Paul C. H. Li

Artesunate derived from traditional Chinese medicine induces DNA damage and repair.

Artesunate is a semisynthetic derivative from artemisinin, a natural product from the Chinese herb Artemisia annua L. It exerts antimalarial activity, and, additionally, artemisinin and its derivatives are active against cancer cells. The active moiety is an endoperoxide bridge. Its cleavage leads to the formation of reactive oxygen species and carbon-centered radicals. These highly reactive molecules target several proteins in Plasmodia, which is thought to result in killing of the microorganism. DNA damage induced by artemisinins has not yet been described. Here, we show that artesunate induces apoptosis and necrosis. It also induces DNA breakage in a dose-dependent manner as shown by single-cell gel electrophoresis. This genotoxic effect was confirmed by measuring the level of gamma-H2AX, which is considered to be an indication of DNA double-strand breaks (DSB). Polymerase beta-deficient cells were more sensitive than the wild-type to artesunate, indicating that the drug induces DNA damage that is repaired by base excision repair. irs1 and VC8 cells defective in homologous recombination (HR) due to inactivation of XRCC2 and BRCA2, respectively, were more sensitive to artesunate than the corresponding wild-type. This was also true for XR-V15B cells defective in nonhomologous end-joining (NHEJ) due to inactivation of Ku80. The data indicate that DSBs induced by artesunate are repaired by the HR and NHEJ pathways. They suggest that DNA damage induced by artesunate contributes to its therapeutic effect against cancer cells.

Microfluidic DNA microarray analysis: A review

Microarray DNA hybridization techniques have been used widely from basic to applied molecular biology research. Generally, in a DNA microarray, different probe DNA molecules are immobilized on a solid support in groups and form an array of microspots. Then, hybridization to the microarray can be performed by applying sample DNA solutions in either the bulk or the microfluidic manner. Because the immobilized probe DNA binds and retains its complementary target DNA, detection is achieved through the read-out of the tagged markers on the sample target molecules. The recent microfluidic hybridization method shows the advantages of less sample usage and reduced incubation time. Here, sample solutions are confined in microfabricated channels and flow through the probe microarray area. The high surface-to-volume ratio in microchannels of nanolitre volume greatly enhanced the sensitivity as obtained with the bulk solution method. To generate nanolitre flows, different techniques have been developed, and this including electrokinetic control, vacuum suction and syringe pumping. The latter two are pressure-driven methods which are more flexible without the need of considering the physicochemical properties of solutions. Recently, centrifugal force is employed to drive liquid movement in microchannels. This method utilizes the body force from the liquid itself and there are no additional solution interface contacts such as from electrodes or syringes and tubing. Centrifugal force driven flow also features the ease of parallel hybridizations. In this review, we will summarize the recent advances in microfluidic microarray hybridization and compare the applications of various flow methods.

Microfluidic lab-on-a-chip for chemical and biological analysis and discovery

Introduction Micromachining Methods Micromachining of Silicon Micromachining of Glass Micromachining of Fused Quartz (or Fused Silica) Micromachining of Polymeric Chips Metal Patterning World-to-Chip Interface Microfluidic Flow Liquid Pumping Methods Microfluidic Flow Control Sample Introduction Electrokinetic Injection Hydrodynamic Injection Other Sample Injection Methods Sample Pre-concentration Sample Stacking Extraction Porous Membrane Other Pre-concentration Methods Separation Gas Chromatography (GC) Capillary Electrophoresis (CE) Chromatographic Separations Coupled Separations Detection Methods Optical Detection Methods Electrochemical (EC) Detection Mass Spectrometry (MS) Other Detection Methods Applications to Cellular/Particle Analysis Retention of Cells and Particles Studies of Cells in a Flow Other Cell Operations Applications to Nucleic Acids Analysis Nucleic Acids Extraction and Purification Nucleic Acids Amplification DNA Hybridization Other Nucleic Acid Applications Applications to Protein Analysis Immunoassay Protein Separation Enzymatic Assays Appendix Problem Sets Introduction Micromachining Microfluidic Flow Sample Introduction Sample Preconcentration Separation Detection Cellular Analysis Nucleic Acid Analysis Protein Analysis References Glossary

Micellar electrokinetic capillary chromatographic separation and fluorescent detection of amino acids derivatized with 4-fluoro-7-nitro-2,1,3-benzoxadiazole.

Another method has been developed for the separation of amino acids (1 min derivatization plus 22 min separation) by micellar electrokinetic capillary chromatography (MECC) with laser-induced fluorescence detection. Interestingly enough, such work has never been performed on essential amino acids derivatized by 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F). Fifteen L-amino acid standards were labelled with NBD-F at 60 degrees C for 1 min, and separated in a buffer system containing 20 mM borate, 25 mM sodium cholate, 10 mM Brij 35 and 2.5% methanol. Methanol was employed to expand the MECC migration time window; whereas Brij 35 was used to improve the fluorescence intensity of amino acid derivatives. This method also indicates that bile salt is effective for MECC separation of ionic analytes. Surprising though, improvements in resolution, sensitivity and speed for amino acids analysis are obtained in this work, which are not initially apparent in just employing another derivatizing reagent. Under optimal conditions, 15 amino acids were separated in a short 22 min analysis time, the shortest ever reported, and detection limits of nanomolar concentration and attomole mass were obtained. Furthermore, RSDs of migration time and peak height are better than 1% and 1.8%, respectively, again the smallest ever reported in the literature.

Optimization of a microfluidic microarray device for the fast discrimination of fungal pathogenic DNA

A microfluidic microarray device, which has been developed for parallel DNA detection, is now further optimized for more rapid and sensitive DNA detection and for the single-base-pair discrimination of two fungal pathogenic PCR products. Two poly(dimethylsiloxane) (PDMS)-based microfluidic chips consist of radial and spiral microchannels in which flexible probe creation and convenient sample delivery have been achieved by centrifugal pumping. The microarray hybridizations occurred at the cross sections within the spiral channels intersecting the preprinted radial probe lines. The centrifugal pumping method showed advantages over the vacuum suction method in terms of parallel solution delivery and less signal variations between replicate samples. The effect of microchannel depth was studied, and hybridization time is predictable at a certain rotation speed. Cy5 dye labels were proved to show much higher hybridization efficiency as well as less photobleaching effect as compared with the fluorescein dye labels used in our previous work. With these optimized conditions, the method was applied to the detection of three fungal pathogenic polymerase chain reaction (PCR) products with a sample load of 0.2 ng (in 1 microl). Furthermore, the single-base-pair discrimination between the PCR products of two relevant Botrytis species (B. cinerea and B. squamosa) was achieved in a duration as short as 3 min.

Transport, retention and fluorescent measurement of single biological cells studied in microfluidic chipsElectronic supplementary information (ESI) available: movie clip of cell retention. See http://www.rsc.org/suppdata/lc/b4/b400770k/

Cellular manipulation and fluorescent measurement were performed on two types of biological cells. First, transport and retention of yeast cells were demonstrated on a glass microfluidic chip, which consists of special U-shaped microstructures. These microstructures have the openings parallel to the liquid flow and weirs perpendicular to the flow. These allow the retention of yeast cells in the U-shaped pocket and drainage of liquid over the weirs. Thereafter, the same chip was used to carry out real-time fluorescent measurement for the cellular changes in single Jurkat T cells. In this case, the Jurkat cells were localized inside the straight portion of a microchannel. Fluorescent imaging on the same, single suspension cell was carried out to study two cellular processes occurring in viable cells, (1) the intracellular conversion of fluorescein diacetate (FDA) to fluorescein; (2) the degradation of an inhibitory protein, IkappaB, as involved in the NF-kappaB signalling pathway. In the former, the increase in fluorescent intensity of single Jurkat T cells (due to fluorescein formation) was measured; whereas in the latter, the decrease in the fluorescent intensity of a single transfected Jurkat cell (due to the degradation of the IkappaB-EGFP fusion protein) was monitored. In addition, we employed a Jurkat cell expressed with IkappaB-EGFP to probe any possible action of an herbal compound, isoliquiritigenin (IQ), on the degradation of IkappaB-EGFP. These examples have demonstrated that Jurkat cells remain viable within microfluidic channels for cellular studies and that the microfluidic chip can facilitate monitoring of cellular changes of biological cells at the single cell level and in the same cell.

An acoustic wave sensor incorporated with a microfluidic chip for analyzing muscle cell contraction.

We report the fabrication of a microfluidic chip or lab-on-a-chip integrated with a thickness-shear mode (TSM) acoustic wave sensor for muscle cell analysis. The sensor, essentially an AT-cut quartz crystal, serves as a detector for recording changes in acoustic wave properties occurring in an attached cardiomyocyte (single heart muscle cell) during its contraction and relaxation. Presumably, the changes resulted from alterations in viscoelastic properties (e.g. stiffness) of the cells. The effects of excitation electrode size, the presence of a microfluidic channel plate, and liquid loading on the sensor were first examined. Thereafter, muscle cell contraction analysis upon chemical stimuli were described. The potential of the chip for screening of cardiovascular drugs is discussed.

A rotating microfluidic array chip for staining assays

We have developed a microfluidic method to construct an array on a circular disk for staining assays. In this method, convenient centrifugal liquid pumping has been achieved within the spiral microchannels by disk rotation or spinning. Moreover, the liquids flowing in spiral channels effectively interact with the along-channel intercepted cell trapping holes. Live cells were encapsulated in wet low-melting point agarose along radial strips on the disk. When embedded in agarose, the cells remained viable to interact with, and respond to, test reagents. This method illustrates the potential use of the circular microfluidic chip to construct the cell array, intended for multi-cell multi-reagent tests.

Gold nanoparticle-assisted single base-pair mismatch discrimination on a microfluidic microarray device

Two simple gold nanoparticle (GNP)-based DNA analysis methods using a microfluidic device are presented. In the first method, probe DNA molecules are immobilized on the surface of a self-assembled submonolayer of GNPs. The hybridization efficiency of the target oligonulceotides was improved due to nanoscale spacing between probe molecules. In the second method, target DNA molecules, oligonulceotides or polymerase chain reaction (PCR) amplicons, are first bound to GNPs and then hybridized to the immobilized probe DNA on a glass slide. With the aid of GNPs, we have successfully discriminated, at room temperature, between two PCR amplicons (derived from closely related fungal pathogens, Botrytis cinerea and Botrytis squamosa) with one base-pair difference. DNA analysis on the microfluidic chip avoids the use of large sample volumes, and only a small amount of oligonucelotides (8 fmol) or PCR products (3 ng), was needed in the experiment. The whole procedure was accomplished at room temperature in 1 h, and apparatus for high temperature stringency was not required.

A proposed mechanism of the influence of gold nanoparticles on DNA hybridization.

A combination of gold nanoparticles (AuNPs) and nucleic acids has been used in biosensing applications. However, there is a poor fundamental understanding of how gold nanoparticle surfaces influence the DNA hybridization process. Here, we measured the rate constants of the hybridization and dehybridization of DNA on gold nanoparticle surfaces to enable the determination of activation parameters using transition state theory. We show that the target bases need to be detached from the gold nanoparticle surfaces before zipping. This causes a shift of the rate-limiting step of hybridization to the mismatch-sensitive zipping step. Furthermore, our results propose that the binding of gold nanoparticles to the single-stranded DNA segments (commonly known as bubbles) in the duplex DNA stabilizes the bubbles and accelerates the dehybridization process. We employ the proposed mechanism of DNA hybridization/dehybridization to explain the ability of 5 nm diameter gold nanoparticles to help discriminate between single base-pair mismatched DNA molecules when performed in a NanoBioArray chip. The mechanistic insight into the DNA-gold nanoparticle hybridization/dehybridization process should lead to the development of new biosensors.