SU-8-Based Microchips for Capillary Electrophoresis and Electrospray Ionization Mass Spectrometry

Miniaturization of analytical instrumentation is attracting growing interest in response to the explosive demand for rapid, yet sensitive analytical methods and low-cost, highly automated instruments for pharmaceutical and bioanalyses and environmental monitoring. Microfabrication technology in particular, has enabled fabrication of low-cost microdevices with a high degree of integrated functions, such as sample preparation, chemical reaction, separation, and detection, on a single microchip. These miniaturized total chemical analysis systems (microTAS or lab-on-a-chip) can also be arrayed for parallel analyses in order to accelerate the sample throughput. Other motivations include reduced sample consumption and waste production as well as increased speed of analysis. One of the most promising hyphenated techniques in analytical chemistry is the combination of a microfluidic separation chip and mass spectrometer (MS). In this work, the emerging polymer microfabrication techniques, ultraviolet lithography in particular, were exploited to develop a capillary electrophoresis (CE) separation chip which incorporates a monolithically integrated electrospray ionization (ESI) emitter for efficient coupling with MS. An epoxy photoresist SU-8 was adopted as structural material and characterized with respect to its physicochemical properties relevant to chip-based CE and ESI/MS, namely surface charge, surface interactions, heat transfer, and solvent compatibility. As a result, SU-8 was found to be a favorable material to substitute for the more commonly used glass and silicon in microfluidic applications. In addition, an infrared (IR) thermography was introduced as direct, non-intrusive method to examine the heat transfer and thermal gradients during microchip-CE. The IR data was validated through numerical modeling. The analytical performance of SU-8-based microchips was established for qualitative and quantitative CE-ESI/MS analysis of small drug compounds, peptides, and proteins. The CE separation efficiency was found to be similar to that of commercial glass microchips and conventional CE systems. Typical analysis times were only 30-90 s per sample indicating feasibility for high-throughput analysis. Moreover, a mass detection limit at the low-attomole level, as low as 10E+5 molecules, was achieved utilizing MS detection. The SU-8 microchips developed in this work could also be mass produced at low cost and with nearly identical performance from chip to chip. Until this work, the attempts to combine CE separation with ESI in a chip-based system, amenable to batch fabrication and capable of high, reproducible analytical performance, have not been successful. Thus, the CE-ESI chip developed in this work is a substantial step toward lab-on-a-chip technology.

An automated pipette puller for fabrication of glass micropipettes

Glass micropipettes are versatile probing tools for performing micro-and nano-manipulation tasks. This paper presents the design and development of an automated pipette puller system for fabrication of glass micropipettes. The pipette puller employs a new strategy for fabrication of micropipettes that enables achieving independent control of their taper, tip diameter, and bend-angle, and also facilitates theoretical derivation of simple, approximate relationships between the pipette shape and the pulling parameters. Subsequently, the design and fabrication of the pipette puller is described, which include that of the pipette heating system, the mechanical motion stages, and the control electronics of the pipette puller. The fabricated pipette puller is experimentally evaluated to demonstrate control of the taper, tip diameter, and the bend-angle of the micropipette. Further, the dependence of the taper and tip diameter on the pulling parameters is evaluated and is shown to be in alignment with the proposed theoretical relationships. (C) 2014 AIP Publishing LLC.

The emergence of automated high-frequency flow cytometry: revealing temporal and spatial phytoplankton variability

Phytoplankton observation is the product of a number of trade-offs related to sampling processes, required level of diversity and size spectrum analysis capabilities of the techniques involved. Instruments combining the morphological and high-frequency analysis for phytoplankton cells are now available. This paper presents an application of the automated high-resolution flow cytometer Cytosub as a tool for analysing phytoplanktonic cells in their natural environment. High resolution data from a temporal study in the Bay of Marseille (analysis every 30 min over 1 month) and a spatial study in the Southern Indian Ocean (analysis every 5 min at 10 knots over 5 days) are presented to illustrate the capabilities and limitations of the instrument. Automated high-frequency flow cytometry revealed the spatial and temporal variability of phytoplankton in the size range 1−∼50 μm that could not be resolved otherwise. Due to some limitations (instrumental memory, volume analysed per sample), recorded counts could be statistically too low. By combining high-frequency consecutive samples, it is possible to decrease the counting error, following Poisson’s law, and to retain the main features of phytoplankton variability. With this technique, the analysis of phytoplankton variability combines adequate sampling frequency and effective monitoring of community changes.

Measuring icon complexity: An automated analysis

Measures of icon designs rely heavily on surveys of the perceptions of population samples. Thus, measuring the extent to which changes in the structure of an icon will alter its perceived complexity can be costly and slow. An automated system capable of producing reliable estimates of perceived complexity could reduce development costs and time. Measures of icon complexity developed by Garcia, Badre, and Stasko (1994) and McDougall, Curry, and de Bruijn (1999) were correlated with six icon properties measured using Matlab (MathWorks, 2001) software, which uses image-processing techniques to measure icon properties. The six icon properties measured were icon foreground, the number of objects in an icon, the number of holes in those objects, and two calculations of icon edges and homogeneity in icon structure. The strongest correlates with human judgments of perceived icon complexity (McDougall et al., 1999) were structural variability (r(s) = .65) and edge information (r(s) =.64).

Octopus Standard Automated Perimetry, Pulsar Perimetry, Moorfields Motion Displacement Test and Heidelberg Retinal Tomography in Glaucoma Detection - A Comparison of Diagnostic Accuracy

Purpose: To investigate the accuracy of 4 clinical instruments in the detection of glaucomatous damage. Methods: 102 eyes of 55 test subjects (Age mean = 66.5yrs, range = [39; 89]) underwent Heidelberg Retinal Tomography (HRTIII), (disc area<2.43); and standard automated perimetry (SAP) using Octopus (Dynamic); Pulsar (TOP); and Moorfields Motion Displacement Test (MDT) (ESTA strategy). Eyes were separated into three groups 1) Healthy (H): IOP<21mmHg and healthy discs (clinical examination), 39 subjects, 78 eyes; 2) Glaucoma suspect (GS): Suspicious discs (clinical examination), 12 subjects, 15 eyes; 3) Glaucoma (G): progressive structural or functional loss, 14 subjects, 20 eyes. Clinical diagnostic precision was examined using the cut-off associated with the p<5% normative limit of MD (Octopus/Pulsar), PTD (MDT) and MRA (HRT) analysis. The sensitivity, specificity and accuracy were calculated for each instrument. Results: See table Conclusions: Despite the advantage of defining glaucoma suspects using clinical optic disc examination, the HRT did not yield significantly higher accuracy than functional measures. HRT, MDT and Octopus SAP yielded higher accuracy than Pulsar perimetry, although results did not reach statistical significance. Further studies are required to investigate the structure-function correlations between these instruments.

Differences between the CME fronts tracked by an expert, an automated algorithm, and the Solar Stormwatch project

Observations from the Heliospheric Imager (HI) instruments aboard the twin STEREO spacecraft have enabled the compilation of several catalogues of coronal mass ejections (CMEs), each characterizing the propagation of CMEs through the inner heliosphere. Three such catalogues are the Rutherford Appleton Laboratory (RAL)-HI event list, the Solar Stormwatch CME catalogue, and, presented here, the J-tracker catalogue. Each catalogue uses a different method to characterize the location of CME fronts in the HI images: manual identification by an expert, the statistical reduction of the manual identifications of many citizen scientists, and an automated algorithm. We provide a quantitative comparison of the differences between these catalogues and techniques, using 51 CMEs common to each catalogue. The time-elongation profiles of these CME fronts are compared, as are the estimates of the CME kinematics derived from application of three widely used single-spacecraft-fitting techniques. The J-tracker and RAL-HI profiles are most similar, while the Solar Stormwatch profiles display a small systematic offset. Evidence is presented that these differences arise because the RAL-HI and J-tracker profiles follow the sunward edge of CME density enhancements, while Solar Stormwatch profiles track closer to the antisunward (leading) edge. We demonstrate that the method used to produce the time-elongation profile typically introduces more variability into the kinematic estimates than differences between the various single-spacecraft-fitting techniques. This has implications for the repeatability and robustness of these types of analyses, arguably especially so in the context of space weather forecasting, where it could make the results strongly dependent on the methods used by the forecaster.

Development of an automated DNA purification module using a micro-fabricated pillar chip

We present a fully automated DNA purification module comprised of a micro-fabricated chip and sequential injection analysis system that is designed for use within autonomous instruments that continuously monitor the environment for the presence of biological threat agents. The chip has an elliptical flow channel containing a bed (3.5 × 3.5 mm) of silica-coated pillars with height, width and center-to-center spacing of 200, 15, and 30 µm, respectively, which provides a relatively large surface area (ca. 3 cm²) for DNA capture in the presence of chaotropic agents. We have characterized the effect of various fluidic parameters on extraction performance, including sample input volume, capture flow rate, and elution volume. The flow-through design made the pillar chip completely reusable; carryover was eliminated by flushing lines with sodium hypochlorite and deionized water between assays. A mass balance was conducted to determine the fate of input DNA not recovered in the eluent. The device was capable of purifying and recovering Bacillus anthracis genomic DNA (input masses from 0.32 to 320 pg) from spiked environmental aerosol samples, for subsequent analysis using polymerase chain reaction-based assays.

Cholinesterase measurements with an automated kit

Background the Test-mate kit determines acetylcholinesterase (AChE, EC 3.1.1.7) and hemoglobin content of a drop of blood, displaying enzyme activities normalized to 25degreesC. Previous models produced inconsistent results at different temperatures. This report focuses on the current model, ChE 400, and two instruments of a previous OP model.Methods AChE activities were determined by the Ellman assay, using the three kits and a 96-well microplate reader Temperatures ranged from 10 to 37degreesC. Fetal bovine serum was the source of AChE.Results Normalized activities decreased below 20degreesC in the ChE model and below 25 C in the OP models. Activities of the same serum sample differed between the three Test-mate kits, ranging from 1.03 to 1.49 mumoles/min/ml. Percent errors were greater than with the microplate reader at all temperatures.Conclusions Neither we nor the manufacturer recommend the current Test-mate model for fieldwork. Nevertheless, there have been field measurements with Test-Mate kits, and we recommend that an enzyme activity standard be run in parallel with their use. (C) 2002 Wiley-Liss, Inc.

Cone-beam computed tomography analysis of the apical third of curved roots after mechanical preparation with different automated systems

The present study evaluated by cone-beam computed tomography (CBCT) the apical canal transportation and centralizing ability of different automated systems after root canal preparation. The mesiobuccal canals of maxillary first molars (n=10 per group) were prepared with: GI - reciprocating system with K-Flexofile; GII - reciprocating system with NiTiFlex files; GIII - rotary system with K3 instruments; GIV - rotary system with RaCe instruments. CBCT scans were taken before and after biomechanical preparation up to a #40.02 diameter. Canal transportation was determined by measuring the smallest distance between the inner canal walls and the mesial and distal sides of the root. The centralization ability corresponded to the difference between the measurements from transportation evaluation, using the linear voxel to voxel method of analysis. The mean transportation was 0.06 ± 0.14 mm, with a tendency to deviate to the mesial side of the root (n=22), with no statistically significant difference among the groups (p=0.4153). The mean centralization index was 0.15 ± 0.65 also without statistically significant difference among the groups (p=0.0881). It may be concluded that apical canal transportation and centralization ability were not influenced by the type of mechanical movement and instruments used.

AMS-C14 analysis of graphite obtained with an Automated Graphitization Equipment (AGE III) from aerosol collected on quartz filters

AMS-14C applications often require the analysis of small samples. Such is the case of atmospheric aerosols where frequently only a small amount of sample is available. The ion beam physics group at the ETH, Zurich, has designed an Automated Graphitization Equipment (AGE III) for routine graphite production for AMS analysis from organic samples of approximately 1 mg. In this study, we explore the potential use of the AGE III for graphitization of particulate carbon collected in quartz filters. In order to test the methodology, samples of reference materials and blanks with different sizes were prepared in the AGE III and the graphite was analyzed in a MICADAS AMS (ETH) system. The graphite samples prepared in the AGE III showed recovery yields higher than 80% and reproducible 14C values for masses ranging from 50 to 300 lg. Also, reproducible radiocarbon values were obtained for aerosol filters of small sizes that had been graphitized in the AGE III. As a study case, the tested methodology was applied to PM10 samples collected in two urban cities in Mexico in order to compare the source apportionment of biomass and fossil fuel combustion. The obtained 14C data showed that carbonaceous aerosols from Mexico City have much lower biogenic signature than the smaller city of Cuernavaca.

Automated de novo sequencing of proteins by tandem high-resolution mass spectrometry

A de novo sequencing program for proteins is described that uses tandem MS data from electron capture dissociation and collisionally activated dissociation of electrosprayed protein ions. Computer automation is used to convert the fragment ion mass values derived from these spectra into the most probable protein sequence, without distinguishing Leu/Ile. Minimum human input is necessary for the data reduction and interpretation. No extra chemistry is necessary to distinguish N- and C-terminal fragments in the mass spectra, as this is determined from the electron capture dissociation data. With parts-per-million mass accuracy (now available by using higher field Fourier transform MS instruments), the complete sequences of ubiquitin (8.6 kDa) and melittin (2.8 kDa) were predicted correctly by the program. The data available also provided 91% of the cytochrome c (12.4 kDa) sequence (essentially complete except for the tandem MS-resistant region K13–V20 that contains the cyclic heme). Uncorrected mass values from a 6-T instrument still gave 86% of the sequence for ubiquitin, except for distinguishing Gln/Lys. Extensive sequencing of larger proteins should be possible by applying the algorithm to pieces of ≈10-kDa size, such as products of limited proteolysis.

Automated photomask inspection /

Includes bibliographical references.