Hsien- Chang

An integrated dielectrophoretic chip for continuous bioparticle filtering, focusing, sorting, trapping, and detecting

Multi-target pathogen detection using heterogeneous medical samples require continuous filtering, sorting, and trapping of debris, bioparticles, and immunocolloids within a diagnostic chip. We present an integrated AC dielectrophoretic (DEP) microfluidic platform based on planar electrodes that form three-dimensional (3D) DEP gates. This platform can continuously perform these tasks with a throughput of 3 muLmin. Mixtures of latex particles, Escherichia coli Nissle, Lactobacillus, and Candida albicans are sorted and concentrated by these 3D DEP gates. Surface enhanced Raman scattering is used as an on-chip detection method on the concentrated bacteria. A processing rate of 500 bacteria was estimated when 100 mul of a heterogeneous colony of 10(7) colony forming units ml was processed in a single pass within 30 min.

The role of silicon in osteoblast-like cell proliferation and apoptosis.

The optimal concentration at which Si induces cell functions has not been fully elucidated. In the present study the effects of Si concentration (0-6 mM) on the biological functions of MG63 cells were investigated. Cell proliferation in the presence of 2 mM Si- and 4 mM Si-containing media progressively increased with culture time, whereas that of 6mM Si treated MG63 cells was significantly (P<0.05) reduced. The unusually high Si concentration (6 mM) induced a significant (P<0.05) increase in the sub-G1 phase of cells from the original 3.60% up to 43.01% after culture for 12 h. In contrast, the other lower Si concentration treated MG63 cells in the sub-G1 phase were in the range 3-5% at all culture time points. 4 mM Si treated MG63 cells, but not 6 mM Si treated MG63 cells, showed remarkably enhanced collagen type I (COL I) gene expression and extracellular signal-regulated kinase (ERK) secretion, which were significantly (P<0.05) higher than those in the control medium. The activation of ERK was also stimulated in MG63 cells by 4 mM Si. Cells cultured in the presence of 4 mM Si were found to have calcium matrix formation on day 7 that was 15-fold greater than that in the control medium. The results obtained in this study may be useful in designing calcium silicate-based materials with optimal biological properties.

Rapid (<5 min) identification of pathogen in human blood by electrokinetic concentration and surface-enhanced Raman spectroscopy.

This study reports a novel microfluidic platform for rapid and long-ranged concentration of rare-pathogen from human blood for subsequent on-chip surface-enhanced Raman spectroscopy (SERS) identification/discrimination of bacteria based on their detected fingerprints. Using a hybrid electrokinetic mechanism, bacteria can be concentrated at the stagnation area on the SERS-active roughened electrode, while blood cells were excluded away from this region at the center of concentric circular electrodes. This electrokinetic approach performs isolation and concentration of bacteria in about three minutes; the density factor is increased approximately a thousand fold in a local area of ~5000 μm2 from a low bacteria concentration of 5 × 103 CFU/ml. Besides, three genera of bacteria, S. aureus, E. coli, and P. aeruginosa that are found in most of the isolated infections in bacteremia were successfully identified in less than one minute on-chip without the use of any antibody/chemical immobilization and reaction processes.

A dielectrophoretic chip with a roughened metal surface for on-chip surface-enhanced Raman scattering analysis of bacteria

We present an analysis of the results of in situ surface-enhanced Raman scattering (SERS) of bacteria using a microfluidic chip capable of continuously sorting and concentrating bacteria via three-dimensional dielectrophoresis (DEP). Microchannels were made by sandwiching DEP microelectrodes between two glass slides. Avoiding the use of a metal nanoparticle suspension, a roughened metal surface is integrated into the DEP-based microfluidic chip for on-chip SERS detection of bacteria. On the upper surface of the slide, a roughened metal shelter was settled in front of the DEP concentrator to enhance Raman scattering. Similarly, an electrode-patterned bottom layer fabricated on a thin cover-slip was used to reduce fluorescence noise from the glass substrate. Gram positive (Staphylococcus aureus) and Gram negative (Pseudomonas aeruginosa) bacteria were effectively distinguished in the SERS spectral data. Staphylococcus aureus (concentration of 10(6) CFUml) was continuously separated and concentrated via DEP out of a sample of blood cells. At a flow rate of 1 mulmin, the bacteria were highly concentrated at the roughened surface and ready for on-chip SERS analysis within 3 min. The SERS data were successfully amplified by one order of magnitude and analyzed within a few minutes, resulting in the detection of signature peaks of the respective bacteria.

Effects of altering the Si/Ca molar ratio of a calcium silicate cement on in vitro cell attachment

AIM To examine the effects of altering the Si/Ca molar ratio (6 : 4, 5 : 5, and 4 : 6) of a quick-setting calcium silicate cement on in vitro cell attachment. METHODOLOGY Working time and setting time of three different calcium silicate cements were measured. Alamar Blue was used for real-time and repeated monitoring of cell attachment and proliferation. The Si and Ca ion concentrations of the cell culture medium in the presence of three different calcium silicate cements seeded with MG63 cells were measured. Kinetic immunofluorescent staining of MG63 cells was performed during cell attachment and spreading. Reverse transcription-polymerase chain reaction was employed to determine gene expression in MG63 cells cultured on the cements. One-way analysis of variance was used to evaluate the significance of the differences between the mean values. RESULTS The working time (4-7 min) and setting time (17-24 min) of the cements were shortened with an increase in the Ca content of the calcium silicate powders after mixing the powder with water. In contrast, the higher the Si content in the cement, the more the MG63 cells attached to the cement at all culture time-points, accompanying by the formation of more obvious actin stress fibres. Cell proliferation and differentiation increased significantly (P < 0.05) with an increase in the Si content of the calcium silicate cements. Si ion concentration of the culture medium increased significantly (P < 0.05) with increasing cement Si content and culture time-points. CONCLUSIONS The higher Si content cement enhanced the higher expression of cell attachment, proliferation and differentiation as compared to the lower Si content cement.

Fruiting body of niuchangchih (Antrodia camphorata) protects livers against chronic alcohol consumption damage.

An alcoholic fatty liver disease was induced by drinking water containing 20% (w/w) alcohol. Therapeutic groups were orally administrated dosages of 0.25 g silymarin/kg body weight (BW) and a low dosage of Niuchangchih (Antrodia camphorata) (0.025 g/kg BW) and a high dosage of Niuchangchih (0.1 g/kg BW) per day. Niuchangchih, especially at the high dosage, not only showed a hypercholesterolemic effect (p < 0.05) but also reduced (p < 0.05) hepatic lipids in alcohol-fed rats. Those beneficial effects could be partially attributed to higher (p < 0.05) fecal cholesterol and bile acid outputs, as well as downregulations (p < 0.05) of 3-hydroxy-3-methylglutaryl-CoA reductase, sterol regulatory element-binding protein-1c, acetyl-CoA carboxylase, fatty acid synthase, and malic enzyme gene expressions; meanwhile, there was an upregulation of low-density lipoprotein receptor and peroxisome proliferator-activated alpha gene expression. Besides, Niuchangchih also enhanced (p < 0.05) the liver glutathione, Trolox equivalent antioxidant capacity, and activities of superoxide dismutase, catalase, and glutathione peroxidase and decreased the liver malondialdehyde content, which also partially contributed to the lowered (p < 0.05) serum aspartate aminotransferase levels and no observed lesion in the histological examination of alcohol-fed rats.

The ER Ca2+ sensor STIM1 regulates actomyosin contractility of migratory cells

Summary Stromal interaction molecule 1 (STIM1) is an endoplasmic reticulum (ER) Ca2+ sensor that triggers the store-operated Ca2+ entry (SOCE). The clinical relevance of STIM1 has been highlighted in breast and cervical cancer, but the molecular mechanism by which STIM1 promotes cancer progression remains unclear. This study explores the regulatory mechanisms by which STIM1-dependent Ca2+ signaling controls cancer cell migration. Three different SOCE inhibitors, SKF96365, 2-APB and YM-58483, significantly inhibited cervical cancer cell migration to a similar extent to that of STIM1 silencing. In contrast, STIM1 overexpression significantly enhanced cervical cancer cell migration. Live cell confocal images and three-dimensional tomograms showed that STIM1 formed aggregates and translocated towards the plasma membranes of migratory cells, and this was accompanied by increasing cytosolic Ca2+ spikes. STIM1 silencing also inhibited the recruitment and association of active focal adhesion kinase (pTyr397-FAK) and talin at focal adhesions, indicating the blockade of force transduction from integrin signaling. Epidermal growth factor-induced phosphorylation of myosin II regulatory light chains was abolished by STIM1 knockdown and SOCE inhibition. Dual immunostaining of activated myosin II (pSer19-MLC) and actin revealed that actomyosin formation depended on STIM1-mediated Ca2+ entry. Most importantly, STIM1 expression levels as well as SOCE activity controlled the generation of cell contractile force, as measured by the microfabricated post-array-detector system. These results highlight the unique role of STIM1-dependent Ca2+ signaling in controlling cell migration by the regulation of actomyosin reorganization in conjunction with enhanced contractile forces.

Screening of Antibiotic Susceptibility to β-Lactam-Induced Elongation of Gram-Negative Bacteria Based on Dielectrophoresis

We demonstrate a rapid antibiotic susceptibility test (AST) based on the changes in dielectrophoretic (DEP) behaviors related to the β-lactam-induced elongation of Gram-negative bacteria (GNB) on a quadruple electrode array (QEA). The minimum inhibitory concentration (MIC) can be determined within 2 h by observing the changes in the positive-DEP frequency (pdf) and cell length of GNB under the cefazolin (CEZ) treatment. Escherichia coli and Klebsiella pneumoniae and the CEZ are used as the sample bacteria and antibiotic respectively. The bacteria became filamentous due to the inhibition of cell wall synthesis and cell division and cell lysis occurred for the higher antibiotic dose. According to the results, the pdfs of wild type bacteria decrease to hundreds of kHz and the cell length is more than 10 μm when the bacterial growth is inhibited by the CEZ treatment. In addition, the growth of wild type bacteria and drug resistant bacteria differ significantly. There is an obvious decrease in the number of wild type bacteria but not in the number of drug resistant bacteria. Thus, the drug resistance of GNB to β-lactam antibiotics can be rapidly assessed. Furthermore, the MIC determined using dielectrophoresis-based AST (d-AST) was consistent with the results of the broth dilution method. Utilizing this approach could reduce the time needed for bacteria growth from days to hours, help physicians to administer appropriate antibiotic dosages, and reduce the possibility of the occurrence of multidrug resistant (MDR) bacteria.

A filter-like AuNPs@MS SERS substrate for Staphylococcus aureus detection

An accurate, highly sensitive and rapid identification assay of cells is extremely important in areas such as medical diagnosis, biological research, and environmental monitoring. Laboratory examinations of clinical isolates require time-consuming and complex processes to identify the colony count, with approximately 10(6)-10(8) cells needed for the characterization of strains. In the present study, a highly sensitive SERS filter-like substrate is prepared with AuNPs embedded in mesoporous silica (denoted as AuNPs@MS) synthesized by a simple one-spot method, and an example of its use for the filtration and concentration of analytes from aqueous samples is reported. In an application for Staphylococcus aureus SERS discrimination, the results show that the target cells can be concentrated on the filter-like AuNPs@MS substrates within a few seconds, with much better reproducibility with regard to the SERS spectra that are obtained. The experimental findings suggest that the AuNPs@MS substrate supports much higher intensity with more distinguishable peaks compared to Au/Cr-coated substrate, and the reproducibility is also significantly improved. The substrates investigated in this study generated 900 times more SERS signals at a concentration of 10(6)CFU/mL in the detection of S. aureus on mesoporous silica (Au wt%=0) when using AuNPs@MS with 16 wt% AuNPs. The limitation of this filter-like SERS substrate can be applicable for small volume samples (few to hundred microliter).

Deregulation of AP-1 proteins in collagen gel-induced epithelial cell apoptosis mediated by low substratum rigidity.

In this study, we established that collagen gel, but not collagen gel coating, induced apoptosis exclusively in epithelial cell lines, which indicated that low substratum rigidity might trigger cell apoptosis. To confirm this, we used collagen gels with different rigidities due to cross-linking or physical disruption of collagen fibrils caused by sonication. We found that collagen gel-induced apoptosis was inversely correlated with substratum rigidity. Low substratum rigidity collagen gel-induced apoptosis was neither prevented by Bcl-2 overexpression nor preceded by mitochondrial release of cytochrome c. This suggested that the mitochondrial pathway was not involved in low substratum rigidity-induced apoptosis. Low substratum rigidity activated c-Jun N-terminal kinase (JNK) within 4 h, but it also rapidly down-regulated c-Jun within 1 h and triggered persistent aberrant expression of c-Fos for at least 24 h. Either reduced c-Jun expression or c-Fos overexpression induced apoptosis in several epithelial cells. Inhibiting low substratum rigidity-induced JNK activation prevented aberrant c-Fos expression but only partially blocked low substratum rigidity-induced apoptosis. Taking these results together, we conclude that low substratum rigidity collagen gel induced apoptosis in epithelial cells and that deregulated AP-1 proteins mediated that apoptosis, at least in part.