共查询到20条相似文献，搜索用时 46 毫秒
1.
This paper argues against the moral Turing test (MTT) as a framework for evaluating the moral performance of autonomous systems. Though the term has been carefully introduced, considered, and cautioned about in previous discussions (Allen et al. in J Exp Theor Artif Intell 12(3):251–261, 2000; Allen and Wallach 2009), it has lingered on as a touchstone for developing computational approaches to moral reasoning (Gerdes and Øhrstrøm in J Inf Commun Ethics Soc 13(2):98–109, 2015). While these efforts have not led to the detailed development of an MTT, they nonetheless retain the idea to discuss what kinds of action and reasoning should be demanded of autonomous systems. We explore the flawed basis of an MTT in imitation, even one based on scenarios of morally accountable actions. MTT-based evaluations are vulnerable to deception, inadequate reasoning, and inferior moral performance vis a vis a system’s capabilities. We propose verification—which demands the design of transparent, accountable processes of reasoning that reliably prefigure the performance of autonomous systems—serves as a superior framework for both designer and system alike. As autonomous social robots in particular take on an increasing range of critical roles within society, we conclude that verification offers an essential, albeit challenging, moral measure of their design and performance. 相似文献
2.
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
3.
Russell Blackford 《Ethics and Information Technology》2012,14(1):41-51
We commonly identify something seriously defective in a human life that is lived in ignorance of important but unpalatable
truths. At the same time, some degree of misapprehension of reality may be necessary for individual health and success. Morally
speaking, it is unclear just how insistent we should be about seeking the truth. Robert Sparrow has considered such issues
in discussing the manufacture and marketing of robot ‘pets’, such as Sony’s doglike ‘AIBO’ toy and whatever more advanced
devices may supersede it. Though it is not his only concern, Sparrow particularly criticizes such robot pets for their illusory
appearance of being living things. He fears that some individuals will subconsciously buy into the illusion, and come to sentimentalize
interactions that fail to constitute genuine relationships. In replying to Sparrow, I emphasize that this would be continuous
with much of the minor sentimentality that we already indulge in from day to day. Although a disposition to seek the truth
is morally virtuous, the virtue concerned must allow for at least some categories of exceptions. Despite Sparrow’s concerns
about robot pets (and robotics more generally), we should be lenient about familiar, relatively benign, kinds of self-indulgence
in forming beliefs about reality. Sentimentality about robot pets seems to fall within these categories. Such limited self-indulgence
can co-exist with ordinary honesty and commitment to truth. 相似文献
4.
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
5.
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
6.
Fuchs (2010, 2012) argues that users of social media produce value and surplus value in the Marxian sense. Arvidsson and Colleoni (2012) critique this hypothesis, claiming that Marx's theory of value is irrelevant to the regime of value production on social media platforms in particular and in informational capitalism in general. They claim that the affective relations and financial speculations that generate value on social media are not dependent on labor time. This article critically engages Fuchs, and Arvidsson and Colleoni, by revisiting Marx's theory of value. Contra Fuchs, we argue that audiences do not produce value and surplus value—neither for social nor for mass media. Contra Arvidsson and Colleoni, we argue that so-called affective relations (philia) do not produce value either. Instead we demonstrate that social media generate revenue from four primary sources—by leasing advertisement space to generate advertisement rent, by selling information, by selling services to advertisers, and by generating profits from fictitious capital and speculative windfalls. All four, we argue, can be adequately explained by Marx's theory of value. 相似文献
7.
Each of us has a personal narrative: a story that defines us, and one that we tell about ourselves to our inner and outer worlds. A strong sense of identity is rooted in a personal narrative that has coherence and correspondence (Conway in J Mem Lang 53:594–628, 2005): coherence in the sense that the story we tell is consistent with and supportive of our current version of ‘self’; and correspondence in the sense that the story reflects the contents of autobiographical memory and the meaning of our experiences. These goals are achieved by a reciprocal interaction of autobiographical memory and the self, in which memories consistent with the self-image are reinforced, in turn strengthening the self-image they reflect. Thus, personal narratives depend crucially on the malleable nature of autobiographical memory: a strong sense of self requires that one remember what matters, and forget what does not. Today, anyone who is active online generates a highly detailed, ever—expanding, and permanent digital biographical ‘memory’—memory that identifies where we go, what we say, who we see, and what we do in increasing detail as our physical lives become more and more enmeshed with electronic devices capable of recording our communications, online activities, movements, and even bodily functions. This paper explores the consequences of this digital record for identity, arguing that it presents a challenge to our ability to construct our own personal narratives–narratives that are central to a sense of ‘self’. In the end, the ‘right to be forgotten’ may be, above all else, a psychological necessity that is core to identity—and therefore a value that we must ensure is protected. 相似文献
8.
Michael T. McFall 《Ethics and Information Technology》2012,14(3):221-230
Aristotle??s account of friendship has largely withstood the test of time. Yet there are overlooked elements of his account that, when challenged by apparent threats of current and emerging communication technologies, reveal his account to be remarkably prescient. I evaluate the danger that technological advances in communication pose to the future of friendship by examining and defending Aristotle??s claim that perfect or character-friends must live together. I concede that technologically-mediated communication can aid existing character-friendships, but I argue that character-friendships cannot be created and sustained entirely through technological meditation. I examine text-based technologies, such as Facebook and email, and engage a non-text based technology that poses the greatest threat to my thesis??Skype. I then address philosophical literature on friendship and technology that has emerged in the last decade in Ethics and Information Technology to elucidate and defend my account by contrast. I engage Cocking and Matthews (2000), who argue that friendship cannot be created and sustained entirely through text-based contact, Briggle (2008), who argues that friendship can be created and sustained entirely through text-based contact, and Munn (2012), who argues that friendship cannot be created and entirely sustained through text-based contact but can be created and sustained entirely in immersive virtual worlds. My account discusses a certain kind of friendship, character-friendship, and a certain kind of technology, Skype, that these accounts do not. Examination of these essays helps to demonstrate that character friendship cannot be sustained entirely by technologically-aided communication and that character-friends must live together. 相似文献
9.
Leslie Y. Yeo 《Biomicrofluidics》2009,3(2)
The inaugural conference on Advances in Microfluidics and Nanofluidics was held at the Hong Kong University of Science and Technology on 5–7 January 2009 and brought together leading researchers from across a wide variety of disciplines from North America, Europe, Asia, and Oceania. This Special Topic section forms the second of the two issues dedicated to original contributions covering both fundamental physicochemical aspects of microfluidics and nanofluidics as well as their applications to the miniaturization of chemical and biological systems that were presented at the conference.In the last five years, we have observed rapid growth in the microfluidics and nanofluidics community in Asia, owing largely to the substantial strategic investments by both government and industry in the region to promote the microfabrication and nanotechnology sectors.1 The organization of a regular meeting focusing on activities in the Asia-Pacific rim region was, therefore, timely, particularly to enhance dissemination of research of the highest quality within the region and to promote collaboration between researchers in the Asian community with their counterparts from Europe and the USA.Biomicrofluidics is, therefore, proud to be closely involved with the organization of the first of such conferences, Advances in Microfluidics and Nanofluidics 2009, which was kindly hosted by the Hong Kong University of Science and Technology (HKUST). As reported in the preface to the first of the two issues dedicated to invited reviews and original contributions associated with the conference,2 the meeting, which took place over three days in the breathtaking HKUST campus overlooking Clearwater Bay in Hong Kong, was a tremendous success. Together with our colleagues, the Biomicrofluidics editors are busy putting in place arrangements for a follow-up meeting in January 2011. Given the overwhelming response and positive feedback we’ve had to date, we believe that Advances in Microfluidics and Nanofluidics will form a regular event in the calendar of the Asian microfluidics and nanofluidics community in the future.It was particularly pleasing to observe the translation of fundamental and theoretical work into advanced applied chip-based platforms for a variety of practical chemical and biological applications in the talks presented at the conference. The collection of articles in this second part, in fact, provides a gist of the flavor of the multidisciplinary research spanning the entire fundamental to applied research spectrum, which is exactly the scope which the journal intends to cover.Electrokinetics continues to be a dominant theme in this issue and within the microfluidics and nanofluidics community. The article by Ng et al.3 provides experimental evidence that might put to rest a longstanding area of debate within the electrokinetics community on the role of Faradaic charging in driving electro-osmotic flow, first proposed by Ben and Chang.4 In other electrokinetics papers, the role of interfaces is explored, for example, electrowetting on the superhydrophobic nanostructured surfaces of a lotus leaf5 and droplet manipulation in an immiscible dielectric liquid continuum under an electric field.6In addition, the characterization of the surface charge density of the nanopores etched in organic foils is reported by Xue et al.,7 which provides a deeper understanding of the mechanisms by which ions are transported in nanochannels, whereas Wei and Hsiao8 present a stochastic simulation to model the condensation of linear polyelectrolyte molecules under electric fields, in which they show the marked increase in the mobility of the polyelectrolyte chain during its unfolding in free-solution electrophoresis.Continuing along the theme of numerical simulations, particulate transport in converging-diverging microchannels was studied using a Lagrangian-Eulerian finite-element model,9 and slip arising in Couette flows over superhydrophobic surfaces was studied using a hybrid multiscale simulation that interfaces molecular dynamics simulations in the near-wall region with the continuum fluid model in the bulk.10 In other numerical studies, drop coalescence11 and nanotube transport12 were studied.Complementing these fundamental studies is the use of multiphase flows in microfluidic channels to engineer scaffolds for tissue engineering in which the bubbles trapped in liquid droplets transported in microchannels were employed to produced the pores of the scaffold.13 Other practical microfluidics applications, such as chip-based enhancement of DNA hybridization through a genetic-bead-based protocol14 and an automated ELISA chip for chemical-biological analysis with an enhancement in the detection range and time,15 also constitute papers in this Special Topic section.We hope you enjoy reading the papers in this Special Topic section and that it provides you with a feel for the broad multidisciplinary spectrum across fundamental and applied microfluidic and nanofluidic research that the conference, as well as the journal, intends to span. Do watch out for the conference announcement for the next Advances in Microfluidics and Nanofluidics meeting in 2011 on the Biomicrofluidics website (http://bmf.aip.org)—hope to see you there! 相似文献
10.
M. Jain Kavita Sharma V. P. Sharma 《Indian journal of clinical biochemistry : IJCB》1994,9(2):106-108
Zinc (Zn), copper (Cu), magnesium (Mg) and retinol levels were studied in serum and tissue of the patients with prostatic carcinoma (Ca), benign hyperplasia (BHP) and control subjects. Zinc and retinol levels were low in both serum and tissues of patients with carcinoma, while in BHP, both zinc and retinol level were decreased. Copper levels were high in serum and tissues of both BHP as well as carcinoma patients. Magnesium levels were elevated in both serum and tissues of cancerous patients. The results indicate that Zn, Cu, and Mg metabolism is disturbed in malignancy. 相似文献
11.
A potent hypoglycaemic principle was isolated by us earlier from the seeds of fenugreek. We have now investigated its hypocholesterolemic effect. Hypercholesterolemia was induced in two groups of rabbits (5 each) by feeding orally cholesterol 100 mg/kg/day for one week. From 8th day group I animals (controls) received the same dose of cholesterol for 4 more weeks. Group II animals (treated) were given along with the same dose of cholesterol fenugreek principle at 50 mg/kg/day for 4 weeks. Fenugreek principle not only prevented the elevation of serum cholesterol, (LDL+VLDL)c, triacylglycerols and the ratios of total cholesterol/HDLc and (LDL+VLDL)c/HDLc, but also brought down most of these values. Blood glucose levels were normal. The study shows that the same hypoglycaemic principle has hypocholesterolemic effect also. 相似文献
12.
Ronald Pethig 《Biomicrofluidics》2010,4(2)
This Special Topic section is on dielectrophoresis, a growing area of widespread interest and relevance to the microfluidics and nanofluidics community.There was a time when the arrival of a telegram from the local post office would foreshadow a step-function change in one’s equilibrium. An internet service provider can now deliver the same effect, as illustrated by an unexpected e-mail from Leslie Yeo inquiring if I would “be interested in guest editing a special issue of Biomicrofluidics on recent advances in dielectrophoresis (DEP).” Flattery directed towards vanity can produce interesting results—which I hope this special issue of Biomicrofluidics demonstrates. The rationale for this special issue is the belief of the journal’s Editors (Dr. Chia Chang and Dr. Leslie Yeo) that dielectrophoresis is a growing area of widespread interest and relevance to the microfluidics and nanofluidics community. Papers, both fundamental and applied, were solicited from the leaders working across this broad interdisciplinary area of research. I was delighted by the positive responses of those whose invited contributions appear in this special issue—efforts certainly not motivated by vanity but through enthusiasm for the subject. Some of those invited to contribute were unable to do so because of other demands on their time. Ongoing advances being made in DEP, especially in its various applications, will surely merit another special issue in the future and hopefully include contributions from those unable to do so now.Two of the papers in this special issue address fundamental aspects of dielectrophoresis (DEP), namely the influences on DEP from electrical double-layers and from particle-particle interactions. Consideration of electrical double layers associated with charged particle surfaces is particularly important for nanoparticles because their effective polarizabilities, associated with field-induced dynamics of the counterions and co-ions in the double layer, can dominate over the intrinsic polarizability of the particle itself. This can influence, for example, to what extent the observation of changes in the DEP crossover frequency (marking the transition between positive and negative DEP) can be relied upon in new immunoassays based on the DEP behavior of functionalized nanoparticles. By considering the electrodynamics of double layers, Basuray et al.1 propose a theory to predict how the DEP crossover frequency will vary as a function of particle size and the ionic strength of the suspending electrolyte. In their paper, Sancho et al.2 derive a theoretical model to describe how particle-particle interactions (e.g., “pearl-chaining”) influence the DEP crossover frequency value. This model also describes well the changes in electrorotation and a newly observed precession effect as particles approach each other under the influence of a rotating field.DEP at the nanoscale is also addressed in contributions from the groups of Ralph Hölzel, Junya Suehiro, and Karan Kaler. Thus, Henning et al.3 describe a new method, based on the measurement of capacitance changes between planar microelectrodes, for the automatic acquisition of the DEP properties of nanoparticles without the need for labeling protocols or visual observations. Suehiro4 describes how DEP can be employed as a bottom-up approach for fabricating nanomaterial-based devices such as a carbon nanotube gas sensor and a ZnO nanowire photosensor. Kaler et al.5 describe how the DEP manipulation of miniscule amounts of polar aqueous samples, a method known as liquid-DEP, can be used for on-chip bioassays, such as nucleic acid analysis, and through parallel sample processing offer the potential for conducting automated multiplexed assays. The use of DEP to selectively trap and separate cells has been investigated over many years, and contributions from the groups of Hywel Morgan, Ana Valero, Masau Washizu, and Gerard Markx describe the latest advances and applications. Thomas et al.6 describe a new automated DEP cell trap design for the isolation, concentration, separation, and recovery of human osteoblast-like cells from a heterogeneous population. Recovery of small populations of human osteoblast-like cells with a purity of 100% is demonstrated. A cell-sorting device, based on the opposition of DEP forces that discriminates between cell types according to such properties as their membrane permittivity and cytoplasm conductivity, is described by Valeroet al.7 The versatility of the device is demonstrated by synchronizing a yeast cell culture at a particular phase of the cell cycle. Gel et al.8 describe a DEP-assisted cell trapping method for fusing pairs of cells in an array of micro-orifices. This method produces not only a high yield of viable cell fusants, but also allows for subsequent study of postfusion cell development. Zhu et al.9 describe a DEP-based microfluidic separation system in which dead and active cells can be collected from a given cell suspension, whilst at the same time eluting dormant cells. In the second paper from Gerard Markx’s group, Zhu et al.10 demonstrate that the rate-limiting resuscitation of a colony of dormant bacteria is determined by the diffusion of a resuscitation-promoting factor into the colony interior. This study involved the artificial engineering of different sizes and shapes of bacterial aggregates using DEP forces. Finally, in my own contribution,11 I have attempted to summarize the growing output of DEP publications in terms of their contributions to the theory, technology, and applications of DEP. 相似文献
13.
Jaehoon Chung David Issadore Adeeti Ullal Kyungheon Lee Ralph Weissleder Hakho Lee 《Biomicrofluidics》2013,7(5)
We present a hybrid magnetic/size-sorting (HMSS) chip for isolation and molecular analyses of circulating tumor cells (CTCs). The chip employs both negative and positive cell selection in order to provide high throughput, unbiased CTC enrichment. Specifically, the system utilizes a self-assembled magnet to generate high magnetic forces and a weir-style structure for cell sorting. The resulting device thus can perform multiple functions, including magnetic depletion, size-selective cell capture, and on-chip molecular staining. With such capacities, the HMSS device allowed one-step CTC isolation and single cell detection from whole blood, tested with spiked cancer cells. The system further facilitated the study of individual CTCs for heterogeneity in molecular marker expression.Circulating tumor cells (CTCs) have emerged as an important biomarker in clinical practice as well as in fundamental research.1, 2 CTCs, shed from primary tumors, have been shown to be an early harbinger of tumor expansion and metastasis3 and have been used to predict disease progression, response to treatment, relapse, and overall survival.4, 5, 6 Recent work has shown that CTCs display distinct proteomic and genetic profiles; for example, CTCs in pancreatic cancer, have increased RNA expression of Wnt, implicating this pathway in metastasis.7 Proteomic characterization of proliferative markers such as Ki-67, and hormonal markers such as androgen receptor in prostate cancer, also have been shown to be predictive of treatment outcome.8, 9Despite such clinical potential of CTCs, their routine detection and characterization still remains a significant technical challenge.10 The task requires screening of a large number of cells (e.g., > 107 cells in 10 ml blood) and enrichment of heterogeneous targets against a complex biological background. Two main methods of CTC isolation are typically used: positive and negative selection. In positive selection, CTCs are directly isolated from blood via size-based filtration11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or antibody-based capture.1, 8, 21 Negative depletion reduces abundant blood cells, often by immunomagnetic separation, for downstream CTC enrichment.22 Both approaches have been used for high throughput CTC isolation from whole blood (SI Table 1).23 Each method, however, has its own inherent limitations. Positive enrichment could be biased by its selection criteria (e.g., cell size and cell surface markers). Negative selection, albeit unbiased, often requires complex sample processing (e.g., multiple washing steps for CTC isolation) that could result in cell loss.We hypothesized that both positive and negative selection could be combined in a single platform to enable (1) highly efficient and unbiased CTC purification, and (2) in-situ molecular analyses of collected cells. As a proof-of-concept, we herein describe a hybrid magnetic/size-sorting (HMSS) system that integrates magnetic and size-based isolation into a compact microfluidic chip. The HMSS first uses a magnetic filter to deplete leukocytes through immunomagnetic capture. Samples then pass through a size-sorter region that traps individual cells at predefined locations. Since abundant leukocytes are removed by the magnetic filter, the size-sorter could have a low size cut-off (∼5 μm), which allows for the unbiased capture of even small cancer cells. Furthermore, molecular probes can be introduced to perform on-chip, multiplexed analyses at single-cell resolution. We evaluated the utility of the developed system by capturing and profiling tumor cells in whole blood. The HMSS offers the advantages of both negative and positive selection and thereby differs from the recently reported iChip system24 which can operate only in either a negative or a positive selection mode. 相似文献
14.
We demonstrate a microfluidic device capable of tracking the volume of individual cells by integrating an on-chip volume sensor with pressure-activated cell trapping capabilities. The device creates a dynamic trap by operating in feedback; a cell is periodically redirected back and forth through a microfluidic volume sensor (Coulter principle). Sieve valves are positioned on both ends of the sensing channel, creating a physical barrier which enables media to be quickly exchanged while keeping a cell firmly in place. The volume of individual Saccharomyces cerevisiae cells was tracked over entire growth cycles, and the ability to quickly exchange media was demonstrated.Measuring cell growth is of primary interest to researchers who seek to study the effects of drugs, nutrients, disease, and environmental stress. This has traditionally been accomplished by monitoring the optical transmittance of large ensembles of cells and applying the Beer-Lambert Law.1,2 Such population-scale measurements provide important culture statistics, but averaging obscures the behaviour of individual cells. In addition, these techniques often require cell synchronicity in order to correlate growth with specific points in the cell cycle, but synchronicity typically decays rapidly in many cell lines including Saccharomyces cerevisiae (yeast) cultures.3 Researchers have thus adopted methods that study the growth of individual cells. Quantifying cellular growth is especially challenging since proliferating cells such as yeast or Escherichia coli are irregularly shaped, and will only increase in size by a factor of two.4 Growth will affect the mass, volume, and density of the cell; having access to each of these characteristics is important in obtaining a complete picture of this process. Time-lapse fluorescence microscopy can provide valuable information as to the cell cycle progression of individual cells,5 but 2D optics requires geometric assumptions, and, thus, can provide an incomplete picture of growth.6,7Microfluidic lab-on-chip devices with integrated sensors can provide high-resolution growth tracking of individual cells, either through mass, volume, or density monitoring.4,7,8 Recently, a microfluidic mass sensor was used to track the buoyant mass of individual cells using a suspended microchannel resonator (SMR).4,9 Monitoring growth can also be accomplished by tracking volume using microfluidic volume sensors7 operating on the Coulter principle.10 Trapping can be achieved by either (1) cycling the target back and forth through the sensor (pressure-driven4 and electrokinetic7) or (2) holding a cell in place (posts,11 chevron structure,12 and E-Field13). The former, dynamic approach, allows a single cell to be sampled periodically by reversing flow directions after a cell is detected. Simple in its implementation, this technique also has the ability to compensate for a drifting baseline current resulting from parasitic ionic changes within the sensing channel or other sources of noise. On the other hand, static traps allow cells to be held in place while the buffer is rapidly exchanged.12 The ability to dynamically change cellular growth conditions during an experiment can lead to significant insight into the behaviour of cells in environments of varying salinity,14 oxidative,15,16 or osmotic conditions,17 as well as the effect of nutrients18 and drugs.19In this work, we propose a device capable of tracking growth using high-resolution volume measurements, combining the best attributes of both types of measurement systems; continuous baseline correction and the ability to rapidly exchange cell media. This is accomplished by using a pressure-driven, feedback-based dynamic trap, whereby a cell is cycled back and forth through the sensor within a microfluidic channel. On-chip sieve valves positioned at both ends of the sensing channel are able to selectively capture a cell while the solution is being replaced. As proof of principle, the volume of several individual yeast cells was monitored over the course of their respective growth cycles, and the ability to quantify growth response to media exchange was demonstrated.Devices were fabricated using multilayered soft lithography with polydimethylsiloxane (PDMS) molding.20 The completed device is pictured in Figure 1(a); full fabrication protocols are presented as supplementary material.21 To maximize measurement sensitivity, it is optimal to choose a channel width and height slightly larger than the dimensions of the target cell.22 However, yeast cells are asymmetrically shaped and tend to tumble as they traverse the sensor. Preliminary testing suggested this effect could be mitigated by having cells flow along trajectories far from the electrodes (through buoyancy), where electric field is more uniform. Thus, a channel height of 20 μm was chosen as a compromise. Channel height increases to 28 μm in the wider part of the central and bypass channels, a result of using a mold made out of reflowed photoresist.23 Channel width was set at 25 μm through the sensor, and widens to 80 μm at the sieve valves to facilitate valve actuation, which requires a high width to height ratio.20 The fluidic layer is integrated in a 35 μm thick PDMS spin-coated layer, above which sits a 50 μm tall valve channel in a 4 mm PDMS layer. Tubing connects I1 and I2 to a common inlet vial, V1 and V2 to vials filled with deionised water and O1 and O2 connect to empty vials (not pictured). Inlet pressures I1 and I2, and valve pressures V1 and V2 are controlled with manual regulators (SMC IR2000-N02-R and SMC IR2010-N02-R); outlet pressures are computer-controlled (SMC ITV-1011). This pressure scheme is detailed elsewhere.24 Current pulses caused by transiting particles/cells (Figure 1(d)) were acquired by applying a 50 kHz, 220 mV AC voltage between a pair of electrodes and measuring the drawn current. This frequency is sufficiently elevated to avoid the electrical double layer capacitance at the electrode-electrolyte interface,25 but low enough to avoid sensitivity to cell impedance or substrate.26 The electrical setup used for these experiments has been described previously.24,27 A temperature controller maintains the device at 30 °C.Open in a separate windowFIG. 1.(a) Micrograph of the microfluidic device. Two parallel bypass channels are connected by a sensing channel with sensing electrodes. Pressure is applied at inlets (I1, I2) and outlets (O1, O2) to control flow conditions. Valves (V1, V2) are positioned over each end of the sensing channel. Food coloring is used to highlight the valve (red) and fluidic layers (blue). (b) Flow mode: valves are unpressurized, and cells flow freely through the device. (c) Trapping mode: valves are pressurized to capture a cell within the central channel. Pressure-driven flow cycles the cell back and forth across the sensor. (d)Typical current pulses measured for a yeast cell.The cell capture, media exchange, and detection process occurs as follows. A cell suspension is loaded into the bypass channel and made to flow through the central sensing channel by imposing a pressure gradient (Figure 1(b)). Cells flowing through the sensor are observed optically; once a cell of interest is observed (a cell without a bud), valves are sealed (V1 = V2 = 35 psi). This stops all flow through the sensor, and enables bypass channels to be flushed and replaced with fresh media. After 2 min, valve channels are pressurized to 24 psi where they compress the channel to a sufficient height to physically restrict the passage of yeast cells, while allowing the media to flow through the central channel (Figure 1(c)). The pressure gradient between bypasses causes the media in the central channel to be flushed out, while the target cell is physically trapped. Replacing the media in the central channel takes 2 min. At this stage, a pressure-driven feedback-based dynamic trap can be initiated. In this dynamic trap mode, the pressure settings at O1 and O2 are adjusted to redirect the cell back and forth through the sensor, based on current pulses measured from cells transiting through the sensor. Through custom LabView® software, these outlet pressure settings are feedback-adjusted to maintain a speed of 250 μm/s in both directions at a detection frequency of 30 cells/min (Figure 1(d)). To minimize the effects of channel stretching/shrinking, the sum of pressures at O1 and O2 is held constant. This precaution was taken since the sensing channel structured within the flexible PDMS polymer will alter its geometry based on internal pressure.28 The short central channel ensures steady nutrient replenishment from the bypasses. For example, a glucose molecule takes ∼4 min to diffuse from the bypass to the electrodes. In practice, Taylor-Aris dispersion will reduce this replenishment time considerably. Based on video analysis, 25% of the central channel''s media is replenished every pressure reversal (video presented as supplementary material21). Polystyrene microspheres of 3.9 ± 0.3 μm, 5.6 ± 0.2 μm, and 8.3 ± 0.7 μm (NIST size standards) were used to calibrate the sensor, and obtain the current pulse-to-volume calibration for every solution (supplementary material21). The validity of this calibration method is discussed elsewhere.29 Care was taken to limit trajectory-based variations in signal: the device is positioned with electrodes at the top of the sensing channel, and with the negatively buoyant cells/particles flowing along the bottom. Based on previous experimental and theory work, we found that signal amplitude can vary as much as 3.5 fold for different heights.27 The effect of trajectory on current pulse amplitude has also been reported elsewhere.30,31 In this work, buoyancy is used to ensure that the cell flows along a trajectory at the same distance from the electrodes for every measurement.Saccharomyces cerevisiae (BY4743 Mat a/alpha, genotype: his3Δ1/his3Δ1 leu2Δ0/leu2Δ0 LYS2/lys2Δ0 met15Δ0/MET15 ura3Δ0/ura3Δ0 ade2::LEU2/ade2::URA3) was cultured to exponential phase at 30 °C in an incubator/shaker in yeast bacto-peptone (YPD) with 2% w/v glucose, supplemented with 0.2 M NaCl, 0.05% bovine serum albumin (BSA) and 42 mg/l adenine. Sodium chloride was added to enable the current pulse measurement, at a concentration where cells are viable;32 BSA was used to prevent cell agglomeration; adenine was supplemented since this particular yeast mutant does not produce its own supply. A cell suspension was introduced into the device, from which a cell at the early stages of its cell cycle was captured, and dynamically trapped for 100 min. Three typical cell growth results are shown in Figure 2(a). Since the culture was not synchronized, this leads to variability between “initial” cell volumes: there is a 27% difference in initial volume between the cells identified by red squares and green triangles. This is caused by (1) optical limits, whereby cells chosen for study are not all at the exact same cell cycle stage and (2) differences in the age of the mother cell: the more buds a mother cell has produced, the larger it becomes.33 On average, captured yeast cell demonstrated a doubling time consistent with growth rates under ideal incubator/shaker conditions; nutrient depletion, electric field, and shear stresses are not affecting growth. Optical inspection of budding cells confirms that most growth is occurring at the daughter cell, as expected.33 An elevated signal-to-noise ratio allows for high resolution volumetric measurements (4 μm3); cell asymmetry7 and trajectory variability27,30,31 lead to a relative standard deviation of 6% for cells and 4% for microspheres of similar size. While mass or protein synthesis methods have indicated linear34 or exponential4,6,35,36 growth curves, volume-based methods have suggested sigmoidal patterns.7,37 Prior to daughter cell emergence, and later in the cycle as the daughter cell emerges, volumetric growth rate declines.38 In this work, it is difficult to ascertain with mathematical rigor the shape of the growth profile; however, for each cell, volume increases steadily throughout the growth cycle before declining near the end of the cycle.Open in a separate windowFIG. 2.(a) Growth curves for 3 cells trapped in succession. Simultaneous optical and electrical measurements allow cell cycle stage to be correlated with volume. Pictures of cell corresponding to the red squares are presented in 15 min increments. A cell is cycled through the sensor every 2 s. For clarity, each data point for yeast volume represents the average of data points over a period of 5 min, with standard deviation. (b) Demonstration of an interrupted growth cycle, where YPD + 0.2 M NaCl was replaced with 0.2 M NaCl at 40 min, and then again returned to YPD + 0.2 M NaCl at 80 min. The media exchange process takes 4 min.To demonstrate our ability to easily exchange media while maintaining a trap, the solution was exchanged 40 min into a yeast growth cycle; culture media was replaced with a pure saline solution 0.2 M NaCl + 0.05% BSA, and then replaced again with culture media at 80 min (Figure 2(b)). Cell growth is halted temporarily while in saline solution, before resuming normal growth thereafter. The cell cycle time is extended by this period. The cell volume drifts downward after the initial solution change at 40 min. Though this drift lies within our uncertainty bounds, cellular responses to osmotic shock on similar timescales have been documented elsewhere.39 This result demonstrates an ability to quickly exchange cell media, and observe cellular response.In conclusion, we have demonstrated a microfluidic device capable of maintaining a dynamic, pressure-driven cell trap, which can monitor cellular volume over the cell cycle. Concurrent optical microscopy allows for real-time visual inspection of the cells. In addition, sieve valve integration provides for the exchange of media or the addition of drugs. Such a platform could also be key in cancer cell cytotoxicity assays,40 where growth response to anticancer drugs could be monitored. 相似文献
15.
This paper is devoted to stability analysis of discrete-time delay systems based on a set of Lyapunov–Krasovskii functionals. New multiple summation inequalities are derived that involve the famous discrete Jensen?s and Wirtinger?s inequalities, as well as the recently presented inequalities for single and double summation in [16]. The present paper aims at showing that the proposed set of sufficient stability conditions can be arranged into a bidirectional hierarchy of LMIs establishing a rigorous theoretical basis for the comparison of conservatism of the investigated methods. Numerical examples illustrate the efficiency of the method. 相似文献
16.
Large-library fluorescent molecular arrays remain limited in sensitivity (1 × 106 molecules) and dynamic range due to background auto-fluorescence and scattering noise within a large (20–100 μm) fluorescent spot. We report an easily fabricated silica nano-cone array platform, with a detection limit of 100 molecules and a dynamic range that spans 6 decades, due to point (10 nm to 1 μm) illumination of preferentially absorbed tagged targets by singular scattering off wedged cones. Its fluorescent spot reaches diffraction-limited submicron dimensions, which are 104 times smaller in area than conventional microarrays, with comparable reduction in detection limit and amplification of dynamic range.Commercially available fluorescent micro-arrays based on target labeling, northern blot, or enzyme-linked immunosorbent assay (ELISA) are limited to a detection threshold of 1 to 10 × 106 molecules per fluorescent spot,1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 thus requiring cell culturing or Polymerase Chain Reaction (PCR) amplification for many applications. The low sensitivity is often due to broad illumination, which creates auto-fluorescence noise. Even if point illumination and pin-hole filtering of non-focal plane noise are implemented in a confocal setup, the large and non-uniform fluorescent spots create scattering noise over each 20–100 μm element, which degrades the detection limit.4 Smaller spots can, in theory, be introduced by nano-sprays and nano-imprinting. However, directing the targets to such small areas then becomes problematic. Real-time PCR is, in principle, capable of detecting a single molecule but is limited in its target number5 and is hence slow/expensive for large-library assays. A large-library platform with much better detection limit than the current fluorescent microarrays would transform many screening assays. Ideally, this platform would not use the confocal configuration. Instead, it would direct the target molecules to a submicron spot and illuminate them with a nearby point source that does not require scanning.A promising platform is the optical fiber bundle array,6 with more than 104 fibers and targets, in principle. With its endoscopic configuration, these fiber bundles are most convenient for in situ and real-time biosensing modalities in microfluidic biochips and microfluidic 3-D cell cultures. Consequently, the optical sensing is typically carried out in the transmission mode, with the optical signals transmitted through the optical fibers to a detector. Microwell arrays at the distal end of imaging fiber, with molecular targets captured and transported to the microwells by microbeads, are the most popular among these optical fiber arrays. Although detection limit better than 1 × 106 molecules per bead has been reported, the bar-coded beads limit the target number of this platform.7, 8Our previous work9, 10 has shown that plasmonics at nanotips can enhance local electric field by three orders of magnitude. However, conduction loss and quenching of fluorescence11, 12 by the metal substrates limit the use of plasmonic enhanced fluorescence for large-library assays. Only nano-molar sensitivity has been demonstrated using plasmonics from metal coated nanocone tips.13, 14 In this paper, we will extend the conical fiber array platform not by tip plasmonics but by another optical phenomenon with induced dipoles: singular scattering off dielectric wedges and tips.15 Instead of the surface plasmon resonance on metallic nanostructures,16 field focusing at the cone tip by the dielectric media (the silica fiber) is used to produce a localized and singularly large scattering intensity at the tip. Singular scattering from a wedge or a cone has been known for decades.17, 18 It is only recently that numerical simulation19 has revealed that field focusing by this singular scattering can effect a five-order intensity enhancement that is frequency independent. This intense tip scattering produces a local light source at the tip that does not suffer from conduction loss. Unlike plasmonic metal nanostructures, the dielectric tip would also not quench the fluorescent reporters excited by the light source. In fact, it will help scatter the fluorescent signal, with Rayleigh scattering intensity scaling with respect to wavelength. We hence utilize this phenomenon for diffraction-limit fluorescent sensing/imaging for the first time here.The local light source due to tip scattering minimizes background auto-fluorescence and scattering noise, provided the target molecules preferentially diffuse towards the dielectric vertices. If the targets do not preferentially hybridize with probes at the vertices, there would be significant target loss, with a concomitant loss in sensitivity, because the vertex regions are just a small fraction of the total area. Fortunately, like electromagnetic radiation at the electrostatic limit of the Maxwell equations for sharp (sub-wavelength) vertices,20 the steady-state diffusion of molecules also obey the Laplace equation and so do the DC or AC electric potentials that drive electrophoresis and dielectrophoresis of the molecules.21 Hence, the diffusive, electrophoretic, and dielectrophoretic fluxes of target molecules are also singularly large at the vertices and there will be preferential hybridization there until the tip is saturated. Previously, we have demonstrated preferential diffusive transport of colloids to channel corners22 and dieletrophoretic trapping of bacteria23 and DNA molecules24 around sharp nanostructures like carbon nanotubes. Hence, dielectric nanotips fabricated by low-cost techniques can potentially provide the smallest fluorescent spot, which can preferentially capture target molecules and whose fluorescent image is limited in size only by the diffraction limit, without a confocal configuration.Although the scattering singularity is stronger at the conic tip, the total increase in scattering area of this singularity of measure zero is not as high as that of a sharp wedge, thus rendering the signal relatively weak. We hence employ a well-defined multi-wedged silica cone fabricated by wet-etching, with the wedges introduced by non-uniform stress formed during the fiber assembly process, to produce maximum scattering at the tip where three to four wedges converge (see inset of Fig. Fig.1A).1A). Using the reflection mode to fully exploit this singular scattering to excite fluorescent reporters at the tip and transmit the resulting signal, we report a nanocone array that can detect down to 100 molecules per cone tip with a large dynamic range from femtomolar to nanomolar concentrations. Although quantification for a single target is reported in this preliminary report, multi-target assays can readily be developed.Open in a separate windowFigure 1(A) A SEM image of the silica cone array where the single cone inset image shows three wedges converging into a 10 nm junction at the tip. (B) The optical setup of measurement. (C) The diffraction-limited fluorescent spot images.Amine-modified 35-base oligo-probes were functionalized onto both unetched silica fibers (as a control) and etched conic silica tips. The sample of 35-base ssDNA targets (corresponding to a primer for a segment of the Serotype 2 dengue genome) with a 5′ tagged Cy3 fluorophore was inserted into a microfluidic chip housing the fiber bundle (Fig. (Fig.1B)1B) and left overnight (see the supplementary material25 for exact sequence). After a standard rinsing protocol, fluorescent images were taken with an Olympus IX-71 fluorescent microscope for target concentrations ranging from 1 fM to 1 nM. A typical fluorescent image after hybridization is shown in Fig. Fig.1c,1c, where each micron-sized bright spot corresponds to a single tip in the cone array. The intensity profile shown in the supplementary material25 indicates a fluorescent spot smaller than 1 μm, indicating that the fluorescent light source is sub-wavelength and the resolution is close to diffraction limit. The size of this bright spot at the conic tip does not vary much with respect to the concentration but its intensity does, as shown in Fig. Fig.2A.2A. It was found that for flat fibers, only concentrations higher than 1 nM produced significant signals above the background. However, for etched conic fibers, 10 fM is clearly distinguishable from the background, which indicates that an improvement of sensitivity up to five orders can be realized by simply etching the flat surface into cone arrays. It also suggests very little target loss due to preferential hybridization onto the cone at sub-nM concentrations. We estimated the number of molecules per cone from the total number of molecules in target solution divided by the number of pixels on each fiber (104), which suggests less than 100 molecules per cone for a 10 fM bulk concentration, four orders better than any existing technology.Open in a separate windowFigure 2(A) Fluorescent intensity of etched conic fiber and unetched fiber for different concentrations of target molecules from 1 fM to 1 nM. (B) Fluorescent intensity increases linearly with exposure time. Non-target molecules with 1 μM concentration do not produce significant signal compared to lower concentrations of target molecules such as 1 nM and 10 nM (see the supplementary material25 for details of image analysis).Selectivity of the platform was also examined. Fig. Fig.2B2B presents the fluorescent intensity of the tips for non-target (1 μM) and target (1 nM and 10 nM) at different exposure times, which shows that fluorescent intensity increases linearly with exposure time. Beyond 5 s, saturation of images prevents further increase in the signal. For non-target, the intensity is much lower than 1 nM Target and 10 nM Target, which means non-target do not bind to the probes at the wedged tip as effectively as target molecules. Non-specific binding can be further removed by using more stringent buffers and higher flow rates.26 This platform can be extended to detect 70 000 targets, in theory, by functionalizing different probes onto each cones using localized photochemistry via masking, micro-mirror directed illumination, or direct laser writing. Extension to ELISA type protein assays is also straight forward. Integration of a transmission-mode optical fiber endoscope into a microfluidic biochip and into a 3-D cell culture for real-time monitoring of multiple molecular targets at near-single molecule resolution is currently underway. 相似文献
17.
Leslie Y. Yeo 《Biomicrofluidics》2010,4(1)
This Special Topic section is a compilation of several original contributions covering both fundamental and practical aspects of electrokinetic microfluidic phenomena that were presented during the Electrokinetics and Microfluidics sessions held at the conference.Electrokinetics is currently the mechanism of choice for the manipulation of fluids as well as colloidal and biological particles at microscale and nanoscale dimensions.1 The popularity of electrokinetics is perhaps not so surprising as electrodes are easy to fabricate and embed into microfluidic chips, thus allowing the entire fluid and particle actuation mechanism to be completely integrated into the device. In addition, driving microfluidics with electric fields is relatively straightforward and allows for precise actuation. Nevertheless, considerable challenges remain in understanding the complex mechanisms associated with the hydrodynamics of conducting and dielectric fluids and particles under the influence of electric fields. Concomitantly, there has been an exponential increase in research and development in this field along both fundamental and applied themes in the past five years.This sustained growth in the microfluidics community of electrokinetics research has led to a sequel to the first Electrokinetic Phenomena and Microfluidics session at the 82nd ACS Colloid and Surface Science Symposium in Raleigh, NC, in 2008, and which we hope will now be a regular feature at successive ACS Colloid and Surface Science meetings. This year at the combined 2009 13th International Conference on Surface and Colloid Science (ICSCS) and the 83rd ACS Colloid and Surface Science Symposium in New York, the Electrokinetics and Microfluidics symposium proved to be extremely popular, with three keynote lectures presented by Professor Howard Stone, Professor Hsueh-Chia Chang, and Professor Thomas Healy, and 44 oral presentations. In both 2008 and 2009, Biomicrofluidics has organized a special issue to cover some of the contributions reported at these meetings.2The growing interest in using electric fields to manipulate biological entities such as cells, DNA, and even single molecules is reflected in this year’s collection of papers with dielectrophoretic (DEP) phenomena comprising the bulk of the contributions. In Ref. 3, a new theory to describe Stern layer conductance along the surface of nanocolloids is proposed, forming the basis for the derivation of a more accurate prediction of the DEP crossover frequency. This theory is then employed to determine the conformation and, hence, optimum coverage of oligonucleotides on the surface of nanocolloid functionalized molecular probes during DNA hybridization under the influence of DEP, which can be exploited for biomolecular sensing. Other fundamental DEP papers include the investigation of particle motion under DEP induced optically via a photoconductor, in which Zhu et al.4 characterized the frequency dependence of the motion through the synchronous velocity spectra of the particles, and a numerical study of particle trapping at the throat of converging-diverging microchannels under the influence of negative DEP using a transient arbitrary Lagrangian–Eulerian finite element method.5 A more practical implementation is, on the other hand, reported by Yang et al.6 in which the negative DEP is exploited to separate colorectal cancer cells from other cells in a microfluidic device as a demonstration of a portable cancer detection tool.Continuing along the separation theme, but with regard to DNA separation using pulsed-field gel electrophoresis aided by sparse but regularly ordered microfabricated arrays of nanoposts, is a Brownian dynamics simulation model reported by Ou et al.7 in which DNA channeling, which predicts that the motion of DNA is undisturbed by the presence of arrays for large spacing to DNA equilibrium size ratios and when the field lines are straight, is predicted, consistent with experimental observations. In another fundamental paper, a direct numerical simulation model is presented to predict the current-voltage relationship across conducting pores along cell membranes, which is of fundamental importance in the electroporation process.8We hope that you will enjoy reading the contributions in this special topic and that it encourages you to participate in future Electrokinetics and Microfluidics meetings at the ACS Colloid and Surface Science Symposia, which we definitely hope will continue on a regular basis. 相似文献
18.
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