Zhi-Yong Wu

Preparative isolation of alkaloids from Corydalis bungeana Turcz. by high-speed counter-current chromatography using stepwise elution

High-speed counter-current chromatography (HSCCC) was successfully applied for the preparative separation and purification of alkaloids from Corydalis bungeana Turcz. (Kudiding in Chinese) for the first time. After the measurement of partition coefficient of seven target alkaloids in the nine two-phase solvent systems composed of CHCl(3)-MeOH-(0.1 M; 0.2 M; 0.3 M) HCl (4:1.5:2; 4:2:2; 4:3:2, v/v), CHCl(3)-MeOH-0.2 M HCl (4:2:2, v/v) and CHCl(3)-MeOH-0.3 M HCl (4:3:2, v/v) were finally selected for the HSCCC separation using the first upper phase as the stationary phase and the stepwise elution of the two lower mobile phases. Consequently, sanguinarine (10 mg), corynoline (25 mg), protopine (20 mg), corynoloxine (18 mg), and 12-hydroxycorynoline (8 mg) were obtained from 200 mg of crude alkaloid extracts with purities of 94-99% as determined by HPLC. Their chemical structures were characterized on the basis of (1)H-NMR, (13)C-NMR, and LC-ESI-Q-TOF-MS/MS analyses.

Simultaneous electrokinetic concentration and separation of proteins on a paper-based analytical device

In this report, we introduced an electrokinetic strategy for simultaneous concentration and separation of charged analytes in an open and free standing paper fluidic channel. This was achieved by taking advantage of the field amplified sample stacking (FASS) effect. An electric field gradient was developed in the channel due to the conductivity difference between the background electrolyte (BGE) and sample solution. Through adjustment of the electroosmotic flow level and BGE concentration, charged analytes were not only stacked but also separated in the gradient based on their difference in electrophoretic mobility. Proof of concept was demonstrated firstly by two food dyes, brilliant blue and amaranth, and each dye was about 100-fold concentrated within 300 s. Simultaneous stacking and separation of two colored proteins, bovine hemoglobin and cytochrome c, was also demonstrated visually with the proposed method. Sample consumption was reduced to 5 μL by directly loading the sample onto the open channel, and the separation was completed within 30 s. This method is simple, rapid and easy to operate, which is particularly beneficial to the development of sensitive and multifunctional paper-based analytical devices (PADs) for field testing.

Two orders of magnitude electrokinetic stacking of proteins in one minute on a simple paper fluidic channel

A crucial challenge in development of robust paper-based analytical devices (PADs) is the lack of sensitivity. Electrokinetic stacking is a kind of unique sample preconcentration for low-abundant charged species, which can be integrated to PADs to enhance the sensitivity. In this work, we bring new insights into the mechanism underlying an electrokinetic stacking technique based on a field amplified stacking (FAS) effect implemented on a simple paper fluidic channel. Here, we reveal that the acid–base titration reaction between the cations/anions in the background electrolyte (BGE) and the OH−/H+ ions from water electrolysis plays an essential role in the development of the electric field gradient. This was experimentally confirmed by changing the BGE type, monitoring the current variation and pH characterization. Based on the new knowledge, the paper-based FAS was successfully extended to stack cationic analytes. Eventually, a model protein of bovine hemoglobin (BHb) in its anion form was 150-fold stacked within 60 s. This work shows that the sensitivity of PADs can be greatly enhanced with effective use of this simple and low cost electrokinetic stacking technique.

A thermostat chip of indium tin oxide glass substrate for static polymerase chain reaction and in situ real time fluorescence monitoring

A thermostat chip of indium-tin oxide glass substrate for static chip polymerase chain reaction (PCR) is, for the first time, introduced in this paper. The transparent conductive layer was used as an electro-heating element. Pulse width modulation and fuzzy proportional integration-differentiation algorithm were adopted in the temperature programming of the chip. The temperature distribution was investigated, and a dynamic control precision within +/-2 degrees C was achieved. The highest ramping rates were 37 degrees Cs(-1) for heating and 8 degrees Cs(-1) for cooling with an electric fan. The PCR reaction vials were constructed with polyethylene tubes or poly(dimethylsiloxane) directly on the thermostat chip; the chip had a typical size of 25 mm x 25 mm and a thickness of 1.1mm. Static chip PCR was successfully demonstrated either in a single vial or in an up to 8-parallel array vials. In situ real time fluorescence monitoring during PCR of a lambda DNA fragments (236bp) with SYBR Green I was demonstrated using a blue light emission diode as a light source and a photomultiplier as a detector. The method proposed here is characterized by open access, easy fabrication and low cost. This work could be the basis for developing a portable real time PCR system with disposable chips for point of care tests.

Electrokinetic stacking of electrically neutral analytes with paper-based analytical device

Electrokinetic stacking (ES) is effective for improving sensitivity of paper-based analytical device (PAD) for charged analytes. In this paper, we successfully demonstrated ES of electrically neutral analytes on PAD, and the performance was characterized by smartphone-based colorimetry and fluorescence. Firstly, SDS from cathode reservoir stacked as a micelle band on an open paper fluidic channel by ES, and the target analyte was swept by the micelle. Meanwhile, the probes at the other side were carried by electroosmotic flow (EOF). Eventually, neutral components preloaded on the channel were concentrated as the narrow stacking band. Taking the rhodamine B as a probe, the effects of EOF, background electrolyte concentration and anionic surfactant concentration were investigated. Fluorescence detection of rhodamine B and colorimetric analysis of Sudan III demonstrated the sensitivity enhanced and its potential for the semi-quantitative test. Under the optimized conditions, fluorescence detection limit of 50 nM of rhodamine B was achieved with a linear range of 1.0-10 μM (R2 = 0.99). The colorimetric detection limit for Sudan III was 5.2 μM and the linear range was 5-40 μM (R2= 0.99). Compared with direct analysis without stacking, the signal levels of rhodamine B and Sudan III were increased by 30-fold and 6-fold, respectively. This study showed that with ES, sensitive and rapid PAD detection of electrically neutral analytes could be achieved.

Carrier ampholyte-free isoelectric focusing on a paper-based analytical device for the fractionation of proteins.

Isoelectric focusing plays a critical role in the analysis of complex protein samples. Conventionally, isoelectric focusing is implemented with carrier ampholytes in capillary or immobilized pH gradient gel. In this study, we successfully exhibited a carrier ampholyte-free isoelectric focusing on paper-based analytical device. Proof of the concept was visually demonstrated with color model proteins. Experimental results showed that not only a pH gradient was well established along the open paper fluidic channel as confirmed by pH indicator strip, the pH gradient range could also be tuned by the catholyte or anolyte. Furthermore, the isoelectric focusing fractions from the paper channel can be directly cut and recovered into solutions for post analysis with sodium dodecyl sulfate-polyacrylamide gel electrophoresis and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry. This paper-based isoelectric focusing method is fast, cheap, simple and easy to operate, and could potentially be used as a cost-effective protein sample clean-up method for target protein analysis with mass spectrometry.