Microchamber array based DNA quantification and specific sequence detection from a single copy via PCR in nanoliter volumes

A novel method for DNA quantification and specific sequence detection in a highly integrated silicon microchamber array is described. Polymerase chain reaction (PCR) mixture of only 40 nL volume could be introduced precisely into each chamber of the mineral oil layer coated microarray by using a nanoliter dispensing system. The elimination of carry-over and cross-contamination between microchambers, and multiple DNA amplification and detection by TaqMan chemistry were demonstrated, for the first time, by using our system. Five different gene targets, related to Escherichia coli were amplified and detected simultaneously on the same chip by using DNA from three different serotypes as the templates. The conventional method of DNA quantification, which depends on the real-time monitoring of variations in fluorescence intensity, was not applied to our system, instead a simple method was established. Counting the number of the microchambers with a high fluorescence signal as a consequence of TaqMan PCR provided the precise quantification of trace amounts of DNA. The initial DNA concentration for Rhesus D (RhD) gene in each microchamber was ranged from 0.4 to 12 copies, and quantification was achieved by observing the changes in the released fluorescence signals of the microchambers on the chip. DNA target could be detected as small as 0.4 copies. The amplified DNA was detected with a CCD camera built-in to a fluorescence microscope, and also evaluated by a DNA microarray scanner with associated software. This simple method of counting the high fluorescence signal released in microchambers as a consequence of TaqMan PCR was further integrated with a portable miniaturized thermal cycler unit. Such a small device is surely a strong candidate for low-cost DNA amplification, and detected as little as 0.4 copies of target DNA.     

Rapid Identification of ESKAPE Bacterial Strains Using an Autonomous Microfluidic Device

This article describes Bacteria ID Chips ('BacChips'): an inexpensive, portable, and autonomous microfluidic platform for identifying pathogenic strains of bacteria. BacChips consist of a set of microchambers and channels molded in the elastomeric polymer, poly(dimethylsiloxane) (PDMS). Each microchamber is preloaded with mono-, di-, or trisaccharides and dried. Pressing the layer of PDMS into contact with a glass coverslip forms the device; the footprint of the device in this article is ～6 cm(2). After assembly, BacChips are degased under large negative pressure and are stored in vacuum-sealed plastic bags. To use the device, the bag is opened, a sample containing bacteria is introduced at the inlet of the device, and the degased PDMS draws the sample into the central channel and chambers. After the liquid at the inlet is consumed, air is drawn into the BacChip via the inlet and provides a physical barrier that separates the liquid samples in adjacent microchambers. A pH indicator is admixed with the samples prior to their loading, enabling the metabolism of the dissolved saccharides in the microchambers to be visualized. Importantly, BacChips operate without external equipment or instruments. By visually detecting the growth of bacteria using ambient light after ～4 h, we demonstrate that BacChips with ten microchambers containing different saccharides can reproducibly detect the ESKAPE panel of pathogens, including strains of: Enterococcus faecalis, Enteroccocus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter aerogenes, and Enterobacter cloacae. This article describes a BacChip for point-of-care detection of ESKAPE pathogens and a starting point for designing multiplexed assays that identify bacterial strains from clinical samples and simultaneously determine their susceptibility to antibiotics.     

Pressure-driven perfusion culture microchamber array for a parallel drug cytotoxicity assay

This article reports a pressure-driven perfusion culture chip developed for parallel drug cytotoxicity assay. The device is composed of an 8 x 5 array of cell culture microchambers with independent perfusion microchannels. It is equipped with a simple interface for convenient access by a micropipette and connection to an external pressure source, which enables easy operation without special training. The unique microchamber structure was carefully designed with consideration of hydrodynamic parameters and was fabricated out of a polydimethylsiloxane by using multilayer photolithography and replica molding. The microchamber structure enables uniform cell loading and perfusion culture without cross-contamination between neighboring microchambers. A parallel cytotoxicity assay was successfully carried out in the 8 x 5 microchamber array to analyze the cytotoxic effects of seven anticancer drugs. The pressure-driven perfusion culture chip, with its simple interface and well-designed microfluidic network, will likely become an advantageous platform for future high-throughput drug screening by microchip.     

Effect of materials for micro-electro-mechanical systems on PCR yield

In this study we analyzed the surface properties of different silicon-based materials used for micro-electro-mechanical systems (MEMS) production, such as thermally grown silicon oxide, plasma-enhanced chemical vapor deposition (PECVD)-treated silicon oxide, reactive-ion etch (RIE)-treated silicon oxide, and Pyrex. Substrates were characterized by atomic force microscopy (AFM) and X-ray photoelectron spectroscopy (XPS) to define the surface chemical and morphological properties, and by fluorescence microscopy to directly assess the absorption of the different polymerase chain reaction (PCR) components. By using microchips fabricated with the same materials we investigated their compatibility with PCR reactions, exploiting the use of different enzymes and reagents or proper surface treatments. We established the best conditions for DNA amplification in silicon/Pyrex microdevices depending on the type of device and fabrication method used and the quality of reagents, rather than on the passivation treatment or increment in standard Taq polymerase concentration.     

硅对PCR过程的抑制作用研究

探讨不同氧化程度的硅材料对PCR扩增的抑制作用及其机理。将不同氧化程度的硅纳米颗粒加入PCR反应液中,使其与Taq酶、模板等充分接触,通过离心将硅纳米颗粒沉降在管壁上,取出上清或保留硅纳米颗粒上机扩增,扩增产物采用凝胶电泳法检测。结果表明,随着硅材料表面面积与PCR反应液体积之比的增大,核酸扩增效率将明显下降,并且在所研究的范围内,氧化程度高的硅材料对PCR过程抑制作用更强;通过对抑制作用机理进行初步的实验研究,表明硅材料对PCR反应液中的Taq酶的吸附是导致抑制现象产生的主要原因,而对模板的吸附影响较小;并且,反应管内是否保留硅材料对核酸扩增影响较小,硅材料没有明显的直接化学抑制作用。     

Calorimetric biosensors with integrated microfluidic channels

A microfluidic device capable of measuring real-time enthalpy changes of biochemical reactions and thermal properties of biological fluids is presented in this paper. The device consists of a freestanding microthermopile integrated with a glass microfluidic reaction chamber. The p-type polysilicon/gold microthermopiles fabricated on a 2 μm thick thermally isolated membrane showed a sensitivity of 0.94 V/W and a thermal time constant of less than 100 ms. Although the device is not restricted to enzymatic reactions, in this paper measurements of the heat of reaction from the catalytic action of glucose oxidase, catalase, and urease on glucose, hydrogen peroxide, and urea, respectively, are reported. Reactions were performed in open air using liquid batch testing and in enclosed fluidic reaction chamber by continuous flow experiments. A sensitivity of 53.5 μV/M for glucose, 26.5 μV/M for hydrogen peroxide and 17 μV/M for urea was obtained. Detection limit for glucose in the continuous flow mode is 2 mM (30 pmol). The aim of this work is to demonstrate the potential of the integrated calorimetric microfluidic device for fundamental thermodynamic studies in biochemical reactions. Using arrays of such devices with immobilized enzymes multi-analyte detection can be accomplished and the effects of interferents from competing substrates can be compensated. This paper presents the design, fabrication and initial testing results from such a microthermopile-based thermal biosensor.     

Physicochemical principles of tissue material interactions

Biocompatibility of a material has to be adapted to the specific properties of the locus of application that are the type of tissue and the composition of extracellular fluid or the blood being in contact with the surface. The biocompatibility is beyond that greatly influenced by the design of the medical device which has to be planned close to the material's properties and the function within the body. Physical chemical reactions at and physical properties of the surface which influence the adsorption behavior for biomacromolecules. Conformational or functional changes of f.i. proteins due to physical forces originating from the surface could be the communication messages to the immunological system. The immersion of a material into an aqueous electrolyte leads generally to a space charge layer on both sides of the interface forming the electrical double layer, physically described by the isoelectric point of the materials surface. A numerical example hints on the importance of the double layer structure for the 'communication' between an implant and the surrounding extracellular fluid including beside ions complex structured proteins as biomacromolecules. Biocompatibility depends on the physical structure of the material and physicochemical properties of the interface to the biosystem. The conductivity of the surface film control reactions across the interface with biomacromolecules of the biological environment. Conformational unchanged macromolecules are the prior condition for biocompatibility and controls the attachment and probably also the degree of attachment via adhesion proteins. Later on, when the cells develop tension through the cytoskeleton on these attachment sites, the strength of the integrin adhesion protein-matrix protein interaction might probably prove decisive in differentiation state of the cell. It has been proved by molecular biological methods that an undestroyed oxide layer of anatase on titanium through passivation leaves for instance albumin conformational unchanged.     

Determination of antibodies to mycobacterial antigens in tuberculosis by erythrocyte immunoadsorption with a rosette marker

A solid-phase enzyme immunoassay system for the determination of antibodies to mycobacterial antigens, based on the method of erythrocyte immunoadsorption in microchambers for immunological reactions, has been developed. To detect antibodies specifically bound with the solid-phase antigen, the affinity rosettes of Staphylococcus aureus strain Cowan I, carrying protein A, with erythrocytes conjugated with human gamma globulin have been used. The significant correlation of the titers of 34 sera, determined by means of erythrocyte immunoadsorption, with extinction values obtained in the solid-phase enzyme immunoassay of antibodies to Mycobacterium tuberculosis has been established. The coincidence of the results in 92% of cases has been noted.     

Pattern selection in a cooperative biochemical in vitro amplification system: the role of parasites

Previously, numerical simulations have shown that evolving systems can be stabilized against emerging parasites by pattern formation in spatially extended flow reactors. Hence, it can be argued that pattern formation is a prerequisite for any experimental investigation of the biochemical evolution of cooperative function. Here, we study a model of an experimental biochemical system for the cooperative in vitro amplification of DNA strands and show that emerging parasites can induce a complex pattern formation even when no pattern formation occurs without parasites. In an adiabatic approximation where the cooperative amplification reaction is assumed to adapt fast to slowly emerging parasites, the parasite concentration itself acts as a Steuer parameter for the selection of various complex patterns. Without such an adiabatic approximation only transient patterns emerge. As any species can grow for very low concentrations, the parasite is able to infect the entire reactor and the system is finally diluted out. In the experimental biochemical system, however, the species are individual molecules and the growth of spatially separated, non-infected regions becomes feasible. Hence a cutoff threshold for the minimal concentration is applied. In these simulations the otherwise lethal infection by parasites induces the formation of spatiotemporal spirals, and this spatial structure help the host and parasitoid species to survive together. These theoretical results describe an inherent property of cooperative reactions and have an important impact on experimental investigations on the molecular evolution and complex function in spatially extended reactors. Since the formation of the complex pattern is restricted either to a rather large cutoff value or a special choice of the kinetic parameters, we, however, conclude that the persistence of evolving cooperative amplification is not possible in a simple reaction-diffusion reactor. Experimental set-ups with patchy environments, e.g. biomolecular amplification in coupled microstructured flow chambers or in microemulsion, are eligible candidates for the observation of such a self-organized pattern selection.     

A compliance-independent pressure transducer for biomedical device-tissue interfaces

The measurement of the interface pressure between a biomedical device and part of the human body is useful to improve the performance and safety of such devices during design. Testing of a selection of existing interface pressure transducers has demonstrated that many are dependent on device and tissue compliance. Such a transducer is useful only in an application where it has been calibrated for specific device-tissue compliance combinations. To overcome this limitation, the authors developed an interface pressure transducer whose output signal is not affected by changes in interface compliance. This enables the transducer to quantitatively measure pressure in many applications without the need to calibrate it for varying compliance conditions. Surgical retraction and surgical tourniquets were selected as demonstration applications for the developed transducer, because they represent a wide spectrum of device and tissue characteristics and properties, and are in common use.     

Shaping the dynamics of a bidirectional neural interface

Progress in decoding neural signals has enabled the development of interfaces that translate cortical brain activities into commands for operating robotic arms and other devices. The electrical stimulation of sensory areas provides a means to create artificial sensory information about the state of a device. Taken together, neural activity recording and microstimulation techniques allow us to embed a portion of the central nervous system within a closed-loop system, whose behavior emerges from the combined dynamical properties of its neural and artificial components. In this study we asked if it is possible to concurrently regulate this bidirectional brain-machine interaction so as to shape a desired dynamical behavior of the combined system. To this end, we followed a well-known biological pathway. In vertebrates, the communications between brain and limb mechanics are mediated by the spinal cord, which combines brain instructions with sensory information and organizes coordinated patterns of muscle forces driving the limbs along dynamically stable trajectories. We report the creation and testing of the first neural interface that emulates this sensory-motor interaction. The interface organizes a bidirectional communication between sensory and motor areas of the brain of anaesthetized rats and an external dynamical object with programmable properties. The system includes (a) a motor interface decoding signals from a motor cortical area, and (b) a sensory interface encoding the state of the external object into electrical stimuli to a somatosensory area. The interactions between brain activities and the state of the external object generate a family of trajectories converging upon a selected equilibrium point from arbitrary starting locations. Thus, the bidirectional interface establishes the possibility to specify not only a particular movement trajectory but an entire family of motions, which includes the prescribed reactions to unexpected perturbations.     

A conceptual design for cosmo-biology experiments in Earth's Orbit.

A conceptual design was developed for a cosmo-biology experiment. It is intended to expose simulated interstellar ice materials deposited on dust grains to the space environment. The experimental system consists of a cryogenic system to keep solidified gas sample, and an optical device to select and amplify the ultraviolet part of the solar light for irradiation. By this approach, the long lasting chemical evolution of icy species could be examined in a much shorter time of exposure by amplification of light intensity. The removal of light at longer wavelength, which is ineffective to induce photochemical reactions, reduces the heat load to the cryogenic system that holds solidified reactants including CO as a constituent species of interstellar materials. Other major hardware components were also defined in order to achieve the scientific objectives of this experiment. Those are a cold trap maintained at liquid nitrogen temperature to prevent the contamination of the sample during the exposure, a mechanism to exchange multiple samples, and a system to perform bake-out of the sample exposure chamber. This experiment system is proposed as a candidate payload implemented on the exposed facility of Japanese Experiment Module on International Space Station.     

Mechanisms of seed ageing under different storage conditions for Vigna radiata (L.) Wilczek: lipid peroxidation,sugar hydrolysis,Maillard reactions and their relationship to glass state transition

Two primary biochemical reactions in seed ageing (lipid peroxidation and non-enzymatic protein glycosylation with reducing sugars) have been studied under different seed water contents and storage temperatures, and the role of the glassy state in retarding biochemical deterioration examined. The viability loss of Vigna radiata seeds during storage is associated with Maillard reactions; however, the contribution of primary biochemical reactions varies under different storage conditions. Biochemical deterioration and viability loss are greatly retarded in seeds stored below a high critical temperature (approximately 40 degrees C above glass transition temperature). This high critical temperature corresponds to the cross-over temperature (T(c)) of glass transition where molecular dynamics changes from a solid-like system to a normal liquid system. The data show that seed ageing slows down significantly, even before seed tissue enters into the glassy state.     

Use of solid-phase immunoenzyme analysis for the serodiagnosis of pseudotuberculosis

The diagnostic test system based on the solid-phase enzyme immunoassay (EIA) for the detection of antibodies to Yersinia pseudotuberculosis in the sera of patients with the use of Soviet-made preparations and reagents has been developed. The test has been performed in microchambers for immunological reactions, thus making it possible to decrease the consumption of reagents 10-20 times in comparison with the traditional technique with the use of plates. The results of the titration of 42 sera in EIA and in the passive hemagglutination test (PHAT) are indicative of the presence of positive correlation (r = 0.78; p less than 0.05) between antibody titers in EIA and PHAT. A fourfold or greater increase in antibody titers has been determined by means of EIA in 80% of cases and with the use of PHAT in 55% of cases. The minimum diagnostic titer yielded by EIA has been determined: 1:256.     

High-throughput microfluidic system for long-term bacterial colony monitoring and antibiotic testing in zero-flow environments

In this study, a high-throughput microfluidic system is presented. The system is comprised of seven parallel channels. Each channel contains 32 square-shaped microchambers. After simulation studies on samples loaded into the microchambers, and the solute exchange between the microchambers and channels, the long-term culture of Escherichia coli (E. coli) HB101 in the microchambers is realized. Using the principle that L-arabinose (L-Ara) can induce recombinant E. coli HB101 pGLO to synthesize green fluorescent protein (GFP), the real-time analysis of GFP expression in different initial bacterial densities is performed. The results demonstrate that higher initial loading densities of the bacterial colony cause bacterial cell to enter log-phase proliferation sooner. High or low initial loading densities of the bacterial cell suspension induce the same maximum growth rates during the log-phase. Quantitative on-chip analysis of tetracycline and erythromycin inhibition on bacterial cell growth is also conducted. Bacterial morphology changes during antibiotic treatment are observed. The results show that tetracycline and erythromycin exhibit different inhibition activities in E. coli cells. Concentrations of 3 μg/mL tetracycline can facilitate the formation of long filamentous bacteria with the average length of more than 50 μm. This study provides an on-chip framework for bacteriological research in a high-throughput manner and the development of recombinant bacteria-based biosensors for the detection of specific substances.     

Silicon-based microfabricated microbial fuel cell toxicity sensor

Microbial fuel cells (MFCs) have been used for several years as biosensors for measuring environmental parameters such as biochemical oxygen demand and water toxicity. The present study is focused on the detection of toxic matter using a novel silicon-based MFC. Like other existing toxicity sensors based on MFCs, this device is capable of detecting the variation on the current produced by the cell when toxic compounds are present in the medium. The MFC approach presented in this work aims to obtain a simple, compact and planar device for its further application as a biosensor in the design and fabrication of equipment for toxicity monitoring. It consists on a proton exchange membrane placed between two microfabricated silicon plates that act as current collectors. An array of square 80 μm × 80 μm vertical channels, 300 μm deep, have been defined trough the plates over an area of 6 mm × 6 mm. The final testing assembly incorporates two perspex pieces positioned onto the plates as reservoirs with a working volume of 144 μL per compartment. The operation of the microdevice as a direct electron transfer MFC has been validated by comparing its performance against a larger scale MFC, run under the same conditions. The device has been tested as a toxicity sensor by setting it at a fixed current while monitoring changes in the output power. A drop in the power production is observed when a toxic compound is added to the anode compartment. The compact design of the device makes it suitable for its incorporation into measurement equipment either as an individual device or as an array of sensors for high throughput processing.     

Convection Effects in the BIAcore Dextran Layer: Surface Reaction Model

The BIAcore is a surface plasmon resonance (SPR) device used to measure rate constants, primarily for biochemical reactions. It consists of a flow channel containing one reactant adjoining a dextran gel containing the other. In order to explain anomalous measurements from the device, it has been proposed that some flow penetrates into the dextran layer, thus enhancing transport. A model is presented that accounts for such behavior, and typical velocity fields in the dextran are constructed. The system is analyzed in the limit of the surface reaction model, which corresponds to the limit of thin dextran layers. Asymptotic and singular perturbation techniques are used to analyze association and dissociation kinetics. Linear and nonlinear integral equations result from the analysis; explicit and asymptotic solutions are constructed for physically realizable cases. The results indicate that the effects of such penetration are bound to be small, regardless of the flow model used.     

A New Method for Rapid Screening of End-Point PCR Products: Application to Single Genome Amplified HIV and SIV Envelope Amplicons

PCR is the most widely applied technique for large scale screening of bacterial clones, mouse genotypes, virus genomes etc. A drawback of large PCR screening is that amplicon analysis is usually performed using gel electrophoresis, a step that is very labor intensive, tedious and chemical waste generating. Single genome amplification (SGA) is used to characterize the diversity and evolutionary dynamics of virus populations within infected hosts. SGA is based on the isolation of single template molecule using limiting dilution followed by nested PCR amplification and requires the analysis of hundreds of reactions per sample, making large scale SGA studies very challenging. Here we present a novel approach entitled Long Amplicon Melt Profiling (LAMP) based on the analysis of the melting profile of the PCR reactions using SYBR Green and/or EvaGreen fluorescent dyes. The LAMP method represents an attractive alternative to gel electrophoresis and enables the quick discrimination of positive reactions. We validate LAMP for SIV and HIV env-SGA, in 96- and 384-well plate formats. Because the melt profiling allows the screening of several thousands of PCR reactions in a cost-effective, rapid and robust way, we believe it will greatly facilitate any large scale PCR screening.     

离子液体的性能及应用

离子液体不仅可用作环境友好的“绿色溶剂”,而且在生物合成和有机反应中能表现出特殊的催化、促进效应。在介绍离子液体种类、性质、合成方法的基础上,重点综述离子液体功能化方法、离子液体／超临界CO2体系和其在生物催化反应中应用的最新研究进展。     

Reactions of vitamin A with acceptors of electrons. Interactions with iodine and the formation of iodide

1. The reactions of retinol and retinoic acid with iodine were investigated since knowledge of the chemical reactions of vitamin A with acceptors of electrons may shed light on its biochemical mode of action. 2. Colloidal retinol, but not retinoic acid, reacts with iodine to yield a blue-green complex that rapidly decomposes, giving iodide and an unknown species with lambda(max.) at 870mmu. 3. In addition, both retinol and retinoic acid reduce iodine to iodide by a reaction that does not involve an intermediate coloured complex; this reaction appears to yield unstable carbonium ion derivatives of the vitamin. 4. The presence of water greatly facilitates the production of iodide from vitamin A and iodine. 5. Possible chemical pathways involved in these reactions are discussed. 6. It is suggested that the chemical properties of retinol and retinoic acid that underlie their biochemical behaviour might be apparent only when the molecules are at a lipid-water interface, and that vitamin A might be expected to react with a number of different electron acceptors in vivo.