Lina Geng

In situ photo-immobilised pH gradient isoelectric focusing and zone electrophoresis integrated two-dimensional microfluidic chip electrophoresis for protein separation

AbstractA method is introduced for open-column photo-induced site-selective immobilization of pH gradients in a layer of PEG-methacrylate in a multi-dimensional microfluidic chip for use in electrophoresis. It has several attractive features: (a) mixtures of fluorescently labelled proteins carbonic anhydrase, catalase and myoglobin in their native state can be separated by pH-gradient isoelectric focusing (IEF) and zone electrophoresis (CZE) using integrated 2D chip electrophoresis; (b) compared to strip packing or monolithic photo-immobilization, it overcomes the shortcomings of free carrier ampholyte-based 2D chip electrophoresis in an easy way; (c) larger amount of sample can be loaded into the open column-mode electrophoresis (d) immobilized pH gradients can be re-used and the chip can be recycled; (e) a multilayer 3D pH gradient is established by a layer-by-layer assembly technique to further increase the separation capacity. In our perception, this strategy has a large potential in microfluidic chip-based separation schemes because of its simplicity, separation power, re-usability, and separation capacity. Graphical abstractAn open-column layer-by-layer photo-immobilised pH gradient was introduced into two-dimensional chip electrophoresis with simplicity, reusability, improved separation performance and separation capacity.

Proteoliposome-Based Capillary Electrophoresis for Screening Membrane Protein Inhibitors

A method for screening of monoamine oxidase (MAO) inhibitor was carried out using capillary electrophoresis (CE) based on the interaction of MAO and its substrate kynuramine (Kyn). Bioactive proteoliposome was reconstituted by liposome and MAO and then was applied as the pseudostationary phase (PSP) of CE to mimic the interaction between the enzyme and its substrate. N-prolmrgyl-R-2-heptylamine (R-2-HPA) and rasagiline [N-propargyl-1-(R)-aminoindan], which are two kinds of MAO inhibitors, were added into the running buffers containing proteoliposome. The results showed that the relative migration time ratio (RMTR × 10(-1)) values of Kyn were enhanced from 8.88 to 9.31 with an increase of the concentrations of rasagiline from 10(-6) to 1 mM. However, the RMTR values of Kyn were enhanced from 8.83 to 9.14 with an increase of the concentrations of R-2-HPA from 10(-6) to 1 mM. The RMTR value of Kyn in the presence of rasagiline was larger than that in the presence of R-2-HPA when rasagiline and R-2-HPA were at the same concentration. The results indicated that the interaction between Kyn and MAO was weakened with the increase of the inhibitors. In addition, the results of offline incubation showed that the inhibitions of rasagiline were 100.0, 72.1, 51.8 and 5.4% at the concentration of 1, 10(-2), 10(-4) and 10(-6) mM; moreover, the inhibitions of R-2-HPA were 70.0, 44.9, 4.1 and 0.9% at the concentrations of 1, 10(-2), 10(-4) and 10(-6) mM. The inhibition efficiency of rasagiline was stronger than that of R-2-HPA at the same concentration. Additionally, the interaction between Kyn and liposome was also investigated. This newly developed method might provide a potential tool for screening MAO inhibitor.

Microvalve controlled multi-functional microfluidic chip for divisional cell co-culture

Pneumatic micro-valve controlled microfluidic chip provides precise fluidic control for cell manipulation. In this paper, a multi-functional microfluidic chip was designed for three separate experiments: 1. Different cell lines were dispensed and cultured; 2. Three transfected SH-SY5Y cells were introduced and treated with methyl-phenyl-pyridinium (MPP+) as drug delivery mode; 3. Specific protection and interaction were observed among cell co-culture after nerve damage. The outcomes revealed the potential and practicability of our entire multi-functional pneumatic chip system on different cell biology applications.

Native Protein Separation by Isoelectric Focusing and Blue Gel Electrophoresis-Coupled Two-Dimensional Microfluidic Chip Electrophoresis

The use of microfluidic chip-based two-dimensional separation holds great promise in the proteomics field, given its portability, simplicity, speed, efficiency, and throughput. However, inclusion of sodium dodecyl sulfate, reported to be necessary for increasing protein-resolving capability, was also accompanied by the loss of both protein conformation and biological function. Here, we describe separation of native proteins by introducing blue native gel electrophoresis into isoelectric focusing and gel electrophoresis (IEF/CGE)-coupled protein two-dimensional microfluidic chip electrophoresis. After assessing the influence of various experimental conditions, the best separation ability and reproducibility of blue native IEF/CGE (IEF/BN-CGE) chip electrophoresis achieved until now were demonstrated no matter whether with a simple simulated mixture or with a complex mixture of total Escherichia coli proteins. Finally, instead of theoretical calculations, the image analysis technique was also used for the first time to quantitatively evaluate the actual peak capacities of chip electrophoresis. According to the number of features abstracted in the electrophoresis patterns, the superiority of the IEF/BN-CGE two-dimensional microfluidic chip electrophoresis was then exhibited quantitatively. The high native protein separation performance makes this established chip electrophoresis method possible for further application in widely needed drug screening, analysis of bio-molecular function, and assays of protein–protein interactions.

A novel method to predict protein aggregations using two-dimensional native protein microfluidic chip electrophoresis

As is known, protein aggregation is involved in several diseases such as amyloid aggregation. Control of protein aggregation is also a challenge to protein pharmaceuticals. Thus, efficient analytical tools used to predict protein aggregation in the early potential or intermediate stage in a complex sample are crucial to the understanding and prevention of protein aggregation. However, it is always difficult to study early state and microscopy invisible aggregation. In this study, a simple analytical technology based on isoelectric focusing (IEF) and capillary zone electrophoresis (CZE) coupled two-dimensional microfluidic chip native protein electrophoresis was established to study aggregation before the formation of the visible aggregated particles. With IEF aggregation promotion and CZE separation, α-synuclein aggregation was easily observed through whole channel detection, not only in the whole protein extraction of α-synuclein stably overexpressing PC12 cells, but also of AD rat hippocampi. This method was also used to investigate the effects of two drugs on the inhibition or promotion of protein aggregation in PC12 cells, respectively. Further combined with histogram image analysis, this easy, efficient, robust and quantitative protein aggregation prediction of crude sample demonstrates the speciality and great potential of this new method in disease research, drug development and the biopharmaceutical industry. At the same time, our research also expands the application field of chip electrophoresis because, for the first time, chip electrophoresis was used as a separation based aggregation display tool.

Stress Response to Hypoxia in Wistar Rat: LA, MDA, SOD and Na+-K+-ATPase

To study the time course of oxidative damage and the molecular and cellular mechanisms underlying hypoxia-induced brain damage, some stress responses to hypoxia in rat brain, such as lactic acid(LA) and malondialdehyde (MDA) expression, superoxide dismutase (SOD) and Na + -K + -ATPase activety etc, were analysised. The role of those factors in oxidative stress was discussed too. Results showed that the SOD activity reduced obviously at 15% O2 and the level of MDA raised obviously at 12% O2 , the level of lactic acid increased obviously and the Na + - K + -ATPase activity decreased obviously at 10% O2 respectively. Obviously this gave an inkling that the loss of ion homeostasis might be the result of lipid peroxidation damage. Study demonstrated that hypoxia can cause cellular lipid peroxidation, which in turn can cause inhibition/reduction in the activities of

Aptamer biosensors for microorganism detection

Aptamers are single-stranded oligonucleotides that bind with high specificity and affinity to a target molecule. Aptamer-based biosensors have become a new class of biosensors, which utilized aptamers as highly selective recognition elements as a result of good performance in the capture of target analytes. In this study, aptamer was used as an recognition element of biosensor, and was integrated to microarray chip to realized the detection of L. achidophilus. The fluorescence and transmission image of bacterial cells were obtained in different times magnification field. The results demonstrated that the aptamer biosensor based on microarray chip can be used to detect L. achidophilus by means of FITC labeled bacterial cells.

Normobaric Hypoxia-induced Brain Damage and Mechanism in Wistar Rat

To evaluate normobaric hypoxia-induced brain damage and discuss the mechanism in wistar rat brain injury, the water content of brain , brain infarction area ratio , the level of lactic acid and Na + -K + -ATPase activity were detected. The expression of HIF-lalpha was confirmed using immunohistochemistry. Histopathological changes of brain in rat model due to hypoxia were investigated using hematoxylin and eosin stain (HE). Results showed that the levels of lactic acid were positively correlated with the degree of hypoxia which caused a concentration-dependent decrease in total Na + -K + - ATPase activity. Compared with the control group, brain water content of rat model had significant difference under serious hypoxia condition. Brain infarction ratio increased sharply when lactic acid level increased and Na + -K + -ATPase activity decreased obviously. Histopathological examination confirmed that there was a necrotic feature in one single hippocampal gyrus neuron cell. HIF-lalpha (hypoxia induced factor-lalpha) was induced by hypoxia at 6% 02. Expression of HIF-lalpha enhanced the hypoxia adaptation capability of the rat model through regulating the expression of multiple genes. Lactic acid, Na + -K + -ATPase and HIF-lalpha played an important role in brain injury as a possible mechanism.

Recent advances in microfluidic chip integrated electronic biosensors for multiplexed detection

Utilizing biosensors for multiplexed detection can greatly increase analysis throughput and thus, the amount of information obtained in a single assay. The microfluidic chip, a type of micro-total analysis system (µTAS), has provided a necessary platform for portable and high-throughput biosensors. Biosensors and microfluidic chips are powerful individually, and their super combination is very meaningful for analytical especially for biological applications. In this paper, every kind of microfluidic-chip-integrated electronic biosensors including some emerging technologies for simultaneous detection of multiple analytes are reviewed. Different ways to reduce or avoid cross-talking and more efforts to achieve lab on chip multisensors were also introduced to help readers form a general idea of current developments in different angles.

A signal acquisition and data processing system for LED induced fluorescence detector

A system of the signal acquisition and data processing used for light-emitting diodes induced fluorescence (LED-IF) detection in our laboratory was described in this paper. A high-speed data acquisition module was developed. The function of signal acquisition in real-time and data processing was realized utilizing the graphical programming environment LabVIEW. To reduce the distorted come from the technical limitations of the equipment, low-pass Butterworth filter and wavelet filter were applied in this system. Some FITC-derived amino acids were separated coupled with HPLC. The result had been analyzed and the report was finalized automatically.