Research And Implementation Of Continuous Wave-Based Multiphoton Excitation Fluorescence For Capillary Electrophoresis

Due to rapid development of two decades, capillary electrophoresis (CE) featuresultra-low detectability, high resolution, low time consumption and low sample requirementthat has been currently recognized as a powerful method of choice in chemical andbiomedical analysis, particularly in the field of life sciences. Multi-photon excitationfluorescence (MPEF or MPE) takes advantages of high spatial resolution (sub-fL level),high sensitivity (single molecule), greater penetration depth, low-photo-toxicity andphotobleaching, and widely applied in the biomedical field. MPEF had been recentlyintroduced into CE area by Shear and his colleagues. They firstly constructed a novelCE-MPEF system to spectrally characterize diverse zepto-mole biological molecules ofnative fluorescence such as serotonin, NADH and FAD, and of non-auto fluorescence likesome neurotransmitters as well. Nevertheless, Ti:sapphire mode-locked femto-second laserthat used in their experiments, with the more expensive, complicated operation, themaintenance and operation costs limited its application in CE. In recent years, withadvancement of semiconductor laser technology, near-infrared wavelength and high orultra-high energy laser diode (LD) of cheap price is currently available, and has beenextensively employed to be a ideal multi-photon fluorescence excitation source.In this thesis, continuous wave (CW) with LD based MPE fluorescence detection wasfirstly demonstrated for CE; A different detection configrations for CE-MPEF system wasinvestgated; Further more, a series of related applications were investigated.(1) Based on continuous wave laser diode, a novel CE-MPEF system was established.The results showed that CW-LD that replaced of Ti:sapphire mode-locked femto-secondlaser was feasible as a multi-photon excitation resource of CE-MPEF; Three kinds of tagsthat were most frequently used for molecular labeling, such as small molecular dye,fluorescent protein and quantum dot (Q.D.), were investigated in CW-LD CE-MPEF;Identification of fluorescein isothiocyanate (FITC) labeled amino acids was furtherachieved both in Capillary Zone Electrophoresis (CZE) and Micelle ElectrokineticCapillary Electrophoresis (MEKC) that revealed good prospect of this detection scheme invarious micro-column separation technologies.(2) A unique on-column MPEF strategy for CE was demonstrated. By comparingend-column MPEF, on-column MPEF with square capillary, and on-column MPEF withcylindrical-square dual capillaries system, it was concluded that on-column MPEF usingdual capillaries had advantages of better detectability (2~5 times), instrumental feasibility and separation compatibility, which constituted the key factors responsible for furtherwidespread use potential of CE-MPEF.(3) A commercial CE instrument coupling MEKC with single-photon excited (SPE)fluorescence detection and our novel MEKC-MPEF system has been studied. 21FITC-labeled amino acids were investigated using both systems. Results indicate thatMPEF detection had better resolution than the SPE method. The quantitative data, such asconfidence (γ> 0.99), working linear range (two of magnitude), mass limit detection(zepto-mol), concentration limit detection (μM) and recoveries were inferred. To evaluateour CW-LD CE-MPEF, a commercial amino acid oral liquid product was analyzed. Theresults indicated that our experimental data were comparable with the standard product datasheet from Eight-peak Pharmaceutical Chemistries Co. Ltd with high consistency (relativeerror < 5 %).(4) Our system had been proposed to analyze biogenic amines, such as putrescine,cadaverine, histamine, spermine, spermidine, phenylethylamine and the juice from a carpstored in 48h without water under room temperature. It showed that the detection limit wasfound to beμM-level, with linear range of over two orders of magnitude for quantitation. Itwas well suited for complex systems like bio-sample and food, with advantages of highseparation performance, high detection sensitivity and low cost.(5) Persist organic pollutants (POPs), such as aniline species, pesticide residues, andso on, were analyzed by the novel system. P-toludine, p-ethoxyaniline, pphenylenediamineand aniline were completely separated and detected under optimal derivatization andseparation conditions. To test our method for real-world sample applicability, really lakewater was analyzed.(6) MPEF has the capability of spectral excitation in a broad range. With MPE, it isnow possible to use only one excitation line of a laser to detect complex componentslabeled with various fluorescent dyes. For example, the excitation maxima of enhanced 6fluorescent tags with different excited spectrum. It is impossible to be simultaneouslyexcited using one photon excitation. But with MPEF detection, the 6 fluorescent tags weresimultaneously excited by used this CW-LD CE-MPEF system. Furthermore, with the samesystem, DNA fragments labeled by FITC were investigated. The results figured that thetraditional adopted 4 chromatographies DNA analysis could be simultaneous excitationusing IR light. It showed that this novel system has been employed successfully for DNAanalysis, which indicated a promising future of CE-MPEF method in biomedical analysis.