Academic literature on the topic 'Microchip CE'

Abstract Clinical analysis often requires rapid, automated, and high-throughput analytical systems. Microchip capillary electrophoresis (CE) has the potential to achieve very rapid analysis (typically seconds), easy integration of multiple analytical steps, and parallel operation. Although it is currently still in an early stage of development, there are already many reports in the literature describing the applications of microchip CE in clinical analysis. At the same time, more fully automated and higher throughput commercial instruments for microchip CE are becoming available and are expected to further enhance the development of applications of microchip CE in routine clinical testing. To put into perspective its potential, we briefly compare microchip CE with conventional CE and review developments in this technique that may be useful in diagnosis of major diseases.

Abstract Background: Microchip capillary electrophoresis (CE) is a promising method for chemical analysis of complex samples such as whole blood. We evaluated the method for point-of-care testing of lithium. Methods: Chemical separation was performed on standard glass microchip CE devices with a conductivity detector as described in previous work. Here we demonstrate a new sample-to-chip interface. Initially, we took a glass capillary as a sample collector for whole blood from a finger stick. In addition, we designed a novel disposable sample collector and tested it against the clinical standard at the hospital (Medisch Spectrum Twente). Both types of collectors require <10 μL of test fluid. The collectors contain an integrated filter membrane, which prevents the transfer of blood cells into the microchip. The combination of such a sample collector with microchip CE allows point-of-care measurements without the need for off-chip sample treatment. This new on-chip protocol was verified against routine lithium testing of 5 patients in the hospital. Results: Sodium, lithium, magnesium, and calcium were separated in <20 s. The detection limit for lithium was 0.15 mmol/L. Conclusions: The new microchip CE system provides a convenient and rapid method for point-of-care testing of electrolytes in serum and whole blood.

Abstract Background: Detection of mutations by gel electrophoresis and allele-specific amplification by PCR (AS-PCR) is not easily scaled to accommodate a large number of samples. Alternative electrophoretic formats, such as capillary electrophoresis (CE) and microchip electrophoresis, may provide powerful platforms for simple, fast, automated, and high-throughput mutation detection after allele-specific amplification. Methods: DNA samples heterozygous for four mutations (185delAG, 5382insC, 3867G→T, and 6174delT) in BRCA1 and BRCA2, and homozygous for one mutation (5382insC) in BRCA1 and two mutations (16delAA and 822delG) in PTEN were chosen as the model system to evaluate the capillary and microchip electrophoresis methods. To detect each mutation, three primers, of which one was labeled with the fluorescent dye 6-carboxyfluorescein and one was the allele-specific primer (mutation-specific primer), were used to amplify the DNA fragments in the range of 130–320 bp. AS-PCR was combined with heteroduplex (HD) analysis, where the DNA fragments obtained by AS-PCR were analyzed with the conditions developed for CE-based HD analysis (using a fluorocarbon-coated capillary and hydroxyethylcellulose). The CE conditions were transferred into the microchip electrophoresis format. Results: Three genotypes, homozygous wild type, homozygous mutant, and heterozygous mutant, could be identified by CE-based AS-PCR-HD analysis after 10–25 min of analysis time. Using the conditions optimized with CE, we translated the AS-PCR-HD analysis mutation detection method to the microchip electrophoresis format. The detection of three heterozygous mutations (insertion, deletion, and substitution) in BRCA1 could be accomplished in 180 s or less. Conclusions: It is possible to develop a CE-based method that exploits both AS-PCR and HD analysis for detecting specific mutations. Fast separation and the capacity for automated operation create the potential for developing a powerful electrophoresis-based mutation detection system. Fabrication of multichannel microchip platforms may enable mutation detection with high throughput.

Abstract Background: Electrophoresis on polymeric rather than glass microstructures is a promising separation method for analytical chemistry. Assays on such devices need to be explored to allow assessment of their utility for the clinical laboratory. Methods: We compared capillary and plastic microchip electrophoresis for clinical post-PCR analysis of hepatitis C virus (HCV). For capillary electrophoresis (CE), we used a separation medium composed of 10 g/L hydroxypropyl methyl cellulose in Tris-borate-EDTA buffer and 10 μmol/L intercalating dye. For microchip electrophoresis, the HCV assay established on the fused silica tubing was transferred to the untreated polymethylmethacrylate microchip with minimum modifications. Results: CE resolved the 145-bp amplicon of HCV in 15 min. The confidence interval of the migration time was <3.2%. The same HCV amplicon was resolved by microchip electrophoresis in <1.5 min with the confidence interval of the migration time <1.3%. Conclusion: The polymer microchip, with advantages that include fast processing time, simple operation, and disposable use, holds great potential for clinical analysis.

Capillary electrochromatography (CEC) is a separation technique that hybridizes liquid chromatography (LC) and capillary electrophoresis (CE). The selectivity offered by LC stationary phase results in rapid separations, high efficiency, high selectivity, minimal analyte and buffer consumption. Chip-based CE and CEC separation techniques are also gaining interest, as the microchip can provide precise on-chip control over the experiment. Capacitively coupled contactless conductivity detection (C4D) offers the contactless electrode configuration, and thus is not in contact with the solutions under investigation. This prevents contamination, so it can be easy to use as well as maintain. This study investigated a chip-based CE/CEC with C4D technique, including silicon-based microfluidic device fabrication processes with packaging, design and optimization. It also examined the compatibility of the silicon-based CEC microchip interfaced with C4D. In this paper, the authors demonstrated a nanofabrication technique for a novel microchip electrochromatography (MEC) device, whose capability is to be used as a mobile analytical equipment. This research investigated using samples of potassium ions, sodium ions and aspirin (acetylsalicylic acid).

In microchip electrophoresis (μ-CE), sample injection is generally achieved through cross, double-T, or T-form injector structures. In these reported approaches, the separation efficiency and detection sensitivity of μ-CE is significantly influenced by the shape and size of the sample plug introduced into the separation channel or sample leakage in separation phase. Here, we present a sandwich-injection method for controlling discrete sample injection in μ-CE. This method involves four accessory arm channels in which symmetrical potentials are loaded to form a unique parallel electric field distribution at the intersection of sample and separation channels. The parallel electric field effectuate a virtual wall to confine the shape of a sample plug and depress the spreading of the sample plug at the junction of sample and separation channels, and also prevent sample leakage during separation step. The key features of this method are the ability to inject well-defined sample plugs at the original sample concentration and the ability to control the sample plug size. The virtues of the novel injection technique were demonstrated with numerical models and validated with fluorescence visualizations of electrophoretic experiments.

Flow-gated capillary electrophoresis (CE) is a hybrid of conventional and microchip CE since it employs a fused silica capillary as the separation channel while taking advantage of the well-controlled flow-gated injection, which adds versatility in terms of separation efficiency, analytical throughput, and ease of coupling with sample pretreatment procedures.

The past few years have seen a rapid expansion in interest in the characterization of the entire complement of proteins, or proteome. Micro total analysis systems (μTAS) are an emerging promising method, offering rapid, sensitive and low sample consumption separations. I have demonstrated microchip capillary electrophoresis (CE) devices made of CaF2. New methods have been developed for micromachining enclosed capillaries in CaF2. CE analysis of fluorescently labeled amino acids was used to illustrate bioanalytical applications of these microdevices. Initial on-chip infrared spectroscopy results for qualitative analyte identification were achieved in microfluidic CaF2 channels. I have also shown the evaluation of poly(methylmethacrylate) (PMMA) and thermoset polyester (TPE) microchips for use in protein profiling. To improve separation efficiency and reduce protein adsorption, dynamic coating and poly(ethylene glycol) (PEG) grafting using atom transfer radical polymerization (ATRP) have been used in PMMA microdevices. Proteins, peptides and protein digests have been separated electrophoretically in these PMMA microchips. My results demonstrate that PMMA microdevices should be well suited as microfluidic systems for high performance separations of complex biological mixtures. In-channel ATRP has been developed for the surface modification of TPE microdevices. Characterization indicates that PEG-modified microchannels have much lower and more pH-stable electroosmotic flow, more hydrophilic surfaces and reduced nonspecific protein adsorption. CE of amino acid and peptide mixtures in these PEG-modified TPE microchips had good reproducibility. Phosducin-like protein and phosphorylated phosducin-like protein were also separated to measure the phosphorylation efficiency. My results show that PEG-grafted TPE microchips have broad potential application in biomolecular analysis. Cancer marker analysis is important for medical research and applications. I report a method that can covalently attach appropriately oriented antibodies of interest on monolith surfaces. To reduce nonspecific adsorption, protein solutions were used to effectively block the monolith surface. Selective preconcentration and elution of human chorionic gonadotropin have been performed in my affinity columns, demonstrating that this type of system should have promising applications in cancer marker detection.

Over the past 15 years, research in the field of microfluidics has rapidly gained popularity. By seeking to miniaturize and automate separation-based analysis, microfluidic research seeks to improve current methods through decreased cost, analysis time, and sources of contamination. My work has focused on developing a novel fabrication method, based on standard microfabrication techniques, to create thin-film microfluidic devices. This microfabrication format makes it possible to generate devices that provide high efficiencies, enable mass fabrication, and provide a platform capable of integrating the microfluidic and electronic components necessary for a micro-total analysis system (μ-TAS). Device fabrication combines the processes of photolithography, thermal evaporation, plasma enhanced chemical vapor deposition (PECVD), and wet chemical etching to ultimately provide hollow-core channels. When these microcapillaries are filled with buffer and potentials are applied across them, control of the flow in the channels can be established. By designing intersecting microchannels having an offset “T†geometry, I have been able to inject and electrophoretically separate three fluorescently labeled amino acids and obtain efficiencies of over 2500 theoretical plates. Through the addition of commercially available electroosmotic flow reducing coatings, I have been able to improve the separation of these amino acids, decreasing the run time by approximately 6 fold and increasing the efficiency by as much as 10 fold. Through the use of these coatings I have also been able to carry out electrophoretic separations of three peptides. My most recent work has focused on the polymerization of acrylamide gels in these channels. A method for the selective placement of a gel has been developed using a prepolymer solution with a light-sensitive initiator. Further work to adjust the polymer pore size and interface with ampholyte-containing gels should allow methods such as capillary gel electrophoresis (CGE), preconcentration, and two dimensional (isolectric focusing and CGE) separations to be performed. The development of gel-based analysis methods, along with other fluidic and electrical capacities, should move thin-film microdevices toward the realization of the lab-on-a-chip concept.

A combinação de célula eletroquímica (EC) com a entrada do equipamento de eletroforese capilar (CE), apesar de recente, tem permitido realizar determinação de ânions radicais; pré-concentração eletroquímica de metais pesados, seguida de redissolução, separação e detecção; bem como monitorar produtos carregados formados por oxidação eletrocatalítica de espécies neutras, como álcoois primários e glicerol. Empregando o sistema EC-CE-C4D desenvolvido pelo grupo, a determinação simultânea de cátions, ânions (no contra fluxo) e espécies neutras (detectadas após derivatização eletroquímica) foi demonstrada pela primeira vez, tendo o antisséptico bucal (Listerine® Tartar Control) como amostra real. Embora constante e reprodutível, a conversão dos álcoois primários nos respectivos carboxilatos apresentou rendimento relativamente baixo, ~16%, nas condições anteriormente adotadas, 1,6 V vs. Ag/AgClKCl 3M empregando eletrodo de platina em meio ácido (HNO3 5 mmol L-1 / HCl 1 mmol L-1). Dessa maneira, avaliou-se a oxidação de álcoois primários de cadeia normal (C2 − C5) sobre diferentes materiais de eletrodo (ouro e platina) em diferentes meios (ácido, neutro e alcalino). Os carboxilatos gerados foram monitorados injetando uma alíquota da amostra derivatizada no capilar (50 µm d.i., 45 cm de comprimento e 20 cm efetivo) aplicando 5 kPa durante 5 s, e durante as separações, 30 kV foi aplicado entre as extremidades do capilar preenchido com Tris 30 mmol L-1 / HCl 10 mmol L-1 usado como BGE. Os resultados obtidos com o sistema EC-CE-C4D apontaram maior conversão dos álcoois nos respectivos ácidos carboxílicos em meio ácido, tanto em ouro quanto em platina. Adicionalmente, em eletrodo de ouro a formação dos carboxilatos apresentou certa seletividade não observada sobre platina, favorecendo a conversão dos álcoois de cadeia menor. Noutra vertente, buscando atender as necessidades atuais por metodologias que possibilitem monitorar a eletrooxidação do glicerol em reatores eletroquímicos, desenvolveu-se um método que permitiu determinar simultaneamente o glicerol e alguns de seus possíveis produtos de oxidação neutros, como gliceraldeído e dihidroxiacetona, explorando a formação de complexo carregado com borato (presente no BGE composto por H3BO3 60 mmol L-1 / LiOH 30 mmol L-1), além dos produtos ionizáveis (ácidos carboxílicos) que são comumente analisados por CE. O equipamento de CE utilizado, munido de dois detectores C4D, também permitiu avaliar a interação de alguns ácidos carboxílicos com os modificadores de EOF, Polybrene® e CTAB, empregando MES 30 mmol L-1 / His 30 mmol L-1 como BGE. Seguindo a atual tendência à miniaturização de sistemas analíticos, avaliou-se a possibilidade de construir um sistema EC-CE-C4D miniaturizado. Para isso, um novo método para fabricação de microdispositivos em vidro, baseado em ablação a laser de CO2 assistida por parafina, como alternativa aos dispendiosos métodos de corrosão por via úmida foi desenvolvido. Os dispositivos obtidos por esse método apresentaram canais de perfil semicircular, e as dimensões puderam ser controladas variando a potência e/ou a velocidade de ablação do laser. Contudo, pelos desafios ainda encontrados para se construir um sistema EC-CE-C4D completo em substrato de vidro por ablação a laser de CO2, optou-se por iniciar a miniaturização do sistema EC-CE-C4D com um sistema híbrido em que se aproveita as características mais bem definidas e favoráveis dos tubos capilares de sílica fundida usados em CE convencional. Esse sistema permitiu a determinação quantitativa de metanol na presença de alta concentração de etanol, possibilitando, numa primeira aplicação, realizar o monitoramento da quantidade de metanol e etanol nas frações iniciais coletada durante o processo de destilação fracionada na produção de uísque de milho (moonshine) feito em laboratório. Visto a maior seletividade para conversão dos álcoois de cadeia menor obtidas em eletrodo de ouro e meio ácido, esse foi escolhido para a presente aplicação. As condições que apresentaram melhores resultados no sistema híbrido EC-CE-C4D abrangeram diluição de 100 vezes da amostra em HNO3 2 mmol L-1, eletrooxidação a 1,4 V vs. Ag durante 60 s, injeção eletrocinética no capilar mediante aplicação de 3 kV durante 4 s, e a separação dos carboxilatos realizada aplicando 3 kV entre as extremidades do capilar (50 µm d.i., 15 cm de comprimento com 12 cm efetivo), preenchido com CHES 10 mmol L-1 / NaOH 5 mmol L-1, usado como BGE. A análise das primeiras frações destiladas da \"labmade moonshine\" apresentou um aumento na concentração de etanol (variando de ~80 % a ~100 %) e simultâneo decréscimo da concentração de metanol (variando de 4 % a ~0,1 %). Em suma, avançou-se tanto no leque de aplicações da derivatização eletroquímica hifenizada com a eletroforese capilar como na miniaturização da instrumentação analítica para EC-CE-C4D, favorecendo a disseminação dessa poderosa combinação de três técnicas eletroquímicas.
The direct couple of electrochemical cell (EC) with the inlet of the capillary electrophoresis (CE) equipment, recently demonstrated, has allowed the determination of radical anions; to perform electrochemical preconcentration of traces of heavy metals, followed by stripping, injection, separation and detection; and the generation of charged species by electrochemical oxidation of neutral molecules, e.g. primary alcohols and glycerol. Employing the EC-CE-C4D system developed by our group, the simultaneous determination of cations, anions (in the counter EOF mode) and neutral species (after electrochemical derivatization) was demonstrated for the first time and a mouthwash (Listerine® Tartar Control) was used as a real sample. Although constant and reproducible, the conversion of primary alcohols into carboxylates had a low yield (~16%), under the adopted conditions, 1.6 V vs. Ag/AgClKCl 3M using platinum electrode in acid medium (5 mmol L-1 HNO3 / 1 mmol L-1 HCl). Thus, the yield of carboxylates was studied for the oxidation of alcohols (C2 − C5) on two electrode materials (gold and platinum) in different media (acid, neutral and alkaline). After the electrooxidation step an aliquot of the derivatized sample was automatically injected into the capillary (50 µm i.d., 45 cm in length and 20 cm up to detector) by applying 5 kPa during 5 s. The separation was carried out applying 30 kV between the capillary ends previously filled with 30 mmol L-1 Tris / 10 mmol L-1 HCl BGE. Cyclic voltammograms show higher current density for alcohols oxidation in alkaline medium than in acid one both on gold and platinum electrodes. On the other hand the yields of carboxylic acids were higher in acidic medium. Besides that, only on gold electrode some selectivity for the carboxylate formation was observed favoring the conversion of the short chain alcohols. In order to meet the current needs for methodologies that allow the monitoring of the electrooxidation of glycerol in electrochemical reactors, a method was also developed that allowed the determination of glycerol and some of its possible neutral oxidation products, such as glyceraldehyde and dihydroxyacetone, by exploring the formation of borate complexes (provided in the BGE composed of 60 mmol L-1 H3BO3 / 30 mmol L-1 LiOH), together with ionizable ones like carboxylic acids. The employed CE equipment with two C4D detectors allowed the evaluation of the interaction between some carboxylic acids and the EOF modifiers, Polybrene® and CTAB, using 30 mmol L-1 MES / 30 mmol L-1 His as BGE. Aligned with a current trend of analytical instrumentation, the miniaturized EC-CE-C4D system was attempted. For that, a new method for manufacturing microdevices in glass, based on paraffin-assisted CO2 laser ablation, was developed as an alternative to costly wet-etching methods. The devices obtained by this method presented channels of semicircular profile and the dimensions could be controlled by varying the laser power and/or ablation velocity. Due to remaining challenges in the construction of a complete laser ablated EC-CE-C4D system on glass, a miniaturized system based on a hybrid approach is presented in the thesis, by taking advantage of the more defined and favorable characteristics of the well known fused silica capillary tubes used in CE. This system allowed the quantitative determination of methanol in the presence of high ethanol concentration by taking advantage of the higher yield of short-chain carboxylic acid formation on gold in acidic medium. The first application was the monitoring of the amount of methanol and ethanol in the initial fractions collected during the fractional distillation process in the production of corn whiskey (moonshine) made in the laboratory. The conditions that showed the best results with the hybrid EC-CE-C4D system included a 100-fold dilution of the sample in 2 mmol L-1 HNO3, electrooxidation at 1.4 V vs. Ag for 60 s, electrokinetic injection into the capillary by applying 3 kV for 4 s and separation of the carboxylates carried out under 3 kV between the ends of the capillary (50 µm i.d., 15 cm in length and 12 cm up to detector) previously filled with 10 mmol L-1 CHES / 5 mmol L-1 NaOH, used as BGE. Analysis of the first distilled fractions of labmade moonshine showed an increase in ethanol concentration (ranging from ~ 80% to ~ 100%) and a simultaneous decrease in methanol concentration (ranging from 4% to ~ 0.1%). In short, both the range of applications of electrochemical derivatization hyphenated with capillary electrophoresis as well the miniaturization of analytical instrumentation for EC-CE-C4D were improved, favoring the dissemination of this powerful combination of three electrochemical techniques.

Advances in life science require knowledge of active molecules in complex biological systems. These molecules are often only present for a certain time and at limited concentrations. Integrated micro-analytical tools for sampling, separation and mass spectrometric (MS) detection would meet these requests and are therefore continuously gaining interest. An on-line coupling of analytical functions provides shorter analysis time and less manual sample handling. In this thesis, improved compatibility of microdialysis sampling and multidimensional separations coupled to MS detection are developed and discussed.

Microdialysis was used in vitro for determination of the non-protein bound fraction of the drug ropivacaine. The sampling unit was coupled on-line to capillary column liquid chromatography (LC) followed by ultraviolet or MS detection. For MS detection, the system was extended with a desalting step and an addition of internal standard. A method for MS screening of microdialysates, collected in vivo, was also developed. The method involved sampling and measurements of the chemical pattern of molecules that generally are ignored in clinical investigations. Chemometric tools were used to extract the relevant information and to compare samples from stimulated and control tissues.

Complex samples often require separation in more than one dimension. On-line interfaces for sample transfer between LC and capillary electrophoresis (CE) were developed in soft poly(dimethylsiloxane) (PDMS). MS detection in the LC-CE system was optimised on frequent sampling of the CE peak or on high resolution in mass spectra using time-of-flight (TOF)MS or Fourier transform ion cyclotron resonance (FTICR)MS, respectively. Aspects on electrode positioning in the LC-CE interface led to development of an on-column CE electrode. A successful method for deactivation of the PDMS surface using a polyamine polymer was also developed. The systems were evaluated using peptides and proteins, molecules that are gaining increased attention in bioscience, and consequently also in chemical analysis.

The analysis of low abundant biological molecules is often challenging due to their chemical properties, low concentration and limited sample volumes. Neuropeptides are one group of molecules that fits these criteria. Neuropeptides also play an important role in biological functions, which makes them extra interesting to analyze. A classic chemical analysis involves sampling, sample preparation, separation and detection. In this thesis, an enhanced solid supported microdialysis method was developed and used as a combined sampling- and preparation technique. In general, significantly increased extraction efficiency was obtained for all studied peptides. To be able to control the small sample volumes and to minimize the loss of neuropeptides because of unwanted adsorption onto surfaces, the subsequent analysis steps were miniaturized to a micro total analysis system (µ-TAS), which allowed sample pre-treatment, injection, separation, manipulation and detection.

In order to incorporate these analysis functions to a microchip, a novel microfabrication protocol was developed. This method facilitated three-dimensional structures to be fabricated without the need of clean room facilities.

The sample pre-treatment step was carried out by solid phase extraction from beads packed in the microchip. Femtomole levels of neuropeptides were detected from samples possessing the same properties as microdialysates. The developed injection system made it possible to conduct injections from a liquid chromatographic separation into a capillary electrophoresis channel, which facilitated for advanced multidimensional separations. An electrochemical sample manipulation system was also developed. In the last part, different electrospray emitter tip designs made directly from the edge of the microchip substrate were developed and evaluated. The emitters were proven to be comparable with conventional, capillary based emitters in stability, durability and dynamic flow range. Although additional developments remain, the analysis steps described in this thesis open a door to an integrated, on-line µ-TAS for neuropeptides analysis in complex biological samples.

碩士
輔仁大學
化學系
97
Abstract Photodiode array detector coupled to HPLC typifies authentication of peak by multiple detections. The unrivaled simplicity and ease of HPLC-PAD outcries other examples in peak recognition. On the other hand, the marriage of multiple electrochemical detectors and capillary electrophoresis microchip is less fortunate, because of limit on space. Only the electrodes in the form of thin photolithographic imprints can escape from such curse. However, they are fragile: often lasting no more than a few hundreds of runs. This investigation reports a novel method of chip making to evade the difficulty of fitting four micro electrodes into the limited space at the exit. The novel design, adopting the basic sampling-separation cross pattern, but, divides the stream into two, shortly down the stream from the junction. Further splitting of the two streams into four would create a dendrogram of four branches. The criteria to emulate the single channel with four-electrode detection need channel-to-channel and electrode-to-electrode duplicabilities. The precision inherent in photolithography, therefore the resultant flow reproducibilities, improves the odds of success: a negligible 0.23% (RSD) in eletroosmotic flow. While the duplicabilities among the hand-made electrodes are in the hands of the worker: an unsettled 2.23% (RSD). Fortunately, the inborn differences can be credited to selectivity when put to work as modified electrodes. The bare platinum electrodes tailed to the exits show no selectivity. The electrodes, with one left intact as reference, are coated with carbon paste doped with Cobalt Phthalocyanine, Cobalt oxide, and Ruthenium oxide respectively. The latter’s roles as electrocatalyst are well known: the result in increase in electrodes’ sensing speed is but a nature of course. Therefore, the modified electrodes all give sharper peaks and more theoretical plates toward the solutes of dopamine, epinephrine, tyrosine, 5-hydroxyindoleacetic acid, and ascorbic acid. When applied to identification, the Match Factor, adopted from the algorithm for HPLC-PAD, can scout a hidden peak from a normal looking one. The adulterated peak gives MF=866, significantly below the threshold of 950, while the pure ones all above 990.

碩士
輔仁大學
化學系
97
Capillary electrophoresis microchip, µCE, is a fast analytical tool, but still nowhere near as fast as the array biochip that reaches 96 samples a run. In principle the µCE can be faster than a millisecond for a run. In real life, the acts of sampling and injection that inducing electrodispersion would result in broad peaks. Fast EOF and the incurred band expansion both arising from the surface rich in anionic active sites are ever mutually counteractive. The part of anionic active sites must be tuned down to impede the migrating cations and to tame electrodispersion. Acidifying the capillary surface increases the resolution of the early emergent peaks, but migration time as well. Therefore, there exists an optimum surface pH for every analytical problem. The trained surface can endure several days’ use without noticeable change in migration time. The late emergent broad peaks, apt to introduce great errors in quantitative work, call for an acceleration of migration. The gradient concept commonly practiced in HPLC involving the gradient buffer change is not applicable to the µCE. A gradient packed HPLC column is somewhat similar in approach; except no packing particles in µCE. The linear rising part of the sigmoidal curve that characterizes the feeding solution front is an ideal gradient by nature. The up sloping active sites on surface can be created as follows. As the inactive silanol sites on the acidic surface gradually surrender to the incoming basic solution. It is just a little tricky to match the linear rising part with the column lengthwise, namely optimization. The multitasking chip is an eight-channel µCE with all eight discrete detectors centered on the chip and sharing the waste reservoir. It can perform parallel analysis for eight samples or method development for eight conditions in parallel. The channel-to-channel reproducibility, < 2 % (RSD; n=5) in migration time, is warranted by the precision inherent in photolithography, which is otherwise unavailable. The eight-channel multitasking chip provides the solutions to parallel analysis and to the parallel multiple-parameter method developments. A PC configured homemade multiple Hi-voltage controller manages all the programmed steps.

碩士
輔仁大學
化學系
97
Capillary electrophoresis microchip, μCE, is fast, manageable and low on consumption of samples and reagents. However, the efficiency in real life is less than expected because of the electrodispersion created at the instant of power switch from loading or injection. The incurred band expansion would thus spoil the high efficiency that electroosmotic flow has promised. These two effects ever mutually counteractive are two sides of a sword to the surface rich in anionic active sites that alternately promote either at different stages of a run. The inborn proportion of anionic sites is usually great, from previous contact with the basic solution; it quickens the EOF. As it occurs, the early emergent cationic peaks are fast and sharp but lack adequate resolution. The proportion of anionic active sites must be reduced. A little known trick of trailing the inner surface with traces of polymer can smooth the turbulent flow. It is a long practiced gimmick in fluid dynamics without knowing why until unveiled by the advanced instruments only available in recent years. The maneuver benefits resolution, which would even encourage a radical change of a longer channel with a shorter one for the same efficiency. The choice of polymer is polyethylene oxide for hydrophilicity at ppm level, well below the popular 0.1% for bioapplication. The former involves turbulent taming and active sites tailoring, the latter size sifting. At the optimized conditions of bidirectional flushing with 3 ppm PEO solution for 36 cycles that amounts to 12 minutes. The treatment can double or triple the number of theoretical plate dependent on where it stands on the electropherogram. Nevertheless, the migration times are prolonged and the anionic peaks lagging far behind the neutrals are too broad for quantitative work. The late emergent peaks call for an acceleration of migration. The gradient concept commonly practiced in HPLC involving the gradient change of buffer can help, but is difficult to apply to μCE. In this study, the focus of attention is shifted to the capillary wall. A gradient packed HPLC column is somewhat similar in approach; except there are no packing particles in μCE. The up sloping active sites on surface can be created as follows. The linear rising part of the sigmoidal curve that characterizes the feeding front of the polymer solution is an ideal gradient by nature. From the injecting intersection, as the feeding solution front advances, the silanolate sites on the surface gradually surrender to the incoming traces of polymer. The question is to match the linear rising slope with the column lengthwise: it calls for optimization. For a microchip with an effective 3 cm, a unidirectional feed of 3-ppm PEO solution for 60 seconds can achieve the goal. The peak width at half height improves about one third than that on a basic isoionic surface, but the migration time extends about the same proportion.

Abstract This abstract provides an introduction to the utility of botanical DNA taggants to provide supply chain security for electronic components and to protect against counterfeiting and diversion. A detailed treatment of the science behind Applied DNA Sciences' botanical DNA technology, its applications to semiconductors and microchips, and an overview of DNA analysis by PCR and CE analysis is provided. In addition, information on the evolution of electronic product counterfeiting and inadequate anti-counterfeiting measures is also provided.

A poly(dimethylsiloxane) (PDMS) microfluidic chip-based cartridge was fabricated by sandwiching commercial dialysis membrane and inserting fused-silica capillary into the end of channel according to the principle and structure of a commercial fused-silicon capillary-based cartridge, which can adapt to an IEF analyzer for isoelectric focusing with whole channel imaging detection (IEF-WCID). The novel design of sandwiching membrane in this chip not only eliminated the unfavorable hydrodynamic pressure, leading to poor IEF reproducibility, but made the sample injection much easy. Thus the reproducibility of analysis was very good. The prepared microfluidic chips were applied for qualitative and quantitative analysis of proteins. The six pI markers in the range of 3–10 were separated by IEF under the optimized conditions. The pH gradients exhibited good linear by plotting the pI versus peak position, and the correlation coefficient reached to 0.9994 and 0.9995. The separation of more complicated human hemoglobin control and myoglobin sample could be achieved. By comparison with the separation efficiency obtained on the microfluidic chip and commercial cartridge, the results were similar, which indicates the capillary cartridge may be replaced with the cost-efficient PDMS microfluidic chip. It is anticipated the high throughput analysis can be easily performed on this microfluidic chip patterned multi-channels. The techniques of capillary electrophoresis (CE) have been extensively explored for the chip-based separation. Isoelectric focusing (IEF) as one of high-resolution CE techniques has been widely applied for the separation of zwitterionic biomolecules, such as proteins and peptides. After the samples were focused at their corresponding pIs, the focused zones were mobilized to pass through the detection point for obtaining an electropherogram. This single-point detection imposes extensive restriction for chip-based IEF because a mobilization process requires additional time and lowers resolution and reproducibility of the separation [1]. An alternative is whole-column imaging detection developed by Pawliszyn et al [2] is an ideal detection method for IEF because no mobilization is required, which avoids the disadvantages as mentioned previously. Most microfluidic systems could be fabricated in glass/silicon or polymers in which the channels are defined using photolithography and micromachining. Mao and Pawliszyn [3] have developed a method for IEF on an etched quartz chip following whole-channel imaging detection (WCID). Ren et al [4] presented an integrated WCID system on glass microfluidic chip. However, these materials have some disadvantages such as expensive and fragile and so on. An attractive alternative for fabrication of microfluidic devices is using poly(dimethylsiloxane) (PDMS) as material, which has unique properties such as nontoxic, optical transparent down to 280 nm, elastomeric, hydrophobic surface chemistry Yao et al. [5] designed the glass/PDMS microchip integrated whole-column fluorescence imaging detection for IEF of R-phycoerythrin. Our preliminary studies have successfully developed a PDMS chip-based cartridge for IEF-WCID. It is due to hydrodynamic flow between two reservoirs that the focused zones were mobilized, thus gave poor reproducibility and difficulty in sample infusion. As membranes have been integrated into microchips for microdialysis, protein digestion, solid-phase extraction, desalting, pumping and so on, it could minimize hydrodynamic flow by using membranes as a filter. Although a simple PDMS chip-based cartridge has been successfully fabricated in our labs according to the principle of commercial capillary-based cartridge, it is difficult to introduce the sample into channel for IEF-WCID. As the vacuum was applied in one end of channel for infusing of solution into channel, the lifetime of this chip-based cartridge is shortened. Additionally, the hydrodynamic flow is occurred due to the different heights of anolyte and catholyte in two reservoirs, respectively. The IEF separation was deteriorated by the infusion of anolyte or catholyte, thus leading to poor reproducibility of IEF-WCID analysis. Similar to the hollow fiber in the commercial capillary-based cartridge in which it is aimed to separate the sample in the capillary and electrolytes in the reservoirs, porous membrane was integrated into PDMS chips for decrease of hydrodynamic flow [6]. As a result, integration of dialysis membrane is considered into the design of our new chip-based cartridge. Up to now, many approaches have been described to integrate membranes into glass/quartz or polymeric microfluidic chips. A simple method is direct incorporation by gluing or clamping commercial flat membranes. A major problem of this method is sealing, otherwise, a phenomenon of leakage around the membranes is always occurred due to the capillary force. A novel approach of sandwiching dialysis membrane was developed as schematically indicated in Figure 1. After optimizing IEF conditions, the separation of pI markers was performed on the obtained PDMS microfluidic chip. As exhibited in Figure 2a, six pI markers could be well separated on the PDMS chips patterned the channel of 100 μm deep, 100 μm wide by IEF-WCID. All the peaks were sharp and symmetric, indicating that both EOF and analytes adsorption were completely suppressed by the dynamic coating of PVP. The plots of peak position versus pI of these pI markers suggested good linearity of pH gradient (as shown in Figure 2b). The linear correlation coefficient was 0.9995 (n = 6). As expected to the capillary-based cartridge, the PDMS microfluidic chips could be applied for qualitative and quantitative analysis of proteins. Figure 3a exhibited that human hemoglobin control AFSC contains four known isoforms (HbA, HbF, HbS and HbC) mixed with two pI marker 6.14 and 8.18 were well separated on the PDMS chip by IEF-WCID, indicating the strong separation ability of chip similar to the commercial capillary-based cartridge. According to the linearity of pH gradient, these four isoforms with the pIs of 7.0, 7.1, 7.3 and 7.5, respectively, could be detected. An unknown isoform in human hemoglobin control marked asterisk in Figure 3A observed besides the definite four isoforms A, F, S and C. The myoglobin from horse heart contains two isoforms, whose pIs are 6.8 and 7.2, respectively. It can be seen from Figure 3b that these two isoforms were separated on PDMS chip by IEF-WCID. The peak 1 and 2 could be assigned to the two isoforms according to their pI. The pI of unknown peak marked asterisk could be measured to 6.25.

The study of fluid flow in microchannels is of significant interest due to its application in a wide area of fields ranging from microscale flow injection, cooling of microchips, fuel vaporizer and micro reactors for chemical and biological systems. Design of effective electrokinetic micro reactors requires in-depth understanding of the electrokinetic phenomena and bulk reactions of species in the micro reactor. Although electrokinetic flows are popularly used for applications in the field of capillary electrophoresis (CE) [1], the phenomena of electroosmosis can be conveniently used for bulk transport and mixing of reagents. Electroosmosis occurs when the electrical double layer (EDL) near a solid-liquid interface is created by an external electric field. The uniqueness of electroosmotic flow (EOF) is characterized by plug velocity profile having uniform flow. Devasenathipathy et al. [2] showed that EOF offers a number of significant advantages over conventional pressure driven flow like reduced sample diffusion and controlled sample movement.