Establishment And Application Of Novel Microchip Capillary Electrophoresis System

Capillary electrophoresis(CE)and microchip electrophoresis(MCE)technologies exhibit excellent separation capability and high potential in applications such as biomedical research,drug analysis,environmental monitoring,food quality detection,and other fields.However,the real-world use in routine analyses of CE and MCE technologies are far from our expectations.The reasons for this situation include:The analytical performance is sensitive to many experimental parameters,and their repeatability and reliability may not be good enough for routine analysis;the cost of microchips and instruments is high;the function units of the microchip are frequently not as many as expected;the integration level of CE and MCE detector is low.To improve the practicality of these technologies and accelerate their pace to industry,it is necessary to establish robuster methods of CE and MCE,find more low-cost fabrication methods for microchips,incorporate more functional units onto a microchip and perform multi-mechanism detection for the separation.This dissertation has obtained the following innovative results:(1)The adverse effects of pressure-driven flow in MCE were eliminated and the separation performance of MCE was improved by combining matched liquid volume and surface shape in the reservoirs of microchips with appropriate viscosity of multi-functional polymeric additives in the running buffer.(2)The problem of Joule heating in sub-millimeter MCE was solved by using low conductivity running buffers,and a reliable method for DNA separation by sub-millimeter MCE was established.The use of sub-millimeter channels can eliminate the necessity of filtration of solution and avoid blockage,which greatly simplified the operation and improved the practicality of MCE.The sub-millimeter channels can be easily fabricated by computer numerical controlled(CNC)machines,which can significantly reduce the cost of the microchip fabrication.(3)A new fabrication method of polymer microchips with nanochannels has been developed based on thermal bonding assisted with nano-materials.An online enrichment unit was constructed based on the nanochannels prepared using the proposed method and integrated with the sub-millimeter channels for MCE,which improved the degree of integration.(4)The widely available fused deposition modeling(FDM)3D printing is used for the fabrication of low-cost microchips that can be printed in only one step and ensure no leakage in microchannels.The proposed method is simple,low-cost,and fast.A miniaturized capacitive coupled contactless conductance detection(C⁴D)cell was also fabricated by FDM.The separation of three cations was achieved completed with the3D printed microchip and detector.(5)A"three-in-one"multi-mechanism detector was established to improve the integration degree of the CE detector.The detector showed good detection performance,it is useful for the analysis of complicated samples.With this detector,a cross-calibration method is proposed for the first time,which could effectively correct the migration time drift and peak area variations caused by experimental conditions and improve the repeatability of CE.This dissertation consists of six chapters:In chapter 1,we introduced the development history and basic principles of CE and MCE technology.The materials and fabrication methods of microfluidic chips were also summarized systematically in this chapter.Furthermore,the single and integrated detectors used in CE and MCE were introduced in detail.Finally,based on the factors that affect the robustness and the challenges of CE and MCE in routine applications,we proposed the aim and significance of this dissertation.In chapter 2,we systematically studied the effects of pressure-driven flow on separation performance caused by liquid level and meniscus shape in reservoirs,and unknown pressure in the channel.Through matching the liquid level and meniscus shape and adjusting the solution viscosity of running buffers,the sample band broadening could be eliminated,and the separation performance would be improved.The experimental results showed that the theoretical plate number of sodium fluorescein decreased significantly with the mismatched liquid level in the reservoirs,and the relative standard deviations(RSD)of migration time were up to 12%.Based on the fluidic principles,a theoretical modeling of the optimal liquid volume in reservoirs was performed.The optimal liquid volume obtained from experiments was consistent with the simulated results,indicating that this model could be used for evaluating the appropriate liquid volume in each reservoir.The additional pressure brought by the meniscus could be eliminated when the shape of the liquid surface of each reservoir was consistent.By using hydroxypropyl cellulose as a viscosity regulator,more than 4×10⁵ m^-1 theoretical plate numbers were obtained in the submillimeter channel with sizes of 180?m×120?m(width×depth),350?m×150?m and 450?m×150?m,respectively.In chapter 3,a low-cost microchip with a sub-millimeter channel was fabricated by computer numerical controlled(CNC)machining.Low conductivity buffers were used to solve the Joule heating problem in MCE with sub-millimeter channels.A reliable MCE system with sub-millimeter channels was developed.Combined with laser-induced fluorescence(LIF)detector and viscosity regulation using hydroxypropyl methyl cellulose(HPMC),we realized a rapid and highly sensitive detection of mycoplasma contamination and the real samples of circulating cell-free DNA(cf DNA),which gave a limit of detection(LOD)as low as 2.3 pg·?L^-1.Owing to the significant reduction in cost,ease of operation,and fast separation capabilities demonstrated here,the proposed MCE could be a viable alternative to the slab gel electrophoresis running in most biological laboratories.In chapter 4,nanochannels were fabricated on the microchip with the assistance of graphene oxide(GO)in the thermal bonding.With the introduction of nanochannels into a traditional cross-type microchip as an enrichment unit,the multifunctional microchips for both enrichment and separation were constructed.Nanochannels were formed prepared by direct writing with GO solution as the ink before the thermal bonding at 112?for 10 min.The fabrication method was simple and convenient.Due to the overlap of double electric layers,ion enrichment occurred at one end of nanochannels under the electric field.The factors affecting the enrichment effect were investigated.The results show that a shallower of GO writing layer and a lower buffer concentration were more conducive for higher enrichment efficiency.The microchip with integrated online enrichment and separation units was tested with sodium fluorescein,and an enrichment factor of reached 118 was obtained.In chapter 5,a low-cost microchip and the capacitive coupled contactless conductance(C⁴D)detector were established by using 3D printing technology.Using polylactic acid(PLA)as the material,a microchip with the size of 7 cm×2cm×0.8mm could be printed in 15 min by the fusion deposition method(FDM).By optimizing the printing parameters and conditions,the one-step printing of the microchip with leak-free microchannels was successfully fabricated achieved.The proposed method had the advantages of high speed,low cost,and no extra operation and post-processing in the process of fabrication.As for the C⁴D detection system with the electrode width of 2mm and the electrode gap of 0.3 mm,a LOD of 3?M for K⁺at the excitation frequency of 150 k Hz was obtained.The electrophoretic separation of K⁺,Na⁺and choline ions in sub-millimeter channels were achieved successfully.In chapter 6,a multi-mechanism detector with visible absorption,C⁴D and LIF detectors at the same detection point for CE was designed and fabricated.LIF and visible absorption detectors shared a single laser light source,and the detector is simple in its configuration.The LOD of methyl orange was 3.0?M for visible absorption detector,0.7?M for K⁺for C⁴D,and 0.3–0.8 n M for four amino acids derived with FITC for LIF detector.These results showed that the performance of the integrated detector was equivalent to that of single mechanism detectors.A new method for improving the repeatability of CE was developed based on this multi-mechanism detector.Taking C⁴D detection mode and LIF detection mode as examples,a cross-calibration method was proposed to correct the drift resulting from variations of experimental parameters or capillary surface status.The method used one signal as the reference signal and the other as the sample signal,which significantly improved the repeatability of CE,significantly increased the tolerance of CE for experimental variations.