Designing polymer matrix for microchip-based double-stranded DNA capillary electrophoresis

An optimization strategy for ternary solvent-strength gradient elution RP chromatography is described in which a two-dimensional model of gradient time (2 levels) against ternary proportions of organic modifiers (4 levels) was constructed. From the resolution surface the optimum ratio of organic modifiers could be selected. Excellent retention time and acceptable peak width and resolution simulations were obtained. The separation could be further optimized from the same input data by using a standard one-dimensional model in order to optimize for gradient slope, duration and shape. Excellent retention time and acceptable peak width and resolution simulations were obtained (< 1, 2 and 6% error, respectively).     

Sample preconcentration by field amplification stacking for microchip-based capillary electrophoresis

A microchip structure for field amplification stacking (FAS) was developed, which allowed the formation of comparatively long, volumetrically defined sample plugs with a minimal electrophoretic bias. Up to 20-fold signal gains were achieved by injection and separation of 400 microm long plugs in a 7.5 cm long channel. We studied fluidic effects arising when solutions with mismatched ionic strengths are electrokinetically handled on microchips. In particular, the generation of pressure-driven Poiseuille flow effects in the capillary system due to different electroosmotic flow velocities in adjacent solution zones could clearly be observed by video imaging. The formation of a sample plug, stacking of the analyte and subsequent release into the separation column showed that careful control of electric fields in the side channels of the injection element is essential. To further improve the signal gain, a new chip layout was developed for full-column stacking with subsequent sample matrix removal by polarity switching. The design features a coupled-column structure with separate stacking and capillary electrophoresis (CE) channels, showing signal enhancements of up to 65-fold for a 69 mm long stacking channel.     

A multireflection cell for enhanced absorbance detection in microchip-based capillary electrophoresis devices

The design, fabrication and testing of a photolithographically fabricated, glass-based multireflection absorbance cell for microfluidic devices, in particular microchip-based capillary electrophoresis (CE) systems is described. A multireflection cell was fabricated lithographically using a three-mask process to pattern aluminum mirrors above and below a flow channel in a chip, with 30 microm diameter optical entrance or exit apertures (one in each mirror) positioned 200 microm apart. Source and detector were positioned on opposite sides, and the metal mirrors were made 1 cm square, to reduce stray light effects. Calibration curves using bromothymol blue (BTB) with a 633 nm source (He:Ne laser) were linear to at least 0.5 absorbance units, with typical r2 values of 0.9997, relative standard deviations in the slopes of +/- 1.3%, and intercepts of zero within experimental error. Effective optical pathlengths of 50-272 microm were achieved, compared to single-pass pathlengths of 10-30 microm, corresponding to sensitivity enhancements (i.e., optical path length increase) of 5 to 10-fold over single-pass devices. Baseline absorbance noise varied within a factor of two in almost all devices, depending only weakly on path length. This device can give much higher absorbance sensitivity, and should be much easier to manufacture than planar, glass-based devices previously reported.     

An integrated light emitting diode-induced fluorescence detector for capillary electrophoresis

An integrated light emitting diode (LED)-induced fluorescence detector was described and evaluated. The LED and its related components including lens and interference filter, the optical fiber used to collect fluorescence, and the capillary column are integrated into a substrate block, which eliminates the need of align procedure of the fiber and the capillary. Forty-fold enhancement of sensitivity was obtained compared with our previous work and the detection limit for fluorescein was 5 nM. Application of the detector for the analysis of FITC-labeled Ephedrine extract was demonstrated.     

GUcal: An integrated application for capillary electrophoresis based glycan analysis

Recent emergence in the use of monoclonal antibody therapeutics and other glycoprotein biopharmaceuticals requires high‐throughput, robust, and automated techniques for their glycosylation analysis. Capillary electrophoresis is one of the high‐performance methods of choice; however, while the necessary instrumentation is well developed, the related bioinformatics tools are lacked behind. In this paper, we introduce an integrated toolset dubbed as GUcal, to automatically calculate the glucose unit (GU) values for all sample components of interest in an electropherogram with a concomitant database search for structural assignment. The database comprises CE GUs and suggested structures of N‐glycans released from human IgG. The app is freely available online ( www.lendulet.uni‐pannon.hu/gucal ) and readily facilitates CE‐based glycan analysis.     

Interfacing a microchip-based capillary electrophoresis system with a microwave induced plasma spectrometry for copper speciation

A microchip-based capillary electrophoresis (μCE) system was interfaced with a microwave induced plasma optical emission spectrometry (MIP-OES) to provide copper species separation capabilities. This system uses an extremely low flow demountable direct injection high efficiency nebulizer (D-DIHEN) sited directly at the liquid exit of the chip. A supplementary flow of buffer solution at the channel exit was used to improve nebulization efficiency. A small evaporation chamber has been incorporated into the interface in order to prevent the losses associated with traditional spray chambers, allowing the entire aerosol sample to enter the plasma. Syringe pumps were used to manipulate the flow rate and flow direction of the sample, buffer, and supplementary buffer solution. Sample volumes of 25 nL can be analyzed. With application of an electric field up to 500 V cm⁻¹, species such as Cu(II) and Cu(EDTA)²⁻ were separated in acidic solution within 90 s using a 26 mm long separation channel etched in a glass base. Resolution of the Cu(II) and Cu(EDTA)²⁻ peaks was 1.1 using the chip-based μCE-MIP-OES system.      

Towards a microchip-based chromatographic platform. Part 1: Evaluation of sol-gel phases for capillary electrochromatography

Silica monolithic columns suitable for implementation on microchips have been evaluated by ion-exchange capillary electrochromatography. Two different silica monoliths were created from the alkyl silane, tetramethyl orthosilicate (TMOS), by introducing a water-soluble organic polymer, poly(ethylene oxide) (PEO), with varying molecular weights into the prehydrolyzed sol. Silica monoliths created using 10 kDa PEO were found to have a much more closed gel structure with a smaller percentage of pores in the microm size range than gels created using 100 kDa PEO. Additionally, the size of the mesopores in the 100 kDa PEO monolith was 5 nm, while those in the 10 kDa PEO gel were only 3 nm. This resulted in a strong dependence of the electroosmotic flow (EOF) on the ionic strength of the background electrolyte, with substantial pore flow through the nm size pores observed in the 10 kDa PEO gel. The chromatographic performance of the monolithic columns was evaluated by ion-exchange electrochromatography, with ion-exchange sites introduced via dynamic coating with the cationic polymer, poly(diallyldimethylammonium chloride) (PDDAC). Separating a mixture of inorganic anions, the 10 kDa PEO monolithic columns showed a higher effective capacity than the 100 kDa PEO column.     

Negative pressure pinched sample injection for microchip-based electrophoresis

A simple method for injecting well-defined non-biased sample plugs into the separation channel of a microfluidic chip-based capillary electrophoresis system was developed by a combination of flows generated by negative pressure, electrokinetic and hydrostatic forces. This was achieved by using only a single syringe pump and a single voltage supply at constant voltage. In the loading step, a partial vacuum in the headspace of a sealed sample waste reservoir was produced using a syringe pump equipped with a 3-way valve. Almost instantaneously, sample was drawn from the sample reservoir across the injection intersection to the sample waste reservoir by negative pressure. Simultaneously, buffer flow from the remaining two buffer reservoirs pinched the sample flow to form a well-defined sample plug at the channel intersection. In the subsequent separation stage, the vacuum in headspace of the sample waste reservoir was released to terminate all flows generated by negative pressure, and the sample plug at the channel intersection was electrokinetically injected into the separation channel under the potential applied along the separation channel. The liquid levels of the four reservoirs were optimized to prevent sample leakage during the separation stage. The approach considerably simplified the operations and equipment for pinched injection in chip-based CE, and improved the throughput. Migration time precisions of 3.3 and 1.5% RSD for rhodamine123 (Rh123) and fluorescein sodium (Flu) in the separation of a mixture of Flu and Rh123 were obtained for 56 consecutive determinations with peak height precisions of 6.2% and 4.4% RSD for Rh123 and Flu, respectively.     

On-chip potential gradient detection with a portable capillary electrophoresis system

A portable chip-CE system with potential gradient detection (PGD) was developed and applied to the determinations of alkali metals and alkaloids. The separation efficiency appeared to be satisfactory and nonaqueous capillary electrophoresis (NACE) proved to be applicable to PGD or conductivity detection. The power supplies, separation and detection were built on a device of 3 kg in weight. A branch channel near the end of the separation channel was designed to perform PGD and make the application of relatively high field strength possible. The study is the first report on the application of PGD on the microchip platform. The design of the chip-CE system shows several advantages, such as simplicity, miniaturization and wide applicability.     

A platform method for plasmid isoforms analysis by capillary gel electrophoresis

In the production of novel biological products, plasmids are often engineered into delivery vectors for target genes, which can be used directly as vaccines or as intermediate products for gene/cell therapy. Plasmid DNA exists in several topological forms such as supercoiled, linear, and open circular. As supercoiled plasmid shows the highest efficiency in transfecting eukaryotic cells, the content of supercoiled plasmids becomes an important indicator of plasmid quality. CGE is an effective analysis method for separating different topological structures of plasmids. For the purpose of providing plasmid manufacturers and regulatory agencies with an efficient and readily used tool for monitoring the quality of plasmids, this article identifies the optimal separation and detection conditions of CGE, presents a platform-based plasmid analytical method, and uses plasmid of different sizes to verify the feasibility of this method. In terms of detector, the LIF detector has obvious advantages over the ultraviolet detector in sensitivity and resolution. Using the optimal CE condition (10× gel buffer), baseline separation of different topological forms and impurities can be achieved for different plasmid sizes (5.9, 7.8, 15.4 kb). In addition, 6.5 kb plasmid was used to compare the different separation technologies such as CGE-LIF, ion exchange chromatography and agarose gel electrophoresis. The result shows that CGE-LIF can provide better resolution and quantitation accuracy than ion exchange chromatography and agarose gel electrophoresis. CGE-LIF, as a quick and convenient method to separate and quantify plasmids, has the advantages of high sensitivity, high resolution, and high quantitative accuracy. Therefore, it is ideal for analysis of plasmids with different sizes, and it can also be used as a platform method for manufacturers and regulatory agencies to monitor the purity and stability of plasmids.     

Influence of soluble polymer polyvinylpyrrolidone on separation of small peptides and amino acids by microchip-based capillary electrophoresis

In microchip-based capillary electrophoresis, the resolution and separation efficiency of small peptides and amino acids can be noticeably improved by adding a low molecular weight (30,000) soluble polymer additive, polyvinypyrrolidone in the separation medium. Several separation conditions such as injection time and electrophoretic buffer have been investigated and optimized. Using an electro-stacking scheme, the resolution and separation efficiency of small peptides and amino acids can be enhanced significantly. Under the optimal conditions, the separation of fluorescein isothiocyanate Isomer I-labeled small peptides and amino acids was successfully achieved within 100 s.     

Detection of fuel-oxidizer explosives utilizing portable capillary electrophoresis with wipe-based sampling

Portable analytical instrumentation that can provide an alarm indication for the presence of explosives and related components is critical for the identification of explosives-based hazards and threats. Many explosives incident reports involve an inorganic oxidizer-fuel mixture which can include pyrotechnics, fireworks, flash powders, black powders, black powder substitutes, and improvised or homemade explosives. A portable CE instrument with targeted analysis of common inorganic oxidizer ions, for example, chlorate, perchlorate, and nitrate, was used here as a rapid detection platform. Unlike frequently used gas-phase separation and detection instrumentation such as ion mobility spectrometry (IMS), an automated liquid extraction mechanism is required for CE separation using acetate paper sample collection wipes. Target inorganic oxidizers were inkjet-printed onto sample wipes to investigate instrument response relative to the collected analyte spatial distribution. Overall, analyte signal intensities increased with off-center sample deposition due to improved sample extraction from wipes and no change in response was observed for varied array distributions across wipes. The system demonstrated sub 200 ng detection limits for all target analytes, with further improvement when normalizing to an internal standard.     

An inexpensive, portable and microcontrolled near infrared LED-photometer for screening analysis of gasoline

A microcontrolled, portable and inexpensive photometer is proposed. It uses a near infrared light emitting diode (NIR LED) as radiation source, a PbSe photoresistor as infrared detector and a programmable interrupt controller (PIC) microcontroller as control unit. The detector system presents a thermoresistor and a thermoelectric cooling to control the detector temperature and keep the noise at low levels. The microcontroller incorporated total autonomy on the proposed photometer. As its components are inexpensive and of easy acquisition, the proposed NIR LED-photometer is an economical alternative for chemical analyses in small routine, research and/or teaching laboratories. By being portable and microcontrolled, it also allows carrying out field chemical analyses. The instrument was successfully applied on the screening analysis to verify adulteration in gasoline samples.     

An automatic sampling device for capillary zone electrophoresis

An automatic sampling device has been developed for capillary zone electrophoresis. The sample is introduced to the column electrokinetically by rapidly exchanging buffer for sample in a narrow channel drilled in a plexiglass block. The autosampler is capable, under computer control, of performing multiple sample injections from a large volume sample source (such as a reaction vessel) as the sample concentration changes, and thus presents the possibility of analyzing time-varying processes by CZE. Peak area reproducibility in electropherograms obtained after use of the sampler is less than 1% and efficiency is more than 2.2 × 10⁵ theoretical plates.     

An end-channel amperometric detector for microchip capillary electrophoresis

An end-channel amperometric detector with a guide tube for working electrode was designed and integrated on a home-made glass microchip. The guide tube was directly patterned and fabricated at the end of the detection reservoir, which made the fixation and alignment of working electrode relatively easy. The fabrication was carried out in a two-step etching process. A 30 μm carbon fiber microdisk electrode and Pt cathode were also integrated onto the amperometric detector. The characteristics and primary performance of the home-made microchip capillary electrophoresis (MCCE) were investigated with neurotransmitters. The baseline separation of dopamine (DA), catechol (CA) and epinephrine (EP) was achieved within 80 s. Separation parameters such as injection time, buffer components, pH of the buffer were studied. Relative standard deviations of not more than 6.0% were obtained for both peak currents and migration times. Under the selected separation conditions, the response for DA was linear from 5 to 200 μM and from 20 to 800 μM for CA. The limits of detection of DA and CA were 0.51 and 2.9 μM, respectively (S/N=3).     

Towards a microchip-based chromatographic platform. Part 2: sol-gel phases modified with polyelectrolyte multilayers for capillary electrochromatography.

The potential for using polyelectrolyte multilayers (PEMs) to provide chromatographic functionality on continuous silica networks created from sol-gel chemistry has been evaluated by capillary electrochromatography (CEC). Construction of the PEM was achieved by flushing the column with polyelectrolytes of alternative charge, with variation of the properties of the exposed polyelectrolyte providing a unique means to vary the chromatographic surface. Variation of the exposed polyelectrolyte from poly(diallyldimethylammonium chloride) (PDDAC) to dextran sulfate (DS) allowed the direction of the electroosmotic flow (EOF) to be changed and also provided a means to vary the chromatographic capacity. Variation of negative polymer from DS to poly(styrene sulfonate) (PSS) significantly altered the EOF and the migration of peptides, with both the reversed-phase and ion-exchange capacities increasing. An alternative method for changing the column capacity was to change the thickness of the PEM, which was evaluated by anion-exchange CEC. A 70-80% increase in retention was observed for all anions without any increase in EOF suggesting significant penetration of the analytes through the PEM and interaction with buried charges within the PEM.     

Integrated platform for detection of DNA sequence variants using capillary array electrophoresis

We have developed a highly versatile platform that performs temperature gradient capillary electrophoresis (TGCE) for mutation/single-nucleotide polymorphism (SNP) detection, sequencing and mutation/SNP genotyping for identification of sequence variants on an automated 24-, 96- or 192-capillary array instrument. In the first mode, multiple DNA samples consisting of homoduplexes and heteroduplexes are separated by CE, during which a temperature gradient is applied that covers all possible temperatures of 50% melting equilibrium (Tms) for the samples. The differences in Tms result in separation of homoduplexes from heteroduplexes, thereby identifying the presence of DNA variants. The sequencing mode is then used to determine the exact location of the mutation/SNPs in the DNA variants. The first two modes allow the rapid identification of variants from the screening of a large number of samples. Only the variants need to be sequenced. The third mode utilizes multiplexed single-base extensions (SBEs) to survey mutations and SNPs at the known sites of DNA sequence. The TGCE approach combined with sequencing and SBE is fast and cost-effective for high-throughput mutation/SNP detection.     

Development of an integrated capillary electrophoresis/sensor for L-ascorbic acid detection

A CE/biosensor for measuring ascorbic acid was developed by coupling a polyaniline optical sensor and capillary electrophoresis (CE). The capillary column was partially modified with a thin film of polyaniline redox sensitive material. Ascorbic acid was detected by monitoring the changes in optical absorbance occurring to the polyaniline film upon the reduction reaction. The sensor response (change in optical absorbance at 650 nm) is proportional to the concentration of ascorbic acid over a range of 2.5-250 mg/L and the response range has shown a clear dependence on the characteristics of the polymerized film. High specificity and sensitivity of the present method, low sample consumption, short times of response (ca. 2 min) and the reproducibility of the results demonstrate that the CE/polyaniline-sensor could be further employed in the study of the relation between the content of L-ascorbic acid in body fluids and clinical parameters, e.g., cell ageing.