Zhongqi Xu

High-sensitivity capillary gel electrophoretic analysis of DNA fragments on an electrophoresis microchip using electrokinetic injection with transient isotachophoretic preconcentration

The research adopted a single-channel microchip as the probe, and focused electrokinetic injection combined with transient isotachophoresis preconcentration technique on capillary electrophoresis microchip to improve the analytical sensitivity of DNA fragments. The channel length, channel width and channel depth of the used microchip were 40.5 mm, and 110 and 50 microm, respectively. The separation was detected by CCD (charge-coupled device) (effective length=25 mm, 260 nm). A 1/100 diluted sample (0.2 mg/l of each DNA fragment) of commercially available stepladder DNA sample could be baseline separated in 120 s with S/N=2-5. Compared with conventional chip gel electrophoresis, the proposed method is ideally suited to improve the sensitivity of DNA analysis by chip electrophoresis.

Another Approach Toward over 100 000-Fold Sensitivity Increase in Capillary Electrophoresis: Electrokinetic Supercharging with Optimized Sample Injection

Electrokinetic supercharging (EKS) is a powerful and practical method for multifold in-line concentration of various analytes prior to capillary electrophoresis (CE) analysis. However, a problem of insufficient sensitivity has always existed when trace analyte quantification by EKS-CE is a target, especially when coupled with conventional detectors. Normally this requires a greatly increased amount of analyte injected without separation degradation. In this contribution, we have shown that it is possible to substantially improve analyte loading and hence CE method detectability by modifying sample introduction configuration. The volume of sample vial was increased (from typical 500 μL to 17 mL), the common wire electrode was replaced by a ring electrode, and the sample solution was stirred. With these alterations, more analyte ions are accumulated within the effective electric field during electrokinetic injection and then maintained as focused zones due to transient isotachophoresis. The versatility of the customized EKS-CE approach for sample concentration was demonstrated for a mixture of seven rare-earth metal ions with an enrichment factor of 500 000 giving detection limits at or below 1 ng/L. These detection limits are over 100 000 times better than can be achieved by normal hydrodynamic injection, 1000 times better than the sensitivity thresholds of inductively coupled plasma atomic emission spectrometry (ICP-AES), and even close to those of inductively coupled plasma mass spectrometry (ICPMS).

Sensitive determination of anions in saliva using capillary electrophoresis after transient isotachophoretic preconcentration

A transient isotachophoresis-capillary electrophoresis (tITP-CE) system for the determination of minor inorganic anions in saliva is described. The complete separation and quantification of bromide, iodide, nitrate, nitrite, and thiocyanate has been achieved with only centrifugation and dilution of the saliva sample. In-line tITP preconcentration conditions, created by introduction of the plugs of 5 mM dithionic acid (leading electrolyte) and 10 mM formic acid (terminating electrolyte) before and after the sample zone, respectively, allowed the limits of direct UV absorption detection (at 200 nm) to be up to 50-fold improved as compared with CE without tITP. As a result, nitrate and thiocyanate were still detectable at 4.6 and 3.8 microgl(-1), respectively, in 1000 times diluted saliva. The daily variations of anionic concentrations in saliva samples taken from a smoking health volunteer were discussed based on the results of tITP-CE analysis. It was confirmed that the thiocyanate concentration in saliva noticeably increased after smoking. This is apparently the first report on simultaneous quantification of more than four anionic salivary constituents using CE.

High-sensitivity capillary and microchip electrophoresis using electrokinetic supercharging preconcentration: Insight into the stacking mechanism via computer modeling

This review discusses recent progress in the application of one of the most effective in-line preconcentration techniques used in electrophoresis in capillaries and microchips, electrokinetic supercharging (EKS). Conventionally considered as a transient isotachophoresis (tITP) step put into effect after the electrokinetic sample injection (EKI), EKS presumes that the electrolyte filled into the capillary (or microchip channel) comprises a co-ion acting as a leading ion to stack the injected analytes. Subsequently, to create the tITP state, one needs an additional injection of a suitable terminating ion. As a resulting increase in sensitivity strongly depends on the performance of both EKS stages, two theoretical sections are focused on hints for proper arrangement of EKI and tITP elaborated by means of computer simulation. In particular, factors affecting the injected amount of analytes, different modes of introducing the sample, suitable combinations of leading and terminating ions, and optimization of supporting electrolyte compositions are discussed with an objective to increase the enrichment factors. A comprehensive coverage of recent EKS applications in capillary and microchip electrophoresis, including metal ions, pharmaceuticals, peptides, DNA fragments, and proteins, demonstrates attainable sensitivity enhancements up to two orders of magnitude. This should make this method exportable to other analytes and facilitate its more widespread use to applications that require low limits of detection.

Electrokinetic sample injection for high-sensitivity capillary zone electrophoresis (part 1): Effects of electrode configuration and setting.

Electrokinetic injection (EKI) is usually considered as one of the useful approaches to improve sensitivity of CZE analysis. In the present study, we explored the relationship between electrode position and sample amount injected during EKI process by using 2D computer simulation (CFD‐ACE+) and real experiments, aiming to obtain higher detection sensitivity. Two different models of electrode configuration, a capillary inserted in a hollow electrode and a capillary surrounded by a cylindrical electrode on the reservoir wall, were simulated to evaluate the efficiency of EKI. It was found that analytes, occurring only in an effective potential field, could be introduced into the capillary while the other analytes remain outside of the field because of slow diffusion. Consequently, the longer distance between the electrode and the end of capillary, the higher efficiency of EKI was found by the simulation. This finding was verified by the real CZE analysis of dilute rare‐earth metal ions in a chloride solution (pH almost neutral). In fact, when the distance of Pt electrode and the capillary end in a CE apparatus (an Otsuka CAPI‐3100) was default (ca. 1 mm), LOD of Er was 0.27 μg/L. When the distance was increased to 19.5 mm, the LOD was improved over ten times down to 0.02 μg/L. The LOD achieved is 50‐fold better than that of inductively coupled plasma atomic emission spectrometry (1–2 μg/L for Er).

Electrokinetic sample injection for high‐sensitivity CZE (part 2): Improving the quantitative repeatability and application of electrokinetic supercharging‐CZE to the detection of atmospheric electrolytes

Electrokinetic supercharging (EKS) is defined as a technique that combines electrokinetic sample injection with transient ITP. Quantitative repeatability of EKS‐CZE and the other CE methods using electrokinetic sample injection process is usually inferior in comparison with the CE methods using hydrodynamic or hydrostatic injection. This is due to some effects, such as the temperature change and the convection of the sample solution in the reservoir, as well as the change of the distance between an electrode and a capillary end (Dec). In particular, we have found that the Dec change might most seriously affect the repeatability, especially when the electrode is a thin Pt wire that could be unintentionally bent during sampling. By using a Teflon spacer to fix Dec to 1.1 mm, the RSD of peak area (n=5) was decreased from 20 to 3.4% in EKS‐CZE for several metal cations. This Dec dependence of the sample amount injected was supported by computer simulation using CFD‐ACE+ software. The improved repeatability (down to 5.1% at n=5, averaged RSD for Co2+, Li+, Ni2+, Zn2+ and Pb2+) was also experimentally attained by increasing the Dec to ca. 20 mm, which was also effective to obtain high sensitivity. Since the temperature and the convection effects on the repeatability are comparatively small in a proper laboratory environment, these effects were estimated from the EKS‐CZE experiments using conditions such as warming and agitating the sample solution during EKS process. Finally, EKS‐CZE was applied to the detection of ions from atmospheric electrolytes in high‐purity water exposed to ambient air for 2 h. The microgram per liter levels of anions (chloride, sulfate, nitrate, formate, acetate and lactate) and cations (ammonium, calcium, sodium and magnesium) could be detected using conventional UV detector.

Electrokinetic supercharging preconcentration prior to CGE analysis of DNA: sensitivity depends on buffer viscosity and electrode configuration.

Aiming to high sensitivity DNA analysis by CGE, electrokinetic supercharging (EKS) approach was adopted in this article. EKS is known as an online preconcentration technique that combines electrokinetic sample injection (EKI) with transient ITP (tITP). Herein, two factors of buffer viscosity and electrode configuration were studied to further improve EKS performance. An ultralow‐viscosity Tris‐Boric acid‐EDTA (TBE) buffer solution, consisted of 2% low‐molecular‐weight hydroxypropyl methyl cellulose (HPMC) and 6% mannitol and with pH 8.0 adjusted by boric acid, was applied. The boric acid would make a complex with mannitol and generates borate polyanion, which acts as the leading ion for tITP process. The new electrode configuration, a Pt ring around capillary, was modified on Agilent CE system to lead large amount sample introduction during EKS. The standard DNA sample of φX174/HaeIII digest was used to evaluate the qualitative and quantitative abilities of the proposed strategy. The 170 000‐fold highly diluted sample at concentration of 3.0 ng/mL was enriched by EKS and detected by normal UV detection method. The obtained LOD of the weakest peak of 72 bp fragment was around 7.7 pg/mL, apparently improved more than 10 000‐fold in comparison with conventional CGE with UV detection.

Electrokinetic supercharging with a system-induced terminator and an optimized capillary versus electrode configuration for parts-per-trillion detection of rare-earth elements in CZE.

A further improvement of electrokinetic supercharging (EKS) methodology has been proposed, with the objective to enhance the sensitivity of the conventional CZE‐UV method down to a single‐digit part per trillion (ppt) level. The advanced EKS procedure is based on a novel phenomenon displaying the formation of a zone with an increased concentration of the hydrogen ion, capable to perform the function of a terminator, behind the sample zone upon electrokinetic injection. In combination with a visualizing co‐ion of BGE, protonated 4‐methylbenzylamine, acting as the leading ion, such system‐induced terminator a effected the transient ITP state to efficiently concentrate cationic analytes prior to CZE. Furthermore, to amass more analyte ions within the effective electric field at the injection stage, a standard sample vial was replaced with an elongated vial that allowed the sample volume to be increased from 500 to 900 μL. Alongside, this replacement made the upright distance between the electrode and the capillary tips prolonged to 40.0 mm to achieve high‐efficiency electrokinetic injection. The computer simulation was used for profiling analyte concentration, pH, and field strength in order to delineate formation of the terminator during sample injection. The proposed preconcentration strategy afforded an enrichment factor of 80 000 and thereby the LODs of rare‐earth metal ions at the ppt level, e.g. 0.04 nM (6.7 ng/L) for erbium(III).

Study of a novel sample injection method (floating electrokinetic supercharging) for high-performance microchip electrophoresis of DNA fragments.

Aiming to achieve high‐performance analysis of DNA fragments using microchip electrophoresis, we developed a novel sample injection method, which was given the name of floating electrokinetic supercharging (FEKS). In the method, electrokinetic injection (EKI) and ITP preconcentration of samples was performed in a separation channel, connecting two reservoir ports (P3 and P4) on a cross‐geometry microchip. At these two stages, side channels, crossing the separation channel, and their ports (P1 and P2) were electrically floated. After the ITP‐stacked zones passed the cross‐part, they were eluted for detection by using leading ions from P1 and P2 that enabled electrophoresis mode changing rapidly from ITP to zone electrophoresis (ZE). Possible sample leakage at the cross‐part toward P1 and P2 was studied in detail on the basis of computer simulation using a CFD‐ACE+ software and real experiments, through which it was validated that the analyte recovery to the separation channel was almost complete. The FEKS method successfully contributed to higher resolution and shorter analysis time of DNA fragments on the cross‐microchip owing to more rapid switching from ITP status to ZE separation in comparison with our previous EKS procedure realized on a single‐channel microchip. Without any degradation of resolution, the achieved LODs were on average ten times better than using conventional pinched injection.

Sensitive profiling of biogenic amines in urine using CE with transient isotachophoretic preconcentration.

A transient ITP-CZE system is proposed for the determination of biogenic amines in urine. The complete separation and identification of dopamine, tyramine (TA), tryptamine (T), serotonin, epinephrine, norepinephrine, and normetanephrine have been achieved in a twofold diluted urine sample (in which the analytes were below the LODs by normal CZE). The tITP preconcentration conditions were created by introducing a 30 mM solution of NaOH, containing 0.1% hydroxypropylcellulose (pH 6.5 adjusted with MES), and 0.1 M HCl before and after the sample zone to work as leading and terminating electrolytes, respectively. This allowed the LODs of direct UV absorption detection to be decreased down to the 10(-8) M level that is comparable with the sensitivity thresholds of LIF detection or inline SPE-CE. The RSDs of migration time and peak area for real-biofluid analysis were in the range of 0.1-4.5% and 0.8-16% (n=3), respectively. Quantification of dopamine, TA, T, and serotonin was performed using internal calibration. To the best of our knowledge, this is the first report on probing urinal biogenic amines and their metabolites by tITP-CZE.