and all of the lanthanides, except for Pm whose isotopes are exclusively radioactive). The concentration ranges of property values and informative values were from 1.59% (mass) of K to 0.0139 mg kg(-1) of Cd and from 0.6 mg kg(-1) of Ti to 0.0014 mg kg(-1) of Lu, respectively. Combined relatively standard uncertainties of the property values were estimated by considering the uncertainties of the homogeneity, analytical methods, characterization, calibration standard, and dry-mass correction factor. The range of the relative combined standard uncertainties was from 1.5% of Mg and K to 4.1% of Cd.

Development of low-energy methods for preparing food Nano-emulsions.

The aim of this work was to investigate the effect of sucrose on the phase behavior of vegetable oil/polyoxyethylene sorbitan monooleate (MOPS, Tween 80) and decaglycerol monolaurilester (DGML)/aqueous solution systems to establish low-energy emulsification methods for preparing nano-emulsions suitable for food uses. Phase diagrams were constructed to elucidate the optimal process for preparing the nano-emulsions. It was found that nano-emulsions were obtained when the composition was altered to either cross the sponge phase (L(3)) or lamellar phase (La) in the vegetable oil/MOPS/aqueous solution system or vegetable oil/DGML/aqueous solution system, respectively. The average droplet sizes in the former and latter emulsions were 0.203 µm and 0.165 µm, respectively. The addition of sucrose changed the hexagonal phase in the vegetable oil/MOPS/aqueous solution system into the sponge phase. As a result, the sponge region in the vegetable oil/MOPS/sucrose aqueous solution system occupied a larger area than that in the vegetable oil/MOPS/water system. In contrast, sucrose had no effect on the area of the La region in the vegetable oil/DGML/aqueous solution system. However, the addition of sucrose decreased the amount of emulsifier required to prepare nano-emulsions in both the vegetable oil/MOPS and DGML/aqueous solution systems. Sucrose was confirmed to facilitate the preparation of nano-emulsions in both systems.

A novel approach to simultaneous screening and confirmation of regulated pharmaceutical compounds in dietary supplements by LC/MS/MS with an information-dependent acquisition method.

The commercial success of synthetic phosphodiesterase-5 (PDE-5) inhibitors (viz. sildenafil, vardenafil and tadalafil) for erectile dysfunction (ED) has led to their widespread use as adulterants in dietary supplements (DSs). Reports on adulteration by ED drugs or their analogues in DSs suggest they may cause a serious threat to human health. The problem is becoming more complex as hidden and structurally modified analogues are continuously being reported. To analyse known drugs and their analogues, three commonly used PDE-5 inhibitors, naturally existing icariin and yohimbin, and their 19 analogues were analyzed in this study. They were identified using ion-spray liquid chromatography/tandem mass spectrometry (LC/MS/MS) using multiple reaction monitoring (MRM). This MRM procedure gave a limit of detection of less than 0.02 ng ml(-1) for the 24 compounds, selectivity of fragmentation using MRM for 2.5 - 8.5 min in a single run and peak height repeatability of coefficient of variation of 3.9 - 31.8%. An IDA method using the MRM scans to detect the presence of known analytes was set up and added to a built-in library for screening for PDE-5 inhibitors. These MRM experiments were used to trigger product ion scans using a hybrid quadrupole-linear ion trap instrument. The product ion scan was compared and confirmed by a library search of MS/MS spectra acquired from a reference standard. To search for new analogues of PDE-5 inhibitors, a precursor ion scan of an expected ion m/z 283, which was one of the mass fragments from the analogues of sildenafil or vardenafil, was performed and fragmentation of the precursor ion, by combining a precursor ion scan with automatic confirmation using EPI spectra, was acquired. Of the 37 DSs tested, two were eventually found to be adulterated with yohimbin and vardenafil, respectively. The approach proposed in this study would be valuable in characterizing chemical constituents of drug residues and their analogues with identical chemical substructures from complex natural and synthetic sources in DSs using an information-dependent acquisition-enhanced product ion (IDA-EPI) scan.

A new arginase enzymatic reactor: development and application for the research of plant-derived inhibitors.

This work was dedicated to the development of a new micro immobilized enzyme reactor (IMER) by using an in situ procedure. Arginase was covalently immobilized on an ethylenediamine (EDA) monolithic convective interaction media (CIM) disk (12mm × 3mm i.d.) previously derivatized with glutaraldehyde. The activity of this IMER was investigated by inserting this micro-IMER in a HPLC system. The effect of the arginase inhibitors was evaluated by the simultaneous injection of each inhibitor with the nitro guanidino benzene (NGB) substrate. The relative IC50 values were found in agreement with those derived by the conventional spectrometric method. This arginase micro-IMER system was also used to study the effects of plant-derived products on the arginase activity. The pet ether extract from the stem bark of the plant Ficus glomerata Roxob. and the procyanidin oligomers of cocoa and chocolate inhibit the arginase activity. Our results confirmed the direct effect of some plant extracts on the arginase activity and their interest in therapies for treating several NO-dependent smooth disorders.

Sensitive and selective liquid chromatography-tandem mass spectrometry method for the determination of five ganoderic acids in Ganoderma lucidum and its related species.

The present paper describes a novel, sensitive and selective liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the simultaneous analysis of ganoderic acids C(2), B, A, H, D in Ganoderma lucidum and its related species. Ganoderma samples were prepared using simple ultrasonic extraction. Chromatographic separation was carried out on an Agilent Zorbax XDB C(18) column (250 mm × 4.6 mm i.d., 5µm) with an isocratic mobile phase consisting of acetonitrile, water and formic acid (42:58:0.5, v/v/v). Mass spectrometric detection was achieved by a triple-quadrupole mass spectrometer equipped with an atmospheric pressure chemical ionization (APCI) interface operating in negative and positive ionization mode via a single within-run polarity switching. Quantitation of five ganoderic acids was performed using selective reaction monitoring (SRM) mode. The intra- and inter-day precision was less than 6.2% and the accuracy ranged from 90.0% to 105.7%. The limit of quantification (LOQ) was 20.0-40.0 ng/mL and the limit of detection (LOD) was 3.0-25.0 ng/mL. With this method, low levels of ganoderic acids in the fruiting bodies of Ganoderma sinense and Ganoderma applanatum were accurately quantified for the first time. Importantly, the method allows unequivocal quantification of the five ganoderic acids in the spores and fruiting bodies of Ganoderma lucidum, whereas the previously published methods have lacked the capability. The method presented will be a powerful tool for quality control of Ganoderma lucidum and its related species.

Fast and simple droplet sampling of sap from plant tissues and capillary microextraction of soluble saccharides for picogram-scale quantitative determination with GC-MS.

Soluble saccharides are very important metabolites of the life cycle and synthesis of structural polysaccharide components (cellulose, hemicellulose, pectin, etc.) of cell walls. A new method for droplet sampling of saps from tissues of organisms and manipulation routines in capillaries for extraction, derivation, and partitioning were developed for picogram-scale quantitative determination with gas chromatography-mass spectrometry (GC-MS). Five to ten microliters of sap was sampled with a glass capillary containing ribitol (internal standard). Subsequently, the analytes were acetylated with acetic anhydride and catalyzed by 1-methylimidazole. Finally, the soluble saccharides were qualitatively detected with GC-MS SIM (selective ion monitoring) mode. The linear ranges of the method were up to 1×10(-6) mol/L and the theoretically lowest limits of detection (LOD, s/n≥3) were up to 1×10(-9) mol/L. The method is suitable and applicable to analysis of soluble monosaccharides in fresh tissues and other aqueous samples in wide fields of agriculture, food science, biological sciences, and even medical studies.

Transcriptional profiling by deep sequencing identifies differences in mRNA transcript abundance in in vivo-derived versus in vitro-cultured porcine blastocyst stage embryos.

In vitro embryo culture systems promote development at rates lower than in vivo systems. The goal of this project was to discover transcripts that may be responsible for a decrease of embryo competency in blastocyst-stage embryos cultured in vitro. Gilts were artificially inseminated on the first day of estrus, and on Day 2, one oviduct and the tip of a uterine horn were flushed and the recovered embryos were cultured in porcine zygote medium 3 for 4 days. On Day 6, the gilts were euthanized and the contralateral horn was flushed to obtain in vivo derived embryos. Total RNA was extracted from three pools of 10 blastocysts from each treatment. First and second strand cDNA was synthesized and sequenced using Illumina sequencing. The reads generated were aligned to a custom-built database designed to represent the known porcine transcriptome. A total of 1170 database members were different between the two groups (P < 0.05), and 588 of those had at least a 2-fold difference. Eleven transcripts were subjected to real-time PCR that validated the sequencing. There was an overall decrease in inner cell mass (ICM) and trophectodermal (TE) cell numbers in embryos cultured in vitro; however, no difference in the ICM:TE ratio was found. Interestingly, the transcript SLC7A1 was higher in the in vitro cultured group. This difference disappeared after addition of arginine to the 4-day culture. Illumina sequencing and alignment to a custom transcriptome identified a large number of genes that yield clues on ways to manipulate the culture media to mimic the in vivo environment.

Nanocrystalline diamond microelectrode arrays fabricated on sapphire technology for high-time resolution of quantal catecholamine secretion from chromaffin cells.

The quantal release of oxidizable molecules can be successfully monitored by means of polarized carbon fiber microelectrodes (CFEs) positioned in close proximity to the cell membrane. To partially overcome certain CFE limitations, mainly related to their low spatial resolution and lack of optical transparency, we developed a planar boron-doped nanocrystalline diamond (NCD) prototype, grown on a transparent sapphire wafer. Responsiveness to applied catecholamines as well as the electrochemical and optical properties of the NCD-based device were first characterized by cyclic voltammetry and optical transmittance measurements. By stimulating chromaffin cells positioned on the device with external KCl, well-resolved quantal exocytotic events could be detected either from one NCD microelectrode, or simultaneously from an array of four microelectrodes, indicating that the chip is able to monitor secretory events (amperometric spikes) from a number of isolated chromaffin cells. Spikes detected by the planar NCD device had comparable amplitudes, kinetics and vesicle diameter distributions as those measured by conventional CFEs from the same chromaffin cell.

A tubulin polymerization microassay used to compare ligand efficacy.

We developed tubulin purification strategies that allowed sufficient material to be produced for compound-screening projects. Tubulins were polymerized in the presence of compounds using either turbidometric or fluorescence polymerization assays. IC50 and EC50 values were calculated and used to determine ratios between host and target tubulin (TT) (e.g., IC50-neuronal tubulin/IC50-TT). This ratio can be compared between compounds to identify the ones which are most selective for a particular TT. We found ratios for different compounds ranged from 0.16 to 4.0 between neuronal and cancer cell tubulin indicating that the sequence and posttranslational heterogeneity between these tubulins are sufficient to identify selective ligands for the TT. Likewise, compounds compared between neuronal and fungal tubulin had ratios ranging from 0.03 to 0.60, and compounds compared between neuronal to plant tubulin had ratios ranging from 0.03 to 52. Considering these data, we believe cancer cell tubulin-targeted drugs could be obtained with ratios in excess of 20, herbicides with ratios in excess of 200, and fungicides in excess of 200.