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Chip-based capillary electrophoresis/mass spectrometry determination of carnitines in human urine.

A chip-based capillary electrophoresis/mass spectrometry (CE/MS) system is described for the CE separation and on-line electrospray detection of carnitine and selected acylcarnitines from mixtures of analytical standards as well as extracts of fortified human urine. Chip-based CE/MS experiments in two different laboratories were carried out using a triple-quadrupole mass spectrometer and a quadrupole time-of-flight (QTOF) mass spectrometer, respectively. The glass chips used with both systems were comparably equipped with a microfabricated capillary electrophoresis (CE) channel but with different electrosprayers. The quadrupole chip-based CE/MS experiments employed a miniature coupled microsprayer, which allowed coupling of the microelectrospray process via a micro liquid junction at the exit of the CE capillary channel. Selected ion monitoring (SIM) CE/MS experiments were employed for all of the quadrupole CE/MS work. The QTOF CE/MS full-scan single MS and MS/MS experiments were carried out in another laboratory using accurate mass measurement TOF mass spectrometry techniques. The electrospray process that was employed with the QTOF system differed in that an inserted nanoelectrospray capillary needle was carefully affixed into a flat-bottomed hole that was aligned with the CE channel exit orifice. SIM CE/MS using the described quadrupole system provided acceptable ion current electropherograms from fmole levels from analytical standard solutions of carnitine and acylcarnitines that were manually injected (loaded) onto the chip. In addition, the corresponding electropherograms for human urine fortified with the target carnitine and acylcarnitines at a 10-20 microg/mL (35-124 microM) level were obtained via SIM CE/MS techniques. The measured CE separation efficiency for the SIM CE/MS electropherograms was determined to be 2860 plates (peak width at half-height method or N = 5.54(T/WO.5(2)), and carnitine and three acylcarnitines were separated in less than 48 s. In contrast, using quadrupole-TOF technologies, the same samples could be diluted by a factor of 2-4 to obtain a comparable detector response for the target compounds. In the full-scan, single mass analyzer mode (m/z 150-500), the CE separation efficiency was measured to be 2600 plates, but mass measurement accuracy was less than 5.0 ppm for the quaternary cations. In the CE/MS/MS mode, full-scan collision-induced dissociation (CID) mass spectra were obtained with a mass accuracy of < or =10 ppm for the higher mass ions and < or =27 ppm for the lower mass product ions. These results demonstrate the feasibility for on-chip CE separation and electrospray mass spectrometric detection for these important compounds in synthetic mixtures, as well as in human urine extracts.

Carnitine↗

Immunobiosensor chips for detection of Escherichia coil O157:H7 using electrochemical impedance spectroscopy.

Impedance biosensor chips were developed for detection of Escherichia coli O157:H7 based on the surface immobilization of affinity-purified antibodies onto indium tin oxide (ITO) electrode chips. The immobilization of antibodies onto ITO chips was carried out using an epoxysilane monolayer to serve as a template for chemical anchoring of antibodies. The surface characteristics of chips before and after the binding reaction between the antibodies and antigens were characterized by atomic force microscopy (AFM). The patterns of the epoxysilanes monolayer, antibodies, and E. coli cells were clearly observed from the AFM images. Alkaline phosphatase as the labeled enzyme to anti-E. coli O157:H7 antibody was used to amplify the binding reaction of antibody-antigen on the chips. The biocatalyzed precipitation of 5-bromo-4-chloro-3-indolyl phosphate by alkaline phosphatase on the chips in pH 10 PBS buffer containing 0.1 M MgCl2 increased the electron-transfer resistance for a redox probe of Fe(CN)6(3-/4-) at the electrode-solution interface or the electrode resistance itself. Electrochemical impedance spectroscopy and cyclic voltammetric method were employed to follow the stepwise assembly of the systems and the electronic transduction for the detection of E. coli. The biosensor could detect the target bacteria with a detection limit of 6 x 10(3) cells/mL. A linear response in the electron-transfer resistance for the concentration of E. coli cells was found between 6 x 10(4) and 6 x 10(7) cells/mL.

Biosensing Techniques↗

On-chip hydrodynamic chromatography separation and detection of nanoparticles and biomolecules.

For the first time, on-chip planar hydrodynamic chromatography is combined with UV absorption detection. This technique is suitable for size characterization of synthetic polymers, biopolymers, and particles. Possible advantages of an on-chip hydrodynamic chromatography system over conventional techniques, such as size exclusion chromatography, and field-flow fractionation are fast analysis, high efficiency, reduced solvent consumption, and easy temperature control. The hydrodynamic separations are performed in a planar configuration realized in fused silica using a mixture of fluorescent and nonfluorescent polystyrene particles with sizes ranging from 26 to 155 nm. The planar chip configuration consists of a 1-microm-high, 0.5-mm-wide, and 69-mm-long channel, an integrated 150-pL injection structure, and a 30-microm-deep and 30-microm-wide detection cell, suitable for UV absorption detection. By combination of the separation data obtained in the new fused-silica chip with those obtained using a previously presented planar hydrodynamic chromatography chip, which was realized using silicon and glass microtechnology, a description of the retention and dispersion behavior of planar hydrodynamic chromatography is obtained. Especially the influence of the sidewalls on the dispersion is investigated. Furthermore a hydrodynamic separation within 70 s of several biopolymers is shown in the glass-silicon chip.

Chromatography, Gel↗

High-performance genetic analysis on microfabricated capillary array electrophoresis plastic chips fabricated by injection molding.

We have developed a novel technique for mass production of microfabricated capillary array electrophoresis (mu-CAE) plastic chips for high-speed, high-throughput genetic analysis. The mu-CAE chips, containing 10 individual separation channels of 50-microm width, 50-microm depth, and a 100-microm lane-to-lane spacing at the detection region and a sacrificial channel network, were fabricated on a poly(methyl methacrylate) substrate by injection molding and then bonded manually using a pressure-sensitive sealing tape within several seconds at room temperature. The conditions for injection molding and bonding were carefully characterized to yield mu-CAE chips with well-defined channel and injection structures. A CCD camera equipped with an image intensifier was used to monitor simultaneously the separation in a 10-channel array with laser-induced fluorescence detection. High-performance electrophoretic separations of phiX174 HaeIII DNA restriction fragments and PCR products related to the human beta-globin gene and SP-B gene (the surfactant protein B) have been demonstrated on mu-CAE plastic chips using a methylcellulose sieving matrix in individual channels. The current work demonstrated greatly simplified the fabrication process as well as a detection scheme for mu-CAE chips and will bring the low-cost mass production and application of mu-CAE plastic chips for genetic analysis.

Chemistry Techniques, Analytical↗

Plastic ELISA-on-a-chip based on sequential cross-flow chromatography.

A plastic chip that can perform immunoassays using an enzyme as signal generator, i.e., ELISA-on-a-chip, was developed by incorporating an immunostrip into channels etched on the surfaces of the chip. To utilize an analytical concept of cross-flow chromatography, the chip consisted of two cross-flow channels in the horizontal and vertical directions. In the vertical channel, we placed a 2-mm-wide immunostrip for cardiac troponin I (cTnI), which was identical to a conventional rapid test kit except for the utilization of an enzyme, horseradish peroxidase (HRP), as tracer. An enzyme substrate supply channel and a horizontal flow absorption pad compartment were transversely arranged on each lateral side of the signal generation pad of the strip, respectively. Upon application of a sample containing cTnI, it migrated vertically through the membrane strip by capillary action, and antigen-antibody binding occurred. After 15 min, the horizontal flow was initiated by the addition of a chromogenic substrate solution for HRP into the supply channel and by partial superimposition of the horizontal flow absorption pad onto the signal generation pad. A color signal proportional to the analyte concentration was produced on this pad, measured after 5 min as optical densities using a digital camera-based detector, and quantified by integration of the densities under the peak after normalization. Its calibration curve indicated that the detection limit of the chip was approximately 0.1 ng/mL and its quantification limit was 0.25 ng/mL. In measuring blindly prepared samples, the chip performance correlated with that of a reference system, Beckman Coulter Access, within 2.5-fold discrepancy at the detection limit.

Antibodies↗

Laser-induced fluorescence detection system for microfluidic chips based on an orthogonal optical arrangement.

In this work, a simple LIF detection system based on an orthogonal optical arrangement for microfluidic chips was developed. Highly sensitive detection was achieved by detecting the fluorescence light emitted in the microchannel through the sidewall of the chip to reduce scattered light interference from the laser source. A special crossed-channel configuration, with a 1.5-mm distance from the separation channel to the sidewall of the glass chip, was designed in order to facilitate collection of emitted fluorescence light through the sidewall. The significant difference in intensity distribution of scattered laser light on the chip plane observed in this study was fully exploited to optimize S/N ratio of detected signals by rejection of scattered light, both through systematic measurements and employing ray-tracing simulation. A fluorescence collection angle of 45 degrees in the chip plane gave the best result, with a scattered light intensity 1/38 of that obtained at an angle of 90 degrees. Sodium fluorescein and fluorescein isothiocyanate-labeled amino acids were used as model samples to demonstrate the performance of the LIF system. A detection limit (S/N = 3) of 1.1 pM fluorescein was obtained, which is comparable to that of optimized confocal LIF systems for chip-based capillary electrophoresis. Apart from the high detection power, the system also has the advantages of simple optical structure, compactness, and ease in building.

Journal Article↗

Numeric simulation of heat transfer and electrokinetic flow in an electroosmosis-based continuous flow PCR chip.

Precise design and operational control of the polymerase chain reaction process is key to the performance of on-chip DNA analysis. This research is dedicated to understanding the fluid flow and heat transfer mechanisms occurring in continuous flow PCR chips from the engineering point of view. In this work, a 3-dimensional model was developed to simulate the electrical potential field, the flow field, and the temperature field in an electroosmosis-based continuous flow PCR chip. On the basis of the simultaneous solution to this model, the effects of the channel/chip size, the chip material, and the applied voltage difference on the temperature distribution and control are discussed in detail. The importance of each heat transfer mechanism for different situations is also discussed. It was found that if a larger chip thickness or a material with a lower heat conductivity was used, the temperature in the microfluidic PCR chip would decrease dramatically. The effects of the applied electrical field strength and flow velocity on the temperature distribution, however, are negligible for microchannels with a small cross-sectional area. With bigger channels, the flow direction will affect the temperature distribution in the channel because heat convection will dominate heat transfer.

Computer Simulation↗

Fate of fumonisins during the production of fried tortilla chips.

The fate of fumonisin B(1) (FB(1)), a mycotoxin found in corn, during the commercial manufacture of fried tortilla chips was studied. FB(1) and hydrolyzed FB(1) (HFB(1)) concentrations in four lots of corn and in the masa, other intermediates, liquid and waste byproducts, and fried chips were determined by HPLC. FB(1) concentrations in the masa and chips were reduced significantly, up to 80% in the fried chips, compared to that in the raw corn. HFB(1) was also found in the masa and chips, but at low concentrations compared to FB(1). LC-MS analyses corroborated HPLC findings and further showed the presence of partially hydrolyzed FB(1) (PHFB(1)), which, like HFB(1), was formed during the nixtamalization (cooking/steeping the corn in alkaline water to make masa) step and found predominantly in the cooking/steeping liquid and solid waste. No significant amounts of N-(carboxymethyl)-FB(1) or N-(1-deoxy-D-fructos-1-yl)-FB(1), indicative of fumonisin-sugar adduct formation, were found. Thus, FB(1) is removed from corn and diverted into liquid and waste byproducts during the commercial production of fried tortilla chips. Nixtamalization and rinsing are the critical steps, whereas grinding, sheeting, baking, and frying the masa had little effect.

Carboxylic Acids↗

Specific binding of integrin alphaIIbbeta3 to RGD peptide immobilized on a nitrilotriacetic acid chip: a surface plasmon resonance study.

Nitrilotriacetic acid has been routinely used in protein purification for its high affinity for His-tagged protein in the presence of Ni2+. Here we reported a type of nitrilotriacetic acid chip (NTA-chip) prepared by transferring NTA-DOGS containing a lipid monolayer to a 50 nm thick gold layer deposited on a glass slide. The surface binding ability of His-tagged protein and regeneration of NTA chip were characterized using a synthetic polypeptide P1 (His-His-His-His-His-His-epsilon-aminohexanoic-Gly-Gly-Arg-Gly-Asp-Ser). The effect of divalent cations on integrin binding affinity for RGD ligand was investigated after P1 had been immobilized onto the sensor chip. The results show that the NTA-chip is a useful tool to immobilize His-tagged protein on the chip surface, and can provide a functional orientation for further investigation. The results also show that removing of Ca2+ bound on low affinity sites or adding of Mn2+ can increase the binding ability of integrin.

Lysine↗

An in-vitro evaluation of a dental subtraction radiography system using bone chips on dried human mandibles.

The aim of this in vitro study was to evaluate the ability of digital subtraction radiography to detect small differences in bone density between 2 dental images captured with the Digora radiographic imaging system. 5 dried human mandibles were held in a fixed position and cortical bone chips were superimposed on the alveolar bone between the 1st molar and 2nd premolar teeth. For each mandible, a reference image was obtained without bone chip placement. Subsequently, another image without a bone chip and a series of 10 images with bone chips ranging from 1.2 to 35.3 mg, were recorded. Each of the initial images of the 5 mandibles was then compared to the 11 subsequent images using digital subtraction radiography. The difference in density between the 2 images, at the site between the 1st molar and 2nd premolar, was calculated and represented as a volume equivalent of aluminium. Regression analysis demonstrated a close relationship between the weight of the bone chips and the aluminium equivalent density difference between the 2 radiographs (r2 = 0.96). Good results were obtained with the smaller bone chips that more closely resembled the dimensions of bone changes likely to be of clinical interest (1-8 mg). It is concluded that this subtraction radiography system is suitable for clinical investigations of localised small changes in alveolar bone and for the diagnosis and monitoring of destructive forms of periodontal diseases.

Absorptiometry, Photon↗

Quantitative measurement of serum allergen-specific IgE on protein chip.

Type I allergy is an immunoglobulin E (IgE)-mediated hypersensitivity disease inflicting more than quarter of the world population. In order to identify allergen sources, skin provocation test and IgE serology was performed using allergen extracts. Such process identifies allergen-containing sources but cannot identify the disease-eliciting allergenic molecules. Recently, microarray technology has been developed for allergen-specific IgE detection using rolling circle amplification. This study was carried out to evaluate protein chip technology for the quantitative measurement and limits of sensitivity of multiple allergen-specific IgE by an immunofluorescence assay. Significance of positive calibrators was tested using purified human IgE. Dermatophagoides pteronyssinus (Dp), egg white, milk, soybean, and wheat were used as allergens and human serum albumin as negative control. Sensitivity and clinical efficacy of protein chip were evaluated using allergy immune serum for Dp. The fluorescent intensities for purified human IgE as calibrator were well correlated with the concentrations of human IgE. Two-fold dilution of serum allowed an optimal reaction with Dp (1 mg/ml) at which serum Dp-specific IgE levels by protein chip were compatible with those by UniCap. The sensitivity of protein chip in this study was found at level of 1 IU/ml of IgE. Dp-specific IgE levels by protein chip correlated well with those of UniCap by comparing 10 atopic dermatitis. Additional 18 sera were tested for above multiple antigens other than Dp and significant results were obtained for many antigens as well as Dp. These results indicated that spotting of heterogeneous protein mixture on protein chip and the quantitative measurement of serum allergen-specific IgE levels using immunofluorescence assay can be successfully applied in the clinical laboratory for the diagnosis of allergy and could be applied to diagnosis of autoimmune and infectious diseases

Allergens↗

Interconnected reversible lab-on-a-chip technology.

Interconnected lab-on-a-chip modules with minimal dead volume have been developed resulting in the 'plug and play' concept based upon a reversible bonding process. This paper describes the detail of a chip to chip interconnection method, where devices have been aligned and bonded within 15 min and rapidly disassembled in under 5 min. The transport of fluorescein between the chip modules was used as a model microfluidic system and analysed in order to demonstrate the electrophoretic performance of the device and the interconnected junction. Using this technology, in the future different modules for various applications can be developed and interconnected, depending on the required applications. In addition, this simple but rapid method of chip to chip connection overcomes potential problems associated with integrating incompatible materials on one device.

Journal Article↗

An integrated digital microfluidic lab-on-a-chip for clinical diagnostics on human physiological fluids.

Clinical diagnostics is one of the most promising applications for microfluidic lab-on-a-chip systems, especially in a point-of-care setting. Conventional microfluidic devices are usually based on continuous-flow in microchannels, and offer little flexibility in terms of reconfigurability and scalability. Handling of real physiological samples has also been a major challenge in these devices. We present an alternative paradigm--a fully integrated and reconfigurable droplet-based "digital" microfluidic lab-on-a-chip for clinical diagnostics on human physiological fluids. The microdroplets, which act as solution-phase reaction chambers, are manipulated using the electrowetting effect. Reliable and repeatable high-speed transport of microdroplets of human whole blood, serum, plasma, urine, saliva, sweat and tear, is demonstrated to establish the basic compatibility of these physiological fluids with the electrowetting platform. We further performed a colorimetric enzymatic glucose assay on serum, plasma, urine, and saliva, to show the feasibility of performing bioassays on real samples in our system. The concentrations obtained compare well with those obtained using a reference method, except for urine, where there is a significant difference due to interference by uric acid. A lab-on-a-chip architecture, integrating previously developed digital microfluidic components, is proposed for integrated and automated analysis of multiple analytes on a monolithic device. The lab-on-a-chip integrates sample injection, on-chip reservoirs, droplet formation structures, fluidic pathways, mixing areas and optical detection sites, on the same substrate. The pipelined operation of two glucose assays is shown on a prototype digital microfluidic lab-on-chip, as a proof-of-concept.

Body Fluids↗

An automated electrokinetic continuous sample introduction system for microfluidic chip-based capillary electrophoresis.

An automated and continuous sample introduction system for microfluidic chip-based capillary electrophoresis (CE) was developed in this work. An efficient world-to-chip interface for chip-based CE separation was produced by horizontally connecting a Z-shaped fused silica capillary sampling probe to the sample loading channel of a crossed-channel chip. The sample presentation system was composed of an array of bottom-slotted sample vials filled alternately with samples and working electrolyte, horizontally positioned on a programmable linearly moving platform. On moving the array from one vial to the next, and scanning the probe, which was fixed with a platinum electrode on its tip, through the slots of the vials, a series of samples, each followed by a flow of working electrolyte was continuously introduced electrokinetically from the off-chip vials into the sample loading channel of the chip. The performance of the system was demonstrated in the separation and determination of FITC-labeled arginine and phenylalanine with LIF detection, by continuously introducing a train of different samples. Employing 4.5 kV sampling voltage (1000 V cm(-1) field strength) for 30 s and 1.8 kV separation voltage (400 V cm(-1) field strength) for 70 s, throughputs of 36 h(-1) were achieved with <1.0% carryover and 4.6, 3.2 and 4.0% RSD for arginine, FITC and phenylalanine, respectively (n = 11). Net sample consumption was only 240 nL for each sample.

Electrophoresis, Capillary↗

Ubiquitylation of neuronal nitric-oxide synthase by CHIP, a chaperone-dependent E3 ligase.

It is established that neuronal nitric-oxide synthase (nNOS) is ubiquitylated and proteasomally degraded. The proteasomal degradation of nNOS is enhanced by suicide inactivation of nNOS or by the inhibition of hsp90, which is a chaperone found in a native complex with nNOS. In the current study, we have examined whether CHIP, a chaperone-dependent E3 ubiquitin-protein isopeptide ligase that is known to ubiquitylate other hsp90-chaperoned proteins, could act as an ubiquitin ligase for nNOS. We found with the use of HEK293T or COS-7 cells and transient transfection methods that CHIP overexpression causes a decrease in immunodetectable levels of nNOS. The extent of the loss of nNOS is dependent on the amount of CHIP cDNA used for transfection. Lactacystin (10 microM), a selective proteasome inhibitor, attenuates the loss of nNOS in part by causing the nNOS to be found in a detergent-insoluble form. Immunoprecipitation of the nNOS and subsequent Western blotting with an anti-ubiquitin IgG shows an increase in nNOS-ubiquitin conjugates because of CHIP. Moreover, incubation of nNOS with a purified system containing an E1 ubiquitin-activating enzyme, an E2 ubiquitin carrier protein conjugating enzyme (UbcH5a), CHIP, glutathione S-transferase-tagged ubiquitin, and an ATP-generating system leads to the ubiquitylation of nNOS. The addition of purified hsp70 and hsp40 to this in vitro system greatly enhances the amount of nNOS-ubiquitin conjugates, suggesting that CHIP is an E3 ligase for nNOS whose action is facilitated by (and possibly requires) its interaction with nNOS-bound hsp70.

Acetylcysteine↗

Fumonisin concentration and ceramide synthase inhibitory activity of corn, masa, and tortilla chips.

Nixtamalization removes fumonisins from corn and reduces their amounts in masa and tortilla products. Fumonisin concentrations and potential toxicity could be underestimated, however, if unknown but biologically active fumonisins are present. Therefore, the relative amounts of fumonisins in extracts of fumonisin-contaminated corn and its masa and tortilla chip nixtamalization products were determined with an in vitro ceramide synthase inhibition bioassay using increased sphinganine (Sa) and sphinganine to sphingosine ratio (Sa/So) as endpoints. African green monkey kidney cells (Vero cells ATCC CCL-81) were grown in 1-ml wells and exposed to 4 microl of the concentrated extracts for 48 h. The corn extract inhibited ceramide synthase as Sa (mean = 132 pmol/well) and Sa/So (mean = 2.24) were high compared to vehicle controls (Sa = 9 pmol/well; Sa/So = 0.10). Inhibitory activity (mean Sa = 14-24 pmol/well; mean Sa/So = 0.17-0.28) of the masa and tortilla chip extracts were reduced > or = 80% compared to the corn extract. Results were corroborated in a second experiment in which Sa and Sa/So of the wells treated with masa or tortilla chip extracts were reduced > or = 89% compared to those treated with the corn extract. Masa and tortilla chip FB1 concentrations (4-7 ppm) were reduced about 80-90% compared to the corn (30 ppm) when the materials were analyzed by high-performance liquid chromatography (HPLC). Therefore, nixtamalization reduced both the measured amount of FB1 and the ceramide synthase inhibitory activity of masa and tortilla chips extracts. The results further suggest that the masa and tortilla chip extracts did not contain significant amounts of unknown fumonisins having ceramide synthase inhibitory activity.

Animals↗

A review of factors affecting fat absorption in hot chips.

Consumption of hot chips is a convenience food in most countries. Unfortunately, these are high in fat and contribute to fat-related diseases in societies with a high fat consumption. There is substantial scope through best-practice deep-frying techniques for producing lower fat, high-quality chips. From a review of the literature, the main factors associated with a lower-fat content of chips are thick (>12 mm), straight cut chips; cryogenic freezing methods; low moisture content of potatoes (specific gravity >1.1); frying fat: chip volume ratio of 6:1; frying at optimal temperature (180 to 185 degrees C) during cooking and turning the temperature down (approximately 140 degrees C) and covering the vats during slack periods; vigorously shaking the basket and hanging it over the deep fryer to drain after frying; maintaining the quality of the frying fat by regularly skimming the cracklings, filtering the fat, and topping up the fryer with fresh fat; keeping the fat turnover <5 days; regular cleaning of frying equipment. It is important that all deep frying operators are adequately trained in these techniques. It is also important that the frying medium is low in saturated and trans fatty acids (<20%) because of their effects on blood lipids and low in linolenic acid (<3%) because it is readily degraded. The widespread implementation of best-practice deep-frying would reduce fat content of hot chips and thus lower overall fat consumption.

Absorption↗

Study I: development and criterion validity of the Children's Interview for Psychiatric Syndromes (ChIPS).

OBJECTIVE: To test the psychometric properties of the Children's Interview for Psychiatric Syndromes (ChIPS), a second-generation structured diagnostic interview designed to improve on the methodology provided by first-generation structured interviews, which have increased diagnostic reliability over unstructured clinical interviews. METHOD: Forty-two children hospitalized on a children's inpatient unit were administered the ChIPS and the Diagnostic Interview for Children and Adolescents (DICA). The level of agreement regarding syndrome presence or absence of 15 diagnoses according to the two instruments was assessed using a standard kappa coefficient or a rare kappa coefficient and percentage agreement. RESULTS: Agreement between the two instruments was significant (p < 0.05) for 13 of 14 diagnoses for which either kappa coefficient could be calculated. Percentage agreement was 98% and 100% for the remaining two diagnoses. ChIPS and DICA results also were compared with a psychiatrist's diagnoses. Sensitivity was 80% for ChIPS and 61% for DICA. Specificity was 78% for ChIPS and 87% for DICA. CONCLUSION: ChIPS is proposed as a valid measure of child psychopathology that offers advantages over existing interviews.

Child↗