Chenchen Liu

Electromigration behavior of nucleic acids in capillary electrophoresis under pulsed-field conditions.

We have presented a study focused on the migration pattern of double-stranded DNA (dsDNA) and RNA under pulsed field conditions. By calculating the dependence of nucleic acid mobility on its molecular size in a double logarithm plot, we found that (I) dsDNA molecules proceeded by a sigmoidal migration regime which was probably related to Ogston sieving, transition regime, and reptation model. Furthermore, the transition regime disappeared if DNA was resolved in a higher molecular mass HEC. (II) The migration pattern of RNA was relevant to the denaturant used for separation. When RNA was denatured by acetic acid, its mobility parabolically declined with its molecular size. The mobility was linearly decreased with the molecular size if urea was employed as denaturant. (III) RNA may migrate by Ogston, reptation without orientation mechanism when denatured by urea, whereas these two models were not suitable for RNA if denatured by acetic acid. Even though the electrophoretic conditions of PFCE were varied, the sigmoidal, linear, parabolic migration patterns could still be observed. (IV) Under certain modulation depth, the migration time (Tm) of acetic acid decreased with the increase of average separation voltage (Va), and when RNA denatured in 4.0M urea, Tm showed a linear correlation with Va. (V) The mobility of nucleic acids increased with the growth of artificial temperature in the capillary volume due to the decrease in the viscosity of the polymer. This is the first systematic and comparative research of high molecular mass nucleic acids in PFCE, which provides us deep insight into RNA and DNA migration behavior under pulsed electric field conditions.

Quantification of periodontal pathogens cell counts by capillary electrophoresis.

Gingivitis is a highly prevalent periodontal disease around the worldwide. Porphyromonas gingivalis (P.g), Treponema denticola (T.d) and Tannerela forsythia (T.f) were considered to be three important periodontal pathogens related to gingivitis, and research shows that the counts of periodontal pathogen cells in the patients before, during, and after fixed orthodontic appliance therapy were quite different. We proposed a simple method to extract the periodontal pathogens from the periodontal pocket in this work and demonstrated a new approach to determine periodontal pathogen level based on capillary electrophoresis (CE). After polymerase chain reaction amplification of P.g (197 bp), T.d (311 bp), and T.f (641 bp), it shows that they can rapidly identified by CE within 5 min. The peak area in the eletropherogram is linearly related to the concentration of P.g, T.d, and T.f, and the correlation coefficients R(2) corresponding to them are 0.993, 0.993, and 0.956, respectively. According to this linearly relationship, the estimated concentration of P.g, T.d, and T.f in gingival crevicular fluid from one volunteer was inferred to be about 9.90×10(2), 1.48×10(3), and 9.01×10(2)cells/μl, respectively.

Capillary electrophoresis of a wide range of DNA fragments in a mixed solution of hydroxyethyl cellulose

We carried out capillary electrophoresis of 0.1–10.0 kilo base pair DNA fragments in a mixed hydroxyethyl cellulose (HEC) polymer. The mixed HEC polymer was prepared with different molecular weights (Mw) (90k, 250k, 720k and 1300k). The effects of important parameters, including the ratio of the mixture composition and the concentration of the mixing polymer, on the separation performance were investigated. Results show that these parameters can not only shorten the migration time of DNA without great deterioration in resolution, but they can also decrease the viscosity of the polymer, and thus make it easy to fill the capillary. Finally, we separated φ×174-Hirc II digest and λ-EcoT14 I DNA digest with high resolution in the mixed HEC solution within 18 min.

Rapid identification and quantitation for oral bacteria based on short-end capillary electrophoresis

High-speed capillary electrophoresis (HSCE) is a promising technology applied in ultra-rapid and high-performance analysis of biomolecules (such as nucleic acids, protein). In present study, the short-end capillary electrophoresis coupled with one novel space domain internal standard method (SDIS) was employed for the rapid and simultaneous analysis of specific genes from three oral bacteria (Porphyromonas gingivalis (P.g), Treponema denticola (T.d) and Tannerela forsythia (T.f)). The reliability, reproducibility and accuracy properties of above mentioned SDIS method were investigated in detail. The results showed the target gene fragments of P.g, T.d and T.f could be precisely, fast identified and quantitated within 95s via present short-end CE system. The analyte concentration and the ratio of space domain signals (between target sample and internal standard sample) featured a well linear relationship calculated via SDIS method. And the correlation coefficients R(2) and detection limits for P.g, T.d, T.f genes were 0.9855, 0.9896, 0.9969 and 0.077, 0.114 and 0.098ng/μl, respectively.

Polyethylene Oxide (PEO) and Polyethylene Glycol (PEG) Polymer Sieving Matrix for RNA Capillary Electrophoresis.

The selection of sieving polymer for RNA fragments separation by capillary electrophoresis is imperative. We investigated the separation of RNA fragments ranged from 100 to 10,000 nt in polyethylene glycol (PEG) and polyethylene oxide (PEO) solutions with different molecular weight and different concentration. We found that the separation performance of the small RNA fragments (<1000 nt) was improved with the increase of polymer concentration, whereas the separation performance for the large ones (>4000 nt) deteriorated in PEG/PEO solutions when the concentration was above 1.0%/0.6%, respectively. By double logarithmic plot of mobility and RNA fragment size, we revealed three migration regimes for RNA in PEG (300-500k) and PEO (4,000k). Moreover, we calculated the smallest resolvable nucleotide length (N min) from the resolution length analysis.

Analysis of small interfering RNA by capillary electrophoresis in hydroxyethylcellulose solutions.

The analysis of small interfering RNA (siRNA) is important for gene function studies and drug developments. We employed CE to study the separation of siRNA ladder marker, which were ten double‐stranded RNA (dsRNA) fragments ranged from 20 to 1000 bp, in solutions of hydroxyethylcellulose (HEC) polymer with different concentrations and molecular weights (Mws). Migration mechanism of dsRNA during CE was studied by the mobility and resolution length (RL) plots. We found that the RL depended on not only the concentration of HEC, but also the Mw of HEC. For instance, RL of small dsRNA fragment was more influenced by concentration of high Mw HEC than large dsRNA fragment and RL of large dsRNA fragment was more influenced by concentration of low Mw HEC than small dsRNA fragment. In addition, we found electrophoretic evidence that the structure of dsRNA was more compact than dsDNA with the same length. In practice, we succeeded to separate the glyceraldehyde 3‐phosphate dehydrogenase siRNA in the mixture of the siRNA ladder marker within 4 min.

Gene analysis of multiple oral bacteria by the polymerase chain reaction coupled with capillary polymer electrophoresis.

Capillary polymer electrophoresis is identified as a promising technology for the analysis of DNA from bacteria, virus and cell samples. In this paper, we propose an innovative capillary polymer electrophoresis protocol for the quantification of polymerase chain reaction products. The internal standard method was modified and applied to capillary polymer electrophoresis. The precision of our modified internal standard protocol was evaluated by measuring the relative standard deviation of intermediate capillary polymer electrophoresis experiments. Results showed that the relative standard deviation was reduced from 12.4-15.1 to 0.6-2.3%. Linear regression tests were also implemented to validate our protocol. The modified internal standard method showed good linearity and robust properties. Finally, the ease of our method was illustrated by analyzing a real clinical oral sample using a one-run capillary polymer electrophoresis experiment.

Capillary electrophoresis of RNA in hydroxyethylcellulose polymer with various molecular weights.

Recent research demonstrates that large numbers of long noncoding RNAs (lncRNAs) in mammals exhibit indices of functionality, and thus analysis of longer RNAs is of great significance. In the present work, we investigated the effect of molecular weight on the separation performance of long RNA by capillary electrophoresis (CE). Results demonstrate that (1) low molecular weight of hydroxyethylcellulose (HEC) (90k) favors the separation of short RNA (<1000 nt). The resolution for short RNA was improved and the migration time was linearly extended with the increase of polymer concentration. (2) In the longer chain HEC (250k, 720k and 1300k), the resolution for the small RNA fragment (<1000 nt) became better as the polymer concentration increased, whereas the resolution for the large ones (>3000 nt) deteriorated. (3) Based on logarithmic plot, there exist two migration regimes for RNA in short chain HEC (90k), three regimes in moderate chain HEC (250k and 720k), and four regimes in the long chain HEC (1300k). Such a systematic investigation of long RNAs may be useful for research on lncRNAs in the length range of 100-10,000 nt.

A light-up fluorescent probe for citrate detection based on bispyridinum amides with aggregation-induced emission feature

Citrate is an important intermediate in the citric acid cycle, a vital metabolic pathway for animals, plants and bacteria. It is of great significance to detect its levels in human beings because several diseases may cause the abnormal of citrate. In this paper, a new turn-on fluorescent sensor (TPE-Py) using the classic tetraphenylethylene (TPE) as the aggregation-induced emission (AIE) fluorophore and bipyridinium-based amides as the recognition receptor has been synthesized for the detection of citrate. The probe exhibits good selectivity and sensitivity to citrate with a relatively low detection limit (1.0 × 10-7M). The enhancement of the fluorescence is relevant with the AIE property based on the complexation of TPE-Py with citrate caused by the hydrogen bonding and electrostatic interactions between the bipyridinium diamides and citrate, which has been proved by 1H NMR and mass spectra titration, scanning electronic microscope and dynamic light scattering analyses. More importantly, the quantification of citrate in artificial urine may develop TPE-Py fluorometric probe for the citrate detection in real biosystems.

Quantitative Detection of Ethanol/Acetone in Complex Solutions Using Raman Spectroscopy Based on Headspace Gas Analysis:

This paper demonstrated the quantitative detection of ethanol and acetone mixtures in complex solutions with Raman spectroscopy based on headspace gas analysis. By analyzing the volatile components in the headspace, their concentrations in liquid solutions were determined. We constructed our own Raman spectroscopy system to detect the headspace gas quantitatively over a solution in a sealed vial. The Raman spectra of the headspace gases over standard solutions were standardized for finding the concentrations of ethanol, acetone, and ethanol–acetone in mixture solutions. The results showed that the concentration of a gaseous component in the headspace gas was proportional to its ratio in the liquid solution. We obtained a linear relationship between the spectral intensity of volatile components in headspace and the concentration of the liquid solutions. Then, we analyzed the alcohol concentration in a white wine and a Chinese liquor called Fen Chiew by measuring the Raman spectra of the headspace gas over their liquids. For the river water sample, we also implemented our headspace gas detection with Raman spectra to obtain the concentration of acetone in the river sample. This work demonstrated the facilitation of headspace gas analysis by the qualitative and quantitative determination of volatile substances from liquid samples using Raman spectroscopy.