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

Abstract

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.

DNA aggregation and cleavage in CGE induced by high electric field in aqueous solution accompanying electrokinetic sample injection

“…Figure B shows the potential gradient profile along the capillary center. Obviously there was a very short zone but with very high potential (>600 V/cm) at the capillary end, which corresponded to so‐called system induced terminator . Accordingly, when DNA fragments migrated across this zone, they might possibly be damaged when the applied voltage was high.…”

“…This led to efficient sample introduction from a large volume of the sample (230 μL). The obtained LOD was 7.7 ng/L (for the weakest peak of 72 bp at S/N = 3), so that apparently improved by a factor of more than 10 000‐fold in comparison with conventional CGE .…”

Highly sensitive analysis of nucleic acids using capillary gel electrophoresis with ultraviolet detection based on the combination of matrix field-amplified and head-column field-amplified stacking injection

“…LOD Sensitivity improvement ratio C-FASI 0.13 ng/ml (standard DNA marker) 10 ng/ml (X174-Hae III digest, total concentration) EKS [60] 200 ng/ml (standard DNA marker) C-FASI/EKS = 1538 EKS [61] 200 ng/ml (standard DNA marker and PCR product) C-FASI/EKS = 1538 tITP-FD [62] 50 ng/ml (X174-Hae III digest, total concentration) C-FASI/tITP-FD = 5 EKS [63] 3.0 ng/ml (X174-Hae III digest, total concentration) C-FASI/EKS = 0.3 Sweeping-EKS [64] 0.49 ng/ml (standard DNA marker) C-FASI/Sweeping-EKS = 3.8 Based on this conclusion from the previous report [64] "the LOD of DNA was decreased to 0.49 ng/ml by PAMAM sweeping-EKS. The developed approach improved the enrichment factor by more than 3500 and 30 folds relative to the hydrodynamic injection and conventional FASI, respectively.…”

“…In 2013, Ye and Hirokawa et al introduced the electrokinetic supercharging (EKS) approach that combines electrokinetic sample injection (EKI) with transient ITP (tITP) [63]. Although their sensitivity was slightly better than that of C-FASI (3.0 ng/ml versus 10 ng/ml, respectively), their methodology was not as good for the following reasons: (1) The sensitivity of EKS depends on buffer viscosity and electrode configuration.…”

Capillary electrophoresis based on nucleic acid detection for diagnosing human infectious disease

“…The combination of these two fields requires researchers knowledgeable in both subjects, but many scientists lack such a comprehensive background. The relatively low sensitivity of CE analysis is the second limitation in the field of DNA analysis [136][137][138][139][140]. The double bond formed by the base group in DNA has a low UV-visible absorption, and a normal UV detector has a low sensitivity.…”