共查询到20条相似文献,搜索用时 31 毫秒
1.
This article discusses mechanisms and principles for assignment of moral responsibility to intelligent robots, with special focus on military robots. We introduce the concept autonomous power as a new concept, and use it to identify the type of robots that call for moral considerations. It is furthermore argued that autonomous power, and in particular the ability to learn, is decisive for assignment of moral responsibility to robots. As technological development will lead to robots with increasing autonomous power, we should be prepared for a future when people blame robots for their actions. It is important to, already today, investigate the mechanisms that control human behavior in this respect. The results may be used when designing future military robots, to control unwanted tendencies to assign responsibility to the robots. Independent of the responsibility issue, the moral quality of robots’ behavior should be seen as one of many performance measures by which we evaluate robots. How to design ethics based control systems should be carefully investigated already now. From a consequentialist view, it would indeed be highly immoral to develop robots capable of performing acts involving life and death, without including some kind of moral framework. 相似文献
2.
Research has shown that university technology transfer offices (TTOs) learn through experimentation and failure, and by sharing these experiences with others. There are many barriers to successfully sharing the best practice between TTOs. The Maturity Model (MM) created by Secundo et al. (Meas Bus Excell, 20:42–54, 2016) provides a means by which the performance of a TTO can be better understood to allow for effective sharing of best practices. The aim of this study is to improve and validate the MM to formalize a mechanism through which best practices can be identified and shared between TTOs. This was accomplished by testing the MM in 54 TTOs across Europe and the United Kingdom. Findings regard several improvements of the intangible indicators and the maturity levels of the MM. This research improves the rigor of the MM and formalizes its application as a mechanism for sharing best practices through the Improved MM. 相似文献
3.
Rami Ali 《Ethics and Information Technology》2015,17(4):267-274
Luck (2009) argues that gamers face a dilemma when it comes to performing certain virtual acts. Most gamers regularly commit acts of virtual murder, and take these acts to be morally permissible. They are permissible because unlike real murder, no one is harmed in performing them; their only victims are computer-controlled characters, and such characters are not moral patients. What Luck points out is that this justification equally applies to virtual pedophelia, but gamers intuitively think that such acts are not morally permissible. The result is a dilemma: either gamers must reject the intuition that virtual pedophelic acts are impermissible and so accept partaking in such acts, or they must reject the intuition that virtual murder acts are permissible, and so abstain from many (if not most) extant games. While the prevailing solution to this dilemma has been to try and find a morally relevant feature to distinguish the two cases, I argue that a different route should be pursued. It is neither the case that all acts of virtual murder are morally permissible, nor are all acts of virtual pedophelia impermissible. Our intuitions falter and produce this dilemma because they are not sensitive to the different contexts in which games present virtual acts. 相似文献
4.
Raffaele Rodogno 《Ethics and Information Technology》2016,18(4):257-268
I examine the nature of human-robot pet relations that appear to involve genuine affective responses on behalf of humans towards entities, such as robot pets, that, on the face of it, do not seem to be deserving of these responses. Such relations have often been thought to involve a certain degree of sentimentality, the morality of which has in turn been the object of critical attention (Sparrow in Ethics Inf Technol 78:346–359, 2002; Blackford in Ethics Inf Technol 14:41–51, 2012). In this paper, I dispel the claim that sentimentality is involved in this type of relations. My challenge draws on literature in the philosophy of art and in cognitive science that attempts to solve the so called paradox of fictional emotions, i.e., the seemingly paradoxical way in which we respond emotionally to fictional or imaginary characters and events. If sentimentality were not at issue, neither would its immorality. For the sake of argument, however, I assume in the remaining part of the paper that sentimentality is indeed at play and bring to the fore aspects of its badness or viciousness that have not yet been discussed in connection with robot pets. I conclude that not even these aspects of sentimentality are at issue here. Yet, I argue that there are other reasons to be worried about the wide-spread use of ersatz companionship technology that have to do with the potential loss of valuable, self-defining forms of life. 相似文献
5.
In this research, we are interested in ERP systems which are common information repositories that are aimed at matching the knowledge, practices, and skills that drive the organization in the best possible way. Can the cognitive and hierarchical models coexist within the same project? What is the impact of ERP on the interconnection between communities? To answer these questions, we rely in particular on the work of Levina and Vaast (MIS Quarterly 29(2):335–363, 2005), which underlines that the modes of interaction between CPs must be mediated by the activation of boundary objects and/or the mobilization of boundary spanners. Finally, this leads us to discriminate between two types of ERPPs (hierarchical/cognitive) and to underline the role of the switch in the ERPP success. 相似文献
6.
Iyad Rahwan 《Ethics and Information Technology》2018,20(1):5-14
Recent rapid advances in Artificial Intelligence (AI) and Machine Learning have raised many questions about the regulatory and governance mechanisms for autonomous machines. Many commentators, scholars, and policy-makers now call for ensuring that algorithms governing our lives are transparent, fair, and accountable. Here, I propose a conceptual framework for the regulation of AI and algorithmic systems. I argue that we need tools to program, debug and maintain an algorithmic social contract, a pact between various human stakeholders, mediated by machines. To achieve this, we can adapt the concept of human-in-the-loop (HITL) from the fields of modeling and simulation, and interactive machine learning. In particular, I propose an agenda I call society-in-the-loop (SITL), which combines the HITL control paradigm with mechanisms for negotiating the values of various stakeholders affected by AI systems, and monitoring compliance with the agreement. In short, ‘SITL = HITL + Social Contract.’ 相似文献
7.
Pille Pruulmann-Vengerfeldt 《The Information Society》2013,29(5):303-310
Using Layder's domain theory (1997) as an analytical framework, this article shows how the information society can be measured through various levels of society. Layder's notions of psychobiography, situated activity, social setting, and contextual resources help identify cultural and social indicators for understanding changes in the information society. With the help of empirical indicators for each domain, this article uses the case of Estonia to show that there is often more to the information society than what is captured by traditional measures. This article calls for a context-sensitive approach, which takes into consideration social and cultural indicators. Measurements from all four domains are necessary for understanding the complexity of information-society-related issues. 相似文献
8.
Bipolar membranes (BMs) have interesting applications within the field of bioelectronics, as they may be used to create non-linear ionic components (e.g., ion diodes and transistors), thereby extending the functionality of, otherwise linear, electrophoretic drug delivery devices. However, BM based diodes suffer from a number of limitations, such as narrow voltage operation range and/or high hysteresis. In this work, we circumvent these problems by using a novel polyphosphonium-based BM, which is shown to exhibit improved diode characteristics. We believe that this new type of BM diode will be useful for creating complex addressable ionic circuits for delivery of charged biomolecules.Combined electronic and ionic conduction makes organic electronic materials well suited for bioelectronics applications as a technological mean of translating electronic addressing signals into delivery of chemicals and ions.1 For complex regulation of functions in cells and tissues, a chemical circuit technology is necessary in order to generate complex and dynamic signal gradients with high spatiotemporal resolution. One approach to achieve a chemical circuit technology is to use bipolar membranes (BMs), which can be used to create the ionic equivalents of diodes2, 3, 4, 5 and transistors.6, 7, 8 A BM consists of a stack of a cation- and an anion-selective membrane, and functions similar to the semiconductor PN-junction, i.e., it offers ionic current rectification9, 10 (Figure (Figure1a).1a). The high fixed charge concentration in a BM configuration make them more suited in bioelectronic applications as compared to other non-linear ionic devices, such as diodes constructed from surface charged nanopores11 or nanochannels,12 as the latter devices typically suffers from reduced performance at elevated electrolyte concentration (i.e., at physiological conditions) due to reduced Debye screening length.13 However, a unique property of most BMs, as compared to the electronic PN-junction and other ionic diodes, is the electric field enhanced (EFE) water dissociation effect.10, 14 This occurs above a threshold reverse bias voltage, typically around −1 V, as the high electric field across the ion-depleted BM interface accelerates the forward reaction rate of the dissociation of water into H+ and OH− ions. As these ions migrate out from the BM, there will be an increase in the reverse bias current. The EFE water dissociation is a very interesting effect and is commonly used in industrial electrodialysis applications,15 where highly efficient water dissociating BMs are being researched.16 Also, BMs have also been utilized to generate H+ and OH− ions in lab-on-a-chip applications.2, 17 However, the EFE water dissociation effect diminishes the diode property of BMs when operated outside the ±1 V window, which is unwanted in, for instance, chemical circuits and addressing matrices for delivery of complex gradients of chemical species. The effect can be suppressed by incorporating a neutral electrolyte inside the BM,10, 18 for instance, poly(ethylene glycol) (PEG).2, 6, 7 However, as previously reported,2 the PEG volume will introduce hysteresis when switching from forward to reverse bias, due to its ability to store large amounts of charges. This was circumvented by ensuring that only H+ and OH− are present in the diode, which recombines into water within the PEG volume. Such diodes are well suited as integrated components in chemical circuits for pH-regulation, due to the in situ formed H+ and OH−, but are less attractive if, for instance, other ions, e.g., biomolecules, are to be processed or delivered in and from the circuit. Furthermore, a PEG electrolyte introduces additional patterning layers, making device downscaling more challenging. This is undesired when designing complex, miniaturized, and large-scale ionic circuits. Thus, there is an interest in BM diodes that intrinsically do not exhibit any EFE water dissociation, are easy to miniaturize, and that turn off at relatively high speeds. It has been suggested that tertiary amines in the BM interface are important for efficient EFE water dissociation,19, 20, 21 as they function as a weak base and can therefore catalyze dissociation of water by accepting a proton. For example, anion-selective membranes that have undergone complete methylation, converting all tertiary amines to quaternary amines, shows no EFE water dissociation,19 although this effect was not permanent, as the quaternization was reversed upon application of a current. Similar results were found for anion-selective membranes containing alkali-metal complexing crown ethers as fixed charges.21 Also, EFE water dissociation was not observed or reduced in BMs with poor ion selectivity, for example, in BMs with low fixed-charge concentration5 or with predominantly secondary and tertiary amines in the anion-selective membrane,22 as the increased co-ion transport reduces the electric field at the BM interface. However, due to decreased ion selectivity, these membranes show reduced rectification. In this work, we present a non-amine based BM diode that avoids EFE water dissociation, enables easy miniaturization, and provides fast turn-off speeds and high rectification.Open in a separate windowFigure 1(a) Ionic current rectification in a BM. In forward bias, mobile ions migrate towards the interface of the BM. The changing ion selectivity causes ion accumulation, resulting in high ion concentration and high conductivity. At high ion concentration, the selectivity of the membranes fails (Donnan exclusion failure), and ions start to pass the BM. In reverse bias, the mobile ions migrate away from the BM, eventually giving a zone with low ion concentration and low conductivity. Reverse bias can cause EFE water dissociation, producing H+ and OH- ions. (b) Chemical structures of PSS, qPVBC, and PVBPPh3. (c) The device used to characterize the BMs and the BM1A, BM2A, and BM1P designs. The BM interfaces are 50 × 50 μm.An anion-selective phosphonium-based polycation (poly(vinylbenzyl chloride) (PVBC) quaternized by triphenylphospine, PVBPPh3) was synthesized and compared to the ammonium-based polycation (PVBC quaternized by dimethylbenzylamine, qPVBC) previously used in BM diodes2 and transistors,7, 8 when included in BM diode structures together with polystyrenesulfonate (PSS) as the cation-selective material (Figure (Figure1b).1b). Three types of BM diodes were fabricated using standard photolithography patterning (Figure (Figure1c),1c), either with qPVBC (BM1A and BM2A) or PVBPPh3 (BM1P) as polycation and either with (BM2A) or without PEG (BM1A and BM1P). Poly(3,4-ethylenedioxythiophene):poly(styrenesulfonate) (PEDOT:PSS) electrodes covered with aqueous electrolytes were used to convert electronic input signals into ionic currents through the BMs, according to the redox reaction PEDOT+:PSS− + M+ + e− ↔ PEDOT0 + M+:PSS−.The rectifying behavior of the diodes was evaluated using linear sweep voltammetry (Figure (Figure2).2). The BM1A diode exhibited an increase in the reverse bias current for voltages lower than −1 V, a typical signature of EFE water dissociation,10, 14 which decreased the current rectification at ±4 V to 6.14. No such deviation in the reverse bias current was observed for BM2A and BM1P, which showed rectification ratios of 751 and 196, respectively. In fact, for BM1P, no evident EFE water dissociation was observed even at −40 V (see inset of Figure Figure2).2). Thus, the PVBPPh3 polycation allows BM diodes to operate at voltages beyond the ±1 V window with maintained high ion current rectification.Open in a separate windowFigure 2Linear sweep voltammetry from −4 to +4 V (25 mV/s) for the BM diodes. The inset shows BM1P scanning from −40 V to +4 V (250 mV/s).The dynamic performance of the diodes was tested by applying a square wave pulse from reverse bias to a forward bias voltage of 4 V with 5–90 s pulse duration (Figure (Figure3).3). To access the amount of charge injected and extracted during the forward bias and subsequent turn off, the current through the device was integrated. For BM2A, we observed that the fall time increased with the duration of the forward bias pulse. This hysteresis is due to the efficient storage of ions in the large PEG volume, with no ions crossing the BM due to Donnan exclusion failure.2 As a result, during the initial period of the return to reverse bias, a large amount of charge needs to be extracted in order to deplete the BM. After a 90 s pulse, 90.6% of the injected charge during the forward bias was extracted before turn-off. This may be contrasted with BM1P, where the fall time is hardly affected by the pulse duration, and the extracted/injected ratio is only 15.4% for a 90 s pulse. For this type of BM, the interface quickly becomes saturated with ions during forward bias, leading to Donnan exclusion failure and transport of ions across the BM.4 Thus, less charge needs to be extracted to deplete the BM, allowing for faster fall times and significantly reduced hysteresis.Open in a separate windowFigure 3Switching characteristics (5, 10, 20, 30, 60, or 90 s pulse) and ion accumulation (at 90 s pulse) of the BM2A and BM1P diodes. BM1A showed similar characteristics as BM1P when switched at ±1V (see supplementary material).24Since the neutral electrolyte is no longer required to obtain high ion current rectification over a wide potential range, the size of the BM can be miniaturized. This translates into higher component density when integrating the BM diode into ionic/chemical circuits. A miniaturized BM1P diode was constructed, where the interface of the BM was shrunk from 50 μm to 10 μm. The 10 μm device showed similar IV and switching characteristics as before (Figure (Figure4),4), but with higher ion current rectification ratio (over 800) and decreased rise/fall times (corresponding to 90%/–10% of forward bias steady state) from 10 s/12.5 s to 4 s/4 s. Since the overlap area is smaller, a probable reason for the faster switching times is the reduced amount of ions needed to saturate and deplete the BM interface. In comparison to our previous reported low hysteresis BM diode,2 this miniaturized polyphosphonium-based devices shows the same rise and fall times but increased rectification ratio.Open in a separate windowFigure 4(a) Linear sweep voltammetry and (b) switching performance of a BM1P diode with narrow junction.In summary, by using polyphosphonium instead of polyammonium as the polycation in BM ion diodes the EFE water dissociation can be entirely suppressed over a large operational voltage window, supporting the theory that a weak base, such as a tertiary amine, is needed for efficient EFE water dissociation.17, 18 As no additional neutral layer at the BM interface is needed, ion diodes that operate outside the usual EFE water dissociation window of ±1 V can be constructed using less active layers, fewer processing steps and with relaxed alignment requirement as compared to polyammonium-based devices. This enables the fabrication of ion rectification devices with an active interface as low as 10 μm. Furthermore, the exclusion of a neutral layer improves the overall dynamic performance of the BM ion diode significantly, as there is less hysteresis due to ion accumulation. Previously, the hysteresis of BM ion diodes has been mitigated by designing the diode so that only H+ and OH− enters the BM, which then recombines into water.2 Such diodes also show high ion current rectification ratio and switching speed but are more complex to manufacture, and their application in organic bioelectronic systems is limited due to the H+/OH− production. By instead using the polyphosphonium-based BM diode, reported here, we foresee ionic, complex, and miniaturized circuits that can include charged biomolecules as the signal carrier to regulate functions and the physiology in cell systems, such as in biomolecule and drug delivery applications, and also in lab-on-a-chip applications. 相似文献
9.
Anil Haraksingh Thilsted Vahid Bazargan Nina Piggott Vivien Measday Boris Stoeber 《Biomicrofluidics》2012,6(4)
A flow redirection and single cell immobilization method in a microfluidic chip is presented. Microheaters generated localized heating and induced poly(N-isopropylacrylamide) phase transition, creating a hydrogel that blocked a channel or immobilized a single cell. The heaters were activated in sets to redirect flow and exchange the fluid in which an immobilized cell was immersed. A yeast cell was immobilized in hydrogel and a 4′,6-diamidino-2-phenylindole (DAPI) fluorescent stain was introduced using flow redirection. DAPI diffused through the hydrogel and fluorescently labelled the yeast DNA, demonstrating in situ single cell biochemistry by means of immobilization and fluid exchange.The ability to control microfluidic flow is central to nearly all lab-on-a-chip processes. Recent developments in microfluidics either include microchannel based flow control in which microvalves are used to control the passage of fluid,1 or are based on discrete droplet translocation in which electric fields or thermal gradients are used to determine the droplet path.2, 3 Reconfigurable microfluidic systems have certain advantages, including the ability to adapt downstream fluid processes such as sorting to upstream conditions and events. This is especially relevant for work with individual biomolecules and high throughput cell sorting.4 Additionally, reconfigurable microfluidic systems allow for rerouting flows around defective areas for high device yield or lifetime and for increasing the device versatility as a single chip design can have a variety of applications.Microvalves often form the basis of flow control systems and use magnetic, electric, piezoelectric, and pneumatic actuation methods.5 Many of these designs require complicated fabrication steps and can have large complex structures that limit the scalability or feasability of complex microfluidic systems. Recent work has shown how phase transition of stimuli-responsive hydrogels can be used to actuate a simple valve design.6 Beebe et al. demonstrated pH actuated hydrogel valves.7 Phase transition of thermosensitive poly(N-isopropylacrylamide) (PNIPAAm) using a heater element was demonstrated by Richter et al.8 Phase transition was also achieved by using light actuation by Chen et al.9 Electric heating has shown a microflow response time of less than 33 ms.11 Previous work10 showed the use of microheaters to induce a significant shift in the viscosity of thermosensitive hydrogel to block microchannel flow and deflect a membrane, stopping flow in another microchannel. Additionally, Yu et al.12 demonstrated thermally actuated valves based on porous polymer monoliths with PNIPAAm. Krishnan and Erickson13 showed how reconfigurable optically actuated hydrogel formation can be used to dynamically create highly viscous areas and thus redirect flow with a response time of ~ 2?s. This process can be used to embed individual biomolecules in hydrogel and suppress diffusion as also demonstrated by others.15, 16 Fiddes et al.14 demonstrated the use of hydrogels to transport immobilized biomolecules in a digital microfluidic system. While the design of Krishnan and Erickson is highly flexible, it requires the use of an optical system and absorption layer to generate a geometric pattern to redirect flow.This paper describes the use of an array of gold microheaters positioned in a single layer polydimethylsiloxane (PDMS) microfluidic network to dynamically control microchannel flow of PNIPAAm solution. Heat generation and thus PNIPAAm phase transition were localized as the microheaters were actuated using pulse width modulation (PWM) of an applied electric potential. Additionally, hydrogel was used to embed and immobilise individual cells, exchange the fluid parts of the microfluidic system in order to expose the cells to particular reagents to carry out an in situ biochemical process. The PDMS microchannel network and the microheater array are shown in Figure Figure11.Open in a separate windowFigure 1A sketch of the electrical circuit and a microscope image of the gold microheaters and the PDMS microchannels. The power to the heaters was modulated with a PWM input through a H-bridge. For clarity, the electrical circuit for only the two heaters with gelled PNIPAAm is shown (H1 and V2). There are four heaters (V1-V4) in the “vertical channels” and three heaters (H1-H3) in the “horizontal” channel.The microchannels were fabricated using a patterned mould on a silicon wafer to define PDMS microchannels, as described by DeBusschere et al.17 and based on previous work.10 A 25 × 75 mm glass microscope slide served as the remaining wall of the microchannel system as well as the substrate for the microheater array. The gold layer had a thickness of 200 nm and was deposited and patterned using E-beam evaporation and photoresist lift-off.21 The gold was patterned to function as connecting electrical conductors as well as the microheaters.It was crucial that the microheater array was aligned with an accuracy of ~ 20μm with the PDMS microchannel network for good heat localization. The PDMS and glass lid were treated with plasma to activate the surface and alignment was carried out by mounting the microscope slide onto the condenser lens of an inverted microscope (TE-2000 Nikon Instruments). While imaging with a 4× objective, the x, y motorized stage aligned the microchannels to the heaters and the condenser lens was lowered for the glass substrate to contact the PDMS and seal the microchannels.Local phase transition of 10% w/w PNIPAAm solution in the microchannels was achieved by applying a 7 V potential through a H-bridge that received a PWM input at 500 Hz which was modulated using a USB controller (Arduino Mega 2650) and a matlab (Mathworks) GUI. The duty cycle of the PWM input was calibrated for each microheater to account for differences in heater resistances (25?Ω to 52?Ω) due to varying lengths of on-chip connections and slight fabrication inconsistencies, as well as for different flow conditions during device operation. Additionally, thermal cross-talk between heaters required decreasing the PWM input significantly when multiple heaters were activated simultaneously. This allowed confining the areas of cross-linked PNIPAAm to the microheaters, allowing the fluid in other areas to flow freely.By activating the heaters in sets, it was possible to redirect the flow and exchange the fluid in the central area. Figure Figure22 demonstrates how the flow direction in the central microchannel area was changed from a stable horizontal flow to a stable vertical flow with a 3 s response time, using only PNIPAAm phase transition. Constant pressures were applied to the inlets to the horizontal channel and to the vertical channels. Activating heaters V1-4 (Figure (Figure2,2, left) resulted in flow in the horizontal channel only. Likewise, activating heaters H1 and H2 allowed for flow in the vertical channel only. In this sequence, the fluid in the central microchannel area from one inlet was exchanged with fluid from the other inlet. Additionally, by activating heater H3, a particle could be immobilised during the exchange of fluid as shown in Figure Figure33 (top).Open in a separate windowFigure 2Switching between fluid from the horizontal and the vertical channel using hydrogel activation and flow redirection with a response time of 3 s. A pressure of 25 mbar was applied to the inlet of the horizontal channel and a pressure of 20 mbar to the vertical channel. The flow field was determined using particle image velocimetry, in which the displacement of fluorescent seed particles was determined from image pairs generated by laser pulse exposure. Processing was carried out with davis software (LaVision).Open in a separate windowFigure 3A series of microscope images near heater H3 showing: (1a)-(1c) A single yeast cell captured by local PNIPAAm phase transition and immobilized for 5 min before being released. (2a) A single yeast cell was identified for capture by embedding in hydrogel. (2b) The cell as well as the hydrogel displayed fluorescence while embedded due to the introduction of DAPI in the surrounding region. (2c) The diffusion of DAPI towards the cell as the heating power of H3 is reduced after 15 min, showing a DAPI stained yeast cell immobilized.Particle immobilisation in hydrogel and fluid exchange in the central area of the microfluidic network were used to carry out an in situ biochemical process in which a yeast cell injected through one inlet was stained in situ with a 4′,6-diamidino-2-phenylindole (DAPI) solution (Invitrogen), which attached to the DNA of the yeast cell.18 A solution of yeast cells with a concentration of 5 × 107cells/ml suspended in a 10% w/w PNIPAAm solution was injected through the horizontal channel. A solution of 2μg/l DAPI in a 10% w/w PNIPAAm solution was injected through the vertical channel. A single yeast cell was identified and captured near the central heater, and by deactivating the heaters in the vertical channel, DAPI solution was introduced in the microchannels around the hydrogel. After immobilising the cell for 15 min, the heater was deactivated, releasing the cell in the DAPI solution. This process is shown in Figure Figure33 (bottom). The sequence of the heater activation and deactivation in order to immobilize the cell and exchange the fluid is outlined in the supplementary material.21Eriksen et al.15 demonstrated the diffusion of protease K in the porous hydrogel matrix,19 and it was therefore expected that DAPI fluorescent stain (molecular weight of 350 kDa, Ref. 20) would also diffuse. DAPI diffusion is shown in Figure 3(2b) in which the yeast cell shows fluorescence while embedded in the hydrogel. The yeast cell was released by deactivating the central heater and activating all the others to suppress unwanted flow in the microchannel. As a result, the single cell was fully immersed in the DAPI solution. Immobilization of a single cell allows for selection of a cell that exhibits a certain trait and introduction of a new fluid while maintaining the cell position in the field of view of the microscope such that a biochemical response can be imaged continuously.In summary, a microfluidic chip capable of local heating was used to induce phase transition of PNIPAAm to hydrogel, blocking microchannel flow, and thereby allowing for reconfigurable flow. Additionally, the hydrogel was used to embed and immobilise a single yeast cell. DAPI fluorescent stain was introduced using flow redirection, and it stained the immobilized cell, showing diffusion into the hydrogel. The versatile design of this microfluidic chip permits flow redirection, and is suitable to carry out in situ biochemical reactions on individual cells, demonstrating the potential of this technology for forming large-scale reconfigurable microfluidic networks for biochemical applications. 相似文献
10.
Dingsheng Liu Bejan Hakimi Michael Volny Joelle Rolfs Robbyn K. Anand Frantisek Turecek Daniel T. Chiu 《Biomicrofluidics》2014,8(4)
This paper describes the use of electro-hydrodynamic actuation to control the transition between three major flow patterns of an aqueous-oil Newtonian flow in a microchannel: droplets, beads-on-a-string (BOAS), and multi-stream laminar flow. We observed interesting transitional flow patterns between droplets and BOAS as the electric field was modulated. The ability to control flow patterns of a two-phase fluid in a microchannel adds to the microfluidic tool box and improves our understanding of this interesting fluid behavior.Microfluidic technologies have found use in a wide range of applications, from chemical synthesis to biological analysis to materials and energy technologies.1,2 In recent years, there has been increasing interest in two-phase flow and droplet microfluidics, owing to their potential for providing a high-throughput platform for carrying out chemical and biological analysis and manipulations.3–8 Although droplets may be generated in many different ways, such as with electric fields or extrusion through a small nozzle,9–12 the most common microfluidic methods are based on the use of either T-junctions or flow-focusing geometries with which uniform droplets can be formed at high frequency in a steady-state fashion.13,14 Various operations, such as cell encapsulation, droplet fusion, splitting, mixing, and sorting, have also been developed, and these systems have been demonstrated for a wide range of applications, including cell analysis, protein crystallization, and material synthesis.1–17In addition to forming discrete droplets, where a disperse phase is completely surrounded by a continuous phase, it is also possible in certain situations to have different phases flow side-by-side. In fact, multi-stream laminar flow, either of the same phase or different phases, has been exploited for both biochemical analysis and microfabrication.1,2,18–20 Beads-on-a-string (BOAS) is another potential flow pattern, which has been attracting attentions in microfluidics field. BOAS flow, owing to its special flow structures, may be particularly useful in some applications, such as optical-sensor fabrication.21 In BOAS flow, queues of droplets are connected by a series of liquid threads, which makes them look like a fluid necklace with regular periods.21–25 The BOAS pattern is easily found in nature, such as silk beads and cellular protoplasm, and is often encountered in industrial processes as well, such as in electrospinning and anti-misting.21,22 In general, it is thought that BOAS structure occurs mostly in viscoelastic fluids22 and is an unstable structure, which evolves continually and breaks eventually.21–29Flow patterns determine the inter-relations of fluids in a microdevice and are an important parameter to control. Common methods for adjusting microfluidic flow patterns include varying the fluid flow rates, fluid properties, and channel geometries. Additionally, the application of an electric field can be a useful supplement for adjusting microfluidic flow patterns, although most work in this area has been focused on droplets and in some cases also on multi-stream laminar flows.30–33 Here, in addition to forming droplets and two-phase laminar flow with electro-hydrodynamic actuation, we also observed a new stable flow pattern in a non-viscoelastic fluid, BOAS flow. Such flow patterns may find use in controlling the interactions between droplets, such as limited mixing by diffusion between neighboring droplets.To generate droplets, we used the flow-focusing geometry (Figure 1(a)), in which aqueous phase (water) was flown down the middle channel and droplets were pinched off by the oil phase (1-octanol) from the two side channels at the junction; Figure 1(b) shows the droplets formed after the junction. To apply electric field along the main channel where the droplets were formed, we patterned a pair of electrodes upstream and downstream of the junction (Figure 1(a); for experimental details, please see Ref. 34 for supplementary material). The average electric field strength may be calculated from the voltages applied and the distance (1.7 mm) between the two electrodes. When a high voltage was applied along the channel between the two electrodes, the aqueous-oil interface at the flow-focusing junction became charged and behaved like a capacitor. As a result, more negative charges were drawn back upstream towards the positive electrode, and left behind more positive charges at the aqueous-oil interface, which then became encapsulated into the aqueous droplets dispersed in the oil phase.Open in a separate windowFIG. 1.(a) Schematic of the setup. (b) Micrograph showing droplet generation in a flow-focusing junction. The scale bar represents 40 μm.The positively charged aqueous-oil interface was stretched under an applied electric field, and by adjusting the voltage and/or the two-phase flow-rate ratio, we found interestingly that various flow patterns emerged. We tested different combinations of applied voltages and flow-rate ratios and found that most of them resulted in similar flow patterns and transitions between flow patterns.Figure Figure22 illustrates the effects of varying the applied voltages on droplets at a fixed liquid flow rate. With increasing electric-field strength and force, we found it was easier for the aqueous phase to overcome interfacial tension and form droplets. For example, as the voltage increased from 0.0 kV to 0.8 kV (average field strength increased from 0 to 0.47 V/μm), droplet-generation frequencies became slightly higher, and the formed droplets were smaller in volume. Additionally, droplets gradually became more spherical in shape at higher voltages.Open in a separate windowFIG. 2.Images showing the effects of applied voltage on droplet shape and flow pattern. Oil-phase flow rate, 0.5 μl/min; aqueous-phase flow rate, 0.2 μl/min. The scale bar represents 40 μm.As the voltage increased further (e.g., up to 1.0 kV in Figure Figure3),3), the distance between neighboring droplets became smaller, and the aqueous-oil interface at the junction was stretched further toward the downstream channel. At a threshold voltage (1 kV here with corresponding average field strength of 0.59 V/μm), the tip of the aqueous-oil interface would catch up with the droplet that just formed, and the tip of the interface of this newly captured droplet would in turn catch up with the interface of the droplet that formed before it. Consequently, a series of threads would connect all the droplets flowing between the two electrodes, thus resulting in a BOAS flow pattern.Open in a separate windowFIG. 3.Series of images showing the reversibility and synchronicity of a transitional flow pattern between droplets and BOAS (bead-on-a-string). Voltage applied, 1.00 kV (corresponding field strength of 0.59 V/μm); oil-phase flow rate, 0.5 μl/min; aqueous-phase flow rate, 0.2 μl/min. The scale bar represents 40 μm.At voltages near the threshold value, the flow pattern was not stable, but oscillated between droplets flow and BOAS flow. Figure Figure33 is a series of images captured by a high-speed camera that show the flow in this transition region. In Figures 3(a) and 3(b), the string of BOAS became thinner over time, and then the BOAS broke into droplets (Figures 3(c) and 3(d)). The newly formed droplets, however, were not stable either. Thin liquid threads would appear and then connect neighboring droplets, and a new switching period between discrete droplets and BOAS would repeat (Figures 3(e)–3(h)). In addition to this oscillation and reversibility, the flow pattern had a synchronous behavior: all the droplets appeared connected simultaneously by liquid threads or were separated at the same time.When the voltage reached 1.3 kV, which corresponded to an average field strength of 0.76 V/μm, a stable BOAS flow was obtained (Figure 4(a)). BOAS structures are thought to be present mostly in viscoelastic fluids,22 because viscoelasticity is helpful in enhancing the growth of beads and in delaying breakup of the string; thus, the viscoelastic filament has much longer life time than its Newtonian counterpart. Here, with the help of electric field, regular BOAS structures are realized in a non-viscoelastic fluid (water) in microchannels.Open in a separate windowFIG. 4.(a) Micrograph showing BOAS flow in a channel. (b) Profile of the top-half of the BOAS flow recorded continuously at a cross-section (shown in Figure 4(a)) of a channel. Voltage applied, 1.30 kV (corresponding field strength of 0.76 V/μm); oil-phase flow rate, 0.5 μl/min; aqueous-phase flow rate, 0.2 μl/min. The scale bar represents 40 μm.Microenvironment and electric fields alter the common evolution of BOAS structure observed in macroscopic or unbound environments. The BOAS structure formed in our experiments is not a stationary pattern, but a steady-state flowing one. Electric-field force prevents liquid strings from breaking between beads, and thus plays a similar role as elastic force in viscoelastic fluids. Figure 4(b) shows the dynamic BOAS profile, obtained at a fixed plane (shown in Figure 4(a)) perpendicularly across the channel as the BOAS structure passed through it. Droplets and liquid-thread diameters were nearly constant during the sampling time. The longer term experiments (over 3 min) showed there were slight variations of the two diameters in time, but the essential BOAS structure still remained qualitatively the same as a whole.When the voltage was further increased, the string diameter became larger and the droplet diameter became smaller. Because of the low flow-rate ratio (0.4) between the aqueous phase and oil phase used in the experiment depicted in Figure Figure4,4, the flow did not further develop into a multi-stream laminar flow, as would be expected at a higher voltage, and instead became unstable and irregular. When the flow-rate ratio was increased to 1.0 and the voltage was adjusted to 3.0 kV (corresponding field strength of 1.76 V/μm), we observed a stable multi-stream laminar flow (Figure (Figure5).5). The aqueous stream flowed in the channel center surrounded by the oil phase on the sides. This experiment showed that higher electric-field strengths alone would not give rise to another stable flow pattern (i.e., multi-stream laminar flow), but a suitable flow-rate ratio of aqueous phase to oil phase is required for the formation of stable two-phase laminar flow.Open in a separate windowFIG. 5.Micrograph showing multi-stream two-phase laminar flow in the channel. Voltage applied, 3.00 kV (corresponding field strength of 1.76 V/μm); oil-phase flow rate, 0.5 μl/min; aqueous-phase flow rate, 0.5 μl/min. The scale bar represents 40 μm.The flow patterns we observed may be described by a phase diagram (Figure (Figure6),6), which depends on two dimensionless numbers: capillary number, Ca = μaUa/σ, and electric Bond number, Boe = E2(εD/σ). Ca and Boe describe the ratio of viscous force to interfacial tension force and the ratio of electric-field force to interfacial tension force, respectively. Here, μa (1 mPa s), σ (8.5 mN/m), ε (7.1 × 10−10 F/m), E, Ua, and D are, respectively, the aqueous-phase viscosity, aqueous-oil interfacial tension, aqueous-phase permittivity, electric field strength, aqueous-phase velocity, and the hydraulic diameter of the channel at the junction. Figure Figure66 shows clearly that at higher Ca, flow pattern changes gradually from droplet to BOAS and to multi-stream laminar flow with increasing Boe, which indicates the increasing importance of the electric-field force compared with the interfacial tension force. At lower Ca, flow pattern and transition show similar trend with increasing Boe as in the higher Ca case, except that multi-stream laminar flow is not observed. The relatively higher viscous force at higher Ca may be necessary for transitioning to the multi-stream laminar flow regime. In addition, Figure Figure66 shows that the BOAS window at the lower Ca is smaller than that at the higher Ca.Open in a separate windowFIG. 6.Phase diagram showing different flow patterns in the Ca and Boe space. Hollow symbols: oil-phase flow rate, 0.5 μl/min; aqueous-phase flow rate, 0.5 μl/min. Solid symbols: oil-phase flow rate, 0.5 μl/min; aqueous-phase flow rate, 0.2 μl/min.In summary, we showed the ability to use electric fields to generate and control different flow patterns in two-phase flow. With the aid of an applied field, we were able to generate BOAS flow patterns in a non-viscoelastic fluid, a pattern that typically requires a viscoelastic fluid. The BOAS structure was stable and remained as long as the applied electric field was on. We also report transitional flow patterns, those between droplets and BOAS exhibited both good reversibility as well as synchronicity. And with a suitable flow-rate ratio between the two phases, BOAS flow could be transitioned into a stable two-phase laminar flow by applying a sufficiently high field strength. Finally, a phase diagram was presented to describe quantitatively the flow-pattern regimes using capillary number and electric Bond number. The phenomena we report here on the properties of two-phase flow under an applied electric field may find use in developing a different approach to exert control over droplet based or multi-phase laminar-flow based operations and assays, and also aid in understanding the physics of multi-phase flow. 相似文献
11.
Plasmonic hot spots, generated by controlled 20-nm Au nanoparticle (NP) assembly, are shown to suppress fluorescent quenching effects of metal NPs, such that hair-pin FRET (Fluorescence resonance energy transfer) probes can achieve label-free ultra-sensitive quantification. The micron-sized assembly is a result of intense induced NP dipoles by focused electric fields through conic nanocapillaries. The efficient NP aggregate antenna and the voltage-tunable NP spacing for optimizing hot spot intensity endow ultra-sensitivity and large dynamic range (fM to pM). The large shear forces during assembly allow high selectivity (2-mismatch discrimination) and rapid detection (15 min) for a DNA mimic of microRNA.Irregular expressions of a panel of microRNAs (miRNA) in blood and other physiological fluids may allow early diagnosis of many diseases, including cancer and cardiovascular diseases.1 However, quantifying all relevant miRNAs (out of 1000), with similar sequences over 22 bases2 and large variations in expression level (as much as 100 fold) at small copy numbers, requires a new molecular diagnostic platform with high-sensitivity, high-selectivity, and large dynamic range. Current techniques for miRNA profiling, such as Northern blotting,3 microarray-based hybridization,4 and real-time quantitative polymerase chain reaction5 are expensive and complex. A simple and rapid miRNA array would allow broad distribution of molecular diagnostic devices for cancer and chronic diseases, eventually into homes for frequent prescreening of many diseases.At their low concentrations in untreated samples, optical sensing of miRNA is most promising. Plasmonically excited Raman scattering (SERS) and fluorescence sensors from metallic nanoparticles (NPs) or surfaces have enhanced the sensitivity of optical molecular sensors by orders of magnitude.6, 7, 8, 9 However, probe-less SERS sensing or fluorescent sensing of unlabeled targets are insufficiently specific for miRNA targets in heterogeneous samples. Plasmonic detection is also very compatible with FRET probes whose donor dye offers small light sources to excite fluorescently labelled targets upon hybridization.7, 10A particular family of FRET reporters does offer label-free sensing: hairpin oligo probes whose end-tagged fluorophores are quenched by the Au NP to which they are functionalized.11 The fluorescent signal is only detected when the hairpin is broken by the hybridizing target nucleic acid or protein (for an aptamer probe), and the more rigid paired segment separates the end fluorophore from the quenching surface to produce a fluorescent signal. It is often hoped that plasmonics on the metal surface will enhance the intensity to overcome the quenching effect, if the linearized hairpin is within the NP plasmonic penetration length. However, since fluorescent quenching decays slowly (linearly) with fluorophore-metal spacing10 whereas the plasmonic intensity decays exponentially from a flat surface, careful experimentation shows that quenching dominates and the hairpin probe actually produces a larger intensity on non-metallic surfaces,10 on which it can not function as a label-free probe. Hence, only μM limit-of-detection (LOD) has been achieved with this technique on single NPs or on flat metal surfaces,12 with expensive laser excitation and confocal detection.Plamonic hot spots formed between metal nanostructures and sharp nanocones can further amplify the plasmonic field.13, 14 The hot spot intensity decays algebraically with respect to the separation or cone tip distance and hence should dominate the linear decay of the metal quenching effect at some optimum separation.15 It is hence possible that plasmonic hot spots may allow much lower LOD with inexpensive optical instruments—ideally light-emitting diode light source and miniature camera. However, the dimension of the gaps, cones, and wedges needs to be at nanoscale, and the cost is now transferred to fabrication of such hot-spot substrates like bow-ties, double crescents, bull-eyes, etc.16 Low-cost wet-etching techniques for addressable nanocones that sustain converging plasmonic hot spots17 have been reported but the fabricated nanocones are often too non-uniform to allow precise quantification. NP monolayers have been shown to exhibit plasmonic hot spots and fluorescence enhancement.18, 19 However, the enhancement only occurs within a range of spacing between aggregated NPs, which is difficult to control and the location or even the existence of the hotspots are not known a priori.Higher sensitivity is expected if a minimum number of NPs are used in an assembly at a known location and if the NP assembly can produce crystal-like aggregates with controllable NP spacing. Induced DC and AC NP dipoles (related to dielectrophoresis) have been used to assemble NP crystals by embedded micro-electrodes to provide the requisite high field.20, 21 The resulting NP crystals are ideal for plasmonic hot spots, since the spacing of the regimented NP crystal can be controlled by the applied voltage. Conic nanocapillaries22, 23 will be used here for such field-induced NP assembly because the submicron-tip can focus the electric field into sufficient high intensity for NP assembly without embedded-electrodes. Because the field is highest at the tip due to field focusing, the micron-sized crystal would be confined to a small volume, which will be shown to be less than typical confocal volumes, at a known location. So long as the hotspots are regimented, the quantification of target molecules is determined by the total fluorescent intensity and is hence insensitive to the exact geometry of the nanocapillary.Fluorescent microscope equipped with tungsten lamp light source and normal CCD camera from Q Imaging were used for simultaneous optical and ion current measurements, as shown in Fig. Fig.1a.1a. The nanocapillaries were pulled from commercial glass capillaries using laser-assisted capillary puller. SEM image of a typical pulled glass nanocapillary in Fig. Fig.1b1b shows an inner diameter of 111 nm and cone angle of 7.3°. The capillary was inserted into a Polydimethylsiloxane chip with two reservoirs. The 20 nm Au NPs, functionalized with fluorescently labelled dsDNA, were injected into the base reservoir. With SEM imaging (Fig. S3 in the supplementary material24), the functionalized DNA is found to prevent NP aggregation even in high ionic-strength Phosphate buffered saline buffer. The NP solution is then driven into the capillary through the tip by applying a positive voltage. Fig. Fig.1c1c shows the ion current evolution over 2 h at +1 V packing voltage. The ion current increases rapidly in the first 10 min, then at a much slower rate. The rise of current indicates assembly of conductive Au NP assembly at the tip. This was confirmed by the strong fluorescence signal at the tip region during the packing process (inset of Fig. Fig.1c).1c). The one-micron region (corresponding to roughly an aggregate volume of one attoliter) near the capillary tip shows a fluorescence signal after 1 min and also appeared as a dark spot in the transmission image (supplementary material, Fig. S124). This spot darkens with longer packing time but does not grow in size, consistent with the monotonically increasing ion current with increased packing density of the NP assembly. As contrast, a strong fluorescence appeared after only 1 min of packing, but the signal became weaker after 15 min (supplementary material, Fig. S124). This reduction in fluorescence is not due to bleaching of fluorophores because we took 2 images in 15 min at 5 s exposure each and control experiments show significant bleaching only beyond an exposure time of 100 s (see supplementary material).24 Instead, the non-monotonic dependence of the fluorescence intensity with respect to time is because of the optimal hotspot spacing for highest plasmonic intensity at about 5–20 nm,25, 26, 27 which is reached at about 10 min.Open in a separate windowFigure 1Plasmonic hotspots generated at the tip of a nano-capillary. (a) Schematic of the experimental set up. (b) SEM image of glass nanocapillary shows opening at the tip with a diameter of 111 nm. (c) Current evolution during packing of fluorescently labeled gold particles at +1 V. Inset shows strong fluorescence only after 1 min of packing.The FRET probe is designed to exploit the plasmonic hotspot.24 We first electrophoretically drove the target molecules in the tip side reservoir into the nano-capillary by applying a negative voltage of −1 V. During this process, the targets are trapped within the capillary and hybridize with the hairpin probes on the Au NP in the nanocapillary. Fluorescence of the unquenched hybridized probes is too weak to be detected by our detector as shown in Fig. Fig.2b.2b. A reverse positive voltage of +1 V was then applied to the capillary to pack the Au NPs to the tip. Due to plasmonic hot spots of aggregated gold nanoparticles, the fluorescence signal is significantly enhanced at the tip and can be detected by our CCD camera, as shown in Fig. Fig.2c2c.Open in a separate windowFigure 2(a) Schematics of designed hairpin probe on gold particle. (b) Before packing gold particles, probe fluorescence signal was too weak to be detect. (c) After packing for 3 minutes, a strong fluorescence signal appears at the NP aggregate. (d) Normalized intensity (average of all pixels above a threshold (15 au) normalized with respect to the average over all pixels (with 0-250 au)) as a function of packing voltage for different samples. Black, 1 nM target ; blue, 10 pM target; purple, 10 nM 2-mismatch non-target. (e) Intensity dependence on target concentration. Measured normalized intensity before packing (black) and after packing (red), for three independent experiments with different nano-capillaries at each concentration. NT stands for non-target at 10 nM as a reference.For the same packing time, the fluorescence intensity increases initially but saturates after 10 min time of trapping (supplementary material, Fig. S2(a)24). Over 10 min of trapping with a negative voltage, we found the fluorescence intensity exhibits a maximum at a packing time of 3 min (supplementary material, Fig. S2(b)24). In later experiments, we used 10 min trapping time and 3 min packing time as standards.Fig. Fig.2d2d shows the fluorescence intensity is sensitive to the positive packing voltage at different concentration of target and non-target molecules. For target samples (1 nM and 10pM), the optimal voltage is about 1 V. We suspect that with larger voltage, the NPs are packed too tightly such that the NP spacing is smaller than the optimal distance for plasmonic hotspots. The fluorescence intensity for a nontarget with two mismatches is 7 times lower than the target even with a 10 times higher concentration (10 nM). Moreover, the optimal voltage for the non-target miRNA is reduced to 0.5 V instead 1 V for the target miRNA. Strong shear during electrophoretic packing has probably endowed this high selectivity.20Using the protocol above, the LOD and dynamic range of the target was determined (Fig. (Fig.2e).2e). The intensity at each concentration is measured with three independent experiments with different nanocapillaries to verify insensitivity with respect to the nanocapillary. The intensity increases monotonically with respect to the concentration from 1fM to 1pM. Beyond 1pM, the fluorescence signal saturates, presumably because all hairpin probes at the tip have been hybridized. At 1 fM, the fluorescent intensity is still well above the background measured from the non-target sample. Note both auto-fluorescence of gold nanoparticles and free diffusing non-target DNA molecules contribute to the background. Given the volume of tip side reservoir (∼50 μl), there are about 30 000 target molecules in the reservoir at 1 fM. However, with a short 10 min trapping time, we estimate only a small fraction of these molecules, less than 100, have been transferred from the tip reservoir into the nanocapillary. 相似文献
12.
S. Biswas A. Bhelwa A. Itoop S. Biswal N. Nath 《Indian journal of clinical biochemistry : IJCB》1994,9(1):9-12
Membrane hydrophobicity and slalidase activity of normal Poly morphonuclear Leucocyte were significantly enhanced when incubated with DSF. As a consequence, internalisation ofE. coli andS. aureus (opsonised or unopsonised) were greatly dimnished, internalisation ofE. coli being higher in either category. Although, increase in hydrophobicity of the membrane correlated well with the time of decrease of particle internalisation (both at 30 min.), enhancement of sialidase activity did not coincide with the said alterations. 相似文献
13.
This research reports an improved conjugation process for immobilization of antibodies on carboxyl ended self-assembled monolayers (SAMs). The kinetics of antibody/SAM binding in microfluidic heterogeneous immunoassays has been studied through numerical simulation and experiments. Through numerical simulations, the mass transport of reacting species, namely, antibodies and crosslinking reagent, is related to the available surface concentration of carboxyl ended SAMs in a microchannel. In the bulk flow, the mass transport equation (diffusion and convection) is coupled to the surface reaction between the antibodies and SAM. The model developed is employed to study the effect of the flow rate, conjugating reagents concentration, and height of the microchannel. Dimensionless groups, such as the Damköhler number, are used to compare the reaction and fluidic phenomena present and justify the kinetic trends observed. Based on the model predictions, the conventional conjugation protocol is modified to increase the yield of conjugation reaction. A quartz crystal microbalance device is implemented to examine the resulting surface density of antibodies. As a result, an increase in surface density from 321 ng/cm2, in the conventional protocol, to 617 ng/cm2 in the modified protocol is observed, which is quite promising for (bio-) sensing applications.Microfluidics have been implemented in various bio-medical diagnostic applications, such as immunosensors and molecular diagnostic devices.1 In the last decade, a vast number of biochemical species has been detected by microfluidic-based immunosensors. Immunosensors are sensitive transducers which translate the antibody-antigen reaction to physical signals. The detection in an immunosensor is performed through immobilization of an antibody that is specific to the analyte of interest.2 The antibody is often bound to the transducing surface of the sensor covered by self-assembled monolayers (SAMs). SAMs are organic materials that form a thin, packed and robust interface on the surface of noble metals like that of gold, suitable for biosensing applications.3 Thiolic SAMs have a “head” group that shows a high affinity to being chemisorbed onto a substrate, typically gold. The SAMs'' carboxylic functional group of the “tail” end can be linked to an amine terminal of an antibody to form a SAM/antibody conjugation.3,4 The conjugation process is usually accomplished in the presence of carbodiimides, such as 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC). A yield increasing additive, N-Hydroxysuccinimide (NHS), is often used to enhance the surface loading density of the antibody.4,5A typical reaction for coupling the carboxylic acid groups of SAMs with the amine residue of antibodies in the presence of EDC/NHS is depicted in Figure Figure11.4 NHS promotes the generation of an active NHS ester (k2 reaction path). The NHS ester is capable of efficient acylation of amines, including antibodies (k3 reaction path). As a result, the amide bond formation reaction, which typically does not progress efficiently, can be enhanced using NHS as a catalyst.4Open in a separate windowFIG. 1.NHS catalyzed conjugation of antibodies to carboxylic-acid ended SAMs through EDC mediation (Adapted from G. T. Hermanson, Bioconjugate Techniques, 2nd. Edition. Copyright 2008 by Elsevier4). EDC reacts with the carboxylic acid and forms o-acylisourea, a highly reactive chemical that reacts with NHS and forms an NHS ester, which quickly reacts with an amine (i.e., antibody) to form an amide.A number of groups have studied EDC/NHS mediated conjugation reactions such as the ones depicted in Figure Figure1.1. The general stoichiometry of the reaction involves a carboxylic acid (SAM), an amine (antibody), and EDC to produce the final amide (antibody conjugated SAM) and urea. However, the recommended concentration ratio of the crosslinking reagents inside the buffer, i.e., the ratio of EDC and NHS with respect to adsorbates and each other, varies from one study to another.6 The kinetics of the reactions outlined in Figure Figure11 have also been investigated,4,6–8 but only in the absence of NHS for EDC or carboxylic acids in aqueous solutions.8 A relatively recent experimental study verified the catalytic role of the yield-increasing reagent N-hydroxybenzotriazole (HOBt), which acts similarly to NHS.7 In this study, the amide formation rate (k3 reaction path, Figure Figure1)1) was found to be dependent on the concentration of the carboxylic acid and EDC in the buffer solution, and independent of the amine and catalyst reagent concentration. The same group also showed that the amide bond formation kinetics is controlled by the reaction between the carboxylic acid and the EDC to give the O-acylisourea, which they marked as the rate-determining step (k1 reaction path, Figure Figure11).The k1 reaction path, or the conjugation reaction, is usually a lengthy process and takes between 1 and 3 h.4,9 Compared to k1, the k2 and ?k3 reactions are considerably faster. Microfluidics has the potential to enhance the kinetics of these reactions using the flow-through mode.10,11 This improvement occurs because while conventional methods rely only on diffusion as the primary reagent transport mode, microfluidics adds convection to better replenish the reagents to the reaction surfaces. However, there are many fundamental fluidic and geometrical parameters that might affect the process time and reagents consumption in a microfluidics environment, such as concentration of antibodies and reagents, flow rate, channel height, and final surface density of antibodies. A model that studies the kinetics of conjugation reaction against all these parameters would therefore be helpful for the optimization of this enhanced kinetics.There are a number of reports on numerical examination of the kinetics of binding reactions in microfluidic immunoassays.12–15 All these models developed so far couple the transport of reagents, by diffusion and convection, to the binding on the reaction surface. Myszka''s model assumes a spatially homogeneous constant concentration of reagents throughout the reaction chamber, thus fails to describe highly transport-limited conditions due to the presence of spatial heterogeneity and depletion of the bulk fluid from reagents.16,17 In transport-limited conditions, the strength of reaction is superior to the rate of transport of reagents to the reaction surface.18,19 More recently, the convection effects were included in a number of studies, describing the whole kinetic spectrum from reaction-limited conditions to transport-limited reactions.20–22 Immunoreaction kinetics has also been examined with a variety of fluid driving forces, from capillary-driven flows,20 to electrokinetic flows in micro-reaction patches,21 pressure-driven flows in a variety of geometric designs.22 Despite these comprehensive numerical investigations, the fundamental interrelations between the constitutive kinetic parameters, such as concentration, flow velocity, microchannel height, and antibody loading density, have not been studied in detail. In addition, the conjugation kinetics has not yet been exclusively examined.In this paper, a previous model for immunoreaction is modified to study the antibody/SAM conjugation reaction in a microfluidic system. Model findings are used to examine the process times recommended in the literature and possible modification scenarios are proposed. The new model connects the convective and diffusive transport of reagents in the bulk fluid to their surface reaction. The conjugation reaction is studied against fluidic and geometrical parameters such as flow rate, concentration, microchannel height and surface density of antibodies. Damköhler number is used to compare the reaction and fluidic phenomena present and justify the kinetic trends observed. Model predictions are discussed and the main finding on possible overexposure of carboxylates to crosslinking reagents, in conventional protocols, is verified by comparing the resultant antibody loading densities obtained using a quartz crystal microbalance (QCM) set up. The results demonstrate an improved receptor (antibody) loading density which is quite promising for a number of (bio-) sensing applications.23,24 Major application areas include antibody-based sensors for on-site, rapid, and sensitive analysis of pathogens such as Bacillus anthracis,23
Escherichia coli, and Listeria monocytogenes, and toxins such as fungal pathogens, viruses, mycotoxins, marine toxins, and parasites.24 相似文献
14.
Ryan Tonkens 《Ethics and Information Technology》2012,14(2):137-149
The emerging discipline of Machine Ethics is concerned with creating autonomous artificial moral agents that perform ethically
significant actions out in the world. Recently, Wallach and Allen (Moral machines: teaching robots right from wrong, Oxford
University Press, Oxford, 2009) and others have argued that a virtue-based moral framework is a promising tool for meeting this end. However, even if we
could program autonomous machines to follow a virtue-based moral framework, there are certain pressing ethical issues that
need to be taken into account, prior to the implementation and development stages. Here I examine whether the creation of
virtuous autonomous machines is morally permitted by the central tenets of virtue ethics. It is argued that the creation of
such machines violates certain tenets of virtue ethics, and hence that the creation and use of those machines is impermissible.
One upshot of this is that, although virtue ethics may have a role to play in certain near-term Machine Ethics projects (e.g.
designing systems that are sensitive to ethical considerations), machine ethicists need to look elsewhere for a moral framework
to implement into their autonomous artificial moral agents, Wallach and Allen’s claims notwithstanding. 相似文献
15.
Wang Zhao Li Zhang Wenwen Jing Sixiu Liu Hiroshi Tachibana Xunjia Cheng Guodong Sui 《Biomicrofluidics》2013,7(1)
A microfluidic device was successfully fabricated for the rapid serodiagnosis of amebiasis. A micro bead-based immunoassay was fabricated within integrated microfluidic chip to detect the antibody to Entamoeba histolytica in serum samples. In this assay, a recombinant fragment of C terminus of intermediate subunit of galactose and N-acetyl-D-galactosamine-inhibitable lectin of Entamoeba histolytica (C-Igl, aa 603-1088) has been utilized instead of the crude antigen. This device was validated with serum samples from patients with amebiasis and showed great sensitivity. The serodiagnosis can be completed within 20 min with 2 μl sample consumption. The device can be applied for the rapid and cheap diagnosis of other infectious disease, especially for the developing countries with very limited medical facilities.Entamoeba histolytica is the causative agent of amebiasis and is globally considered a leading parasitic cause of human mortality.1 It has been estimated that 50 × 106 people develop invasive disease such as amebic dysentery and amebic liver abscess, resulting in 100 000 deaths per annum.2, 3 High sensitive diagnosis method for early stage amebiasis is quite critical to prevent and cure this disease. To date, various serological tests have been used for the immune diagnosis of amebiasis, such as the indirect fluorescent antibody test (IFA) and enzyme-linked immunosorbent assay (ELISA).We have recently identified a 150-kDa surface antigen of E. histolytica as an intermediate subunit (Igl) of galactose and N-acetyl-D-galactosamine-inhibitable lectin.4, 5 In particular, it has been shown that the C-terminus of Igl (C-Igl, aa 603-1088) was an especially useful antigen for the serodiagnosis of amebiasis. ELISA using C-Igl is more specific than the traditional ELISA using crude antigen.6 However, the ELISA process usually takes several hours, which is still labor-intensive and requires experienced operators to perform. More economic and convenient filed diagnosis methods are still in need, especially for the developing countries with limited medical facilities.Among all the bioanalytical techniques, microfluidics has been attracting more and more attention because of its low reagent/power consumption, the rapid analysis speed as well as easy automation.7, 8, 9, 10, 11 Especially with the development of the fabrication technique, microfluidics chip can include valves, mixers, pumps, heating devices, and even micro sensors, so many traditional bioanalytical methods can be performed in the microfluidics. Qualitative and quantitative immune analysis on the microfluidic chip was successfully proved by plenty of research with improved sensitivity, shorten reaction time, and less sample consumption.8, 10, 11, 12, 13, 14, 15, 16, 17 Moreover, with the intervention of other physical, chemical, biology, and electronic technology, microfluidic technique has been successfully utilized in protein crystallization, protein and gene analysis, cell capture and culturing and analysis as well as in the rapid and quantitative detection of microbes.13, 14, 15, 16, 17, 18, 19, 20Herein, we report a new integrated microfluidic device, which is capable of rapid serodiagnosis of amebiasis with little sample consumption. The microfluidic device was fabricated from polydimethysiloxane (PDMS) following standard soft lithography.21, 22 The device was composed of two layers (shown in Figure Figure1)1) including upper fluidic layer (in green and blue) and bottom control layer (in red).Open in a separate windowFigure 1Structure illustration of microfluidic chip.To create the fluidic layer and the control layer, two different molds with different patterns have fabricated by photolithographic processes. The mold to create the fluidic channels was made by positive photoresist (AZ-50 XT), while the control pneumatic mold was made by negative photoresist (SU8 2025). For the chip fabrication, the fluidic layer is made from PDMS (RTV 615 A: B in ratio 5:1), and the pattern was transferred from the respective mold. The control layer is made from PDMS (RTV 615 A:B in ratio 20:1). The two layers were assembled and bonded together accurately, and there is elastic PDMS membrane about 30 μm thick between the fluidic layer channels and control layer.21, 22 The elastic membrane at the intersection can deform to block the fluid inside the fluidic channels, functioning as valves under the pressures introduced though control channels. There are two types of channels in fluidic layer, the rectangular profiled (in green, 200 μm wide, 35 μm thick) channel and round profiled channels (in blue, 200 μm wide, 25 μm center height). Because of the position of the valves on the fluidic channels, two types of valves (Figure (Figure2a)2a) were built, working as a standard valve and a sieve valve. The standard valves (on blue fluidic channels) can totally block the fluid because of the round profile of fluidic channel; the sieve valve can only half close because of the rectangular profile. The sieve valve can be used to trap the microspheres (beads) filled inside the green fluidic channels, while letting the fluid pass through. By this sieve valve, a micro column (in green) is constructed, where the entire ELISA reaction happens. The micrograph of the fabricated micro device is shown in Figure Figure2b.2b. The channels were filled with food dyes in different colors to show the relative positions of the channels. The pressures though different control channels are individually controlled by solenoid valves, connected to a computer through relay board. By programming the status (on/off) of various valves at different time periods, all the microfluidic chip operation can be digitally controlled by the computer in manual, semi-automatic, or automatic manner.Open in a separate windowFigure 2(a) Structure illustration of micro column, standard valve and sieve valve; (b) photograph of the microfluidic chip.To validate this device, 12 patient serum samples were collected. Sera from 9 patients (Nos. 1–9) with an amebic liver abscess or amebic colitis were used as symptomatic cases. The diagnosis of these patients was based on their clinical symptoms, ultrasound examination (liver abscess) and endoscopic or microscopic examination (colitis). We also identified the clinical samples using PCR amplification of rRNA genes.24 As negative control, sera obtained from 3 healthy individuals with no known history of amebiasis were mixed into pool sera. The serum was positive for E. histolytica with a titer of 1:64 (borderline positive), as determined by an indirect fluorescent-antibody (IFA) test.23, 24 In our previously study, the sensitivity and specificity of the recombinant C-Igl in the ELISA were 97% and 99%.6, 25 In the current study, the serodiagnosis of amebiasis was also examined by ELISA using C-Igl.26 The cut-off for a positive result was defined as an ELISA value > 3 SD above the mean for healthy negative controls27 (shown in Figure Figure3).3). The seropositivity to C-Igl was 100% in patients with amebiasis.Open in a separate windowFigure 3ELISA reactivity of sera from patients against C-Igl. ELISA plate was coated with 100 ng per well of C-Igl. Serum samples from patients and healthy controls were used at 1:400 dilutions. The dashed line indicates the cut-off value. Data are representative of results from three independent experiments.In the diagnosis process with microfluidic chip, the 4 micro immuno-columns filled with C-Igl-coated microspheres were the key components of the device. The C-Igl was prepared in E. coli as inclusion bodies. After expression, the recombinant protein was purified and analyzed by SDS-PAGE. The apparent molecular mass was 85 kDa.26The immune-reaction mechanism is illustrated in Figure Figure4.4. The anti-His monocolonal antibody was immobilized onto the microspheres (beads, 9 μm diameter) coated with protein A. The C-Igl was then immobilized onto the beads through the binding between the His tag and C-Igl. For the diagnosis, the microspheres immobilized with C-Igl and blocked by 5% BSA were preloaded into the columns for the rapid analysis of the patient serum samples. Generally, serum samples which were diluted 100 times were first loaded into the reaction column and incubated at room temperature for 5 min. After being washed by PBS buffer, FITC-conjugated goat anti-human polyclonal antibody was added into the column for 4 min incubation. The fluorescence image can be collected by the fluorescence microscope after the micro column was washed with PBS buffer. From loading diluted serum samples into column to collecting fluorescence images, the total time to complete the immunoassay is less than 10 min. The final fluorescence results were analyzed by Image Pro Plus 6.0.Open in a separate windowFigure 4Schematic representation of the ELISA in the chip.Different reaction conditions have been investigated to find the optimized ones. For each patient, 2 μl sample is enough for the analysis. The designed microfluidic chip with 4 micro columns is capable for 4 parallel analyses at the same time. More micro columns can be integrated into the device if more parallel tests are needed.Different incubating time for the diagnosis has also been investigated and no significant difference has been found for various time periods. It is enough to incubate the chip for only 5 min. The total diagnosis time for one sample is less than 10 min. The detection result appeared as the fluorescence intensity of the reaction column. As shown in Figure Figure5,5, the negative sample showed relatively low fluorescence intensity, because little FITC-conjugated goat anti-human polyclonal antibody could attach to the surface of microspheres; on the contrast, the positive sample showed much brighter fluorescence. The fluorescence intensity can be transferred to digital data (Table Sample Average scores Standard deviation 1 33 790 368 2 23 269 271 3 39 598 307 4 7784 52 5 21 222 197 6 38 878 290 7 22 437 227 8 36 295 334 9 41 024 396 Negative 200 32