| PrefaceRemarkable progress has been made in the development of microfabricated systems for biological and chemical analyses in recent years. An integrated mi-crofluidic device incorporates many of the necessary components and functionality of the typical room - sized laboratory on a small chip. These devices or " lab -on -chip" systems lead to some benefits, including inherent miniaturization, rapid heat and transfer, better efficiency, easy automation, and improved portability and disposability.Capillary electrophoresis on a microchip is a new technology that promises a way to achieve fast and highly efficient separations in microscale analytical devices. It offers several advantages over traditional gel electrophoresis, the most important of which are high speed, high resolution, the small volume sample requirements, low reagent consumption, and sensitivity. With reduction of size of channels the properties of channel walls are getting probably more important than originally thought.Surface chemistry is of great importance in chip - based microfluidic devices especially in highly miniaturized and integrated systems due to high surface area - to - volume ratio. Glass is a popular material for the preparation of chips. It has good optical properties with low fluorescence background. Silanol groups on the channel surfaces enable strategies developed for surface modification of fused - silica capillaries to be applied here. Stabilization of EOF is of great importance in glass microchip. Accordingly stabilization of EOF is not only impor-tant in order to improve the precision of migration times but is necessary for a robust injection process with predefined potentials in order to avoid back leakage of the sample and to insure injection of a reproducible sample plug. The stability of EOF in repetitive runs is, however, often unsatisfactory. To overcome this problem, excessive handling and etching steps are often applied in order to recondition the surface of mictrofluidic chips and to stabilize the EOF, alternatively disposable devices are used.MicroChannel coating can be separated into two major categories: dynamic coatings and permanent coatings. Dynamic coating is the easiest way for surface modification. For dynamic coating dissolved surface — active compounds are utilized, which adsorb strongly at the surface. But the coating cant stabilize for a long time leading to the limitation of its application. Permanent coatings are often regarded as the most effective way for surface modification in order to reduce analyte — wall interactions and to modify the EOF. A permanent wall coating is usually more difficult to prepare than a dynamic wall coating. However, it performance is usually better so it is suibable for high - resolution separations that may be difficult or impossible with a dynamic coating.In this work, we developed a highly active coating method for glass microchips using silanol and HEC as a dynamic coating agent, and discussed its effect on the modified microchannels s ability and the reproducibility of DNA samples separation. The performance of the coated chips at different phases of the coating process was studied by consecutive electrophoretic separations with LIF detection using a $X174 - HaelH DNA digest sample. To examine the applicability, we explored three DNA fluorescence dyes for separation of X174 — Haelll DNA digest sample. The results showed the electrophoresis process with coated chips could have ideal ability to separation and reproducibility.Materials and MethodsMicrochips were fabricated by using standard photolithography and wet chemical etching techniques. The etched plate and the cover plate were bonded together via fusion bonding. The sieving matrix, which consists of 1.5 % HECsolution that was prepared by adding the appropriate amount of polymer to the 1 x Tris - borate - EDTA(TBE) , and the intercalating dye was added to the solution. The 1 xTBE consisting of 1. 5 %HEC solution was pumped into the channels during storage to proceed static absorptive coating. The chip channels were filled with silanol to process silanization. After that, the 1 x TBE consisting of 1.5% HEC solution was injected through reservoir to fill the chip channels. A home - build LIF detection device and a PMT detector were employed for detection. A diode laser was used as excitation source. A home - build high - voltage power unit was used for on - chip sample injection and electrophoresis separation. The electrophoresis conditions were optimized before saw about the repro-ducibility. The PCR products about 144 bp were separated efficiently in this system . The applicability of the coating method was tested in three detective system including YOYO - 1, EB and TO - PRO - 3.ResultsThe glass chips immediately filled with the sieving matrix after fusion bonding and failed to show any signals. After static treatment of the channel walls with the 1 x TBE consisting of 1. 5 % HEC solution, no improvement in performance was observed. First meaningful results were observed after chips were si-lanized. The performance was steadily improved, 9 out of 11 fragments were detected. But the resolution for the 271/281bp and 1078/1353bp fragments was not ideal. After silanized, the channels were filled up the 1 xTBE consisting of 1.5% HEC solution to process a static treatment. The performance was steadily improved. Optimum and stabilized performance was achieved. All 11 fragments were observable with the more ideal resolution for the 271/281bp and 1078/ 1353bp fragments. The electrophoresis conditions were optimized, including concentration of HEC was 1.5% and the strength of electric field was 100V/ cm. The coating chips could preferably achieve the reproducibility of all of the fragments, which approved the microchannel wall had stable nature after having silanization and static treatment process. It also demonstrated that the PCR products could be detected effectively in the coating chips. The chips dealed withthis method could complete the separation of all 11 fragments in YOYO - 1, EB, TO -PRO -3 detective systems.ConclusionA high performing and reproducible procedure to coat glass microchip channels has been developed. Further application of the analysis system in diagnosis and mutation detection can be anticipated. |
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