Disposable Pipette Tips Research Articles

Selective Electroanalytical Method for the Determination of Roxarsone in Poultry Feed and Litter

An extremely sensitive and selective voltammetric method was developed for the determination of roxarsone (ROX) at a newly modified carbon paste microelectrode in Britton–Robinson buffer (BRB) at pH 2.0. The electrode is based on a disposable plastic pipette tip filled with carbon paste based on a mixture of graphite powder and Amberlite XAD-4 (5:1, w/w). The experimental parameters, such as pH, type of the buffer, mode of voltammetry, accumulation potential, accumulation time, and stirring rate were optimized. The reduction peak current dependences were linear for the concentration of ROX from 2.0 to 100 μM. The method showed reproducible results with RSD (n = 11) of <2.0 %. The limit of detection (LOD) and limit of quantification (LOQ) were 0.53 and 1.76 μM, respectively. ROX showed that there was no apparent surface passivation indicating the suitability of the modified electrode for ROX detection. The developed method was successfully applied for the determination of ROX in poultry feed and poultry litter collected from various localities of Hyderabad.

Glucosinolates in collard greens grown under three soil management practices

Glucosinolates (GSLs, β-D-thioglucoside-N-hydroxysulfates) are polar compounds present in varying amounts in members of the Brassicaceae family. They suppress soil-borne pests due to the biofumigant properties of the highly toxic isothiocyanates present in Brassica vegetables. The objectives of this investigation were to: (1) assess variation in GSLs concentrations among collard plants grown under three soil management practices: sewage sludge (SS) mixed with native soil, chicken manure (CM) mixed with native soil, and no-mulch (NM) native soil, (2) quantify GSLs concentrations in collard roots, leaves, and stems at harvest for potential use of their crude extracts in plant protection, and (3) assess myrosinase activity in soil amended with CM and SS mixed with native soil. Separation of GSLs was accomplished by adsorption on a DEAE-Sephadex ion exchange resin using disposable pipette tips filled with DEAE, a weak base, with a net positive charge when ionized and exchange anions such as GSLs (hydrophilic plant secondary metabolites). Quantification of total GSLs was based on inactivation of collard endogenous myrosinase and liberation of the glucose moiety from the GSLs molecule by addition of standardized myrosinase and colorimetric determination of the liberated glucose moiety. Across all treatments, SS and CM increased soil organic matter content from 2.2% in native soil to 4.2 and 6.5%, respectively. GSLs concentrations were significantly greater in collard leaves (30.9 µmoles g−1 fresh weight) compared to roots and stems (7.8 and 1.2 µmoles g−1 fresh weight), respectively. Leaves of collard grown in soil amended with SS contained the greatest concentrations of GSLs compared to leaves of plants grown in CM and NM treatments. Accordingly, leaves of collard plants grown in soil amended with SS could play a significant role in sustainable agriculture as alternative tools for soil-borne disease management in conventional and organic agriculture.

Extraction of trace elements in human fluids using cation exchange resin (chelex-100) columns for ICP-MS analysis

A successful metal ion extraction method was developed using cation exchange resin packed disposable micro pipette tip columns for the extraction of trace metal ions in human fluids. Using this method, concentrations of Mn, Ni, Cu, Zn, Cd and Pb ions in human urine were determined by inductively coupled plasma mass spectroscopy (ICP-MS). The main objective of this study was to develop a technique to overcome spectral interferences in ICP analysis of trace elements in biological fluids mainly due to salts present in the samples. The amount of resins packed to the column, amount of eluent needed to extract trace elements, and particle size of the resin were examined to determine conditions for metal extraction. The validity of this technique was evaluated by calculating recoveries of trace element concentrations of above elements in certified standard urine samples and by using the technique for trace metal spiked urine samples. The results revealed that Pb, Cd, Zn, Cu and Mn had good recoveries (about 95%) while Ni &amp; Co had about 80% recovery. It was also found that the recoveries of trace elements depended on the concentrations of relevant elements in the samples.Journal of the University of Ruhuna 2013 1(2): 23-30

Realizing nano electrospray ionization using disposable pipette tips under super atmospheric pressure

Nano electrospray with a solution flow rate below 100 nL min(-1) is usually performed using a metallized pulled glass capillary with an inner diameter tip of <10 μm. In this paper, we demonstrated that it was possible to achieve a stable solution flow rate of ~10 nL min(-1) for an aqueous solution using a disposable plastic pipette tip with an inner diameter of 100 μm as an ESI emitter. This pipette tip (gel loading tip) was originally designed for the loading of sample onto the gel in gel electrophoresis. The inner and outer diameters were much larger than those of a nanoESI emitter, but the low solution flow rate could be sustained by conducting the electrospray under a super-atmospheric gas pressure. The high pressure condition ensured the absence of electrical discharge which was essential for stable electrospray of aqueous solution. ESI with this low cost tip was equally as sensitive as the standard nanoESI emitter and capable of producing a long lasting signal from a small amount of sample liquid. Due to a small initial droplet size under the low flow rate regime, ESI with the present method exhibited the property of nanoESI where the ion suppression of the hydrophilic analyte by the hydrophobic compound present in the solution was greatly reduced. Furthermore, owing to its larger inner diameter, the tip relatively easily avoided the clogging problem often encountered when dealing with "dirty" samples when compared with a nanoESI emitter made from a fine glass capillary.

Multiresidue pesticides analysis in soils using modified QuEChERS with disposable pipette extraction and dispersive solid‐phase extraction

QuEChERS original method was modified into a new version for pesticides determination in soils. The QuEChERS method is based on liquid-liquid portioning with ACN and was followed by cleanup step using dispersive SPE and disposable pipette tips. Gas chromatographic separation with MS detection was carried out for pesticides quantification. The method was validated using recovery experiments for 36 multiclass pesticides. Mean reco-veries of pesticides at each of the four spiking levels between 10-300 μg/kg of soil ranged from 70-120% for 26 pesticides with RSD values less than 15%. The method achieved low limit of detection less than 7.6 μg/kg. Matrix effects were observed for 13 pesticides. Matrix effects were compensated by using matrix-matched calibration. The method was applied successfully using d-SPE or DPX in the analysis of the pesticides in soils from organic farming and integrated pest management.

Droplet centrifugation, droplet DNA extraction, and rapid droplet thermocycling for simpler and faster PCR assay using wire-guided manipulations

A computer numerical control (CNC) apparatus was used to perform droplet centrifugation, droplet DNA extraction, and rapid droplet thermocycling on a single superhydrophobic surface and a multi-chambered PCB heater. Droplets were manipulated using “wire-guided” method (a pipette tip was used in this study). This methodology can be easily adapted to existing commercial robotic pipetting system, while demonstrated added capabilities such as vibrational mixing, high-speed centrifuging of droplets, simple DNA extraction utilizing the hydrophobicity difference between the tip and the superhydrophobic surface, and rapid thermocycling with a moving droplet, all with wire-guided droplet manipulations on a superhydrophobic surface and a multi-chambered PCB heater (i.e., not on a 96-well plate). Serial dilutions were demonstrated for diluting sample matrix. Centrifuging was demonstrated by rotating a 10 μL droplet at 2300 round per minute, concentrating E. coli by more than 3-fold within 3 min. DNA extraction was demonstrated from E. coli sample utilizing the disposable pipette tip to cleverly attract the extracted DNA from the droplet residing on a superhydrophobic surface, which took less than 10 min. Following extraction, the 1500 bp sequence of Peptidase D from E. coli was amplified using rapid droplet thermocycling, which took 10 min for 30 cycles. The total assay time was 23 min, including droplet centrifugation, droplet DNA extraction and rapid droplet thermocycling. Evaporation from of 10 μL droplets was not significant during these procedures, since the longest time exposure to air and the vibrations was less than 5 min (during DNA extraction). The results of these sequentially executed processes were analyzed using gel electrophoresis. Thus, this work demonstrates the adaptability of the system to replace many common laboratory tasks on a single platform (through re-programmability), in rapid succession (using droplets), and with a high level of accuracy and automation.

Conception through Build of an Automated Liquids Processing System for Compound Management in a Low-Humidity Environment

Boehringer Ingelheim's Automated Liquids Processing System (ALPS) in Ridgefield, Connecticut, was built to accommodate all compound solution-based operations following dissolution in neat DMSO. Process analysis resulted in the design of two nearly identical conveyor-based subsystems, each capable of executing 1400 × 384-well plate or punch tube replicates per batch. Two parallel-positioned subsystems are capable of independent execution or alternatively executed as a unified system for more complex or higher throughput processes. Primary ALPS functions include creation of high-throughput screening plates, concentration-response plates, and reformatted master stock plates (e.g., 384-well plates from 96-well plates). Integrated operations included centrifugation, unsealing/piercing, broadcast diluent addition, barcode print/application, compound transfer/mix via disposable pipette tips, and plate sealing. ALPS key features included instrument pooling for increased capacity or fail-over situations, programming constructs to associate one source plate to an array of replicate plates, and stacked collation of completed plates. Due to the hygroscopic nature of DMSO, ALPS was designed to operate within a 10% relativity humidity environment. The activities described are the collaborative efforts that contributed to the specification, build, delivery, and acceptance testing between Boehringer Ingelheim Pharmaceuticals, Inc. and the automation integration vendor, Thermo Scientific Laboratory Automation (Burlington, ON, Canada).

Background bisphenol A in experimental materials and its implication to low-dose in vitro study

In vitro low-dose studies are important to understand the mechanisms of bisphenol A (BPA) action. BPA doses used in current in vitro studies varied considerably, and doses as low as 10 −15 M have been reported. The actual doses of BPA used in the in vitro low-dose studies were rarely checked analytically, and the background BPA levels in experimental materials, which will determine the lowest BPA dose to be used, should be investigated or considered. In this study, the background BPA levels in various materials typically used in in vitro low-dose studies for BPA were investigated. Background BPA levels from the use of disposable pipettes and pipette tips were low (<0.20 ng mL −1 or 0.88 nM). BPA was also detected in several commercial buffer solutions at levels close to the method limit of quantification (LOQ) (0.02 ng mL −1; 0.088 nM). However, BPA was detected in all cell culture media obtained from various sources at levels ranging from 0.080 to 4.26 ng mL −1 (or 0.35 to 19 nM) with an average of 0.83 ng mL −1 (3.5 nM). We suggest that culture media used for low-dose BPA studies should be analysed for background BPA levels prior to use, and the medium with the lowest BPA levels should be used.

A Simplified Method for Quantifying Cell Migration/Wound Healing in 96-Well Plates

Cell migration plays a key role in both normal physiological and pathological conditions. The study of cell migration and its underlying mechanisms is of great significance in various fields of research, including basic biology and pharmaceutical development. The cell migration or scratch wounding assay is an easy and economical in vitro method that allows researchers to assess a large number of testing compounds. Even though this simple assay has been used for decades, researchers are still trying to modify such experimental protocols and wounding devices. In this study, an 8-channel mechanical "wounder" was designed for performing a cell migration assay, particularly in a 96-well culture plate format. With special designs of a guiding bar and adjustable pins for use with disposable pipette tips, this wounder confined the scratch area within the center of each well to ensure a perfect contact between the pins and the well surface. As a result, this mechanical wounder produces a uniform denudation of a cell monolayer in a 96-well plate with a wound size of around 600 microm. Using this improved wounding device, the effects of epidermal growth factor and DL-alpha-difluoromethylornithine on the reepithelialization of rat intestinal epithelial cells (IEC-6) and serum on the wound recovery of human umbilical vein endothelial cells were demonstrated. This wounder facilitates cell migration study and can be applicable for multiple sample analysis.

Voltammetric Determination of 4‐Nitrophenol Using a Novel Type of Silver Amalgam Paste Electrode

AbstractA differential pulse voltammetric (DPV) method was developed for the determination of 4‐nitrophenol (4‐NP) at a newly developed silver amalgam paste electrode (AgA‐PE) in Britton–Robinson buffer pH 3.0. The electrode is based on a disposable plastic pipette tip filled with paste amalgam based on a mixture of mercury and fine silver powder (9 : 1, w/w). The experimental parameters, such as pH of Britton–Robinson buffer and activation and regeneration potential of the electrode surface were optimized. The reduction peak current dependences were linear for the concentration of 4‐NP from 0.2 to 100 μM. The method showed reproducible results with RSD (n=45) of 1.7%. The limit of determination (LOD) was 0.3 μM. The method was successfully applied for the direct determination of 4‐NP in drinking water.

Screening Brassica species for glucosinolate content

Glucosinolates (GSLs), a group of compounds found in Brassica plants, are toxic to some soil-borne plant pathogens because of the toxicity of their hydrolysis products, isothiocyanates. Other phytochemicals found in Brassica plants, such as phenols and ascorbic acid, may compliment the activity of GSLs. A survey of Brassica accessions from the national germplasm repository was conducted to identify potential cover crops that could be soil-incorporated for use as biofumigants. Ten Brassica accessions that demonstrated relative cold tolerance, rapid maturity, and superior biomass production were selected. The selected accessions were grown under three climatic conditions (fall greenhouse, winter high tunnel, and spring field) to investigate whether growing conditions affect their GSL, phenol, and ascorbic acid content. The selected accessions included seven accessions of Brassica juncea (Indian mustard), one of Brassica napus (oil seed rape), one of Brassica campestris (field mustard), and one of Eruca sativa (arugula). Separation of GSLs from the selected Brassica accessions was achieved using ion-exchange sephadex in disposable pipette tips. Quantification of total GSLs was based on inactivation of the endogenous thioglucosidase and liberation of the glucose moiety from the GSL molecule by addition of standardized thioglucosidase (myrosinase) and colorimetry. GSL concentration of greenhouse, high tunnel, and field-grown shoots (leaves and stems) averaged 24, 40 and 76 μ moles g− 1 fresh weight, respectively. Accessions of B. juncea generally had the highest GSL content. A comparison of accessions revealed that Ames 8887 of B. juncea contained the greatest GSL concentration, but had the lowest biomass yield and ascorbic acid concentration, in part because phytochemical concentration tended to be negatively correlated with biomass yield. More promising was B. juncea accession ‘Pacific Gold’ which coupled high biomass yield with above-average GSL production, but had low phenol and ascorbic acid concentration. We concluded that environmental stress on growing plants can increase the concentration of GSLs, ascorbic acid, and total phenols in Brassica shoots, but does not increase yields of these phytochemicals per unit area.

Petri dish PCR: laser-heated reactions in nanoliter droplet arrays

We report high-speed real-time PCR performed on an unmodified disposable polystyrene Petri dish. The reaction cycle relies solely on an infrared laser for heating; no conventional heater is required. Nanoliter droplets of PCR mixture as water-in-oil emulsions printed in an array format served as individual PCR microreactors. A simple contact printing technique was developed to generate a large array of uniform sized nanoliter droplets using disposable pipette tips. Printed droplets showed variation of less than 10% in volume and the oil/water/polystyrene interface formed a compact droplet microreactor approximately spherical in shape. The uniform droplet array was used to optimize the laser power required for the two heating steps of PCR, annealing/extension and melting, while the ambient conditions were at room temperature. The optical heating allows for an extremely fast heating rate due to the selective absorption of the infrared laser by PCR buffer only and not the oil or polystyrene Petri dish, allowing completion of 40 amplification cycles in approximately 6 minutes. The quantitative assay capability of the system is also presented and discussed.

Small Molecule Activator of the Human Epithelial Sodium Channel

The epithelial sodium channel (ENaC), a heterotrimeric complex composed of alpha, beta, and gamma subunits, belongs to the ENaC/degenerin family of ion channels and forms the principal route for apical Na(+) entry in many reabsorbing epithelia. Although high affinity ENaC blockers, including amiloride and derivatives, have been described, potent and specific small molecule ENaC activators have not been reported. Here we describe compound S3969 that fully and reversibly activates human ENaC (hENaC) in an amiloride-sensitive and dose-dependent manner in heterologous cells. Mechanistically, S3969 increases hENaC open probability through interactions requiring the extracellular domain of the beta subunit. hENaC activation by S3969 did not require cleavage by the furin protease, indicating that nonproteolyzed channels can be opened. Function of alphabetaG37Sgamma hENaC, a channel defective in gating that leads to the salt-wasting disease pseudohypoaldosteronism type I, was rescued by S3969. Small molecule activation of hENaC may find application in alleviating human disease, including pseudohypoaldosteronism type I, hypotension, and neonatal respiratory distress syndrome, when improved Na(+) flux across epithelial membranes is clinically desirable.

Effects of Mouse and Human Lipocalin Homologues 24p3/lcn2 and Neutrophil Gelatinase–Associated Lipocalin on Gastrointestinal Mucosal Integrity and Repair

The lipocalin superfamily, including the mouse and human homologues 24p3/lcn2 and neutrophil gelatinase-associated lipocalin, show great functional diversity including roles in olfaction, transportation, and prostaglandin synthesis in mammals. Their potential role in maintaining gastrointestinal mucosal integrity and repair is, however, unclear. Changes in 24p3/lcn2 expression in the mouse gut in response to various noxious agents were examined using Northern blot, in situ hybridization, and immunohistochemistry. Effects of recombinant 24p3/lcn2 on proliferation ([3H]-thymidine uptake), and restitution (cell-wounding migration) were assessed using human colonic HT29 and HCT116 cells. In addition, the effects of recombinant 24p3/lcn2 on the amount of gastric damage were assessed in rats treated with indomethacin (20 mg/kg) and restraint. Marked up-regulation of expression of 24p3/lcn2 was seen throughout the gut in response to indomethacin or dextran sodium sulfate treatment. Expression was increased particularly in the surface epithelial cells and infiltrating inflammatory cells. Proliferation and restitution assays in the presence of recombinant wild-type sequence neutrophil gelatinase-associated lipocalin, wild-type cys(98)-24p3/lcn2, and mutant ala98-24p3/lcn2 showed that all 3 peptides caused a 3- to 4-fold increase in promigratory activity (P < .01 vs control) but did not influence proliferation. The administration of wild-type cys98-, or mutant ala98-24p3/lcn2 (25 and 50 microg/kg/h, respectively), given via the subcutaneous route, both caused similar reductions in the rat gastric damage model (60% reduction at highest dose, P < .01 vs control), although oral administration was ineffective. 24p3/lcn2 facilitates mucosal regeneration by promoting cell migration.

A fully automated robotic system for purification of polyhistidine‐tagged proteins

A protein isolation method that results in rapid and high yield proteins is essential for protein sample preparation in proteomics. A magnetic bead-based automated method to purify both soluble and insoluble 6x histidine-tag (His-tag) proteins from cell lysates has been developed employing PSS Magtration® technology. Magtration® is a PSS patented technique that traps magnetic particles against the sidewall of a disposable pipette tip, resulting in superior washing and recovery compared to other magnetic particle-separation systems. We have extended this technology from immunoassay and nucleic acid purification to His-tag protein purification using Magtration® 12GC robotic system with commercially available Ni2+ and Co2+ magnetic beads. Unlike other automated protein purification systems, Magtration®12GC allows for high volumes (up to 800 μl) of initial sample, and can run up to 12 samples at once. Experimental conditions including buffer compositions, buffer pH and various His-tag protein binding magnetic beads have been studied and optimized for the robotic purification on the Magtration® system. A two-step elution protocol has been developed for effective removal of nonspecific proteins resulting in highly pure His-tag target proteins. The yield and purity of the His-tag protein from the automated system are more successful in terms of efficiency and reproducibility compared to manual protocols due to more thorough washing, complete separation between the magnetic beads and supernatant, and consistent robotic operation.

Fully Automated Magnetic Labeling and Separation of Hematopoietic Cells from Multiple Samples.

Laboratory process automation is an important requirement for streamlining and standardizing technical procedures. Despite the extensive use of magnetic cell separation, only the latter steps in these procedures have been automated. Currently magnetic cell labeling is done manually followed by automated magnetic separation (e.g. AutoMACS and Isolex). Additionally, current technology only allows for processing of a single sample at a time. Our objective was to develop a fully automated system to magnetically separate multiple blood and bone marrow samples. The major barrier to automation of cell labeling is that these procedures typically require a centrifugal wash step, which is relatively expensive to automate and requires bulky equipment. We had previously developed a magnetic cell labeling/separation system call EasySep® (Stemcell Technologies) which does not require a centrifugal wash step. We have now fully automated EasySep® and present the RoboSep™ instrument which magnetically labels and separates 4 samples at once, with up to 2×109 total cells per sample or 8×109 total cells. The instrument operates in a standard biosafety hood and uses sterile disposable pipette tips to ensure aseptic operation and avoid cross-contamination between samples. Standardized automation protocols have been developed for both positive and negative selection. With positive selection, the desired cells are magnetically labeled and then purified by a sequence of magnetic wash steps. With negative selection, unwanted cells are magnetically labeled and then depleted. To demonstrate the suitability of RoboSep™ for automated positive selection of hematopoietic progenitors and stem cells, we performed CD34+ cell selection from previously frozen cord blood (CB) and mobilized peripheral blood (MPB). For the CB separations, the CD34+ cell content was enriched from 1.2±0.4% to 96.6±3.1% with a recovery of 45±9% (n=9, mean ± 1 SD). For the MPB separations the CD34+ cell content was enriched from 0.7±0.1% to 96.7±3.1%, with a recovery of 45±13% (n=4). To test RoboSep in negative selection we used an EasySep® antibody cocktail depleting cells that express any of CD2, CD3, CD11b, CD11c, CD14, CD16, CD19, CD24, CD56, CD66b, and glycophorin A to isolate hematopoietic progenitors from bone marrow (BM) and MPB. CB separations required the addition of anti-CD41 to the antibody cocktail for depletion of platelets. The table below shows results for negative selection from BM, CB and MPB. Manual separations performed in parallel with the above automated separations showed comparable purity and recovery, indicating that we have succeeded in automating both positive and negative selection procedures. The RoboSep instrument processes up to 4 tissue samples at once and provides the opportunity to isolate multiple cell subsets from the same sample by combining positive and negative selection methods in a single automated procedure.Negative Selection Results (Mean± 1 SD)Sample% CD34+ in start% CD34+ in enriched% Recovery CD34+ cellsFold-enrichment of total BFU-E, CFU-GM, CFU-GEMM% recovery of total BFU-E, CFU-GM, CFU-GEMMCB (n=2)1.567.4503638MPB (n=2)1.150.0455041BM (n=4)4.7±3.147.5±7.5N.A.47±1071±13N.A. Not Available

The ADVIA Centaur infectious disease assays: a technical review.

Bayer Healthcare, Diagnostics Division, has developed several new several new assays for hepatitis B, hepatitis C and HIV 1/O/2 on the ADVIA Centaur Immunoassay system. The panel for detection of markers for hepatitis B infection includes assays for hepatitis B surface antigen (HBsAg) and HBsAg. Confirmatory, antibodies to hepatitis B surface antigen (anti-HBs), IgM and IgG antibodies to hepatitis B core antigen (anti-HBc Total) and IgM antibodies to hepatitis B core antigen (anti-HBc IgM). The hepatitis B panel is complemented by third generation assays for anti-HCV and anti-HIV that concomitantly detects HIV-1 (including group O) and HIV-2. The features of the ADVIA Centaur and the design of the assays combine to deliver state-of-the-art performance. Features, such as disposable sample pipette tips to prevent sample carryover, clot detection and management to verify sample addition, reagent integrity checks to assure accurate reagent addition, auto repeat capabilities and the capacity to maintain up to 30 assays onboard at 2-8 degrees C all contribute to the systems's utility for infectious disease testing. Furthermore, the flexibility of the ADVIA Centaur has allowed for selection of formats to provide optimal assay performance. The anti-HBs, anti-HBc Total and anti-HIV 1/O/2 assays are antigen-bridging assays. The anti-HBc IgM assay is a class-capture two step assay configured for detection of IgM anti-HBc antibodies during acute HBV infection. The anti-HCV assay is an indirect assay utilizing streptavidin-coated microparticles preformed with a combination of recombinant and synthetic peptide antigens from the NS2, NS4, NS5 and core regions of the HCV genome. The HBsAg assay is a sandwich assay with an incubation time of 28 minutes and the HBsAg Confirmatory assay is a fully automated neutralization assay. The availability of these assays on the ADVIA Centaur Immunoassay system enables laboratories to consolidate other immunoassays along with the infectious disease assays for workstation operation and efficiencies.

Critical Comparison of Four Simple Adaptors for Flow Amperometric and Stripping Voltammetry with Mercury Drop Electrodes in Batch Cells

AbstractElectrochemical batch cells with mercury drop electrodes, MDEs, are readily available from a number of producers and widely used in electroanalysis for polarography, voltammetry and stripping voltammetry. To increase sample throughput and reduce reagent and sample consumption, the tendency is to couple the MDE with flow analysis systems (e.g., FIA, SIA or BIA). Many special flow cells for MDEs are described in the literature, but it is easier to convert existing commercial batch cells to flow operation. To assess the performance of such flow adaptors, four models were chosen because they are directly fitted to the glass capillary of any MDE, light enough not to impair the mechanical drop knocker and can be effortlessly built in the laboratory. They were all found adequate for flow amperometry and stripping voltammetry, with differences in sensitivity, flow rate dependence, response time, drop stability, tolerance to bubbles, uncompensated resistance and recontamination of disposed analyte from the electrolytic bath. Two of them, a simple L‐shaped PTFE tube and an upstream nozzle holder made from a disposable pipette tip, gather a larger set of desirable features.

Automated chip-based nanoelectrospray-mass spectrometry for rapid identification of proteins separated by two-dimensional gel electrophoresis.

We report a method using a fully automated chip-based nanoelectrospray system for two-dimensional (2-D) gel sample analyses with mass spectrometric detection. The automated nanoelectrospray system, consisting of the NanoMate and electrospray ionization (ESI) chip, serves as both an autosampler and nanoESI source. This infusion system aspirates samples from a 96-well plate using disposable pipette tips and then delivers these samples sequentially to an ESI chip. This chip is a fully integrated monolithic device consisting of a 10x10 array of nozzles. The automated nanoelectrospray system is easily controlled through software, permitting the user to select the number of samples to be analyzed, the volume of sample to aspirate, the spray voltage, and analysis time. The system offers all the advantages of conventional nanoelectrospray plus automated, high-throughput analyses without analyte carryover. The system was used for a protein identification study of 2-D gel spots of both Escherichia coli and yeast crude cell extracts. The identification of 50 spots from E. coli crude cell extract and 27 spots from yeast extract is presented, demonstrating the powerful combination of the automated nanoESI system, the Thermo Finnigan LCQ Deca ion-trap mass spectrometer, and SEQUEST search software. In addition, the effects of silver staining and colloidal Coomassie blue staining of 2-D gel spots on the detection sensitivity and protein sequence coverage are compared and discussed. Furthermore, the comparison results using the multiwell microscale preparation kit versus manual extraction for in-gel samples are presented.

Microcolumns with self-assembled particle frits for proteomics

LC-MS-MS experiments in proteomics are usually performed with packed microcolumns employing frits or outlets smaller than the particle diameter to retain the packing material. We have developed packed microcolumns using self-assembled particles (SAPs) as frits that are smaller than the size of the outlet. A five to one ratio of outlet size to particle diameter appears to be the upper maximum. In these situations the particles assembled into an arch over the outlet like the stones in a stone bridge. When 3 microm particles were packed into a tapered column with an 8 microm outlet, two particles bridged the outlet with 0.3 pl dead volume and perfect success rate. In peptide analysis by LC-MS, the peak width at half height was normally less than 6 s, compared to 12 s without SAPs. The LC-MS-MS system provided 37% sequence coverage (21 matched peptides) for a tryptically-digested sample of 10 fmol bovine serum albumin. We also describe application of the SAP principle to make disposable pipette tip columns with short pieces of fused-silica capillary as the outlet.