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PubMed2005

The manufacture of miniature Cheddar-type cheeses from milks with different fat globule size distributions.

A novel 2-stage gravity separation scheme was developed for fractionation of raw, whole bovine milk into fractions enriched in small (SFG) or large (LFG) fat globules. The volume mean diameter of fat globules in SFG, LFG or control (CTRL) milk was 3.45, 4.68 and 3.58 microm, respectively. The maximum in storage modulus (index of firmness) decreased with increasing fat globule size for rennet-induced gels formed from SFG, LFG or CTRL milks. Miniature (20 g) Cheddar cheeses were manufactured using each of the 3 milks. There were no significant (P > 0.05) differences in the pH, moisture and fat in dry matter levels between cheeses made using any of the 3 milks, however, the fat content of the cheese made using SFG milk was approximately 1% lower than that of cheese made using LFG or CTRL milk in each of the 2 trials. Image analysis of confocal scanning laser micrographs of the cheeses illustrated that the star volume of fat globules in the cheeses decreased significantly (P < or = 0.05) as the size of fat globules in the milks used for cheesemaking was reduced. This indicates that it is possible to manipulate the size distribution of fat globules in Cheddar cheese by adjusting the fat globule size distribution of the milk used for cheese-making. The concentration of free fatty acids (FFA) increased in all cheeses during ripening. At 120 d of ripening, the concentration of FFA varied significantly (P < or = 0.05 and P < or = 0.001 for trials 1 and 2, respectively) with fat globule size, with cheeses made in trial 2 from LFG, SFG or CTRL milks having total FFA levels of 3391, 2820 and 2612 mg/kg cheese, respectively.

Animals↗
PubMed1993

A miniaturized biotyping system for strain discrimination in Escherichia coli.

A two-tier miniaturized scheme of eight tests was devised for biotyping strains of Escherichia coli in microwell plates. Primary biotypes were defined by positive and negative reactions in tests for fermentation of raffinose, sorbose, dulcitol and 2-deoxy-D-ribose and for decarboxylation of ornithine when read after specified periods of incubation; subtypes were identified within primary biotypes according to results in secondary tests for rhamnose fermentation, lysine decarboxylation and motility. The method gave reproducible results on different occasions of testing. Among 100 E. coli strains from various sources, 26 of the 32 possible primary biotypes and 56 full biotypes, as defined by results in both primary and secondary tests, were identified, thus demonstrating a high index of strain discrimination (D = 0.98). The scheme is recommended as a simple, reliable, inexpensive and efficient method of differentiating strains of E. coli.

Bacterial Typing Techniques↗
PubMed2000

A parallel miniature cylindrical ion trap array

A small mass spectrometer array is described in which each element is a cylindrical ion trap (CIT). The array contains four CITs, each having an inner radius of 2.5 mm, arranged in parallel and operated using a single electronics system under common conditions for trapping and mass analysis. By using an array of identically sized traps, higher ion capacity can be achieved than with a single miniature CIT, but the advantage of lower power and voltage requirements associated with the smaller ion trap is maintained. Overall signal intensity of the array of four traps is compared with that of a two-element CIT array to demonstrate the increased ion capacity of larger arrays. Resolution for m-dichlorobenzene is shown to be approximately 180 (full width at half-maximum), with no significant loss in resolution as a result of using multiple CITs. The detection of 5 x 10(-9) Torr partial pressure of krypton in argon with a signal-to-noise ratio of approximately 30 for the most abundant isotope is shown, demonstrating the applicability of the device for process gas monitoring. Also, a preliminary spectrum from a 10-CIT array with each element having an inner radius of 1.5 mm is reported.

Journal Article↗
PubMed2000

A miniaturized DNA amplifier: its application in traditional Chinese medicine.

A Si-based miniaturized device for the polymerase chain reaction (PCR) has been developed. The device has Pt temperature sensors and heaters integrated on top of the reaction chamber for real-time accurate temperature sensing and control. Reaction temperature of the device is digitally controlled to achieve a temperature accuracy of +/-0.025 degrees C. The effects of PCR protocol optimization on the amplification performance of the surface-passivated chip reactor have been investigated in detail and quantitatively compared with that of the conventional thermal cycler. In this study, four traditional Chinese medicine genes including Fritillaria cirrhosa, Cartharmus tinctorius, Adenophora lobophilla, and Stephania tetrandra are used as model template. With appropriate chamber surface treatment (chlorotrimethylsilane/polyadenylic acid or SiO2 coatings), the device demonstrates amplification as efficient as that in the conventional thermal cycler at optimized MgCl2 concentration. The amplified DNA has concentration higher than 27 ng/microL, which is sufficient for subsequent on-chip analyses and detection. Experimental results reveal the importance of inclusion of BSA for an efficient amplification in the SiO2-passivated device and the excellent reusability of the device with a simple cleaning step.

DNA, Plant↗
PubMed2001

Electrospray device for coupling microscale separations and other miniaturized devices with electrospray mass spectrometry.

A miniaturized ion sprayer device is described which is suitable for coupling with chip-based analytical separation devices, multiwell plates, or surfaces containing residues of prepared samples. Two versions of a similar device are described. A "microsprayer" device suitable for coupling to the terminal edge of a capillary electrophoresis (CE) chip is constructed from modified 1/16-in. HPLC fittings. This microsprayer employs a free-standing liquid junction formed via continuous delivery of a flow (2-6 microL/min) of suitable solvent which carries the CE effluent through a pneumatically assisted electrospray (ion spray) needle positioned in front of an atmospheric pressure ionization (API) mass spectrometer. A related but larger "minisprayer" device is also described which employs the same features as the microsprayer, but with an extended sampling capillary tube which can reach into the depths of 96-, 384-, and 1536-multiwell plates containing either sample solutions or dried sample residues. The minisprayer may be positioned in front of an API ion sampling orifice and the multiwell plate positioned stepwise from sample to sample for analysis of trace samples contained in the wells. The resulting infusion-ion spray mass spectrometric analyses can provide sequential analysis of previously prepared biological samples containing small drug compounds, proteins, and related compounds. This same device is also shown to be useful for sampling from a surface containing trace level compounds of biological interest. Results are shown that demonstrate microscale separations and selected ion monitoring (SIM) capillary electrophoresis/mass spectrometry (CE/MS) detection of berberine and palmatine using the microsprayer. SIM ion spray determination of a 2 ng/microL solution of berberine contained as a dry residue in the bottom of a 384-well plate as well as full-scan electrospray mass spectra for low-picomole levels of cytochrome c contained in a 1536-well microtiter plate are shown. The respective micro- and minisprayer devices provide a simple yet effective means of transferring trace-level samples either from a microscale or chip-based separation device as well as samples contained in multiwell plates which are increasingly employed in high-throughput applications in the pharmaceutical industry.

Journal Article↗
PubMed2001

A miniaturized liquid core waveguide-capillary electrophoresis system with flow injection sample introduction and fluorometric detection using light-emitting diodes.

A novel miniaturized capillary electrophoresis (CE) system is described where a Teflon AF-coated silica capillary serves both as the separation channel and as a transversely illuminated liquid core waveguide. This device uniquely uses flow injection (FI)-based split-flow sample introduction through a falling-drop interface. An H-channel structure fixed on a microscope glass slide utilizes a horizontal separation capillary with tubular sidearms on each end that serve as inlet and outlet flow-through electrode reservoirs. The inlet reservoir also functions as a falling-drop interface for coupling to the FI system. A blue LED is used as excitation source. A large-core optical fiber takes the emitted fluorescence to an inexpensive PMT with two layers of green plastic used for optical filtering. No focusing arrangement is needed. Continuous FI introduction of a series of 30-microL samples containing a mixture of of fluorescein isothiocyanate (FITC)-labeled amino acids allowed a throughput rate up to 144 samples/ h, with approximately 2% carryover and good precision (3.2% RSD). Baseline separation was achieved for FITC-labeled arginine, phenylalanine, glycine, and FITC in sodium tetraborate buffer (pH 9.5) with plate heights of 5.4-5.5 microm and plate numbers of 2.34 x 10(4)-2.37 x 10(4) under electrical field strengths of 214 V/cm for injection and 500 V/cm for separation (14-cm capillary, 48-microm i.d.). Detection limits (S/N = 3) were 1.3 microM for arginine and 1.9 microM for phenylalanine and glycine.

Amino Acids↗
PubMed2001

Miniaturization of a homogeneous fluorescence immunoassay based on energy transfer using nanotiter plates as high-density sample carriers.

The miniaturization of a homogeneous competitive immunoassay to a final assay volume of 70 nL is described. As the sample carrier, disposable plastic nanotiter plates (NTP) with dimensions of 2 x 2 cm2 containing 25 x 25 wells, corresponding to approximately 15,000 wells on a traditional 96-well microtiter plate footprint, were used. Sample handling was accomplished by a piezoelectrically actuated micropipet. To reduce evaporation while pipetting the assays, the NTP was handled in a closed humid chamber and cooled to the point of condensation. To avoid washing steps, a homogeneous assay was developed that was based on energy-transfer (ET). As a model system, an antibody-based assay for the detection of the environmentally relevant compound, simazine, in drinking water was chosen. Antibodies were labeled with the long-wavelength-excitable sulfoindocyanine dye Cy5 (donor), and a tracer was synthesized by labeling BSA with a triazine derivative and the acceptor dye Cy5.5. At low analyte concentrations, the tracer was preferably bound to the antibody binding sites. As a result of the close proximity of Cy5.5 and Cy5, an efficient quenching of the Cy5 fluorescence occurred. Higher analyte concentrations led to a progressive binding of the analyte to the antibody binding sites. The increased Cy5 fluorescence was determined by using a scanning laser-induced fluorescence detector. The limit of detection (LOD), using an antibody concentration of 20 nM, was 0.32 microg/L, or 1.11 x 10(-16) mol of simazine. In comparison, the LOD of the 96-well microtiter-plate-based ET immunoassay (micro-ETIA) was 0.15 microg/L, or 1.87 x 10(-13) mol. The LOD of the optimized micro-ETIA at 1 nM IgG, was 0.01 microg/L.

Antibodies, Monoclonal↗
PubMed2002

Miniature cylindrical ion trap mass spectrometer.

A miniature cylindrical ion trap mass spectrometer is described, and preliminary data are presented. Functionality and performance of laboratory-scale instruments have been maintained to the extent possible in this battery-operated mass spectrometer. Capabilities include tandem mass spectrometry experiments. Custom-designed electronic components include the RF scanning and amplification system, data acquisition components, and lens power supplies, as well as a custom-software application. Direct leak and membrane introduction inlet systems are used for sample introduction. A mass/charge range of approximately 250 Th with unit mass resolution has been demonstrated.

Journal Article↗
PubMed2003

Miniaturized tris(2,2'-bipyridyl)ruthenium(II) electrochemiluminescence detection cell for capillary electrophoresis and flow injection analysis.

The design and performance of a miniaturized chip-type tris(2,2'-bipyridyl)ruthenium(II) [Ru(bpy)3(2+)] electrochemiluminescence (ECL) detection cell suitable for both capillary electrophoresis (CE) and flow injection (FI) analysis are described. The cell was fabricated from two pieces of glass (20 x 15 x 1.7 mm), and the 0.5-mm-diameter platinum disk was used as working electrode held at +1.15 V (vs silver wire quasi-reference), the stainless steel guide tubing as counter electrode, and the silver wire as quasi-reference electrode. The performance traits of the cell in both CE and FI modes were evaluated using tripropylamine, proline, and oxalate and compared favorably to those reported for CE and FI detection cells. The advantages of versatility, sensitivity, and accuracy make the device attractive for the routine analysis of amine-containing species or oxalate by CE and FI with Ru(bpy)3(2+) ECL detection.

Journal Article↗
PubMed2004

Radionuclide imaging of miniaturized chemical analysis systems.

We propose radionuclide imaging as a valuable tool for the study of molecular interactions in miniaturized systems for chemical analysis. Sensitive and quantitative imaging can be performed with compounds labeled with short-lived positron-emitting radionuclides, such as (11)C and (68)Ga, within selected parts of the system. Radionuclide imaging is not restricted to transparent materials since the relatively energetic positrons can penetrate high optical density materials. Experimentally, a radiotracer is introduced into the object of study, which is subsequently placed on a phosphor storage plate. After exposure, the plate is scanned with a laser and a digital, quantitative image can be reconstituted. To demonstrate the concept, three types of microstructures suited for integration in chemical analysis systems were imaged with (11)C- and (68)Ga-labeled tracers. The influence of factors such as geometry of the object and type of radionuclide on resolution and sensitivity was investigated. The resolution ranged from 0.9 to 2.7 mm (fwhm). Measuring low amounts of radioactivity in the three structures, 2-20 Bq could be detected, which corresponded to 2.3-500 amol or 2.4-110 pM tracer. The imaging approach was applied to study analyte concentration and sample dilution effects on the performance of a capillary extraction column integrated in an automated LC-ESI-MS system. The utility of the technique was further illustrated by imaging of microchannels in a zeonor plastic compact disk and in a poly(dimethylsiloxane) material for the study of nonspecific peptide adsorption.

Carbon Isotopes↗
PubMed2005

Atmospheric pressure ionization in a miniature mass spectrometer.

A miniature cylindrical ion trap mass spectrometer featuring an atmospheric pressure interface allowing atmospheric pressure chemical ionization and electrospray ionization is described together with its analytical performance characteristics. The vacuum system, ion optics, mass analyzer, control electronics system, and detection system have all been designed and built in-house. The design is based upon a three-stage, differentially pumped vacuum system with the instrument capable of being interfaced to many types of atmospheric pressure ionization sources. Ions are transferred through home-built ion optics, and instrument control is achieved through custom-designed electronics and LabView control software. Corona discharge ionization and electrospray ionization sources are implemented and used to allow the analysis of both gaseous- and solution-phase samples during the characterization of the instrument. An upper mass/charge limit of approximately 450 Th with unit resolution was achieved using a 2.5-mm-internal radius cylindrical ion trap as the mass analyzer. The specificity of the instrument can be increased by employing the MS/MS capabilities of the ion trap and has been demonstrated for nitrobenzene. Limits of detection for the trace analysis in air of the chemical warfare agent simulant methyl salicylate (1.24 ppb) and for nitrobenzene (629 pptr) are achieved. The dynamic range of the instrument is currently limited to approximately 2 orders of magnitude by saturation of the detection electronics. Isolation and collision-induced dissociation efficiencies in MS/MS experiments both greater than 50% are reported. Electrospray/nanospray data are presented on solutions including 100 microM (D,L)-arginine, 10 microM (-)-ephedrine, and 10 microM lomefloxacin.

Journal Article↗
PubMed2005

Toxin detection by a miniaturized in vitro protein expression array.

A novel concept for toxin detection is presented that is based on the inhibitory effects of certain toxins on protein synthesis. Biological synthesis of proteins is called protein expression, which consists of the steps of DNA transcription and protein translation. In the toxin detection concept, a group of proteins are expressed simultaneously in a device consisting of an array of miniaturized wells. The expression yields of these proteins are inhibited by presence of a toxin. Differential inhibitory effects of each toxin on the expression of proteins in wells result in a response pattern (or signature) of the array device, which can be used as a tool for detection and identification of known and unknown agents. The concept has been demonstrated by in vitro protein expression of three proteins, including green fluorescent protein (GFP), chloramphenicol acetyltransferase (CAT), and luciferase. The production yields of these proteins in a cell-free medium were inhibited differentially by two toxin simulants, tetracycline (TC) and cycloheximide (CH). We confirmed that TC has an inhibitory effect on the production of GFP and CAT in the E. coli expression system whereas CH has a negligible effect. Moreover, the degree of TC's inhibitory effect on GFP is different from that on CAT. We also observed an opposite disparity; TC has a negligible inhibitory effect on the luciferase production in the rabbit reticulocyte expression system, whereas CH has a significant inhibitory effect. In addition, we obtained a response pattern in a 3 x 4 array device; the results suggest the feasibility of toxin detection based on the mechanisms of toxin actions. The device and method are also likely applicable to high-throughput drug screening, evident from the fact that TC is an antibiotic medicine.

Animals↗
PubMed2006

Fully integrated miniature device for automated gene expression DNA microarray processing.

A DNA microarray with 12,000 features was integrated with a microfluidic cartridge to automate the fluidic handling steps required to carry out a gene expression study of the human leukemia cell line (K562). The fully integrated microfluidic device consists of microfluidic pumps/mixers, fluid channels, reagent chambers, and a DNA microarray silicon chip. Microarray hybridization and subsequent fluidic handling and reactions (including a number of washing and labeling steps) were performed in this fully automated and miniature device before fluorescent image scanning of the microarray chip. Electrochemical micropumps were integrated into the cartridge to provide pumping of liquid solutions. The device was completely self-contained: no external pressure sources, fluid storage, mechanical pumps, mixers, or valves were necessary for fluid manipulation, thus eliminating possible sample contamination and simplifying device operation. Fluidic experiments were performed to study the on-chip washing efficiency and uniformity. A single-color transcriptional analysis of K562 cells with a series of calibration controls (spiked-in controls) to characterize this new platform with regard to sensitivity, specificity, and dynamic range was performed. The device detected sample RNAs with a concentration as low as 0.375 pM. Experiment also showed that the performance of the integrated microfluidic device is comparable with the conventional hybridization chambers with manual operations, indicating that the on-chip fluidic handling (washing and reaction) is highly efficient and can be automated with no loss of performance. The device provides a cost-effective solution to eliminate labor-intensive and time-consuming fluidic handling steps in genomic analysis.

Bacteriophage lambda↗
PubMed2006

Miniature differential mobility spectrometry using atmospheric pressure photoionization.

Positive and negative ion spectra have been obtained with a miniature differential mobility spectrometer equipped with a photoionization source operating at atmospheric pressure. With benzene as a dopant, providing C6H6+ as reactant ion, protonated molecular ions and proton-bound dimer ions were obtained with dimethyl methylphosphonate and butanone. The spectra obtained from gas chromatographic injections of aromatic hydrocarbons, benzene, toluene, and the xylenes, produced the molecular ions when the moisture level was very low, but at a high level the hydrated proton was also present. Possible mechanisms for the formation of protonated products are discussed. Negative ions were produced from electron capture by sulfur hexafluoride using benzene or acetone as dopant. Photoionization of nitrogen dioxide led to the formation of the nitrate ion whose yield was a nonlinear function of concentration. The use of a suitable dopant enhanced ion formation by up to 2 orders of magnitude, and limits of detection in both the positive and negative modes were all at the sub ppm(v) level. The study makes a strong case for the use of a photoionization source as an alternative to the radioactive 63Ni source.

Journal Article↗
PubMed2006

Isotachophoresis in free-flow using a miniaturized device.

For the first time, we report a miniaturized approach for isotachophoresis employing the technique of free-flow electrophoresis. Using a micromachined separation chamber with a volume of 200 nL, a sample mixture of fluorescein, eosin G, and acetylsalicylic acid was separated, stacked, and concentrated in less than a minute. Additionally, an isotachophoretic separation of a reaction mixture of myoglobin and fluoresceinisothiocyanate as a fluorescence label has shown the potential of this method for on-line sample preparation.

Journal Article↗
PubMed2006

Miniaturization and automation of an internally cooled coated fiber device.

The internally cooled coated fiber device was miniaturized to allow its direct introduction into a gas chromatography injector, while maintaining a reasonable lifetime of the septum. The device was robust, and its fiber, which was accommodated in an 18-gauge needle, was reproducibly used for more than 100 injections without any coating failure. The fiber temperature was controlled within 5 degrees C of the preset value by use of a temperature controller, a solenoid valve, and stainless steel tubings with different inner diameter. The device was mounted and used on the CTC CombiPAL autosampler with minor modifications, such as enlarging the hole of the needle guide of the autosampler and coupling the temperature control system of the device to the autosampler through a logic circuit. The device was validated with the back equilibration of hydrocarbons preloaded in the fiber in air. The automation of the internally cooled coated fiber device provided the feasibility of high throughput for the analysis of analytes in complex matrixes that required simultaneous heating of the sample matrixes and cooling of the fiber coating.

Journal Article↗
PubMed1996

Miniature sodium-selective ion-exchange optode with fluorescent pH chromoionophores and tunable dynamic range.

An extension into the fluorescence mode of ion-exchange optodes is described, allowing miniaturization and its concomitant benefits. A micrometer-size, fluorescent fiber-optic sodium sensor is described, based on a highly sodium-selective, crown ether-capped calix[4]arene ionophore, capable of ratiometric operation. Three sensor configurations are given, employing different lipophilic, fluorescent pH chromoionophores (Nile Blue derivatives), demonstrating the ability to improve the detection limit and tune the dynamic range to the desired region of interest. Two of the sensors are of special interest in that their working ranges lie within those desired for measuring intracellular cytosolic or blood levels of sodium at the respective physiological pH. These optodes have excellent sodium selectivity, with other physiologically relevant cations (e.g., potassium, calcium, and magnesium) being highly discriminated. Three simple mathematical relationships are given for the three experimentally used fluorescent signal mechanisms (intensity, intensity ratios, and inner-filter or energy transfer effects), permitting visualization on a single graph and enabling direct comparison of the different sensors' optical responses on a common platform. Finally, these optodes measure the sample's sodium activity, rather than the concentration, provided that the sample's pH is measured simultaneously by another sensor, such as a glass electrode.

Fiber Optic Technology↗
PubMed1996

Miniaturized biosensors employing electropolymerized permselective films and their use for creatine assays in human serum.

Miniaturized, disposable amperometric biosensors for determination of creatinine in human serum are described. The base electrodes are fabricated using micro-electronics techniques, to build a multilayer film structure on a polyimide foil. By using a thin electropolymerized film of poly(1,3-diaminobenzene), the electrochemical interferences from ascorbate, urate, acetaminophen, and other oxidizable species are greatly diminished. The multienzyme system (creatininase, creatinase, sarcosine oxidase) is immobilized on top of the permselective layer using cross-linking of the proteins with glutaraldehyde. The electropolymerization conditions for obtaining almost ideal permselectivity of the inner layer are defined, as well as the optimal enzyme layer preparation. A composite polymeric outer membrane [Nafion + poly-(2-hydroxy-ethyl methacrylate) is used for diffusion control and to protect the enzyme layer from fouling. The reagentless planar sensors for creatinine and creatine have fast response time (t95 = 1 min), linear response up to 1.2 mM in batch-type and 2.0 mM in flow injection analysis and a detection limit of 10-20 muM. They are applied in a differential setup for creatinine assay in control and hospital human serum samples and are suitable for incorporation in a portable analyzer.

Biosensing Techniques↗