| capillary electrophoresis obtained the exclusive attention and application since it's formed due to its qualities of high efficiency,high-speed,microscale,automation high-throughput,clean without pollution.but the limited light detection dimension and the confined length which results in small sample injection volume cause its low detection sensitivity,especially on the condition of using the common UV detector,the problem of low sensitivity become the crux for the application of capillary electrophoresis。Therefore,the research referring to the problem of sensitivity had became a hot topic,among them,the techniques for sample preconcentration became a focus because of its easy-operation,low-cost and high sensitivity effect. And field-amplified became the most widely used technique between them due to its mature theory foundation,simple operation and high sensitivity result.Nowadays,in the field of DNA analysis by CE-UV,the problem of sensitivity becomes prominent.In addition to the double bond formed by basic group of DNA has lower ultraviolet-vis absorption and normal UV detector has lower sensitivity,the intrinsic low detection sensitivity drawback of CE that the limited light absorption dimension and the confined length which results in small sample injection volume also restrain the application of DNA analysis by CE-UV greatly.Resulting in the area of DNA analysis by CE, fluorescence detection become the solution for sensitivity by most researchers。However,it demands sample labeling with a fluorescent marker or intercalating dyes,time-consuming,costs high,most dyes have toxicity(such as EB)and fluorescence detector has a high price,also the fluorescence detection electropherograms certainly lead to the trouble of the integration of data owing to the most widely used UV electropherograms. As the result, fluorescence detection hasn't achieved the extensive application yet.Therefore,this experiment enhanced the CGE-UV detection sensitivity for DNA by field-amplified stacking,enabled trace DNA analysis by CE-UV accomplished simply and conveniently and layed the foundation of enlarging the application of CE-UV in the field of molecular biology. The results of this article as follows:1. a review that included preconcentration techniques based on the electrophoresis such as field-amplified stacking,pH-mediated stacking,Transient Isotachophoresis preconcentration,acid stacking, transient moving chemical reaction boundary method etc and techniques based on the chromatograph such as Sweeping,single-drop microextraction,Solid-Phase Extraction,etc.they also include new developing techniques according to new theories and new designs such as Anion/Cation-selective exhaustive injection and sweeping,Transient Trapping-release,Micelle Collapse,moving interaction boundary,Glycerol-salt mediated stacking,cellulose acetate-coated porous joint,etc.all of them was helpful for the solution of the sensitivity in capillary electrophoresis and facilitated its application in the field of analytical chemistry.2. enhancing the UV detection sensitivity of capillary electrophoresis for DNA by water-plug field-amplified stacking injection.the known concentration of DNA marker were as standard samples,TE buffer diluted the standard samples gardiently,inserted a water-plug(0.5psi,20s)before pressure injection,the pressure injection time was prolonged until 0.5psi,990s without deteriorating the resolution,compared to normal pressure injection(0.5psi,10s)and extended pressure injection(0.5psi,90s),the sensitivity was enhanced 94.4 folds and 8.2 folds respectively. 1ng/μL was the lowest detection overall concentration of DNA,80ng/mL(S/N=3)was the detection limit,the result was 87.5 folds better than the former researcher'detection limit of 7ng/μL.3. Considering that water-plug field-amplified stacking pressure injection improved the volume of injection,however,the prolongation of injection time and the extended sample plug existing in capillary caused the increase of analysis time,meanwhile,the sensitivity effect was not so good.So gave a try of enhancing the UV detection sensitivity of capillary electrophoresis for DNA by matrix field-amplified. Matrix field-amplified reduced the limit of DNA to below 40ng/mL。Compared to pressure injection(0.5psi,90s)and water-plug field-amplified pressure injection(0.5psi,20s water,990s), the sensitivity was enhanced 65 folds and 8 folds respectively.At the same concentration, Electrokinetic injection had a better sensitivity than pressure injection. Whereas the sensitivity was still lower simply by matrix field-amplified(Electrokinetic injection time can not beyond 30s).there were the troubles such as that the interface of sample solution and BGE was disrupted easilily,sample plug,sample solution was disrupted easilily under high field intensity,etc.They would interrupted the efficiency like that broad peak,limited electrokinetic injection time,the bad repetitiveness and so on. the mismatch of EOF between low ion strength sample solution and BGE solution also influenced the sensitivity in matrix field-amplified.For example,the unsteady of injection electric current,the distorted and broad band,baseline drift etc.As the consequence,the combination of matrix field-amplified and head-column field-amplified injection was as means for the purpose of overcoming the troubles.4. The combination of matrix field-amplified and head-column field-amplified injection was as means for CGE-UV analyses to enhance the sensitivity further. And then we validated the method's reliability by analyzing the PCR product without purification.the Results were that the dilution of DNA reached 400 thousand folds,compared with electrokinetic injection(10KV,10s)and pressure injection(0.5psi,90s),on the premise that without deteriorating the width and shape of peaks,extended the electrokinetic injection to 420s,the ion strength of sample solution reduced to 1.5%Tris-Hcl(TE),the sensitivities of head-column field-amplified improved 28,56 folds respectively and 3760,7548 folds elevation after the combination of matrix field-amplified and head-column field-amplified injection.The detection limit of DNA reduced to 0.1ng/mL(S/N=3,CGE-FASI-UV) that approximated the DNA detection limit of 0.09ng/mL which was reported recently by XU Z(CGE—tITP—LIF). At the same time,the analyses of PCR product certified the extremely high sensitivity for the method(50477,33354 folds sensitivity elevation for normal pressure injection and normal electrokinetic injection respectively).the conclusion verified that the combination of matrix field-amplified and head-column field-amplified had a higher sensitivity effect. Meanwhile,in order to assess the stability and the ability of quantification in the combination of field-amplified technique, (2%TE as the sample matrix ion strength,0.5psi,20s water-plug,10KV,210s injection) as the fixed conditions,the results revealed that it had a good linear range (0.4-20ng/mL),correlation coefficient (0.9992) Intraday and interday RSD were 3.65%,2.83%,1.67% and 4.95%,3.06%,5.84% respectively,the RSD of peak area and migration time of PCR sample were 3.86% and 0.28% respectively,showed the good qualitative and quantitative effects of the technique.This technique was easily operated,high sensitivity,was also applicable for analysis of trace DNA samples without purification and had an extremely high applicable value in practical application. it certainly will promote the application of trace DNA analysis by capillary electrophoresis... |
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