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In basic cell biology research and drug discovery, it is important to rapidly introduce genes, proteins or drug compounds into cells without permanent damage. Here, we report a three dimensional SU-8 micro-well structure sandwiched with an indium tin oxide (ITO) electrode-covered slide from the top and an individually addressable array of microelectrodes on the bottom to allow parallel delivery of exogenous molecules into various cells in a spatially specific manner. A positive dielectrophoretic force was selectively applied by energizing appropriate electrodes to capture the dispersed cells at the bottom electrode, while the micro-wells were designed to confine cells in situ when the positive dielectrophoretic force is removed. The combination of spatial positive dielectrophoresis (pDEP) and micro-wells made it possible to construct cell microarrays with specific patterns. Once the cells become attached to the electrodes, different plasmids can be introduced sequentially for selective electroporation. The present cell arraying-assisted electroporation chip integrates a pDEP-assisted cell positioning function with selective electroporation to provide a simple and efficient method for gene transfer. This platform is ideal for high throughput screening of compounds in parallel and thus holds promise for applications in cellular and molecular research.

We have established a mobile phone-assisted microarray decoding platform for signal-enhanced mutation detection. A large amount of single-stranded DNA (ssDNA) was obtained by combining symmetric PCR and magnetic isolation, and ssDNA prepared with magnetic bead as label was further allowed to hybridize against the tag-array for decoding purpose. High sensitivity and specificity was achieved with the detection of genomic DNA. When simultaneously genotyping nine common mutations associated with hereditary hearing loss, the detection limit of 1 ng genomic DNA was achieved. Significantly, a mobile phone was also used to record and decode the genotyping results through a custom-designed imaging adaptor and a dedicated mobile phone software. A total of 51 buccal swabs from patients probably with deafness-related mutations were collected and analyzed. The genotyping results were all confirmed by fluorescence-based laser confocal scanning and direct DNA sequencing. This mobile phone-assisted decoding platform provides an effective but economic mutation detection alternative for the future quicker and sensitive detection of virtually any mutation-related diseases in developing and underdeveloped countries.

Aims: The aim of the study was to investigate the feasibility of a continuous reactor for psychrophilic anaerobic wastewater treatment by using the sludge from cold natural environment. Methods and Results: Six sludge samples (S1–S6) were collected from different cold natural locations to select sludge with high anaerobic microbial activity under low temperatures. After a 225-day incubation, the maximum specific methane production rate of a waterfowl lake sediment (S1) at 15°C (70·5 mLCH4 gVSS−1 day−1) was much higher than all other samples. S1 was thus chosen as the seed sludge for the reactor treating synthetic brewery wastewater at 15°C, by immobilizing the micro-organisms on polyurethane foam carriers. The chemical oxygen demand (COD) removal efficiency reached over 80% after 240-day operation at an organic loading rate of 5·3 kg m−3 day−1, and significant enrichment of biomass was observed. Clone libraries of the microbial communities in the inoculum had high diversities for both archaea and bacteria. Along with a decrease in microbial community diversities, the dominant bacteria (79·5%) at the end of the operation represented the phylum Firmicutes, while the dominant archaeon (41·5%) showed a similarity of 98% with the psychrotolerant methanogen Methanosarcina lacustris. Conclusions: The possibility of using anaerobic micro-organisms from cold environments in anaerobic wastewater treatment under psychrophilic conditions is supported by these findings. Significance and Impact of the Study: This study enriches the theory on microbial community and the application on anaerobic treatment of sludge from cold natural environments.

The abuse of antibiotic drugs during animal production remains a worldwide problem and the subsequent detection of the residues of various drugs present at low concentrations in complex biological matrices poses significant analytical challenges. The present study outlines a practical biochip assay system to identify antibiotic residues in different animal tissue extracts. The system uses a simple but efficient multiresidue sample extraction procedure to isolate the antibiotic residues which were then identified directly using high-affinity monoclonal antibodies presented in a competitive immunoassay with conjugated antibiotic hapten-chips. The hapten-chip can analyze six samples each for eight antibiotics on a single chip within 3 h. The analytical results with both artificial positive standard samples and the incurred samples show that the antibody hapten-chip system has a comparable accuracy and a similar sensitivity to a standard ultra performance liquid chromatography–mass spectrometry (MS)/MS assay. In conclusion, an effective analytical screening system based on antibody hapten-chip was developed for detecting multiple antibiotic residues from multiple samples.

Aims: To investigate the effects of low temperatures on the performance and microbial community of anaerobic wastewater treatment. Methods and Results: An expanded granular sludge bed (EGSB) reactor was employed to treat synthetic brewery wastewater at 20 and 15°C. Reactor performance was represented by chemical oxygen demand (COD) removal efficiency, while the microbial community was analysed using denaturing gradient gel electrophoresis (DGGE) and clone technology. When the hydraulic retention time (HRT) was maintained at 18 h, COD removal efficiencies above 85% were obtained at both 20 and 15°C, with influent COD concentrations up to 7300 and 4100 mg l−1, respectively. At 15°C, the COD removal efficiency was more easily manipulated by increasing the influent COD concentration. DGGE and clone results for both temperatures revealed that Methanosaeta and Methanobacterium were two dominant methanogens, and that the majority of the eubacterial clones were represented by Firmicutes. When the temperature decreased from 20 to 15°C, both archaeal and eubacterial communities had higher diversity, and the proportion of Methanosaeta (acetate-utilizing methanogens) decreased markedly from 60·0% to 49·3%, together with an increase in proportions of hydrogen-utilizing methanogens (especially Methanospirillum). Conclusions: The feasibility of psychrophilic anaerobic treatment of low and medium strength organic wastewaters was demonstrated, although lower temperature could significantly affect both reactor performance and the anaerobic microbial community. Significance and Impact of the Study: The findings enrich the theory involving the microbial community and the application of anaerobic treatment in a psychrophilic environment.

Neuronal activity-dependent gene transcription is a key feature of long-lasting synaptic strengthening associated with learning and memory, as well as activity-dependent neuroprotection. To comprehensively determine the molecular alterations, we carried out genome-wide microarray analysis in cultured rat cortical neurons treated with specific pharmacological agents, a model with alterations in neuronal activity, which were monitored by multi-site electrophysiological recordings. Of the approximately 27,000 genes, the expression of 248 genes was strongly changed in response to enhanced activity. These genes encompass a large number of members of distinct families, including synaptic vesicle proteins, ion channels, signal transduction molecules, synaptic growth regulators, and others. Two subsets of these genes were further confirmed to be specifically induced by Ca2+ influx through N-methyl-D-aspartate (NMDA) receptors and L-type voltage-gated Ca2+ channels (VGCCs). In addition, those genes dynamically regulated by the enhanced activity were also elucidated, as well as those candidate genes associated with synaptic plasticity and neuroprotection. Our findings therefore would help define the molecular mechanisms that occur in response to neuronal activity and identify specific clusters of genes that contribute to activity-dependent and Ca2+-inducible modulation of brain development and function. J. Cell. Physiol. 212: 126–136, 2007. © 2007 Wiley-Liss, Inc.

Microelectrode arrays (MEAs) provide a means to investigate the electrophysiological behavior of neuronal systems through the measurements from neuronal culture preparations. Changes in activity patterns of neuronal networks are usually detected by applying neural chemicals. Because of the difficulties of fabricating the arrays, and the delicate and less reliable properties of cortical neurons, MEA-based systems with cortical neuronal networks for neurophamacological applications are technically difficult, therefore restricting their utility. Here, we report a new approach to the development of such MEA-based system with sensitive and durable MEAs conveniently fabricated and the culture conditions optimized. Upon growth differentiation, cortical neurons, cultured directly on MEAs, reach a developmentally stable and reliable activity state. With this system, we monitored the global spontaneous activities of neuronal networks and demonstrated the fine discrimination for specific substances and unique property of cortical neurons, which validated both the applicability and necessity of such system in pharmacological bioassay.

A protein microarray system for detection and quantification of nine prohibited drugs in serum is described. Chemically modified slides were chosen as the microarray substrates because of their suitable for drug-BSA printing. The developed protein microarray was able to preserve the biological function of the haptens, when immobilized on the microarray surface and demonstrated binding with their corresponding antibodies. The microarray could also be used for quantitative analysis when mouse IgG was chosen as an internal control for data processing. There was no qualitative difference between the results obtained using the protein microarray and ELISA. The protein microarray technology should be applicable to performing, simultaneously, large scale screening tests for many different analytes in serum.

Developing new methods and technologies in order to pattern neurons into regular networks is of utmost scientific interest in the field of neurological research. An efficient method here is developed for trapping neurons and constructing ordered neuronal networks on bioelectronic chips by using arrayed negative dielectrophoretic (DEP) forces. A special bioelectronic chip with well defined positioning electrode arrays was designed and fabricated on silicon substrate. When a high frequency AC signal was applied, the cell positioning bioelectronic chip (CPBC) is able to provide a well-defined non-uniform electric field, and thus generate negative DEP forces. The parameters, such as size of positioning electrode, conductivity of working solution, amplitude and frequency of power signal and cell concentration, were investigated to optimize the performance of the CPBC. When the neuron suspension was added onto the energized bioelectronic chip, the neurons were immediately trapped and quickly formed the predetermined pattern. Neurons may adhere and then be cultured directly on the CPBC, and show good neuron viability and neurite development. The formation of the ordered neuronal networks after two-week culture demonstrates that negative dielectrophoretic force assisted construction of ordered neuronal networks is effective, and it could be used to assist in monitoring functional activities of neuronal networks.

Recently, the analysis of single-nucleotide polymorphisms has attracted much attention. Although many techniques have been reported, new methods with high resolving power, low-cost and fast speed are still in demand. We present a fast SNP detection scheme using chip-based temperature gradient capillary electrophoresis to separate the homoduplex and heteroduplex PCR products which contain one or two SNP sites. The total time of a single run was only 8 minutes.

Yi Lv,1–3,* Lingqin Zeng,1–3,* Guanbin Zhang,2 Youchun Xu,1–3, Ying Lu,1–3, Keith Mitchelson,1,2 Jing Cheng,1–3,* Wanli Xing1–3,* 1Medical Systems Biology Research Center, Department of Biomedical Engineering, Tsinghua University School of Medicine, Beijing, China; 2National Engineering Research Center for Beijing Biochip Technology, Beijing, China; 3The State Key Laboratory of Biomembrane and Membrane Biotechnology, Tsinghua University, Beijing, China *These authors contributed equally to this work Abstract: Dielectrophoresis (DEP) can be used to noninvasively measure the dielectric state of the cell, and this data can be used to monitor cell health or apoptosis. In this study, we followed events associated with cytosine arabinoside (Ara-C)-induced apoptosis in NB4 cells using DEP analysis. Our data showed that the membrane capacitance of NB4 cells decreases from 9.42 to 7.63 mF/m2 in the first 2 hours following treatment with Ara-C, and that this decreased capacitance persists for >12 hours. Additionally, cytoplasmic conductivity decreases from 0.217 to 0.190 S/m within 2 hours of Ara-C treatment; this level is maintained for a short period of time before decreasing. We also investigated these events molecularly at the level of gene expression using microarray analysis and showed that the expression of genes related to membrane capacitance and cytoplasmic conductivity change dramatically as early as 2 hours post-Ara-C treatment, and further demonstrated a temporal relationship between the dielectric properties and key events in apoptosis. This study, integrating physical electrical properties of the cell membrane and cytoplasm with those of conductivity-related gene networks, provides new insights into the molecular mechanisms underlying the initiation of apoptosis, establishing a systematic foundation for DEP application in follow-up drug screening and development of medicines for treating leukemia. Keywords: apoptosis, dielectrophoresis, gene expression profiling, leukemia

Here, we report a novel method for the fabrication of polydimethylsiloxane microdevices with complicated 3-D structures, such as concave and crater shapes, using an easily machined polymethyl methacrylate mold combined with a one-step molding process. The procedure presented here enables rapid preparation of complex 3-D microstructures varying in shape and dimensions. To regulate embryoid body (EB) formation, we fabricated a microfluidic device with an array of concave microwells and found that EBs growing in microwells maintained their shape, viability, and a high degree of homogeneity. We believe that this novel method provides an alternative for rapid prototyping, especially in fabricating devices with curved 3-D microstructures.

The reconstruction of segmental bone defects remains an urgent problem in the orthopaedic field, and bone morphogenetic protein-2 (BMP-2) is known for its potent osteoinductive properties in bone regeneration. In this study, chitosan microspheres (CMs) were prepared and combined with absorbable collagen sponge to maintain controlled-release recombinant human bone morphogenetic protein-2 (rhBMP-2). The rhBMP-2-loaded composite scaffolds were implanted into 15 mm radius defects of rabbits and the bone-repair ability was evaluated systematically. CMs were spherical in shape and had a polyporous surface, according to SEM images. The complex scaffold exhibited an ideal releasing profile in vitro. The micro-computed tomographic analysis revealed that the rhBMP-2-loaded composite scaffold not only bridged the defects as early as 4 weeks, but also healed the defects and presented recanalization of the bone-marrow cavity at 12 weeks. These results were confirmed by x-ray. When compared with other control groups, the composite scaffold group remarkably enhanced new bone formation and mechanical properties, as evidenced by bone mineral content evaluation, histological observations and biomechanical testing. Moreover, the biocompatibility and appropriate degradation of the composite scaffold could be obtained. All of these results clearly demonstrated that the composite scaffold is a promising carrier of BMP-2 for the treatment of segmental bone defects.

Cell migration is crucial in many physiological and pathological processes including embryonic development, immune response and cancer metastasis. Traditional methods for cell migration detection such as wound healing assay usually involve physical scraping of a cell monolayer followed by an optical observation of cell movement. However, these methods require hand-operation with low repeatability. Moreover, it's a qualitative observation not a quantitative measurement, which is hard to scale up to a high-throughput manner. In this article, a novel and reliable on-chip cell migration detection method integrating surface chemical modification of gold electrodes using self-assembled monolayers (SAMs) and real-time cellular impedance sensing is presented. The SAMs are used to inhibit cell adherence forming an area devoid of cells, which could effectively mimic wounds in a cell monolayer. After a DC electrical signal was applied, the SAMs were desorbed from the electrodes and cells started to migrate. The process of cell migration was monitored by real-time impedance sensing. This demonstrates the first occurrence of integrating cellular impedance sensing and wound-forming with SAMs, which makes cell migration assay being real-time, quantitative and fully automatic. We believe this method could be used for high-throughput anti-migratory drug screening and drug discovery.

There had been little progress in development of the theoretical basis of rectangular chromatography columns until Spangler made great progress by using a more exact model than Golay's. Unfortunately, there was a deficiency in his calculations, which led to a conclusion inconsistent with the previous theories. In this paper, a simpler formula with defined variables was first established to calculate the mean permeability coefficient for a rectangular GC column. A formula was also established to calculate the height equivalent to a theoretical plate (HETP) for a rectangular column based on this work and the correction of Spangler's theory. By comparing both our predictions and Spangler's predictions with Golay's, respectively, we could demonstrate that our theory is more exact. Further, one parameter (A) was found to be not monotonous. This finding leads to the conclusion that the square column has the highest performance among all the rectangular-shaped columns used for chromatography, and that a width/depth ratio of around three is desirable if the column is used for mixing reactants in lab-on-a-chip systems, instead of for chromatography. The conclusions are applicable not only for gas but also for liquid chromatography columns.

A sample preparation method for gas chromatography using a two-phase, laminar flow extraction PDMS/glass chip has been developed. A stable two-phase laminar interface was obtained by surface modification, and the organic extraction phase and the aqueous sample phase were separated effectively when the two-phase laminar flows exit the chip. Experiments were conducted on the chip to extract ephedrine from aqueous solution. Good reproducibility was obtained over the entire range of ephedrine concentration using the extraction chips (CV range 2.7%-4.5%). Effects of salt and solvent on extraction efficiency were studied.

It has been widely considered that the global warming was induced by the increasing atmospheric concentrations of carbon dioxide, which has a significant impact on the sustainable development of the human society. Injecting carbon dioxide into depleted oil/gas reservoir has been developed to reduce carbon dioxide emission by the sequestration of CO 2 underground, which is also considered as one promising approach to enhance oil recovery simultaneously. The evaluating of CO 2 sequestration in oil reservoir needs highly accurate experimental data and models about the thermodynamic characters of CO 2 + oil mixture. Density is one of key physical characters for simulating the diffusion and migration of carbon dioxide under the oil/gas reservoir. Research on volumetric behaviour is helpful to understand the expansion of the liquid phases in CO 2 + oil mixture. In this paper, decane is investigated instead of petroleum because of the similar thermal physical property. The measurements have been performed at pressures from 12 MPa to 18 MPa and temperatures from 313.15 K to 343.15 K at four compositions x1 (x1 is the mole fraction of carbon dioxide) = 0.8690, 0.9135, 0.9430 and 0.9818 by a magnetic suspension balance (MSB). The accuracy of the experimental quantities presented in this work are T/ K = ± 0.01, m/ g = ± 10-5 respectively. One circulation pump (AKICO, Japan) is equipped to accelerate the dissolution of CO 2 in decane. Some conclusions can be obtained from the experiment as follow: (1) the dissolution of CO 2 increases the density of decane, the density of CO 2 + decane solution increases with increasing pressure and decreases with increasing temperature. (2) the slope of density with respect to pressure increases with increasing CO 2 mole fractions while the slope with respect to temperature decreases with increasing CO 2 mole fractions. (3) the crossover phenomenon appears at different compositions and the crossover pressure increases with temperature. (4) the excess molar volumes of this binary mixture are negative and show negative increasing with temperature for the data reported in this work. The GERG - 2008 model is used to predict density of CO 2 + decane solution, which takes temperature, pressure, and CO 2 concentration into account. An empirical equation is also used to predict the density of this binary mixture. The predictions show that the empirical equation is in good agreement with the experimental data over the experimental range. The measurement and the model used in this work supplies the basic experimental data and theoretical analysis to CO 2 sequestration and enhance oil recovery.

Injecting carbon dioxide into oil/gas fields has been regarded as a feasible way to reduce the global warming, at the same time it could enhance oil recovery. The density of CO 2 + oil is a key parameter for carbon dioxide capture and storage. Dodecane has similar thermal physical properties with petroleum. The aim of the work is to reveal the relationship between the density and the pressure and the temperature for the carbon dioxide (x) and dodecane (1-x). It was measured by the magnetic suspension balance (MSB) with temperature from 313.15 K to 353.15 K and pressure from 8 to 18 MPa at different CO 2 mole fractions: x = 0, 0.2497, 0.5094, 0.7576 and 0.8610. The experimental results show that the density of CO 2 + dodecane mixture increases with the increasing pressure and decreases with the increasing temperature. It is found that the density of the mixture increases with the increasing concentration of CO 2 firstly and then decreases at high mole fractions. The variation trend is similar with that of CO 2 + decane/tridecane mixtures. BWRS equation of state was established based on the experimental data, which agrees well with the density of CO 2 + dodecane.