A valveless rotary microfluidic device for multiplex point mutation identification based on ligation-rolling circle amplification.

Genetic variations such as single nucleotide polymorphism (SNP) and point mutations are important biomarkers to monitor disease prognosis and diagnosis. In this study, we developed a novel rotary microfluidic device which can perform multiplex SNP typing on the mutation sites of TP53 genes. The microdevice consists of three glass layers: a channel wafer, a Ti/Pt electrode-patterned resistance temperature detector (RTD) wafer, and a rotary plate in which twelve reaction chambers were fabricated. A series of sample injection, ligation-rolling circle amplification (L-RCA) reaction, and fluorescence detection of the resultant amplicons could be executed by rotating the top rotary plate, identifying five mutation points related with cancer prognosis. The use of the rotary plate eliminates the necessity of microvalves and micropumps to control the microfluidic flow in the channel, simplifying the chip design and chip operation for multiplex SNP detection. The proposed microdevice provides an advanced genetic analysis platform in terms of multiplexity, simplicity, and portability in the fields of biomedical diagnostics.

A fully integrated microdevice for biobarcode assay based biological agent detection.

An integrated microdevice, consisting of a micropump, a passive mixer, a magnetic separation chamber, and a microcapillary electrophoretic channel, was constructed for biobarcode assay based multiplex biological agent detection in a sample-to-answer-out manner within 30 min with high sensitivity.

Combination of biobarcode assay with on-chip capillary electrophoresis for ultrasensitive and multiplex biological agent detection.

Early diagnosis of biological agents is of paramount importance to prevent the casualties and fatal disease in human during bioterrorism or biological warfare. In this study, we reported an efficient and sensitive multiplex biological agent detection method based on the DNA biobarcode assay and the micro-capillary electrophoresis (µCE) technology. Monoplex as well as multiplex pathogen identification was performed using five targets including Bacillus anthracis, Francisella tularensis, Yersinia pestis, Vaccinia virus and Botulinum toxin A. Through the DNA biobarcode assay process, the magnetic microparticle-pathogen-polystyrene microbead complexes were formed, and the FAM labeled single stranded barcode DNA could be released from the complexes upon denaturation. Different lengths of a barcode DNA were designed to designate each pathogen, so that the specific peak elution time in the capillary electrophoresis on a chip allows us to distinguish the target with high accuracy within 3 min. We improved the assignment accuracy of the peak in the electropherogram by adding two bracket ladders. Owing to the abundant amount of barcode DNAs, the presence of B. anthracis, F. tularensis, Y. pestis, Vaccinia virus was confirmed with a limit of detection of 50CFU/mL, while Botulinum toxin A was analyzed even at a concentration of 12.5 ag/mL. Multiple pathogen detection was also successfully conducted in a phosphate buffered saline (PBS) as well as a serum medium with background of other pathogens. Thus, our analytical platform based on the biobarcode assay and on-chip CE analysis provides rapid, sensitive, multiplex, and accurate biological agent identification.

Highly sensitive detection of cancer cells based on the DNA barcode assay and microcapillary electrophoretic analysis.

Considering rarity of circulating tumor cells (CTCs) in human blood, the development of highly sensitive detection techniques for cancer cells is crucial for prediction, diagnosis, and prognosis of cancers. In this study, we propose an advanced cellular detection method by combining a biobarcode assay and microcapillary electrophoresis (µCE) technology. While the DNA biobarcode assay can provide ultrasensitive and multiplex detection platforms, the µCE chip can analyze barcode DNAs with high speed and accuracy according to the DNA size. We designed the barcode DNA size as 20 bp for indicating the expression of epithelial cell adhesion molecules (EpCAM) biomarkers and 30 bp for assigning CDX2 expression which is specific for colorectal cancer cells with addition to two bracket ladders (15 and 45 bp). Using MCF-7 (breast cancer) and SW620 (colorectal cancer) as models, we conducted a biobarcode assay and analyzed the resultant biobarcode DNA on the µCE chip. We could detect the 20 bp CE peak in the electropherogram even with ten MCF-7 and SW620 cells in a volume of 200 µL, thereby demonstrating the highly sensitive detection of cancer cells. We furthermore identified the type of colorectal cancer by observing two positive peaks (20 bp for EpCAM and 30 bp for CDX2) in the µCE analysis.

An integrated allele-specific polymerase chain reaction-microarray chip for multiplex single nucleotide polymorphism typing.

An integrated allele-specific polymerase chain reaction (AS PCR) and microarray chip has been developed for multiplex single nucleotide polymorphism (SNP) typing on a portable genetic analyzer instrumentation. We applied the integrated PCR-microarray system for on-site Hanwoo (Korean indigenous beef cattle) identification. Eleven sets of primers were designed, among which ten sets of primers targeted ten SNP loci to discriminate Hanwoo from the imported beef cattle and one primer set was used as a positive PCR control. The AS PCR for multiplex SNP typing was conducted on a glass-based microchip consisting of four layers: a microchannel plate for microfluidic control, a Pt-electrode plate for a resistance temperature detector (RTD), a poly(dimethylsiloxane) (PDMS) membrane and a manifold glass for micropump and microvalve function. The resultant AS PCR products were mixed with a hybridization buffer in a micromixer channel through the micropumping operation, and then the microarray assay was performed in the downstream process. Eleven duplicate probes were spotted in a glass slide, which was connected at the end of the micromixer channel unit. When the mixed solution was injected into the disposable microarray chip, pneumatically actuated micropumping was executed to speed up the hybridization process by inducing the convective flow. The fluorescence signals on each spot were monitored by a miniaturized fluorescence scanner, and the Hanwoo was verified by detecting the number of fluorescent spots with three or fewer among eleven. An integrated portable PCR-microarray genetic analysis microsystem was first demonstrated for rapid, accurate, and on-site multiplex SNP typing to differentiate animal species.

Three-dimensional graphene oxide nanostructure for fast and efficient water-soluble dye removal.

In this study, we demonstrated the potential of graphene nanomaterials as environmental pollutant adsorbents by utilizing the characteristics of ultralarge surface area and strong π-π interaction on the surface. We generated a three-dimensional (3D) graphene oxide sponge (GO sponge) from a GO suspension through a simple centrifugal vacuum evaporation method, and used them to remove both the methylene blue (MB) and methyl violet (MV) dyes which are main contaminants from the dye manufacturing and textile finishing. The efficiency and speed of dye adsorption on a GO sponge was investigated under various parameters such as contact time, stirring speed, temperature, and pH. The adsorption process shows that 99.1% of MB and 98.8% of MV have been removed and the equilibrium status has been reached in 2 min. The 3D GO sponge displays adsorption capacity as high as 397 and 467 mg g(-1) for MB and MV dye, respectively, and the kinetic data reveal that the adsorption process of MB and MV dyes is well-matched with the pseudo second-order model. The MB and MV adsorption on the 3D GO sponge involved in endothermic chemical adsorption through the strong π-π stacking and anion-cation interaction with the activation energy of 50.3 and 70.9 kJ mol(-1), respectively. The 3D GO sponge has demonstrated its high capability as an organic dye scavenger with high speed and efficiency.