Ring-Ling Chien

Field amplified sample injection in high-performance capillary electrophoresis

Abstract A simple on-column concentration technique in high-performance capillary electrophoresis (HPCE) is reported. In conventional electro-injection in HPCE, samples are prepared in a buffer solution which has the same concentration as that inside the capillary column. The amount of ions injected into the column under this condition is limited. By preparing samples in a low-conductivity solution, e.g., water, and injecting the sample solution electroosmotically into the column, one can achieve a field enhancement at the injection point. The amount of ions injected will then be proportional to this enhancement factor. However, if one samples by switching the column directly from the high-conductivity buffer reservoir to the low-conductivity sample solution, the buffer boundary at the end of the column is disturbed and the electric field at the injection point might not be amplified properly. By injecting a short plug of water before sample introduction, one can provide a high electric field strength from the beginning of the injection. Several hundred-fold enhancements in the amount of injection were confirmed experimentally.

Field-amplified polarity-switching sample injection in high-performance capillary electrophoresis

Abstract In conventional electro-injection in high-performance capillary electrophoresis, although one is able to inject both positive and negative ions into the column, the number of negative ions injected is rather limited because of their movement against the electric field, assuming the column wall is negatively charged. If one simply reverses the polarity of the field, the electroosmotic flow will deter all positive and most of negative ions from injecting into the column. In the case of field-amplified sample injection, where samples are prepared in a low-conductivity buffer and injected electrically into the column, the number of positive ions injected is porportional to the field enhancement factor at the injection point. The negative ions will not be enhanced, but will be pushed away from the column by this high field strength. However, since the electroosmotic velocity of the bulk solution is much slower than the electrophoretic velocity of sample ions under the enhanced field, one can inject and concentrate both positive and negative ions into the column by switching the polarity of the electrodes at the proper time. Furthermore, one can also achieve selected charge discrimination.

Improvement in the method of sample stacking for gravity injection in capillary zone electrophoresis

A method that allows capillary electrophoresis to be used as a microconcentrating technique is presented. An 85-fold improvement in the amount of material that can be injected into a capillary column without loss of resolution is shown. The method can be used for negative- and positive-charged species but it cannot be used for both species simultaneously.

Methods For Calculating the Internal Temperature of Capillary Columns During Capillary Electrophoresis

Abstract The internal temperature, the buffer viscosity and the efficiency of heat removal from a silica capillary can be calculated by measuring the differential electroosmostic mobility at a low voltage and a high voltage. the calculated temperature is plotted vs the power generated by the electrophoretic instrument and yields a linear relationship when the power is below 3.0 W. the temperature can also be calculated using the conductivity of the solution. the two methods provide the same temperature, which compares well with literature values. A rule of thumb for a quick calculation of internal temperatures is that a power of 0.1 W

Controlling Data Quality and Reproducibility of a High-Sensitivity Immunoassay Using Isotachophoresis in a Microchip

This report describes a method of controlling the sensitivity and reproducibility of a microchip-based immunoassay by using isotachophoresis to preconcentrate the antigen and antibody prior to binding. Gel electrophoresis separation is coupled to the preconcentration step to separate the immunocomplex products formed. The system employs a quartz-based LabChip that automates the metering, preconcentration, reaction, separation, and detection. The system also uses a handoff mechanism that switches the immunocomplex from the stacking mode to the separation mode. We show that the handoff timing affects the data quality and repeatability of the electropherograms, and we demonstrate an automatic handoff mechanism to precisely control the signal intensity and separation of peaks of interest. In so doing, the automatic handoff mechanism also improves the reproducibility of the assay. When applied to the homogeneous liquid-phase detection of alpha-fetoprotein, a common tumor marker, the system shows a greater than 200-fold stacking of specific analytes of interest.

On-chip quantitative PCR using integrated real-time detection by capillary electrophoresis

Quantitative PCR (qPCR) has been widely used for the detection and monitoring of a variety of infectious diseases. PCR and CE were integrated into a microfluidic chip that was designed to achieve rapid real‐time amplicon sampling, separation, and quantitation without requiring various probes. A novel chip design allows the overlapped execution of PCR and CE, minimizing the time required for CE analysis after each PCR cycle. The performance of the on‐chip qPCR method was demonstrated using a 45‐minutes model assay protocol for the phiX174 bacteriophage, and the multiplexing capability of the method was demonstrated by adding a second target, E. coli genomic DNA, to the model assay. The results indicate good sensitivity, reproducibility, and linearity over the tested assay range, 50 to 2 × 104 copies/25 μL reaction. Based on this performance, the on‐chip qPCR method should be applicable to a wide variety of infectious disease detection and monitoring assays with the addition of suitable sample preparation protocols.

Simultaneous Hydrodynamic and Electrokinetic Flow Control

A system capable to provide simultaneously multiple pressure and voltage controls to microfluidic chips is developed. Several new separation concepts to perform enzymatic assays using combination of hydrodynamic flow and electrokinetic flow in high-throughput-screening (HTS) are described.

High-Efficiency Separation in Microfluidic Devices for High-Throughput Screening of Kinases

A new approach for performing on-chip mobility shift assay on a LabChip device has been developed. The separation of PKA product and substrate was conducted on a microfluidic chip using a multi-port pressure/voltage controller. The product is selectively extracted from the flowing mixture and detected downstream by controlling the pressure and voltage at the two waste wells. This approach significantly reduces the background fluorescence signal and permits detection of conversions as low as a few percent, thus increasing the number of kinases that can be assayed.

Ultraviolet Absorbance Spectroscopy in a 3-Dimensional Microfluidic Chip

The strength of absorbance by a compound is linearly dependent on the path length of the detection cell. Because of this, performing high sensitivity absorbance measurements on samples in microfluidic chips, where channel depths are roughly 10–50 µM, is extremely challenging. There have been several attempts to overcome this inherent difficulty, including 1) coupling optical fibers into the chip and directing light along a section of the channel [1], and 2) creating a multi-reflection cell [2]. We have taken another approach, which is to construct a chip containing a three-dimensional fluid path. In our chips, the section of the fluid path running perpendicular to the plane of the chip forms a detection cell with a path length equal to 720 µm, allowing for highly sensitive absorbance measurements.

Development of An On-Chip Cell Sorting System Designed for Cloning Mammalian Cells with Functional, Heterologously Expressed Surface Receptors

Cell separation technology has become an important tool in cell biology allowing for the analysis and subsequent cultivation of specific cell subsets. This technique is especially applicable to transfected cells, where only a small proportion of the cells may be expressing the antigen of interest. As an example, efficient selection of high-producing subclones during gene amplification of recombinant Chinese Hamster Ovary can be performed using cell sorting [1].