Fundamental Studies of Rapidly Fabricated On-Chip Passive Micromixer for Modular Microfluidics.

Micromixers play an important role in many modular microfluidics. Complex on-chip mixing units and smooth channel surfaces ablated by lasers on polymers are well-known problems for microfluidic chip fabricating techniques. However, little is known about the ablation of rugged surfaces on polymer chips for mixing uses. This paper provides the first report of an on-chip compact micromixer simply, easily and quickly fabricated using laser-ablated irregular microspheric surfaces on a polymethyl methacrylate (PMMA) microfluidic chip for continuous mixing uses in modular microfluidics. The straight line channel geometry is designed for sequential mixing of nanoliter fluids in about 1 s. The results verify that up to about 90% of fluids can be mixed in a channel only 500 µm long, 200 µm wide and 150 µm deep using the developed micromixer fabricating method under optimized conditions. The computational flow dynamics simulation and experimental result agree well with each other.

Dual-opposite multi-walled carbon nanotube modified carbon fiber microelectrode for microfluidic chip-capillary electrophoresis determination of methyl parathion metabolites in human urine.

Methyl parathion (MP) is a highly toxic organophosphate and its exposure may lead to substantial adverse effects to human health. The existence of 4-nitrophenol (4-NP) in the form of free phenol, glucuronide (4-NP-G) or as a sulfate ester (4-NP-S) can be used as biomarkers to assess the duration and extent of MP exposure. In this work, a MC-CE device incorporating post-CE amperometric detection using multi-walled carbon nanotubes (MWNTs) modified carbon fiber microelectrode (CFME) was fabricated and assessed for simultaneous determination of 4-NP, 4-NP-G, and 4-NP-S in human urine. The detection sensitivity and stability was greatly enhanced by the modification of MWNTs. The capability of the MC-CE device with dual MWNTs modified CFME for detecting impurity was assessed and reliability established by high recoveries from 95 to 97% for spiked MP biomarkers. The method developed is shown to provide a simple, sensitive, and reliable means for monitoring 4-NP, 4-NP-G, and 4-NP-S in human urine.

Determination of taurine in human tear fluid by capillary electrophoresis with indirect amperometric detection based on electrogenerated bromine.

A new method for the determination of taurine was developed based on indirect amperometric detection after capillary electrophoresis. A serial dual-electrode detector comprising an on column Pt film electrode (upstream electrode) and an end column Pt microdisk electrode (downstream electrode) was utilized to conduct the indirect amperometric detection. Bromide is oxidized to bromine at upstream electrode and reduced back to bromide at downstream electrode. Since taurine can react with bromine quantitatively and rapidly, its concentration can therefore be determined by the decrease of the current for bromine reduction at the downstream electrode. Principal experimental parameters governing the analytical performance were investigated and optimized. Under the optimal conditions, taurine can be baseline separated from interfering amino acids and the detection limit of 0.18 µM was obtained with a linear correlation coefficient of 0.999 over the concentration range of 0.5-60 µM. The developed method has been successfully applied in the determination of taurine in human tear fluid. The taurine level obtained was in good agreement with previous reports and recoveries for taurine spiked ranged from 92-95% with relative standard deviations within 4.6%, demonstrating the reliability of the developed method in the determination of taurine in human tear fluid.

A serial dual-electrode detector based on electrogenerated bromine for capillary electrophoresis.

A new serial dual-electrode detector for CE has been designed and fabricated for postcolumn reaction detection based on electrogenerated bromine. A coaxial postcolumn reactor was employed to introduce bromide reagent and facilitate the fabrication of upstream generation electrode by simply sputtering Pt film onto the outer surface of the separation capillary. Bromide introduced could be efficiently converted to bromine at this Pt film electrode and subsequently detected by the downstream Pt microdisk detection electrode. Analytes that react with bromine could be determined by the decrease of bromine reduction current at the downstream electrode resulting from the reaction between analytes and bromine. The effects of serial dual-electrode detector working conditions including electrode potentials, bromide flow rate, and bromide concentration on analytical performance were investigated using glutathione (GSH) and glutathione disulfide (GSSG) as test analytes. Under the optimal conditions, detection limits down to 0.16 µM for GSH and 0.14 µM for GSSG (S/N = 3) as well as linear working ranges of two orders of magnitude for GSH and GSSG were achieved. Furthermore, the separation efficiency obtained by our dual-electrode detector design was greatly improved compared with previous reported design. The developed method has been successfully applied to determine the GSH and GSSG impurity in commercial GSH supplement.

2-D t-ITP/CZE determination of clinical urinary proteins using a microfluidic-chip capillary electrophoresis device.

To replace the time-consuming sample pretreatment procedure, a microfluidic chip-CE device incorporating on-chip sample desalting/preconcentration with transient isotachophoresis (ITP)/capillary zone electrophoresis (CZE) was fabricated to perform sequential on-chip sample pretreatment and CE determination of four urinary proteins in clinical samples. On-chip sample desalting, clean-up and analyte preconcentration enable removing interfering sample matrix prior to transferring analytes to separation capillary for transient ITP/CZE determination. Four important urinary proteins transferrin, ß2-microglobulin, human serum albumin (HSA) and immunoglobulin G (IgG), investigated were shown to achieve quantitation limits sufficiently high to meet medical requirements, sensitivity enhancement up to 40-fold and detection limits down to 0.3, 0.05, 0.6, 0.5 mg/L, respectively. Compared to the stacking effect, the use of a large sample size was found to be the major factor for sensitivity enhancement. The method reliability is established by close to 100% recoveries and statistical agreement of results from the method developed with currently used clinical radio-immunoassay method for all four proteins investigated. Moreover, an assay time of less than 10 min is needed in the method developed as compared to 7 h for the radio-immunoassay method.

Capillary electrophoresis with LIF detection for assessment of mitochondrial number based on the cardiolipin content.

Cardiolipin is a mitochondria-specific phospholipid known to be intimately involved with numerous mitochondrial functions. Accordingly, quantitative determination of cardiolipin provides a valuable aspect for assessing mitochondrial content and function. The current study was undertaken to develop a simple and reliable method for direct analysis of cardiolipin with particular application for the assessment of mitochondrial number of HepG2 cells. The method presented is based on the online 10-N-nonyl acridine orange (NAO) interaction with cardiolipin using CE with LIF detection. An aqueous-organic solvent system composed of 10% H(2) O-40% methanol-50% ACN (all v/v) containing 20 µM NAO provides both short analysis time within 2 min and a definite fluorescence enhancement at 525 nm for NAO-cardiolipin complex as compared with NAO alone. Under the optimum condition, a calibration curve between the peak area and the concentration of cardiolipin was established in the range of 0.1-200 µM with a correlation coefficient of 0.9955. The detection limit is 9 nM. The proposed method was successfully applied to the analysis of cardiolipin in mitochondria from HepG2 cells. A new biochemical method estimating the mitochondrial number per cell was developed and used together with the proposed method for cardiolipin per cell measurement and cardiolipin per mitochondrion reported before.

Determination of acrylamide in potato crisps by capillary electrophoresis with quantum dot-mediated LIF detection.

Acrylamide or 2-propenamide (AAM), a water-soluble toxic contaminant, has recently caused health concern after it was found in food products made by high-temperature cooking. Due to its weak UV absorption and electrochemically inactive state, common analytical methods do not have sufficient sensitivity to meet the World Health Organization requirement. A LIF detection method mediated by water-soluble CdTe quantum dots capped with mercaptopropyl acid (MPA) is thus developed for AAM quantitation. The optimized conditions are as follows: 30 mmol/L SDS, 0.1 mmol/L quantum dot, and 40 mmol/L phosphate buffer solution at pH 8.0 under 18 kV run voltage with LIF detection at 473 nm excitation and 568 nm fluorescence. The linear quantitation range for AAM was found to vary from 1.0 to 100 mg/kg and a detection limit (S/N=2) at 0.1 mg/kg, showing sufficient sensitivity to meet the maximum AAM specified by the Joint Food and Agriculture Organization/World Health Organization Expert Committee for potato crisps. Recoveries for potato crisps sample spiked with 10, 20, and 100 mg/kg AAM were found to vary between 90 and 95% with RSD <5.7% (n=3).

Capillary electrophoresis with immobilized quantum dot fluorescence detection for rapid determination of organophosphorus pesticides in vegetables.

Based on the highly sensitive and selective fluorescence enhancement of water-soluble CdTe/CdS core-shell quantum dots (QD) by organophosphorus pesticides (OPs such as mevinphos, phosalone, methidathion and diazinon), a simple, rapid and selective method is developed using CE with QD/LIF detection (473 nm excitation/532 nm fluorescence) to determine OPs in vegetable samples. The method enables the use of a simple pretreatment procedure based only on solvent extraction and eliminates the use of a time-consuming SPE step. A novel procedure is developed to immobilize QD onto the inside capillary surface via the formation of a silane coupling mercaptopropyltrimethoxysilane network. Under optimized CE conditions, baseline separation for all four OPs was observed within 12 min. The CE-QD/LIF method was shown to have a detection limit from 50 to 180 µg/kg, working ranges 0.1-30 mg/kg, recoveries 88.7-96.1% and repeatability (RSD, n=3) 0.36-0.75% for migration time and 2.9-5.7% for peak height. For tomato samples, the detection limits were more than ten times lower than maximum residue levels specified by the Codex Alimentarius Commission for all four OPs investigated. The method thus satisfies the need for a simple, quick and selective method to determine residual OPs in complex vegetable matrix as required by the increasingly strict regulations for health protection introduced in recent years.

L-cysteine-capped CdTe quantum dots as a fluorescence probe for determination of cardiolipin.

This paper described the investigation of surface-modified quantum dots (QDs) as a fluorescence probe for the detection of cardiolipin. A single-step method for preparation of non-toxic and photo-stable cadmium telluride (CdTe) QDs capped by L-cysteine in aqueous solution was developed. The prepared QDs were characterized by high-resolution transmission electron microscopy, X-ray diffraction spectrometry, Fourier transform infrared spectrometry and spectrofluorometry. These functional QDs were used as a fluorescence probe for cardiolipin determination based on the fluorescence quenching. The optimum fluorescence intensity was found to be at pH 7.4 with QDs concentration of 4 x 10(-5) mol L(-1). The effect of other phospholipids on the intensity of CdTe QDs showed a low interference response. Under optimized conditions, the quenched fluorescence intensity was linear with the concentration of cardiolipin in the range of 1.33 x 10(-7) - 10.4 x 10(-7) mol L(-1) (r = 0.9976) and a detection limit (S/N = 3) of 18.5 nmol L(-1). The proposed method was applied to the determination of cardiolipin content of HepG2 cell samples before and after oxidative stress with satisfactory results.

Separation and determination of metal cations in milk and dairy products by CE.

To prevent casein adsorption and improve between-run repeatability, a CE procedure is developed for cation analysis in milk using a modified imidazole/alpha-hydroxyisobutyric acid (HIBA) BGE system to operate at pH 6 to match the pH of milk and elevate imidazole concentration to enhance its buffer action. The procedure is shown to produce a fast, economic and efficient method for cation separation in milk with only simple dilution. Upon direct hydrodynamic injection of diluted milk sample at 8 cm for 30 s in an uncoated column with a BGE consisting of 10 mM imidazole, 10 mM HIBA and 10% methanol at pH 6.0 under +18 kV, baseline separation was achieved for K(+), Na(+), Ca(2+), Mg(2+), Mn(2+), Cd(2+), Co(2+), Ni(2+)and Zn(2+). Agreeable results at 95% confidence level were obtained using CE and inductively coupled plasma-atomic emission spectrometry (ICP-AES) for milk samples after protein removal. Baseline-resolved peaks for essential minerals were obtained for fresh and non-refrigerated reconstituted milks. Long-term stability was demonstrated by repeated determinations without rinsing and improvement in repeatability was shown by rinsing with 60 mM SDS in BGE. Information on metal speciation useful for nutritional assessment was obtained from CE to complement the ICP methods.

Flow analysis coupled with PQC/DNA biosensor for assay of E. coli based on detecting DNA products from PCR amplification.

A flow-through PQC/DNA biosensor system is developed by combining sequential flow polymerase chain reaction (PCR) products denaturing prior to piezoelectric quartz crystal (PQC) detection via hybridization of ssDNA. The PQC/DNA biosensor is fabricated based on complex formation of neutravidin/biotinylated probe in 0.2M NaCl in TE buffer (10mM Tris, 1mM EDTA, pH 7.5). Results show that the coating fabricated provides a desirable quality with satisfactory performance. Its application for Escherichia coli detection under controlled flow at 0.02 mL/min for denaturing PCR products and 10 mL/min for transferring solution between reactors and delivering samples to detector to reduce rehybridization leads to significant improvement in repeatability (R.S.D.<6%, n=5) and sensitivity (DeltaF=34 Hz/1000 E. coli cells) as compared to existing manual method (R.S.D.=19%, n=5 and DeltaF=26 Hz/1000 E. coli cells, respectively). Down to 23 E. coli cells are detected, satisfying the HKEPD requirements for E. coli count in beach water.

Piezoelectric quartz crystal sensor for rapid analysis of pirimicarb residues using molecularly imprinted polymers as recognition elements.

A new piezoelectric quartz crystal (PQC) sensor using molecularly imprinted polymer (MIP) as sensing material has been developed for fast and onsite determination of pirimicarb in contaminated vegetables. Three MIPs particles have been prepared by conventional bulk polymerization (MIP-B) and precipitation polymerization in either acetonitrile (MIP-P1) or chloroform (MIP-P2). MIP-P2, with uniform spherical shape and mean diameter at about 50 nm, has shown the best performance as the sensing material for PQC sensor. The sensor fabricated with MIP-P2 can achieve a steady-state response within 5 min, a very short response time as compared to MIPs-coated PQC sensor reported in the literature. The sensor developed exhibits good selectivity (low response to those pesticides with similar structures to pirimicarb, such as atrazine, carbaryl, carbofuran and aldicarb) and high sensitivity to pirimicarb with a linear working range from 5.0 x 10(-6) to 4.7 x 10(-3) mol L(-1) (following a regression equation (r=0.9988) of -DeltaF=0.552+1.79 x 10(6) C), a repeatability (R.S.D., n=5) of 4.3% and a detection limit (S/N=3, n=5) of 5 x 10(-7) mol L(-1). The MIP-coated PQC sensor developed is shown to provide a sensitive and fast method for onsite determination of pirimicarb in aqueous extract from contaminated vegetables with satisfactory recoveries from 96 to 103% and repeatability (R.S.D., n=5) from 4.6 to 7.1% at pirimicarb concentrations ranging from 8.0x10(-6) to 2.0 x 10(-4) mol L(-1).

Variable selection for discriminating herbal medicines with chromatographic fingerprints.

When discriminating herbal medicines with pattern recognition based on chromatographic fingerprints, typically, the majority of variables/data points contain no discrimination information. In this paper, chemometric approaches concerning forward selection and key set factor analysis using principal component analysis (PCA), unweighted and weighted methods based on the inner- and outer-variances, Fisher coefficient from the between- and within-class variations were investigated to extract representative variables. The number of variables retained was determined based on the cumulative variance percent of principal components, the ratio of observations to variables and the factor indicative function (IND). In order to assess the methods for variable selection and criteria levels to determine the number of variables retained, the original and reduced datasets were compared with Procrustes analysis and a weighted measure of similarity. Moreover, the tri-variate plots of the first three PCA scores were used to visually examine the reduced datasets in low dimensional space. Herbal samples were finally discriminated by use of Bayes discrimination analysis with the reduced subsets. The case study for 79 herbal samples showed that, the methods of forward selection associating the variables with the loadings closest to 0 and key set factor analysis were preferable to determine the representative variables. Procrustes analysis and the weighted measure were not indicative to extract representative variables. High matching between the original and reduced datasets did not suggest high prediction accuracy. Visually examining the PC1-PC2-PC3 scores projection plots with the reduced subsets, not all the herb samples could be separated due to the complexity of chromatographic fingerprints.

Capillary zone electrophoresis characterization of low molecular weight heparin binding to interleukin 2.

A method based on capillary zone electrophoresis (CZE) was used to study the interaction between low molecular weight heparin (LMWH) and interleukin 2 (IL-2). The results showed that the increase of the concentration of LMWH led to the decrease of the peak height and the increase of the peak width of IL-2, but the peak areas were kept constant. The binding constant of IL-2 with LMWH was calculated as 1.2 x 10(6)M(-1) by Scatchard analysis, which is in good agreement with the results found in the references using enzyme-linked immunosorbent assay (ELISA). The results demonstrated that the interaction between IL-2 and LMWH is of fast on-and-off kinetic binding reaction. CZE might be used to study not only slow on-and-off rates interactions, but also fast on-and-off rates ones. The binding constant can be calculated easily, and the method can be applied to study a wide range of heparin-protein interactions.

Rapid authentication of ginseng species using microchip electrophoresis with laser-induced fluorescence detection.

Ginseng is one of the most expensive Chinese herbal medicines and the effectiveness of ginseng depends strongly on its botanical sources and the use of different parts of the plants. In this study, a microchip electrophoresis method coupled with the polymerase chain reaction (PCR)-short tandem repeats (STR) technique was developed for rapid authentication of ginseng species. A low viscosity hydroxypropyl methylcellulose (HPMC) solution was used as the sieving matrix for separation of the amplified STR fragments. The allele sizing of the amplified PCR products could be detected within 240 s or less. Good reproducibility and accuracy of the fragment size were obtained with the relative standard deviation for the allele sizes less than 1.0% (n=11). At two microsatellite loci (CT 12, CA 33), American ginseng had a different allele pattern on the electropherograms compared with that of the Oriental ginseng. Moreover, cultivated and wild American ginseng can be distinguished on the basis of allele sizing. This work establishes the feasibility of fast genetic authentication of ginseng species by use of microchip electrophoresis.

Simultaneous and ultrarapid determination of reactive oxygen species and reduced glutathione in apoptotic leukemia cells by microchip electrophoresis.

A microchip electrophoresis method coupled with laser-induced fluorescence (LIF) detection was established for simultaneous determination of two kinds of intracellular signaling molecules (reactive oxygen species, ROS, and reduced glutathione, GSH) related to apoptosis and oxidative stress. As the probe dihydrorhodamine-123 (DHR-123) can be converted intracellularly by ROS to the fluorescent rhodamine-123 (Rh-123), and the probe naphthalene-2,3-dicarboxaldehyde (NDA) can react quickly with GSH to produce a fluorescent adduct, rapid determination of Rh-123 and GSH was achieved on a glass microchip within 27 s using a 20 mM borate buffer (pH 9.2). The established method was tested to measure the intracellular ROS and GSH levels in acute promyelocytic leukemia (APL)-derived NB4 cells. An elevation of intracellular ROS and depletion of GSH were observed in apoptotic NB4 cells induced by arsenic trioxide (As(2)O(3)) at low concentration (1-2 microM). Buthionine sulfoximine (BSO), in combination with As(2)O(3) enhanced the decrease of reduced GSH to a great extent. The combined treatment of As(2)O(3) and hydrogen peroxide (H(2)O(2)) led to an inverse relationship between the concentrations of ROS and GSH obtained, showing the proposed method can readily evaluate the generation of ROS, which occurs simultaneously with the consumption of the inherent antioxidant.

Genotyping the -6A/G functional polymorphism in the core promoter region of angiotensinogen gene by microchip electrophoresis.

Angiotensinogen (AGT) gene has been regarded as one of the candidate genes for essential hypertension. In our study, the role of AGT gene as a putatively predisposing gene for hypertension was evaluated by genotyping a A (-6) G polymorphism in the core promoter region in 123 patients with essential hypertension and 103 healthy controls. A microchip electrophoresis method coupled with polymorphism chain reaction (PCR)-restriction fragment length polymorphism (RFLP) assay was used for genotyping the A (-6) G single-nucleotide polymorphism. The separation and detection of the digested PCR amplicons were completed just in 280 s or less. The genotype frequency fulfilled the criteria of the Hardy-Weinbery equilibrium (X2 = 3.067, P > 0.05). The results showed a higher frequency of the -6 A allele (0.70) in the normotensive subjects, which is higher than those reported in Germany (0.47) and Czech (0.40) populations, but similar to that found in Japanese populations (0.73). The frequencies of genotype AA, AG, and GG were 0.46, 0.49, and 0.05 in hypertensive subjects, and 0.44, 0.53, and 0.03 in control subjects. There is no significant difference in the distributions of the genotype and allele between the two groups (X2 = 0.88, P > 0.05; X2 = 0.024, P > 0.05). These findings differ from some of the results obtained in other ethnic groups, indicating the potential importance of ethnic origin in the assessment of genetic risk identifiers for a complex disease.

Determination of volatile components in ginger using gas chromatography-mass spectrometry with resolution improved by data processing techniques.

Ginger is widely used as either a food product or an herbal medicine in the world. In this paper, a method was developed for determining volatile components in essential oils from both dried and fresh ginger by use of gas chromatography-mass spectrometry (GC-MS) and chemometric approaches. With the resolution improvement by chemometric methods upon two-dimensional data from GC-MS, the drifting baseline can be corrected. In addition, the peak purity can be assessed and the number of chemical components and their stepwise elution in the peak clusters can be identified. The peak clusters investigated are then resolved into pure chromatograms and related mass spectra for each of the components involved. Finally, with the pure chromatograms and related mass spectra obtained, the chemical components can be qualitatively identified based on the similarity searches in the MS databases and the chromatographic retention times. Quantitative determination can be conducted using the overall volume integration approach. The results showed that 140 and 136 components were separated and that 74 and 75 of them were tentatively identified, which accounted for about 62.82 and 47.11% of the total relative content for dried and fresh ginger, respectively. In comparison with the chromatographic fingerprints of essential oils from dried and fresh ginger, 60 of the volatile components determined match with each other. The study demonstrated that the use of chemometric resolution based on two-dimensional data can mathematically enhance the separation ability of GC-MS and assist qualitative and quantitative determination of chemical components separated from complicated practical systems such as foods, herbal medicines, and environmental samples.

Correction of retention time shifts for chromatographic fingerprints of herbal medicines.

In this study, the combination of chemometric resolution and cubic spline data interpolation was investigated as a method to correct the retention time shifts for chromatographic fingerprints of herbal medicines obtained by high-performance liquid chromatography-diode array detection (HPLC-DAD). With the help of the resolution approaches in chemometrics, it was easy to identify the purity of chromatographic peak clusters and then resolve the two-dimensional response matrix into chromatograms and spectra of pure chemical components so as to select multiple mark compounds involved in chromatographic fingerprints. With these mark components determined, the retention time shifts of chromatographic fingerprints might be then corrected effectively. After this correction, the cubic spline interpolation technique was then used to reconstruct new chromatographic fingerprints. The results in this work showed that, the purity identification of the chromatographic peak clusters together with the resolution of overlapping peaks into pure chromatograms and spectra by means of chemometric approaches could provide the sufficient chromatographic and spectral information for selecting multiple mark compounds to correct the retention time shifts. The cubic spline data interpolation technique was user-friendly to the reconstruction of new chromatographic fingerprints with correction. The successful application to the simulated and real chromatographic fingerprints of two Cortex cinnamomi, fifty Rhizoma chuanxiong, ten Radix angelicae and seventeen Herba menthae samples from different sources demonstrated the reliability and applicability of the approach investigated in this work. Pattern recognition based on principal component analysis for identifying inhomogenity in chromatographic fingerprints from real herbal medicines could further interpret it.

Analysis of volatile components from Cortex cinnamomi with hyphenated chromatography and chemometric resolution.

In this paper, the combination of hyphenated chromatography and chemometric resolution was investigated as a method to qualitatively and quantitatively determine volatile components in Cortex cinnamomi from four main producing areas. With the help of chemometric resolution approaches, whether the chromatographic elution of chemical components is featured by "first-in-first-out" or embedded peaks could be determined. Upon this useful information obtained, the matrix data generated by hyphenated chromatography could be uniquely resolved into pure chromatogram and spectrum of each chemical component involved followed by qualitative and quantitative analysis. The results obtained in this work showed that, 94, 88, 93 and 89 volatile components were separated and 63, 60, 60 and 58 of them qualitatively and quantitatively determined representing about 93.39, 93.62, 92.03 and 92.59% of the total relative content, respectively. The combination of hyphenated chromatography with chemometric resolution could greatly enhance the chromatographic separation and spectral qualitatively determination ability so as to qualitatively and quantitatively detect many more volatile components and improve the analysis accuracy.