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Biological & Pharmaceutical Bulletin 2013Assessing microbiological quality assurance by monitoring bacteria in various sources of freshwater used for human consumption, recreation, and food preparation is...
Assessing microbiological quality assurance by monitoring bacteria in various sources of freshwater used for human consumption, recreation, and food preparation is important for a healthy life. Bacterial number and their community structure in freshwater should be determined as quickly as possible, and "real-time" and "on-site" microbiological methods are required. In this study, we examined the protocol for microchip-based terminal restriction fragment length polymorphism (T-RFLP) analysis, which uses microchip electrophoresis for rapid microbial community analysis. The availability of microchip-based T-RFLP was compared with conventional T-RFLP analysis, which uses a capillary electrophoresis system, with freshwater samples (spring water, river water, groundwater, and hydroponics solution). The detection limit of targeted bacteria by on-chip T-RFLP analysis was 1% (10(3) cells/mL). The fragment sizes determined by the two analysis methods were highly correlated (r(2)=0.98). On-chip T-RFLP analysis was completed within 15 min. T-RFLP profiles of nine hydroponics solution samples were analyzed by multidimensional scaling. Considerable changes and stability in bacterial community structure during hydroponic culture were detected by both analyses. These results show that on-chip T-RFLP analysis can monitor changes in bacterial community structure, as well as conventional T-RFLP analysis. The present results indicate that on-chip T-RFLP analysis is an effective tool for rapid and "on-site" bacterial community profiling in freshwater environments, as well as freshwater used for medical and industrial purposes.
Topics: Bacillus cereus; DNA, Bacterial; Drinking Water; Environmental Monitoring; Fresh Water; Groundwater; Microchip Analytical Procedures; Polymorphism, Restriction Fragment Length; Pseudomonas aeruginosa; Water Pollutants
PubMed: 23902975
DOI: 10.1248/bpb.b13-00118 -
Lab on a Chip Mar 2012We present a microfluidic cell-culture chip that enables trapping, cultivation and release of selected individual cells. The chip is fabricated by a simple hybrid...
We present a microfluidic cell-culture chip that enables trapping, cultivation and release of selected individual cells. The chip is fabricated by a simple hybrid glass-SU-8-PDMS approach, which produces a completely transparent microfluidic system amenable to optical inspection. Single cells are trapped in a microfluidic channel using mild suction at defined cell immobilization orifices, where they are cultivated under controlled environmental conditions. Cells of interest can be individually and independently released for further downstream analysis by applying a negative dielectrophoretic force via the respective electrodes located at each immobilization site. The combination of hydrodynamic cell-trapping and dielectrophoretic methods for cell releasing enables highly versatile single-cell manipulation in an array-based format. Computational fluid dynamics simulations were performed to estimate the properties of the system during cell trapping and releasing. Polystyrene beads and yeast cells have been used to investigate and characterize the different functions and to demonstrate biological compatibility and viability of the platform for single-cell applications in research areas such as systems biology.
Topics: Cells, Immobilized; Electrophoresis, Microchip; Microfluidic Analytical Techniques; Molecular Dynamics Simulation; Nanospheres; Polystyrenes; Yeasts
PubMed: 22193373
DOI: 10.1039/c2lc20911j -
Electrophoresis Dec 2011The high structural variation of glycan derived from glycoconjugates, which substantially increases with the molecular size of a protein, contributes to the complexity... (Review)
Review
The high structural variation of glycan derived from glycoconjugates, which substantially increases with the molecular size of a protein, contributes to the complexity of glycosylation patterns commonly associated with glycoconjugates. In the case of glycoproteins, such variation originates from the multiple glycosylation sites of proteins and the number of glycan structures associated with each site (microheterogeneity). The ability to comprehensively characterize highly complex mixture of glycans has been analytically stimulating and challenging. Although the most powerful MS and MS/MS techniques are capable of providing a wealth of structural information, they are still not able to readily identify isomeric glycan structures without high-order MS/MS (MS(n) ). The analysis of isomeric glycan structures has been attained using several separation methods, including high-pH anion-exchange chromatography, hydrophilic interaction chromatography and GC. However, CE and microfluidics CE (MCE) offer high separation efficiency and resolutions, allowing the separation of closely related glycan structures. Therefore, interfacing CE and MCE to MS is a powerful analytical approach, allowing potentially comprehensive and sensitive analysis of complex glycan samples. This review describes and discusses the utility of different CE and MCE approaches in the structural characterization of glycoproteins and the feasibility of interfacing these approaches to MS.
Topics: Animals; Electrophoresis, Capillary; Electrophoresis, Microchip; Glycoproteins; Humans; Mass Spectrometry; Polysaccharides
PubMed: 22180203
DOI: 10.1002/elps.201100342 -
Lab on a Chip Aug 2011We describe an integrated microfluidic device (μFlowFISH) capable of performing 16S rRNA fluorescence in situ hybridization (FISH) followed by flow cytometric detection...
We describe an integrated microfluidic device (μFlowFISH) capable of performing 16S rRNA fluorescence in situ hybridization (FISH) followed by flow cytometric detection for identifying bacteria in natural microbial communities. The device was used for detection of species involved in bioremediation of Cr(vi) and other metals in groundwater samples from a highly-contaminated environmental site (Hanford, WA, USA). The μFlowFISH seamlessly integrates two components: a hybridization chamber formed between two photopolymerized membranes, where cells and probes are electrophoretically loaded, incubated and washed, and a downstream cross structure for electrokinetically focusing cells into a single-file flow for flow cytometry analysis. The device is capable of analyzing a wide variety of bacteria including aerobic, facultative and anaerobic bacteria and was initially tested and validated using cultured microbes, including Escherichia coli, as well as two strains isolated from Hanford site: Desulfovibrio vulgaris strain RCH1, and Pseudomonas sp.strain RCH2 that are involved in Cr(vi) reduction and immobilization. Combined labeling and detection efficiencies of 74-97% were observed in experiments with simple mixtures of cultured cells, confirming specific labeling. Results obtained were in excellent agreement with those obtained by conventional flow cytometry confirming the accuracy of μFlowFISH. Finally, the device was used for analyzing water samples collected on different dates from the Hanford site. We were able to monitor the numbers of Pseudomonas sp. with only 100-200 cells loaded into the microchip. The μFlowFISH approach provides an automated platform for quantitative detection of microbial cells from complex samples, and is ideally suited for analysis of precious samples with low cell numbers such as those found at extreme environmental niches, bioremediation sites, and the human microbiome.
Topics: Bacteria; Flow Cytometry; In Situ Hybridization, Fluorescence; Microfluidic Analytical Techniques; RNA, Ribosomal, 16S; Water Microbiology
PubMed: 21755095
DOI: 10.1039/c1lc20151d -
International Journal of Molecular... 2011This study presents an experimental and numerical investigation on the use of high-resolution injection techniques to deliver sample plugs within a capillary...
This study presents an experimental and numerical investigation on the use of high-resolution injection techniques to deliver sample plugs within a capillary electrophoresis (CE) microchip. The CE microfluidic device was integrated into a U-shaped injection system and an expansion chamber located at the inlet of the separation channel, which can miniize the sample leakage effect and deliver a high-quality sample plug into the separation channel so that the detection performance of the device is enhanced. The proposed 45° U-shaped injection system was investigated using a sample of Rhodamine B dye. Meanwhile, the analysis of the current CE microfluidic chip was studied by considering the separation of Hae III digested ϕx-174 DNA samples. The experimental and numerical results indicate that the included 45° U-shaped injector completely eliminates the sample leakage and an expansion separation channel with an expansion ratio of 2.5 delivers a sample plug with a perfect detection shape and highest concentration intensity, hence enabling an optimal injection and separation performance.
Topics: DNA; Deoxyribonucleases, Type II Site-Specific; Electrophoresis, Microchip; Microfluidic Analytical Techniques
PubMed: 21747696
DOI: 10.3390/ijms12063594 -
Proceedings of the National Academy of... Apr 2011The metastatic invasion of cancer cells from primary tumors to distant ecological niches, rather than the primary tumors, is the cause of much cancer mortality [Zhang...
The metastatic invasion of cancer cells from primary tumors to distant ecological niches, rather than the primary tumors, is the cause of much cancer mortality [Zhang QB, et al. (2010) Int J Cancer 126:2534-2541; Chambers AF, Goss PE (2008) Breast Cancer Res 10:114]. Metastasis is a three-dimensional invasion process where cells spread from their site of origin and colonize distant microenvironmental niches. It is critical to be able to assess quantitatively the metastatic potential of cancer cells [Harma V, et al. (2010) PLoS ONE 5:e10431]. We have constructed a microfabricated chip with a three-dimensional topology consisting of lowlands and isolated square highlands (Tepuis), which stand hundreds of microns above the lowlands, in order to assess cancer cell metastatic potential as they invade the highlands. As a test case, the invasive ascents of the Tepui by highly metastatic PC-3 and noninvasive LNCaP prostate cancer cells were used. The vertical ascent by prostate cancer cells from the lowlands to the tops of the Tepui was imaged using confocal microscopy and used as a measure of the relative invasiveness. The less-metastatic cells (LNCaP) never populated all available tops, leaving about 15% of them unoccupied, whereas the more metastatic PC-3 cells occupied all available Tepuis. We argue that this distinct difference in invasiveness is due to contact inhibition.
Topics: Cell Line, Tumor; Humans; Male; Microchip Analytical Procedures; Neoplasm Invasiveness; Neoplasm Metastasis; Prostatic Neoplasms
PubMed: 21474778
DOI: 10.1073/pnas.1102808108 -
ISRN Veterinary Science 2011The etiology of feline injection-site sarcomas remains obscure. Sarcomas and other tumors are known to be associated with viral infections in humans and other animals,...
The etiology of feline injection-site sarcomas remains obscure. Sarcomas and other tumors are known to be associated with viral infections in humans and other animals, including cats. However, the available evidence suggests that this is not the case with feline injection-site sarcomas. These tumors have more in common with sarcomas noted in experimental studies with laboratory animals where foreign materials such as glass, plastics, and metal are the causal agent. Tumors arising with these agents are associated with chronic inflammation at the injection or implantation sites. Similar tumors have been observed, albeit infrequently, at microchip implantation sites, and these also are associated with chronic inflammation. It is suggested that injection-site sarcomas in cats may arise at the administration site as a result of chronic inflammation, possibly provoked by adjuvant materials, with subsequent DNA damage, cellular transformation, and clonal expansion. However, more fundamental research is required to elucidate the mechanisms involved.
PubMed: 23738095
DOI: 10.5402/2011/210982 -
Analytical Sciences : the International... 2010In this paper, we describe DNA detection experiments using our two original technologies, power-free microchip and laminar flow-assisted dendritic amplification (LFDA),...
In this paper, we describe DNA detection experiments using our two original technologies, power-free microchip and laminar flow-assisted dendritic amplification (LFDA), which were previously applied to immunoassays. A microchip was fabricated by combining a poly(dimethylsiloxane) (PDMS) part having microchannel patterns and a glass plate modified with probe DNA. We carried out two kinds of experiments: the detection of 21-base biotinylated target DNA and the detection of single-nucleotide polymorphism (SNP) in 56-base unlabeled target DNA by sandwich hybridization with biotinylated probe DNA. For both of the experiments, the necessary solutions were injected into microchannels not by an external power source, but by air dissolution into the PDMS part. After a hybridization reaction, the LFDA was started by injecting FITC-labeled streptavidin and biotinylated anti-streptavidin antibody onto the reaction site. With a detection time of 20 min, the limit of detection (LOD) for the biotinylated target was 2.2 pM, and the LOD for the SNP was 10-30 pM, depending on the SNP type.
Topics: Biotinylation; DNA; DNA Probes; Dimethylpolysiloxanes; Fluorescein-5-isothiocyanate; Humans; Immunoassay; Lab-On-A-Chip Devices; Nucleic Acid Hybridization; Polymorphism, Single Nucleotide; Streptavidin
PubMed: 20953047
DOI: 10.2116/analsci.26.1053 -
Analytical Biochemistry Jan 2011Protein phosphorylation is a critical posttranslational modification that affects cell-cell signaling and protein function. However, quantifying the relative...
Label-free liquid chromatography-tandem mass spectrometry analysis with automated phosphopeptide enrichment reveals dynamic human milk protein phosphorylation during lactation.
Protein phosphorylation is a critical posttranslational modification that affects cell-cell signaling and protein function. However, quantifying the relative site-specific changes of phosphorylation occupancies remains a major issue. An online enrichment of phosphopeptides using titanium dioxide incorporated in a microchip liquid chromatography device was used to analyze trypsin-digested human milk proteins with mass spectrometry. The method was validated with standards and used to determine the dynamic behavior of protein phosphorylation in human milk from the first month of lactation. α-Casein, β-casein, osteopontin, and chordin-like protein 2 phosphoproteins were shown to vary during this lactation time in an independent manner. In addition, changes in specific regions of these phosphoproteins were found to vary independently. Novel phosphorylation sites were discovered for chordin-like protein 2, α-lactalbumin, β-1,4-galactosyl transferase, and poly-Ig (immunoglobulin) receptor. Coefficients of variation for the quantitation were comparable to those in other contemporary approaches using isotopically labeled peptides, with a median value of 11% for all phosphopeptide occupancies quantified.
Topics: Automation; Chromatography, High Pressure Liquid; Female; Humans; Lactalbumin; Lactation; Milk Proteins; Milk, Human; Phosphopeptides; Phosphorylation; Tandem Mass Spectrometry; Titanium
PubMed: 20804719
DOI: 10.1016/j.ab.2010.08.031 -
PloS One Dec 2009Site-specific protein labeling or modification can facilitate the characterization of proteins with respect to their structure, folding, and interaction with other...
BACKGROUND
Site-specific protein labeling or modification can facilitate the characterization of proteins with respect to their structure, folding, and interaction with other proteins. However, current methods of site-specific protein labeling are few and with limitations, therefore new methods are needed to satisfy the increasing need and sophistications of protein labeling.
METHODOLOGY
A method of protein C-terminal labeling was developed using a non-canonical split-intein, through an intein-catalyzed trans-splicing reaction between a protein and a small synthetic peptide carrying the desired labeling groups. As demonstrations of this method, three different proteins were efficiently labeled at their C-termini with two different labels (fluorescein and biotin) either in solution or on a solid surface, and a transferrin receptor protein was labeled on the membrane surface of live mammalian cells. Protein biotinylation and immobilization on a streptavidin-coated surface were also achieved in a cell lysate without prior purification of the target protein.
CONCLUSIONS
We have produced a method of site-specific labeling or modification at the C-termini of recombinant proteins. This method compares favorably with previous protein labeling methods and has several unique advantages. It is expected to have many potential applications in protein engineering and research, which include fluorescent labeling for monitoring protein folding, location, and trafficking in cells, and biotinylation for protein immobilization on streptavidin-coated surfaces including protein microchips. The types of chemical labeling may be limited only by the ability of chemical synthesis to produce the small C-intein peptide containing the desired chemical groups.
Topics: Amino Acid Sequence; Animals; Biotinylation; CHO Cells; Cell Survival; Cricetinae; Cricetulus; Fluorescent Dyes; Humans; Immobilized Proteins; Inteins; Maltose-Binding Proteins; Mass Spectrometry; Molecular Sequence Data; Peptides; Periplasmic Binding Proteins; Proteins; Receptors, Transferrin; Staining and Labeling; Trans-Splicing
PubMed: 20027230
DOI: 10.1371/journal.pone.0008381