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Three-dimensional (3D) paper-based microfluidics, which is featured with high performance and speedy determination, promise to carry out multistep sample pretreatment and orderly chemical reaction, which have been used for medical diagnosis, cell culture, environment determination, and so on with broad market prospect. However, there are some drawbacks in the existing fabrication methods for 3D paper-based microfluidics, such as, cumbersome and time-consuming device assembly; expensive and difficult process for manufacture; contamination caused by organic reagents from their fabrication process. Here, we present a simple printing-bookbinding method for mass fabricating 3D paper-based microfluidics. This approach involves two main steps: (i) wax-printing, (ii) bookbinding. We tested the delivery capability, diffusion rate, homogeneity and demonstrated the applicability of the device to chemical analysis by nitrite colorimetric assays. The described method is rapid (<30 s), cheap, easy to manipulate, and compatible with the flat stitching method that is common in a print house, making itself an ideal scheme for large-scale production of 3D paper-based microfluidics., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

We developed an on-chip DNA analysis method, in which free-solution transient isotachophoresis (FstITP) were coupled with CGE. Using chloride ions in the sample matrix and HEPES in the background electrolyte, respectively, as the leading and terminating ions, dsDNAs were isotachophoretically preconcentrated in free-solution and then separated in sieving polymer. The coupling of FstITP and CGE enabled higher separation efficiency due to higher preconcentration rate in free-solution. The FstITP-CGE analysis offered adjustable signal intensities by varying sample injection time. With the maximum sample loading volume, the LOD of the FstITP-CGE analysis was determined to be 0.24 ng/mL by confocal laser-induced fluorescence detection. The FstITP-CGE method is simple, robust and flexible, thus well suited to the analysis of highly saline DNA samples at different concentration level.

We present a sensitive, simple and robust on-chip transient ITP (TITP) method for the analysis of polymerase chain reaction (PCR) samples. This TITP analysis was performed on a poly (methyl methacrylate) microchip with cross structure. The PCR sample was injected into a 4 mm free-solution buffer channel to increase sample loading. When applied separation voltage, the sample ions were stacked by TITP using chloride ions in the sample matrix as the leading ions and HEPES in the background electrolyte as the terminating ions. The stacked sample plug was then separated by zone electrophoresis (ZE) in hydroxypropylmethyl cellulose polymer. The whole operation was carried out in single background electrolyte. Sample injection, preconcentration / separation were performed continuously with the sequential switching of voltage. By injection of the sample into the free-solution buffer channel, sampling bias was avoided and the injection time was reduced. With this TITP method, the detection sensitivity was improved without loss of resolution, a 20-fold signal enhancement was achieved and the average limit of detection was estimated to be 0.24 pg/microL (S/N = 3). The TITP method is simple, fast and sensitive, thus is well suited to direct analysis of the highly saline PCR samples.

We exploited a PDMS-quartz hybrid microchip with a thin PDMS membrane on the concave detection window for UV absorbance detection. The thickness of the PDMS membrane is about 100 mum, with high UV transmittance. As compared to a PDMS-quartz hybrid chip with a common detection window, the proposed one exhibited over an one order of magnitude sensitivity enhancement, and an about two orders of magnitude S/N increase for gastrodin (p-hydroxymethylphenyl-beta-D-glucopyranoside). In addition, the limit of the detection wavelength has been extended from 240 to 210 nm, which is otherwise impossible for a traditional PDMS-quartz hybrid microchip. This kind of microchip has the potential for a large range of applications in an integrated microfluidic system with UV detection.

Microfabricated capillary array electrophoresis (micro-CAE) was applied to study the interaction between minor groove binder netropsin and a non-selfcomplementary 12 mer double stranded oligodeoxynucleotide: d(CCCCTATACCGC).d(GCGGTATAGGGG). ESI-MS was used to provide an independent verification of the microchip electrophoresis derived data. Simultaneous parallel quantitative assay of multiple samples was performed in a single run (<50 s) on the self-developed micro-CAE device. The binding constant and stoichiometry calculated from Scatchard plot were (2.88 +/- 0.23)x10(5) M(-1) and 1:1, respectively. The values showed a good quantitative agreement with the results determined by ESI-MS and those using other methods reported in the literature.

A quartz microchip integrated isotachophoretic (ITP) preconcentration with zone electrophoresis (ZE) separation was fabricated using a novel multi-point pressure method featured in normal temperature and lower pressure during bonding process. ITP followed by subsequential ZE of two flavonoids, quercetin and isorhamnetin on the microchip was performed consecutively on the homemade microfluidic workstation with UV detection, resulting in a decreased detectable concentration of 32-fold, compared to the ZE mode only, and their detection limits decreased down to 0.2 microg/mL and 1.2 microg/mL, respectively.

The interactions between granulocyte colony-stimulating factor (G-CSF) and dextran sulfate (DS) with different chain lengths and sulfate contents were studied by capillary zone electrophoresis. It was found that DS with a molecular mass of 500 kDa (DS500) could bind to G-CSF and the binding constant and binding sites were determined using Scatchard plot to be 1.17 x 10(6) M(-1) and 3, respectively. DS with a molecular mass of 40 kDa also had the affinity to G-CSF and the binding constant and binding sites were 1.01 x 10(6) M(-1) and 8, respectively. However, DS with a molecular mass of 8 kDa and the non-sulfated saccharide, dextran, had no affinity to G-CSF. The results indicate that the interactions between G-CSF and DS are dependent on the chain lengths and sulfate contents of the saccharides. In addition, the effects of G-CSF-binding DS on a G-CSF-dependent leukemia cell line were investigated using biological methods. Results show that DS500 plus G-CSF has potential therapeutic effect on cancers because their combination could inhibit the growth and induce the differentiation of the leukemia cells.

Angiotensinogen (AGT) gene has been regarded as one of the candidate genes for essential hypertension. In our study, the role of AGT gene as a putatively predisposing gene for hypertension was evaluated by genotyping a A (-6) G polymorphism in the core promoter region in 123 patients with essential hypertension and 103 healthy controls. A microchip electrophoresis method coupled with polymorphism chain reaction (PCR)-restriction fragment length polymorphism (RFLP) assay was used for genotyping the A (-6) G single-nucleotide polymorphism. The separation and detection of the digested PCR amplicons were completed just in 280 s or less. The genotype frequency fulfilled the criteria of the Hardy-Weinbery equilibrium (X2 = 3.067, P > 0.05). The results showed a higher frequency of the -6 A allele (0.70) in the normotensive subjects, which is higher than those reported in Germany (0.47) and Czech (0.40) populations, but similar to that found in Japanese populations (0.73). The frequencies of genotype AA, AG, and GG were 0.46, 0.49, and 0.05 in hypertensive subjects, and 0.44, 0.53, and 0.03 in control subjects. There is no significant difference in the distributions of the genotype and allele between the two groups (X2 = 0.88, P > 0.05; X2 = 0.024, P > 0.05). These findings differ from some of the results obtained in other ethnic groups, indicating the potential importance of ethnic origin in the assessment of genetic risk identifiers for a complex disease.

A poly(dimethylsiloxane) (PDMS) microfluidic chip surface was modified by multilayer-adsorbed and heat-immobilized poly(vinyl alcohol) (PVA) after oxygen plasma treatment. The reflection absorption infrared spectrum (RAIRS) showed that 88% hydrolyzed PVA adsorbed more strongly than 100% hydrolyzed one on the oxygen plasma-pretreated PDMS surface, and they all had little adsorption on original PDMS surface. Repeating the coating procedure three times was found to produce the most robust and effective coating. PVA coating converted the original PDMS surface from a hydrophobic one into a hydrophilic surface, and suppressed electroosmotic flow (EOF) in the range of pH 3-11. More than 1,000,000 plates/m and baseline resolution were obtained for separation of fluorescently labeled basic proteins (lysozyme, ribonuclease B). Fluorescently labeled acidic proteins (bovine serum albumin, beta-lactoglobulin) and fragments of dsDNA phiX174 RF/HaeIII were also separated satisfactorily in the three-layer 88% PVA-coated PDMS microchip. Good separation of basic proteins was obtained for about 70 consecutive runs.

We have developed a new experimental system based on a microfluidic chip to determine severe acute respiratory syndrome coronavirus (SARS-CoV). The system includes a laser-induced fluorescence microfluidic chip analyzer, a glass microchip for both polymerase chain reaction (PCR) and capillary electrophoresis, a chip thermal cycler based on dual Peltier thermoelectric elements, a reverse transcription-polymerase chain reaction (RT-PCR) SARS diagnostic kit, and a DNA electrophoretic sizing kit. The system allows efficient cDNA amplification of SARS-CoV followed by electrophoretic sizing and detection on the same chip. To enhance the reliability of RT-PCR on SARS-CoV detection, duplex PCR was developed on the microchip. The assay was carried out on a home-made microfluidic chip system. The positive and the negative control were cDNA fragments of SARS-CoV and parainfluenza virus, respectively. The test results showed that 17 positive samples were obtained among 18 samples of nasopharyngeal swabs from clinically diagnosed SARS patients. However, 12 positive results from the same 18 samples were obtained by the conventional RT-PCR with agarose gel electrophoresis detection. The SARS virus species can be analyzed with high positive rate and rapidity on the microfluidic chip system.