Robotic nucleic acid isolation using a magnetic bead resin and an automated liquid handler for biological agent simulants

The events that occurred following the mailing of Bacillus anthracis-laced envelopes through the postal system has highlighted the need to perform biological screening on large numbers of environmental samples. High-throughput screening that relies on integrated robotic systems to speed analysis has been undertaken to handle the surge in samples requiring testing in events involving weapons of mass destruction. These automated screening systems require DNA extraction methods capable of handling environmental samples that contain inhibitors and have target organisms at low concentrations. This study describes the development of a method for the detection of the biological warfare agent simulants Erwinia herbicola and Bacillus subtilis var. niger spores using paramagnetic bead-based resin with an automated liquid handler and environmental samples.     

Detection of 4-chlorobenzoate using immobilized recombinant Escherichia coli reporter strains

A test for the detection of 4-chlorobenzoic acid (4-CBA) based on reporter microorganisms is developed. A 1.7-kb DNA fragment, upstream of the dehalogenase operon from Arthrobacter sp. strain SU, is fused to the promoterless luciferase operon of Vibrio fischeri. This reporter construct is introduced into two different Escherichia coli strains (UTL2 and RFM443), which specifically respond to the presence of 4-CBA with light emission. The development and optimization of an alginate-based immobilization procedure of the microorganisms in microtiter plates is described. In this format, 4-CBA can be detected in the concentration range of 113 μM–3.6 mM. The membrane leaky mutant UTL2 of E. coli transformed with the reporter construct improves the detection limit to 28 μM when 150 mM KNO₃ is added to the medium.     

A wireless, remote-query sensor for real-time detection of Escherichia coli O157:H7 concentrations

This paper describes the real-time detection of Escherichia coli O157:H7 concentrations using a remote-query (wireless, passive) magnetoelastic sensor. The resonance frequency of a liquid immersed magnetoelastic sensor, measured through magnetic field telemetry, changes mainly in response to bacteria adhesion to the sensor and the liquid properties (viscosity, density, elasticity, etc.) of the culture medium. In the described application, during its growth and reproduction we find E. coli consumes nutrients from a liquid culture medium that decreases the solution viscosity, and in turn changes the resonance frequency of the medium-immersed magnetoelastic sensor. Using the described technique we are able to directly quantify E. coli O157:H7 concentrations of 2 × 10² to 3 × 10⁶ cells ml⁻¹, and quantify the effect of gentamycin sulfate injection (GSI) on proliferation of the bacteria. The lack of any physical connections between the sensor and the monitoring electronics facilitates aseptic operation, and makes the sensor platform ideally suited for monitoring bacteria from within, for example, sealed food containers.     

Detection and discrimination of coliform bacteria with gas sensor arrays

Electronic noses, which are used for characterizing complex vapors and aromas, may be useful for detection of bacterial contamination or diagnosis of infections, if minimal standards of selectivity and sensitivity can be met. A culture of Enterobacter aerogenes is readily discriminated from an Escherichia coli strain using principal components analysis (PCA) of data generated by an array of eight quartz microbalance (QMB), eight metal oxide semiconductor (MOX), and four electrochemical gas sensors. Two strains of E. coli were not discriminated under identical conditions. Retaining headspace air in a sealed vial containing growing bacteria results in an enhancement of sensitivity, so that a concentration of bacteria of about 5×10⁸/ml may be both detected and distinguished from other species. Improvements in sensitivity to levels useful for practical applications will require enhancement of sensors, sampling system, and pattern classification.     

A wireless biosensor using microfabricated phage-interfaced magnetoelastic particles

A micro-scale, free standing, wireless biosensor has been developed using magnetoelastic particles composed of an amorphous iron–boron binary alloy. Upon the application of an external magnetic field, these particles exhibit a characteristic resonance frequency, determined by their size and mass, due to the phenomena of magnetoelasticity. The particles are produced using the microelectronic fabrication techniques of photolithography and physical vapor deposition (sputtering). The biosensor is formed by coating the magnetoelastic particle with a thin layer of gold and immobilizing a biomolecular recognition element (bacteriophage) on the surfaces. Bacteriophage genetically engineered to bind Bacillus anthracis spores was used in this set of experiments as the detection probe. Once these targeted spores come into contact with the biosensor, the phage will bind selectively with only that pathogen, thereby increasing the particle's mass and causing a shift in the resonance frequency. Due to the magnetic nature of the sensing platform, this resonance frequency shift may be detected remotely by a wireless scanning device, presenting a distinct advantage over other techniques. A good correlation between the actual number of spores bound to the sensors and the calculated attached mass, based upon resonance frequency shifts, was obtained from the experiments.     

基于Profibus-DP现场总线的多功能色谱分离装置控制系统设计

为了实现多功能色谱分离装置的精准化控制,加速模拟移动床色谱纯化分离速度,设计基于Profibus-DP现场总线的多功能色谱分离装置控制系统;以STM32F103微处理器作为底层控制设备,连接Profibus-DP协议芯片、VPC3与RS-485接口电路,转化从站地址电路与设定应用模块,利用模拟移动床控制回路,完成Profibus-DP现场总线的硬件电路设计;建立梯度SMB色谱,采用异步切换方式,控制变浓度进料的实际操作行为,分离处理模拟移动床多功能色谱,结合多功能色谱分离装置的控制原理,运用气相色谱法,分析色谱分离过程中的基质效应形式,实现多功能色谱分离装置控制系统软件设计;实验结果表明,基于Profibus-DP现场总线控制系统的模拟移动床色谱纯化分离速度较快,能够有效满足多功能色谱分离装置精准化控制的操作需求.     

Immobilization of antibodies in micropatterns for cell detection by optical diffraction

Optical diffraction at biochemically microstructured surfaces has been investigated for the label-free in situ detection of cells. The new sensor concept is based on regular arrays of covalently coupled antibodies, which selectively bind cells from solution. Due to the adsorption process, changes are imposed on the intensity distribution of the diffracted light, which can serve to quantify the amount of adsorbed cells. For the formation of such microstructures, different classical film preparation techniques were transferred to a mesoscopic scale by the use of microcontact printing (μCP). Alternatively, receptors were functionalized with thiol groups prior to the immobilization process and directly printed onto the gold surface. Compared to imprinting of non-functionalized proteins on gold, a better replication of the micropatterns could be obtained. Additionally, a significantly lower amount of defects was observed than for the classical coupling techniques. Using such microstructures, first experiments on the detection of Escherichia coli bacteria were performed. Diffraction patterns have been observed for concentrations equal or higher than 10⁶ cells/ml. In time dependent experiments, diffraction spots occurred after 30 – 90 min or 10 – 20 min, depending on whether non-specific cell adsorption or specific binding to anti-E. coli IgG was studied. A first quantitative analysis of the diffraction patterns shows that the total amount of diffracted light increases with increasing incubation time.     

Application of radial basis function and feedforward artificial neural networks to the Escherichia coli fermentation process

Radial basis function and feedforward neural networks are considered for modelling of the recombinant Escherichia coli fermentation process. The models use industrial on-line data from the process as input variables in order to estimate the concentrations of biomass and recombinant protein, normally only available from off-line laboratory analysis. The models performances are compared by prediction error and graphical fit using results obtained from a common testing set of fermentation data.     

A portable,hand-powered microfluidic device for sorting of biological particles

Manually hand-powered portable microfluidic devices are cheap alternatives for point-of-care diagnostics. Currently, on-field tests are limited by the use of bulky syringe pumps, pressure controller and equipment. In this work, we present a manually operated microfluidic device incorporated with a groove-based channel. We show that the device is capable to effectively sort particles/cells by manual hand powering. First, the grooved-based channel with differently sized polystyrene particles was characterized using syringe pumps to study their distributions under various flow rate conditions. Afterward, the particle mixtures were sorted manually using hand power to verify the capability of this device. Finally, the manually operated device was used to sort platelets from peripheral blood mononuclear cells (PBMCs). The platelets were collected with a purity of ~ 100%. The purity of PBMCs was enhanced from 0.8 to 10.4% after multiple processes which results in an enrichment ratio of 13.8. During the process of manual hand pumping, the flow fluctuation caused by unstable injection will not influence the sorting performance. Due to its simplicity, this manually operated microfluidic chip is suitable for outfield settings.     

Classifying molecular sequences using a linkage graph with their pairwise similarities

This paper presents a method for classifying a large and mixed set of uncharacterized sequences provided by genome projects. As the measure of sequence similarity, we use similarity score computed by a method based on the dynamic programming (DP), such as the Smith-Waterman local alignment algorithm. Although comparison by DP based method is very sensitive, when given sequences include a family of sequences that are much diverged in evolutionary process, similarity among some of them may be hidden behind spurious similarity of some unrelated sequences. Also the distance derived from the similarity score may not be metric (i.e., triangle inequality may not hold) when some sequences have multi-domain structure. To cope with these problems, we introduce a new graph structure called p-quasi complete graph for describing a family of sequences with a confidence measure. We prove that a restricted version of the p-quasi complete graph problem (given a positive integer k, whether a graph contains a 0.5-quasi complete subgraph of which size k or not) is NP-complete. Thus we present an approximation algorithm for classifying a set of sequences using p-quasi complete subgraphs. The effectiveness of our method is demonstrated by the result of classifying over 4000 protein sequences on the Escherichia coli genome that was completely determined recently.     

Development and characterization of a capillary-flow microfluidic device for nucleic acid detection

We have developed a capillary flow-driven microfluidic biosensor to meet the needs of diagnostics for resource-limited areas. The device combined elements of lateral flow assays and microfluidic technology resulting in a hybrid with benefits of both formats. The biosensor was achieved by bonding two pieces of polymethyl methacrylate with channels ablated by a CO₂ laser, and enclosing an absorbent pad. The channels were UV/ozone treated to increase hydrophilicity which enabled capillary flow. The absorbent pad allowed for continuous flow in the channels once filled. The application of biosensor was demonstrated by detection of DNA with a sandwich assay. The target DNA was hybridized with nucleic acid modified magnetic beads as well as Ru(bpy) ₃ ²⁺ doped silica nanoparticles. Fluorescent signals were quantified in a holder fabricated to fit in a fluorescent microtiter plate reader. The capillary flow microfluidic was capable to detect 1?pmol target. The assay format which features rapid analysis and does not require the use of pumps could allow for inexpensive point of care diagnostics in the future.     

基于经济运行的一种交流电机节能装置

介绍一种自行研制开发的WRJB-45/380型微电脑控制电机多功能节电保护器,该装置是一个微电脑控制的相位、电流双闭环反馈系统。由晶闸管自动跟踪供电,能随时跟踪负载的变化大小,自动调节供电电压;避免电能的浪费,大大提高电机运行效率,最大限度地提高了电机的有功功率的比率,并且具有短路、过载度保护功能,真正做到一机多用,是电机理想的控制设备。该节能装置是企业能量降损、提高经济效益的理想选择,可广泛应用在石油、橡胶、冶金、矿山等领域,具有良好的市场前景,其经济效益也是相当可观的。     

DNA assembly on streptavidin modified surface: A study using quartz crystal microbalance with dissipation or resistance measurements

Quartz crystal microbalance techniques with either dissipation monitoring (QCM-D) or resistance measurement (RQCM) are employed to study the viscoelastic properties and the conformation of DNA molecular films assembled on gold electrodes modified by streptavidin. Two strategies are used for streptavidin immobilization on QCM gold electrode, namely (1) adsorption on biotin-containing thiol treated surface and (2) adsorption on biotinylated BSA (b-BSA) treated surface. The combinational analysis of frequency change (Δf) and energy losses (i.e. ΔD in QCM-D and ΔR in RQCM) shows that a double-stranded DNA (ds-DNA) film is more dissipative than a single-stranded DNA (ss-DNA) film, giving a bigger ΔD/Δf or ΔR/Δf value. Moreover, we affirm that the flexibility of the DNA film (or the viscoelasticity) is DNA surface density dependent. With these characterizations we demonstrate that the dissipation measured by the D factor of the QCM-D and the resistance (R) of the RQCM correlates with each other. We also evaluate the performance of streptavidin films immobilized using the biotin-thiol method and biotin-BSA method using RQCM. The simultaneously measured frequency (Δf) and resistance changes (ΔR) suggest that the streptavidin film assembled on the biotin-thiol treated surface is a better platform, being more efficient in capturing DNA that ensures a better sensitivity for DNA hybridization detection. To facilitate the discussion, surface plasmon resonance spectroscopy technique is used to monitor the assembly processes and to provide complementary results. The understanding of the basic DNA film properties would be of significance in DNA biosensor- and DNA chip-based studies.     

Characteristics of a label-free piezoelectric immunosensor detecting Pseudomonas aeruginosa

A label-free immunosensor system detecting a psychrophylic bacterium, Pseudomonas aeruginosa was developed as follows. Four types of anti-P. aeruginosa antibody were individually chemisorbed onto one-side gold electrodes of piezoelectric quartz crystals according to a thiolated antibody coupling procedure initiated with a thiol-cleavable heterobifunctional cross-linker, sulfosuccinimidyl-6-[3-(2-pyridyldithio)propionamido]hexanoate. A flow-type biosensor system was operated optimally at 0.2 M sodium potassium phosphate, pH 7.2 with a minimal matrix effect and the selected flow rate for it was 0.155 ml/min. A biosensor response was detected by measuring a steady-state resonant frequency shift after the response time around 8 min. The frequency shifts obtained were quite specific according to the antibody types and P. aeruginosa strains. The biosensor responses to varying concentrations of the P. aeruginosa cells ranging from 1.3×10⁷ to 1.3×10⁸ CFU/ml were determined as 17–176 Hz and a linear calibration curve (r=0.942) was obtained by plotting the responses in a double-logarithmic scale. The selectivity of the biosensor between P. aeruginosa and Xanthomonas spp. which both belong to the aerobic pseudomonads was, however, not so good owing to the property of the antibody used. The sensor chip could be reused at least seven times without an appreciable decrease in sensitivity.     

Miniaturized devices for isothermal DNA amplification addressing DNA diagnostics

Microfluidics is an emerging technology enabling the development of lab-on-a-chip systems for clinical diagnostics, drug discovery and screening, food safety and environmental analysis. Currently, available nucleic acid diagnostic tests take advantage of polymerase chain reaction that allows exponential amplification of portions of nucleic acid sequences that can be used as indicators for the identification of various diseases. At the same time, isothermal methods for DNA amplification are being developed and are preferred for their simplified protocols and the elimination of thermocycling. Here, we present a low-cost and fast DNA amplification device for isothermal helicase dependent amplification implemented in the detection of mutations related to breast cancer as well as the detection of Salmonella pathogens. The device is fabricated by mass production amenable technologies on printed circuit board substrates, where copper facilitates the incorporation of on-chip microheaters, defining the thermal zone necessary for isothermal amplification methods.     

基于MQTT协议与开源硬件的智能监控系统

实现一种采用MQTT协议、通过安卓/iOS APP对基于开源硬件-WRTnode的移动智能监控设备(如智能设备,机器人等)进行远程控制的方案.采用开源MQTT服务器框架-mosquitto作为MQTT broker,通过APP发布控制指令主题,监控设备订阅该主题并根据控制指令执行操作,监控设备发布需要上报的监控数据主题,APP订阅该主题后将接收到监控数据.通过双向的发布-订阅模式,实现多功能智能监控过程.     

Is DNA computing viable for 3-SAT problems?

Adleman reported how to solve a 7-vertex instance of the Hamiltonian path problem by means of DNA manipulations. After that a major goal of subsequent research is how to use DNA manipulations to solve NP-hard problems, especially 3-SAT problems. Lipton proposed DNA experiments on test tubes to solve 3-SAT problems. Liu et al. reported how to solve a simple case of 3-SAT using DNA computing on surfaces. Lipton's model of DNA computing is simple and intuitive for 3-SAT problems. The separate (or extract) operation, which is a key manipulation of DNA computing, only extracts some of the required DNA strands and Lipton thinks that a typical percentage might be 90. But it is unknown what would happen due to imperfect extract operation. Let p be the rate, where 0<p<1. Assume that for each distinct string s in a test tube, there are 10^l (l=13 proposed by Adleman) copies of s and that extracting each of the required DNA strands is equally likely. Here, the present paper will report, no matter how large l is and no matter how close to 1 p is, there always exists a class of 3-SAT problems such that DNA computing error must occur. Therefore, DNA computing is not viable for 3-SAT.     

Automated BIST for sequential logic synthesis

An automated built-in self-test (BIST) technique for general sequential logic is described that can be used directly at all levels of testing from device testing through system diagnostics. The technique selectively replaces existing system memory elements with BIST flip-flop cells, which it then connects to form a circular chain. Data are compacted and test patterns are generated simultaneously. The approach has been incorporated in a system for behavioral model synthesis to implement BIST in VLSI devices based on standard cells and in circuit packs based on PLDs, automatically. Seven production VLSI devices have been implemented with this automated BIST approach. Area overhead was between 6% and 19% for a fault coverage of 90%+ with the BIST capability alone     

A distributed device diagnostics system utilizing augmented reality and 3D audio

Augmented reality (AR), combining virtual environments with the perception of the real world, can be used to provide instructions for routine maintenance and error diagnostics of technical devices. The Rockwell Science Center (RSC) is developing a system that utilizes AR techniques to provide “X-ray vision” into real objects. The system can overlay 3D rendered objects, animations, and text annotations onto the video image of a known object. An automated speech recognition system allows the user to query the status of device components. The response is given as an animated rendition of a CAD model and/or as auditory cues using 3D audio. This diagnostics system also allows the user to leave spoken annotations attached to device modules as ASCII text. The position of the user/camera relative to the device is tracked by a computer-vision-based tracking system using fiducial markers. The system is implemented on a distributed network of PCs, utilizing standard commercial off-the-shelf components (COTS).     

Rapid detection of bacterial cell from whole blood: Integration of DNA sample preparation into single micro-PCR chip

A novel bacterial cell detection method from blood samples has been developed for molecular diagnostics. Functional integration of DNA sample preparation into polymerase chain reaction (PCR) chip enabled detection of pathogenic bacterial cells in a single microchip. Surface-modified micropillars possessing affinity for bacterial cells were fabricated inside a PCR chip, and reaction conditions were optimized to render the microchip with high surface-to-volume ratio PCR-compatible. After bacterial cells were captured on the micropillars from whole blood and PCR inhibitors were washed out, PCR mixture was injected to allow real-time amplification of DNA extracted from the isolated cells. Cell enrichment effect produced by volume reduction from large initial sample to small micro-PCR chip chamber led to increased detection sensitivity. Moreover, the developed method from sample preparation to detection of bacterial cells from whole blood took less than 1 h. These results demonstrated that the surface-modified pillar-packed microchip would be a practical approach for integration into Lab-On-a-Chip (LOC) to enable point-of-care genetic analysis.