Label-free detection of DNA hybridization using gold-coated tapered fiber optic biosensors (TFOBS) in a flow cell at 1310 nm and 1550 nm

We had previously reported the detection of a model protein bovine serum albumin (BSA) using antibody-immobilized tapered fiber optic biosensors (TFOBS) at 1310 nm and 1550 nm under stagnant and flow conditions. Because of recent interest in pathogen detection based on DNA, in this work we explore the application of these sensors for the detection of single stranded DNA (ssDNA). We show that it is feasible to directly detect the hybridization of a 10-mer ssDNA to its complementary strand immobilized on the sensor surface. Detection was performed under flow conditions because flow reduces non-specific binding to sensor surface, eliminates optical transmission changes due to mechanical movements, and allows for instantaneous switching of samples when needed.

TFOBS were fabricated with waist diameters of 5–10 μm and total lengths of 1000–1200 μm. The taper regions were coated with 50 nm of gold and housed in a specially constructed holder which served as a flow cell. The TFOBS was immobilized with 15-mer ssDNA with a C₆ extension and a thiol group, which attaches to Au1 1 1 sites. Then, the complementary 10-mer ssDNA samples were allowed to flow in from low to high concentration (750 fM to 7.5 nM) and the resulting transmission changes were recorded. It is shown that 750 fM of complementary DNA can be detected. This sensor was able to distinguish between complementary DNA from DNA with a single nucleotide mismatch in the middle position.     

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.     

Interfacial hybridization kinetics of oligonucleotides immobilized onto fused silica surfaces

Fused silica optical fibers have been used in an intrinsic mode optical configuration as biosensors for fluorescence based detection of hybridization of nucleic acids. In this work, the kinetics of hybridization of single-stranded oligonucleotides that were covalently immobilized were studied. The probe DNA was dT₂₀, and the target was Fluorescein-labeled non-complementary (dT₂₀) or complementary (dA₂₀) oligonucleotide. Chronofluorimetric monitoring of the adsorption and hybridization processes was used to investigate oligonucleotide films of different density, in different salt concentrations, at temperatures of 25 and 40 °C, with the concentration of the target DNA being 0.005–0.1 μM. Mathematical models based on first- and second-order Langmuir adsorption have been examined to describe both the adsorption and the hybridization processes. Experimental data were processed using the models, and the hybridization kinetics were calculated. Hybridization kinetics on these optical fiber DNA sensors was found to be up to three orders faster than results presented for a number of other experiments using different immobilization chemistries.     

Preparation of an electrochemical PNA biosensor for detection of target DNA sequence and single nucleotide mutation on p53 tumor suppressor gene corresponding oligonucleotide

Development of an electrochemical biosensor based on peptide nucleic acid (PNA) probe for detection of target DNA sequence and single nucleotide mutation in p53 tumor suppressor gene corresponding oligonucleotide using methylene blue (MB) as an electrochemical indicator is described. The interaction between MB and short sequence of p53, one of the most important tumor suppressor genes due to its dysfunction in the majority of human cancers, was studied by differential pulse voltammety (DPV). Probe modified electrode was prepared by self-assembled monolayer (SAM) formation of thiolated PNA molecules on the surface of gold electrode (GE). The hybridization of PNA probe with target DNA was performed in solution to form PNA-DNA hybrid on the surface of the GE. A significant increase in the reduction signal of MB was observed upon hybridization of the probe with the complementary DNA. The selectivity of the biosensor was studied using noncomplementary oligonucleotides. Furthermore, our results confirmed the ability of the sensor to detect single base mismatch in the sample oligonucleotide. The influence of probe concentration on the effective discrimination against noncomplementary sequence and point mutation was also investigated. Diagnostic performance of the biosensor is described and the detection limit is found 6.82 × 10⁻¹⁰ M. The electrochemical impedance spectroscopy was also employed to further investigate the sensor function.     

Detection of 3,4-methylenedioxymethamphetamine (MDMA, ecstasy) by displacement of antibodies

A molecular layer with low non-specific binding enabling determination of low concentrations of 3,4-methylenedioxymethamphetamine (MDMA) by the displacement of antibodies has been developed. Antibody Fab′-fragments at various concentrations have been site-directly immobilised on gold and intercalated with a hydrophilic non-ionic polymer that reduces non-specific binding. Bovine serum albumin conjugated with MDMA and various concentrations of anti-MDMA antibodies were bound to the layer. The amount of conjugates and antibodies bound was dependent on the amount of Fab′-fragments in the layer. Antibodies were also bound to the conjugates physisorbed directly onto the gold surface and in mixtures with the polymer or with a lipoamide. A high displacement of antibodies was observed by surface plasmon resonance (SPR) on interaction of MDMA with the different layers in buffer solution. No displacement could, however, be observed in saliva with the pure conjugate layer because of a high non-specific binding of proteins. When the conjugates were coupled to the surface through the antibody Fab-fragment/polymer layer, MDMA concentrations as low as 0.02 ng mL⁻¹ (0.14 nM) could easily be detected in buffer. In diluted saliva the lowest limit of detection was 0.4 ng mL⁻¹ enabling determination of drugs from saliva with a cut-off concentration of 2 ng mL⁻¹. The molecular layer of antibody Fab′-fragments and polymer thus shows great potential for binding conjugates and antibodies that can be displaced on the interaction with very low concentrations of small-sized molecules. A low non-specific binding is guaranteed by the presence of the hydrophilic polymer.     

Composing web services enacted by autonomous agents through agent-centric contract net protocol

ContextAgents are considered as one of the fundamental technologies underlying open and dynamic systems that are largely enabled by the semantic web and web services. Recently, there is a trend to introduce the notion of autonomy empowered by agents into web services. However, it has been argued that the characteristics of autonomy will make agents become available intermittently and behave variedly over time, which therefore increase the complexity on devising mechanisms for composing services enacted by autonomous agents.ObjectiveIn this work, we propose an extension to Contract Net protocol, called Agent-centric Contract Net Protocol (ACNP), as a negotiation mechanism with three key features for composing web services enacted by autonomous agents.Method(1) A matchmaking mechanism embedded in a middle agent (as a service matchmaker) for discovering web services that are available intermittently is presented based on the concept of agent roles; (2) A selection algorithm based on risk-enabled reputation model (REAL) embedded in a manager agent (as a service composer) is introduced to serve a basis for selecting web services with variant performance; and (3) A negotiation mechanism between a manager agent and contractor agents (as atomic services) is devised and enables both a service composer and the atomic services to request, refuse or agree on adapting changes of services.ResultsThe problem of assembling a computer is discussed in this paper.ConclusionIt is increasingly recognised that web services would become more autonomous by introducing diverse agent technologies to better constitute more complex systems in open and dynamic environments. As web service technologies are best exploited by composite services, it is imperative to devise mechanisms for composing services of autonomy.     

Electrochemical DNA biosensor for the detection of interaction between di[azino-di(5,6-azafluorene)-κ-NN′]dichlormanganous and DNA

A new Mn(II) complex of MnL₂Cl₂ (L = azino-di(5,6-azafluorene)-κ²-NN′) was synthesized and utilized as an electrochemical indicator for the determination of hepatitis B virus (HBV) based on its interaction with MnL₂Cl₂. The electrochemical behavior of interaction of MnL₂Cl₂ with salmon sperm DNA was investigated on glassy carbon electrode (GCE). In the presence of salmon sperm DNA, the peak current of [MnL₂]²⁺ was decreased and the peak potential was shifted positively without appearance of new peaks. The binding ratio between [MnL₂]²⁺ and salmon sperm DNA was calculated to be 2:1 and the binding constant was 3.72 × 10⁸ mol² L⁻². The extent of hybridization was evaluated on the basis of the difference between signals of [MnL₂]²⁺ with probe DNA before and after hybridization with complementary sequence. Control experiments performed with non-complementary and mismatch sequence demonstrated the good selectivity of the biosensor. With this approach, a sequence of the HBV could be quantified over the range from 1.76 × 10⁻⁸ to 1.07 × 10⁻⁶ mol L⁻¹, with a linear correlation of r = 0.9904 and a detection limit of 6.80 × 10⁻⁹ mol L⁻¹. Additionally, the binding mechanism was preliminarily discussed. The mode of interaction between MnL₂Cl₂ and DNA was found to be primary intercalation binding.     

基于抗体包被纳米金和铜配合物共固定修饰电极的癌胚抗原安培免疫传感器研究

研制了基于抗体包被化学镀纳米金(AuNPs)和[Cu(bpy)2(ONO)]NO3配合物(CuL)共固定修饰玻碳电极(GCE)的安培免疫传感器,并用于血清中癌胚抗原(CEA)的检测。首先将GCE电极表面氧化形成羧基,进而键合上乙二胺。将此胺化电极浸泡在CuL和化学镀金溶液后,CuL可通过π-π堆积作用吸附到GCE表面,并在电极表面还原成30~50nm的纳米金层(GCE|CuL/AuNPs)。将上述电极浸泡在CEA抗体(anti CEA)溶液中,利用AuNPs固定anti CEA,并通过辣根过氧化物酶(HRP)封闭剩余的AuNPs位点,由此构建了一类快速检测CEA的无试剂安培免疫传感器(GCE|CuL-AuNPs/anti CEA-HRP)。其中CuL作为电子媒介体对过氧化脲(CP)有催化还原作用,而且HRP可增强这种作用。当该传感器在37℃下,含CEA的pH6.5PBS溶液中温育30min后,随着温育液中CEA浓度的增加,电极表面形成的免疫复合物也增加,导致CuL对CP的催化电流下降。电流下降百分比1%与CEA浓度在0.1~80ng/mL之间成线性关系,检测限为0.052ng/mL(3σ)。由于采用化学镀法可...     

Concentration-enhanced rapid detection of human chorionic gonadotropin (hCG) on a Au surface using a nanofluidic preconcentrator

Here, we report a new method of concentration-enhanced binding kinetics for a rapid immunoassay screening test on a gold surface in a poly(dimethylsiloxane) (PDMS) microfluidic chip format. The use of alkylthiolate self-assembled monolayers on gold surfaces of a PDMS-glass microchip resulted in accelerated binding kinetics of Human chorionic gonadotropin (hCG) at an electrokinetic trapping zone. We used a PBS solution (buffer concentration ~ 150 mM), not a dibasic buffer system (~10 mM), for the dynamic preconcentrating operation and the preconcentration of cy3 labeled streptavidin onto biotinylated Au surface revealed that the binding kinetics of the protein were linearly proportional to the concentration profile of the preconcentration plug. We showed rapid detection of hCG in the clinical range with a shorten assay time of 10 min. Also, we demonstrated that the amount of sample needed were detection was decreased from ~4 mL to ~25 μL in the standard serum tests. The enhanced binding kinetics between hcG Ag-Ab via preconcentration showed good feasibility for use in a rapid immunoassay screening test.     

Multi-agent programming contest 2017: BusyBeaver team description

This paper describes the team BusyBeaver, that participated in and won the Multi-Agent Programming Contest 2017. Its strategy is based on dividing agents into three static groups modeling the work chain of buying, assembling and delivering items. The team is coordinated by a centralized agent doing most of the high-level planning, usually using greedy algorithms and specialized heuristics. There is a heavy focus on proactively buying and assembling some items, in order to quickly complete upcoming jobs.     

基于树状高分子的DNA电化学传感器对禽流感病毒的检测

将G4 PAMAM固定在玻碳电极表面,然后通过共价作用固定禽流感病毒探针ssDNA-1,以[Co(phen)3]3+为指示剂,采用示差脉冲伏安法和交流阻抗法对DNA电化学生物传感器进行了表征.结果发现,通过与双链dsDNA作用的[Co(phen)3]3+的峰电流信号的变化,可以识别和定量检测溶液中互补的禽流感病毒DNA片段.经过条件优化,该法测定DNA的浓度线性范围为1.3×10-9～6.5×10-8 mol/L,最低检测限为9.2×10-10 mol/L.     

Integrate software agents and CORBA in computational grid

This paper presents an Agent-based Computational Grid (ACG), which applies the concept of CORBA and agent to computational grid. The ACG system is used to implement a uniform higher level management of the computing resources and services on the Grid, and provide users with a consistent and transparent interface for accessing such services. In ACG grid, grid services are implemented by CORBA or by grid agent. Grid agents and CORBA objects will interact with each other to achieve user's service request. Our solution is the creation of a bridge between the CORBA and grid agents. The solution provides with the opportunity of considering an agent as a CORBA service and accessing CORBA services even from a grid agent. Thus, in AGC grid, existing legacy systems can be easily exploited as grid services. In this paper, firstly, the features of ACG grid are described, and then the design and implementation are given. Finally, some conclusions are given.     

Development of an integrated CMOS DNA detection biochip

This paper presents a CMOS DNA detection biochip using an electrical detection method with self-assembly multilayer gold nanoparticles (AuNPs). Each measuring spot of this biochip consists of three major parts; a pair of electrodes with a nanogap, a current amplifier circuit, and a heater with an embedded temperature sensor. The biochip is first fabricated by a TSMC (Taiwan Semiconductor Manufacturing Company Ltd.) 0.35 μm 2P4M standard CMOS process. Then, post-CMOS micromachining etch processes are used to expose the surface of the nanogap to test samples for the establishment of multilayer AuNPs through hybridization between single strand DNAs in the samples. The gap distance between a pair of electrodes is 350 nm. Before taking DNA detection measurements, self-assembly monolayer AuNPs is established on the nanogap surface between two microelectrodes. Multilayer AuNPs can be observed if hybridization between single strand DNAs occurs. An approximately 1000-fold increase in electric current between the multilayer AuNPs over the monolayer AuNPs serves an indication of the presence of target DNA in test samples. After integrating the electrodes with an embedded current amplifier, the electric current of multilayer AuNPs is amplified to the order of mA that can be easily measured by a commercial Volt-Ohm-Milliammeter. The heating system with a heating element and a temperature sensor can be used to distinguish single base-pair mismatch hybridization from complementary hybridization for the establishment of multilayer AuNPs. The lowest detectable concentration of target DNA on this biochip is 0.1 nM.     

A novel agent-based concept of household appliances

Mass production, such as white goods manufacturing, is traditionally bound to hierarchical factory-floor procedures and accepts only gradual changes in technology and product architecture. This paper introduces an idea on how to upgrade from classical to network-connected reconfigurable devices. A generic multi-agent architecture was created, derived from belief-desire-and-intention (BDI) agents. It covers all types of white goods in the form of rational home assistant, and enables reconfiguration of agent-based household appliances during the design, production and implementation phases. The introduced concept involves a multi-agent architecture which utilizes distributed processing power at different levels: higher-level agents run on more powerful devices than embedded appliance’s controllers, personal assistant (PDAs), or Windows or Linux based personal computers (PCs). PDAs can run a single agent, for example a GUI agent, whereas the embedded controllers execute lower-level device (embedded) agents. In this way, all the appliance’s basic functionality, such as its hardware units (e.g., electrical motors, valves, heaters, etc.), are initially simulated by auxiliary agents running together with higher-level agents on a PC or PDA. Using this simulator in the design phase, all vital functions and capabilities of the agent-based appliance under development are thoroughly tested first. Afterwards, the agents that simulate the device’s hardware units and environment are simply replaced by the communication to the corresponding device units. In such way, any new functionality or device’s behaviour can be upgraded any time just by adapting the core of the multi-agent architecture on the PC and individual agents on the PDA or the embedded agents in appliances. A thorough design and implementation cycle of the proposed solution using two freeware development tools is also described, i.e., the Prometheus agent design methodology and the agent simulation/execution environment called Jadex. The approach is exemplified by building a simulator of an agent-based household appliance, namely a Multi-agent Washing Assistant as a special instance of rational home assistant.     

A Method for Fabrication of Polycarbonate-Based Bioactive Platforms

Surface-based assays have been used extensively for the functional and structural analysis of biomolecules such as DNA or proteins. These experiments are established by the analysis of binding between acceptor molecules and immobilized receptors on a platform. Site-specific printing of receptor molecules on gold, glass, or polycarbonate (PC) surfaces is conventionally performed by the chemical derivatization of a surface, priming it to covalently bind to subsequently deposited receptor molecules. Unlike conventional methods, we have developed a new fabrication method for bioactive PC surfaces by directly molding PC granules doped with receptor molecules. PC-based receptor molecules were synthesized and commercially available PC granules were doped with these synthesized molecules. In our proof-of-concept study, PC doped with dye 1 (Fig. 1) was used as the receptor molecule. Using an aluminum mold and a hot press machine, PC-based objects were manufactured through compression molding using doped PC granules. Affinity analysis was evaluated by monitoring the localization of a monoclonal antibody elicited against dye 1 to the surface of the molded platforms by fluorescence microscopy. The results illustrated effective binding of an anti-dye 1 monoclonal antibody to the surface, substantiating successful display of assemblies of molecular receptors on the surface through compression molding. Although conventional surface functionalization methods impose limited applications and alter desired opto-mechanical properties of the polymer, our investigation provides a versatile means for the fabrication of bioactive PC-based platforms. It can also be used for engineering and imbedding receptor arrays within three-dimensional objects with applications to the production of opto-medical devices or biosensors.     

Electric field-induced concentration and capture of DNA onto microtips

Simple, high-yield concentration of DNA is important for high-throughput genetic analysis and disease diagnosis. Glass-based microfilters are popular but the process requires centrifugation steps with cumbersome chemical processes. As an alternative, a concentration method using an electric field has been explored previously, but with limited efficiency. In this paper, electric field-induced concentration and capture of DNA are studied by using high-aspect-ratio microtips coated with a gold layer. The microtips are immersed longitudinally into a solution of 100???L containing ??-phage DNA. After DNA concentration using an electric field, the microtips are withdrawn from the solution. Under AC- and biased AC fields, DNA is concentrated by electrophoresis (EP), dielectrophoresis (DEP), and electroosmotic flow (EOF). To reduce capillary effects in the withdrawal process, the microtips are coated with positively charged poly-l-lysine (PLL). The pattern of captured DNA is analyzed by fluorescence microscopy. DEP attracts DNA molecules at the edges of microtips, where the highest gradient of electric field exists. EP attracts DNA onto the surface of microtips following the vectors of an electric field. EOF generates vortexes that deliver DNA onto microtips. Using this method, 85% of DNA is captured on the PLL-coated microtips after three sequential captures. The concentration mechanism can potentially facilitate rapid and simple preparation of DNA for downstream analysis.     

Learning collaboration strategies for committees of learning agents

A main issue in cooperation in multi-agent systems is how an agent decides in which situations is better to cooperate with other agents, and with which agents does the agent cooperate. Specifically in this paper we focus on multi-agent systems composed of learning agents, where the goal of the agents is to achieve a high accuracy on predicting the correct solution of the problems they encounter. For that purpose, when encountering a new problem each agent has to decide whether to solve it individually or to ask other agents for collaboration. We will see that learning agents can collaborate forming committees in order to improve performance. Moreover, in this paper we will present a proactive learning approach that will allow the agents to learn when to convene a committee and with which agents to invite to join the committee. Our experiments show that learning results in smaller committees while maintaining (and sometimes improving) the problem solving accuracy than forming committees composed of all agents.     

Development of an ultrafast quantitative heterogeneous immunoassay on pre-functionalized poly(dimethylsiloxane) microfluidic chips for the next-generation immunosensors

Although the reaction time for antigen-antibody binding has been greatly reduced in microchannels, other processes in heterogeneous immunoassays (HEIs), such as blocking and antigen adsorption have not benefited from miniaturization as a reduction in size to micro dimensions does not increase the speed of these processes significantly. The overall assay time of reported microfluidic HEIs has continued to be limited by these processes. In this study, we successfully develop an ultrafast quantitative HEI with pre-functionalized microfluidic poly(dimethylsiloxane) (PDMS) chips. The protein A functionalized PDMS surface is found to be highly effective in reducing the antigen adsorption time in microchannels. The functionalized surfaces can be stable at least for 2.5 months when stored at 4°C in a buffer solution consisting of 10 mM Tris, 0.05% bovine serum albumin, 0.05% Proclin 300, and 5% glycerol. In addition, the immunosorption process, which is substantially accelerated in micro scale, results in a significant reduction in nonspecific binding. The time of blocking step can therefore be reduced to a minimum or can be eliminated. The overall assay for detecting bovine immunoglobulin G is completed in 19 min with a limit of detection of 3.8 nM. The ultrafast analysis time and superior sensitivity demonstrated by this microfluidic HEI is promising for being used to develop the next-generation immunosensors.     

A rapid polymerisation process realizing 3D biocompatible structures in a microfluidic channel suitable for genetic analysis

Surface probe immobilisation is a complex and time consuming task undertaken prior to microfluidic integration, this requires surface functionalisation, biomolecule spotting, incubation and blocking steps. Traditional bonding techniques (anodic, thermal, etc.) or adhesives (UV cured) used to seal fluidic systems may denature biomolecules due to high temperature or vapour effects, thus bonding techniques such as thin film laminate or PDMS are used to seal systems, with substrate-fluidic alignment required prior to bonding. We propose a technique allowing probe DNA molecules to be immobilised in a sealed microfluidic system using (3D) hydrogel structures without any alignment steps. A prepolymer solution is introduced to the channels where photo-polymerisation is undertaken forming 3D structures covalently attached to the channel surface. We use a photo-initiated prepolymer material poly-ethylene-glycol (PEG) to form structures containing probe DNA. This process is fast compared to conventional biomolecule immobilisation techniques and is also biocompatible, this direct write approach removes overnight immobilisation/incubation of the probe DNA, it also facilitates immobilisation within a sealed fluidic system where conventionally DNA probe spots must be immobilised prior to channel sealing. We consider the transport of target DNA from bulk analyte to the 3D gel structure and evaluate hybridisation within the microfluidic system.     

Protecting mobile-agent data collection against blocking attacks

Full-scale adoption of mobile agent technology in untrustworthy network environment, such as Internet, has been delayed due to several security complexities. The protection of mobile agents against the attacks of malicious hosts is considered a very challenging security problem. It has inspired lot of research interest, but very few measures exist to counter blocking attack where a host with malicious intentions refuses to transmit a mobile agent to the next host. It becomes an important requirement for the agent owner to rescue the data collected by the agent under custody and redeem a loss. In this paper, we present two schemes that rescue the offering results from a malicious host's blocking attack, and make a comparison of their performance from several aspects. Our approach has two new features that previous protocols lack. It allows the proper handling of time-sensitive offers and supports the gradual decision-making execution.