Syringe-vacuum microfluidics: A portable technique to create monodisperse emulsions     

Adam R. Abate  David A. Weitz 《Biomicrofluidics》2011,5(1)

We present a simple method for creating monodisperse emulsions with microfluidic devices. Unlike conventional approaches that require bulky pumps, control computers, and expertise with device physics to operate devices, our method requires only the microfluidic device and a hand-operated syringe. The fluids needed for the emulsion are loaded into the device inlets, while the syringe is used to create a vacuum at the device outlet; this sucks the fluids through the channels, generating the drops. By controlling the hydrodynamic resistances of the channels using hydrodynamic resistors and valves, we are able to control the properties of the drops. This provides a simple and highly portable method for creating monodisperse emulsions.Droplet-based microfluidic devices use micron-scale drops as “test tubes” for biological reactions.^{1, 2, 3} With the devices, the drops are loaded with cells, incubated to stimulate cell growth, picoinjected to introduce additional reagents, and sorted to extract rare specimens.^{4, 5, 6} This allows biological reactions to be performed with greatly enhanced speed and efficiency over conventional approaches: by reducing the drop volume, only picoliters of reagent are needed per reaction, while through the use of microfluidics, the reactions can be executed at rates exceeding hundreds of kilohertz. This combination of incredible speed and efficient reagent usage is attractive for a variety of applications in biology, particularly those that require high-throughput processing of reactions, including cell screening, directed evolution, and nucleic acid analysis.^{7, 8} The same advantages of speed and efficiency would also be beneficial for applications in the field, in which the amount of material available for testing is limited, and results are needed with short turnaround. However, a challenge to using these techniques in field applications is that the control systems developed to operate the devices are intended for use in the laboratory: to inject fluids, mechanical pumps are needed, while computers must adjust flow rates to maintain optimal conditions in the device.^{9, 10, 11, 12} In addition to significantly limiting the portability of the system, these qualities make them impractical for use outside the laboratory. For droplet-based microfluidic techniques to be useful for applications in the field, a general, robust, and portable system for operating them is needed.In this paper, we introduce a general, robust, and portable system for operating droplet-based microfluidic devices. In this system, which we call syringe-vacuum microfluidics (SVM), we load the reagents needed for the emulsion into the inlets of a microfluidic drop maker; using a standard plastic syringe, we generate a vacuum at the outlet of the drop maker,¹³ sucking the reagents through the channels, generating drops, and transporting them to different regions for visualization and analysis. By controlling the vacuum strength and channel resistances using hydrodynamic resistors^{14, 15, 16} and single-layer membrane valves,^{17, 18} we are able to specify the flow rates in different regions of the device and to adjust them in real time. No pumps, control computers, or electricity is needed for these operations, making the entire system portable and of potential use for field applications. To characterize the adjustability and precision of this system, we vary channel resistances and vacuum pressures while measuring the effects on drop size and production frequency. We also show how to use this to form drops of many distinct reagents simultaneously using only a single vacuum syringe.Monodisperse drop formation is the central operation in droplet-based microfluidics but can be quite challenging due to the need for precise, steady pumping of reagents; forming monodisperse drops with controlled properties is thus a stringent demonstration of the effectiveness of a control system. While there are many geometries available for microfluidic drop formation,¹⁹ in this discussion we use a simple cross-junction for its proven ability to form uniform emulsions at high rates of speed,^{20, 21} a schematic of which is shown in Fig. ​Fig.1.1. The devices are fabricated in poly(dimethylsiloxane) (PDMS) using soft lithography.²² The drop formation channels have dimensions of 25 μm in width and 25 μm in height. To enable production of aqueous drops in oil, which are the most useful for biological assays, we require hydrophobic devices, which we achieve using an Aquapel chemical treatment: we flow Aqualpel through the channels for a few seconds, flush with air, and then bake the devices for 20 min at 65 °C. After this treatment, the channels are permanently hydrophilic, as is needed for forming aqueous-in-oil emulsions. To introduce reagents into the device, we use 200 μl plastic pipette tips inserted into the channel inlets. To apply the suction, we use a 10 ml Bectin-Dickenson plastic syringe coupled to the device through a 16 G needle and PE∕5 tubing. The other end of the tubing is inserted into the outlet of the device.Open in a separate windowFigure 1Schematic of the microfluidic drop maker for use with SVM. To form water drops in oil, the device must be hydrophobic, which we achieve by treating the channels with Aquapel. The water and surfactant-containing oil are loaded into pipette tips inserted into the device inlets at the locations indicated. To pump the fluids through the drop maker, a syringe applies a vacuum to the outlet; this sucks the fluids through the drop maker, forming drops. The drops are collected into the suction syringe, where they can be stored, incubated, and reintroduced into a microfluidic device for additional processing.To begin forming drops, we fill the device with HFE-7500 fluorocarbon oil, displacing trapped air bubbles that could restrict flow and interfere with drop formation. Pipette tips containing reagents are then inserted into the device inlets, as shown in Fig. ​Fig.11 and pictured in Fig. ​Fig.2a;2a; during this step, care must be taken to not trap air bubbles under the pipette tips, as they would restrict flow. For the fluids, we use distilled water for the droplet phase and HFE-7500 with the ammonium salt of Krytox 157 FSL at 1.8 wt % for the continuous phase. The suction syringe is then connected to the device outlet; to initiate drop formation, the piston is pulled outward and locked in place with a 1 in. binder clip, as shown in Fig. ​Fig.2a.2a. This expands the air in the syringe, generating a vacuum that is transferred to the device through tubing. Since the inlet reagents are open to the atmosphere and thus maintained at a pressure of 1 atm, this creates a pressure differential through the device that pumps the fluids. As the fluids flow through the cross-channel, forces are generated that create drops, as shown in Fig. ​Fig.2b2b (enhanced online). Due to the very steady flow, the drops are highly monodisperse, as shown in Fig. ​Fig.2c.2c. After they are formed, the drops flow out of the device through the suction tube and are collected into the syringe. Depending on the emulsion formulation, drops may coalesce on the metal needle of the syringe; if so, an Upchurch fitting should be used to couple the tubing instead. The collected drops can be stored in the syringe, incubated, and reintroduced into additional microfluidic devices, as needed for the assay.Open in a separate windowFigure 2Photograph of the microfluidic drop formation device with pipette tips containing emulsion reagents and vacuum syringe for pumping (a). Distilled water is used for the droplet phase and HFE-7500 fluorocarbon oil with fluorinated surfactant for the continuous phase. The vacuum applies a pressure differential through the device that pumps the fluids through the drop maker (b) forming drops. The drops are monodisperse, due to the controlled properties of drop formation in microfluidics (c). The scale bars denote 50 μm (enhanced online).In many biological applications, drop size must be precisely controlled. This is essential, for example, when encapsulating molecules or cells in the drops, in which the number encapsulated depends on the drop size.^{3, 23, 24} With SVM, the drop size can be precisely controlled. Our strategy to accomplish this is motivated by the physics of microfluidic drop formation. In microfluidic devices, the capillary number of the flow is normally small, Ca<0.1; as a consequence, the drop formation physics follows a plugging∕squeezing mechanism, in which the drop size depends on the flow rate ratio of the dispersed-to-continuous phase.^{20, 25} By adjusting this ratio, we can thus control the drop size. To adjust this ratio, we use hydrodynamic resistor channels.^{14, 15, 16} These channels are analogous to electronic resistors in that for a fixed pressure drop (voltage) the flow rate through them (current) is inversely proportional to their resistance. By making the resistors longer or shorter, we adjust their resistance, thereby controlling the flow rate.To use resistors to control the drop size, we place three on the inlets of the cross-junction, at the locations indicated in Fig. ​Fig.3a.3a. In this configuration, the flow rate ratio depends on the resistances of the central and side resistors: shortening the side resistors increases the continuous phase flow rate with respect to the dispersed phase, thereby reducing the ratio and, consequently, the drop size, whereas lengthening it increases the drop size. By varying the ratio, we produce drops over a range of sizes, as shown in Fig. ​Fig.3b3b (enhanced online). The drop size is linear in the resistance ratio, indicating that it is linear in the flow rate ratio, as is expected for plugging∕squeezing drop formation [Fig. ​[Fig.3b3b].^{20, 25} This behavior is identical to that of pump-driven fluidics, demonstrating that SVM affords similar control.Open in a separate windowFigure 3Drop properties can be controlled using resistor channels. The resistors are placed on the inlets of the drop maker at the locations indicated in (a). The resistors enable the flow rates of the inner and continuous phases to be controlled. By varying the length ratio of the inlet resistors, we control the flow rate ratio in the drop maker. This allows the drop volume to be controlled, as shown by drop volume plotted as a function of inlet resistor length ratio in (b); varying this ratio does not significantly affect the drop formation frequency, as shown in (c). By varying the length of the outlet resistor, we control the total flow rate through the device; this allows us to form drops of constant volume, but at a different formation frequency, as shown by the plots of volume and frequency as a function of the inverse of the outlet resistor length in (d) and (e), respectively. The measured hydrodynamic resistance of a resistor channel with water as a function of length is shown as inset into (d) (enhanced online).We can also control the frequency of the drop formation using resistor channels. We place a resistor on the outlet of the device; this sets the total flow rate through the device, thereby adjusting drop frequency, as shown in Fig. ​Fig.3e3e (enhanced online). To confirm that the size and frequency control are independent, we plot size as a function of the outlet resistance and frequency as a function of the resistance ratio [Figs. ​[Figs.3c,3c, ​,3d];3d]; both are constant as a function of these parameters, again demonstrating independent control. Frequency can also be adjusted by changing the strength of the vacuum, which can be accomplished by loading a prescribed volume of air into the syringe before expansion. In this case, the vacuum pressure applied is P_fin=V_in∕V_fin×P_in, where V_in is the initial volume of air in the syringe, V_fin is the volume after expansion, and P_in is the initial pressure, which is 1 atm. By loading a prescribed volume of air into the syringe before connecting it to the device and pulling the piston, the expansion factor can be reduced, thereby lowering the vacuum strength.The flow rates through the microfluidic device depend on the applied pressure differential, which, in turn, depends on the value of the ambient pressure. Since ambient pressure may vary due to differences in altitude, the drop formation may also vary. However, since ambient pressure variations affect the inner and outer phase flows equally, this should alter the total flow rate but not the flow rate ratio. Consequently, we expect it to alter drop formation frequency but not drop size because while the frequency depends on absolute flow rate [as illustrated by Fig. ​Fig.3e],3e], drop size depends on the flow rate ratio [as illustrated in Fig. ​Fig.3b].3b]. Based on normal variations in atmospheric pressure on the surface of the Earth, we expect this to produce differences in the drop formation frequency of ∼25%, for example, when operating a device at sea level compared to at the top of a moderately sized mountain.Resistor channels allow drop properties to be controlled, equivalent to what is possible with pump-driven flow; however, they do not allow real-time control because their dimensions are fixed during the fabrication. Real-time control is often needed, for example, as it is when performing reactions in drops for the first time, in which the optimal drop size is not known. To enable real-time control, we must adjust flow rates, which can be achieved using the fluidic analog of electronic potentiometers. Single-layer membrane valves are analogous fluidic components, consisting of a control channel that abuts a flow channel.^{17, 18} By pressurizing the control channel, the thin PDMS membrane between these channels is deflected laterally, constricting the flow channel, thereby increasing its hydrodynamic resistance and reducing its flow rate.¹⁸ To use these membrane valves to vary drop size, we replace the inlet resistors with inlet valves, as shown in Fig. ​Fig.4a.4a. To set the flow rate through a path, we actuate the valve with a defined pressure. To actuate the valves, we use air-filled syringes: a 1 ml syringe is filled with air and connected to the valve control channel through tubing; an additional component, a three-way stopcock is inserted between the syringe and needle, allowing the pressure to be locked in after optimal actuation conditions are obtained. We use one syringe to control the dispersed phase valves and another to control the continuous phase valves. The valves are pressurized by compressing the air in the syringes to a defined degree using the marked graduations; this is achieved by pressing the piston to a defined graduation mark, compressing the air contained within it, thus increasing pressure. The stopcock is then switched to the off position, locking in the actuation. This simple scheme allows precise actuation of the valves, for accurate, defined flow rates in the drop maker, and controlled drop size, as shown in Figs. ​Figs.4b,4b, ​,4c4c (enhanced online). The drop size can be varied at a rate of several hertz without noticeable loss of control; moreover, changing the drop size does not affect the frequency, indicating that, again, these properties are independent, as shown by the constant drop frequency with varying pressure ratio in Fig. ​Fig.4d4d.Open in a separate windowFigure 4Single-layer membrane valves allow the drop size to be varied in real time to screen for optimal reaction conditions. The valves are positioned on the inner and side inlets, as indicated in (a). By adjusting the actuation pressures of the valves, we vary the flow rates in the drop maker, thereby changing the drop size (b), as shown by the plot of drop volume as a function of the actuation pressure ratio in (c). Varying the inlet resistance ratio does not significantly alter drop formation frequency, as shown by frequency as a function of the pressure ratio in (d). A movie of drop formation during actuation of the valves are available in the supplemental material (Ref. ²⁹). The scale bars denote 100 μm (enhanced online).Another useful attribute of SVM is that it readily lends itself to parallel drop formation²⁶ because the pressure that pumps the fluids through the channels is supplied by the atmosphere and is applied evenly over the whole outer surface of the device. This allows fluids to be introduced at equal pressures from different inlets, for forming drops with identical properties in different drop makers. To illustrate this, we use a parallel drop formation device to emulsify eight distinct reagents simultaneously; the product of this is an emulsion library, consisting of drops of identical size in which different drops encapsulate distinct reagents, useful for certain biological applications of droplet-based microfluidics.⁷ The microfluidic device consists of eight T-junction drop makers.²⁵ The drop makers share one oil inlet and outlet but each has its own inner-phase inlet, as shown in Fig. ​Fig.5.5. The oil and outlet channels are wide, ensuring negligible pressure drop through them, so that all T-junctions are operated under the same flow conditions. A distinct reagent fluid is introduced into the inner phase of each T-junction, for which we use eight concentrations of the dye Alexa Fluor 680 in water. After loading these solutions into the device through pipette tips, a syringe applies the vacuum to the outlet, sucking the reagents through the T-junctions, forming drops, as shown by the magnified images of the T-junctions during drop formation in Fig. ​Fig.5.5. Since the drop makers are identical and operated under the same flow conditions, the drops formed are of the same size, as shown in the magnified images in Fig. ​Fig.55 and in a movie available in the supplemental material.²⁹Open in a separate windowFigure 5Parallel drop formation device consisting of eight T-junction drop makers. The drop makers share a common oil inlet and outlet, both of which are wide to ensure even pressure distribution to all drop makers; support posts prevent these channels from collapsing under the suction. Each drop maker has its own inner-phase inlet, allowing emulsification of a distinct reagent. Since the drop maker dimensions and pressure differentials are constant through all drop makers, the drops formed are of the same size, as shown in the magnified images. The drops are ∼35 μm in diameter.To verify that the dye solutions are successfully encapsulated, we image a sample of the collected drops with a fluorescent microscope. The drops are confined in a monolayer between two glass plates so they can be individually imaged. They are of the same size but have distinct fluorescence intensities, as shown in Fig. ​Fig.6a.6a. To quantify these differences, we measure the intensity of each drop and plot the results as a histogram [see Fig. ​Fig.6b].6b]. There are eight peaks in the histogram, corresponding to the eight dye concentrations, demonstrating that all dyes are encapsulated successfully. The peak areas are also similar, demonstrating that drops of different types are formed in equal amounts due to the uniformity of the parallel drop formation.Open in a separate windowFigure 6Fluorescent microscope image of emulsion library created with parallel T-junction device (a). In this demonstration, eight concentrations of Alexa Fluor 680 dye are emulsified simultaneously, producing an emulsion library of eight elements. The drops are of the same size but encapsulate distinct concentrations of the dye solution, as demonstrated by the eight peaks in the intensity histograms in (b). The scale bar denotes 100 μm.SVM is a simple, accessible, and highly controlled way to form monodisperse emulsions for biological assays. It allows controlled amounts of different reagents to be encapsulated in individual drops, drop size to be precisely controlled, and the ability to form drops of different reagents at the same time, in a parallel drop formation device. These properties should make SVM useful for biological applications of monodisperse emulsions;^{1, 2, 3} the portability of SVM should also make it useful for applications in the field, particularly when no electrical power source is available. The parallel emulsification technique should also be useful for particle templating from drops, in which the particles must be of the same size but composed of distinct materials.^{26, 27, 28, 29}  相似文献   

Shrinking selves in synthetic sites: On personhood in a Walt Disney World     

Charles W. Harvey  Carol Zibell 《Ethics and Information Technology》2000,2(1):19-25

In this essay we show how certain tendencies of theself are enhanced and hindered by technologicallyorganized places. We coordinate a cognitive andbehavioral technology for the control of personalidentity with the technologically totalizedenvironments that we call synthetic sites. Weproceed by describing Mihaly Csikszentmihalyi'sstrategy for intensifying experience and organizingthe self. Walt Disney World is then considered as theexample, par excellence, of a synthetic sitethat promotes ordered experience via self-shrinkage. Finally, we reflect briefly on problems andpossibilities of human life lived in a world that canbe described with increasing accuracy as anarchipelago of synthetic sites.  相似文献   

Design of H2 (H∞)-based optimal structured and sparse static output feedback gains     

Ahmadreza Argha  Li Li  Steven W. Su 《Journal of The Franklin Institute》2017,354(10):4156-4178

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Existence theorem of the quadruple (P, R, F, M): Precision,recall, fallout and miss     

L. Egghe 《Information processing & management》2007

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CNI: Compelled Nonuse of Information     

Ron Houston 《Information processing & management》2011

Purpose

The study reported in this paper reviewed the literatures of information science, psychology, sociology, political science, education, and communication science to analyze Compelled Nonuse of Information (CNI). This study of a behavior defined by its absence (i.e., the not using of information) involved the development of a methodology consisting of an iterative performance of a nine-step heuristic leading to a retroductive recognition of absence, here termed RRA.

Principal results

The study concluded with a hierarchical taxonomy of the mechanisms that compel a person not to use information. The six primary mechanisms are:

1.

Intrinsic somatic (bodily) conditions

2.

Socio-environmental barriers

3.

Authoritarian controls

4.

Threshold knowledge shortfall

5.

Attention shortfall

6.

Information filtering.

Major conclusions

The resultant taxonomy of CNI appears here as a comprehensive checklist with which information workers such as the teacher, librarian, advertiser, politician, or health care professional can respond efficiently and effectively to situations of nonuse of information. For example, a teacher might ask: “Why are students not responding to what I present?” Further, the social implications of any compelled behavior touch the very basis of the social contract, and this paper presents a first step toward understanding the compelled aspects of CNI.  相似文献   

Continuous, weighted Lorenz theory and applications to the study of fractional relative impact factors     

L. Egghe   《Information processing & management》2005,41(6):1330-1359

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Canonical Expansion of a Mixed Bivariate Distribution with Negative Binomial and Gamma Marginals     

P.A. Lee 《Journal of The Franklin Institute》1979,307(6):331-339

For the following mixed bivariate probability distribution between a discrete random variable X and a continuous random variable Λ:

where α, β > 0, 0 < p = 1 ? q < 1, x=0,1,2,...,

a canonical expansion is obtained in terms of the Laguerre and Meixner orthogonal polynomials. The chance mechanisms giving rise to this mixed bivariate distribution are also discussed.  相似文献   

More is different: how aggregation turns on the light     

Philip Ball 《国家科学评论(英文版)》2021,8(6)

The reductionist approach to science seeks to understand the behaviour of systems by studying their individual components. It has been an enormously productive approach, but it is also widely acknowledged now that in some systems the behaviour of interest is an emergent property that cannot be discerned in the separate parts. Biology is replete with such examples, from the flocking of birds to the way metabolic processes in cells rely on a dynamic interplay of proteins and other components.Yet molecular systems do not have to be particularly complex before their properties become more than the sum of the parts. A classic example is the appearance of bulk-like metallic behaviour in small clusters of metal atoms only once they exceed a certain critical size. One of the most striking instances became apparent in 2001, when Ben Zhong Tang of the Hong Kong University of Science and Technology and his co-workers found that heterocyclic silicon-containing molecules called siloles become luminescent as nanoscopic aggregates even though the individual molecules in dilute solution do not emit light [1]. This looked like the opposite of the well-known phenomenon of concentration quenching, in which energy transfer between fluorescent (generally organic) molecules quenches the emission, an effect explained in 1955 [2]. Aggregation-induced ‘switching off’ is intuitively understandable, but ‘switching on’ due to aggregation was more surprising.Yet this effect of ‘aggregation-induced emission’ (AIE), as Tang and colleagues called it, was apparently seen, but not understood, much earlier [3]. In the 1850s, George Stokes noted that some inorganic complexes were fluorescent in the condensed, solid state but not in solution. At first, AIE was seen as a curiosity and deemed likely to be rare. However, subsequent research has shown not only that it is a rather common effect but also that it can be considered just one manifestation of a wide range of behaviours that arise from aggregation—leading to the proposed field of ‘aggregate science’, manifesting at the supramolecular level of small clusters or groups of molecules held together by relatively weak interactions. The field might be considered to illustrate George Whitesides’ notion of a chemistry ‘beyond the molecule’ [4], which bridges disciplines ranging from colloid science to crystal growth, nanotechnology, liquid crystals, photochemistry and molecular biology. At the same time, it echoes the famous insight of physicist Philip Anderson about emergent phenomena and the hierarchical nature of science: ‘More is different’ [5]. An ability to switch properties on and off by controlling intermolecular interactions and aggregation suggests various applications, from optical device technologies to targeted drugs for cancer therapy [6].NSR spoke to Ben Zhong Tang about the origins and possibilities of the field.

NSR: It seems you noticed AIE in 2001 by accident. How did it come about? Tang: Yes, it was serendipity. Development of new light emitters for the fabrication of organic light-emitting diodes was a hot topic at that time. We were trying to make new luminophores [light-emitting molecules] with high efficiencies and novel structures. Attracted by the aesthetically pleasing molecular structures of siloles, I asked my students to prepare various silole compounds. One day, a student told me that he could not see any luminescence when he used a UV lamp to excite the solution of the silole compound he had made. This surprised me, because I myself prepared a silole compound when I was a PhD student and I remember that its crystal was luminescent. I sensed something strange and immediately rushed to the lab. After careful verification and discussion with the student, we concluded that both of us were correct: the silole solution was not luminescent (his observation was right) but the silole powder was emissive (my memory was right). The non-luminescent molecular species in the dilute solution were induced to emit light through formation of aggregates in the solid state. We termed the process aggregation-induced emission or AIE.

A mesoscopic aggregate can have a property that its molecular species does not exhibit at all.—Ben Zhong Tang

Open in a separate windowBen Zhong Tang of the Hong Kong University of Science and Technology, China (Courtesy of Ben Zhong Tang). NSR: The phenomenon seemed to defy conventional expectations. Did you have trouble persuading others—or yourselves!—that it was real? Tang: I initially thought the student might have done something wrong, for the phenomenon he observed was totally unexpected. The common belief in the community of photophysics research is that luminescence from an organic dye generally weakens when its molecules are aggregated, an effect often referred to as aggregation-caused quenching or ACQ. I was shocked when I realized that the silole luminogen was showing an anti-ACQ effect. Still, I felt lucky to encounter something ‘abnormal’. No matter how odd a phenomenon seems, if it can be repeatedly observed, it must be real. We repeated our experiments many times and we were eventually convinced that the AIE effect was true. We had trouble, however, to understand why the silole luminogen behaved in such a way that was diametrically opposed to conventional ACQ. NSR: Are there any historical precedents—experiments in which this effect might have been glimpsed previously, but not recognized as such? Tang: When we published our first AIE paper in 2001, we thought the photophysical effect was unprecedented. However, we gradually found out that similar phenomena had been previously observed by other scientists. For example, in 1853 George Stokes reported in a paper that some inorganic platinocyanide salts ‘are sensitive’ (meaning luminescent in modern terminology) ‘only in the solid state’ but ‘their solutions look like mere water’. Sadly, he didn’t follow it up. Other people have made similar observations in different dye systems, which were, however, not recognized as AIE processes. Partially because of this, we had great difficulty in finding relevant reference papers. As a matter of fact, Stokes’ report, published in the mid-19th century, was not known to us until the middle of 2018. However, we are not surprised by those early works, for we understand that science progresses not in an abrupt but in a continuous way. George Smith articulated this: ‘Very few research breakthroughs are novel. Virtually all of them build on what went on before.’ A discovery is often a happenstance. We happened to have ‘rediscovered’ a very old but largely unnoticed phenomenon. Luckily, we grasped the opportunity to see more and farther by standing on the shoulders of giants.  相似文献   

You cannot tell a book by looking at the cover: Cryptic complexity in bacterial evolution     

Qiucen Zhang  Julia Bos  Grigory Tarnopolskiy  James C. Sturm  Hyunsung Kim  Nader Pourmand  Robert H. Austin 《Biomicrofluidics》2014,8(5)

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Water transport to the core–mantle boundary     

Michael J Walter 《国家科学评论(英文版)》2021,8(4)

Water is transported to Earth''s interior in lithospheric slabs at subduction zones. Shallow dehydration fuels hydrous island arc magmatism but some water is transported deeper in cool slab mantle. Further dehydration at ∼700 km may limit deeper transport but hydrated phases in slab crust have considerable capacity for transporting water to the core-mantle boundary. Quantifying how much remains the challenge.

Water can have remarkable effects when exposed to rocks at high pressures and temperatures. It can form new minerals with unique properties and often profoundly affects the physical, transport and rheological properties of nominally anhydrous mantle minerals. It has the ability to drastically reduce the melting point of mantle rocks to produce inviscid and reactive melts, often with extreme chemical flavors, and these melts can alter surrounding mantle with potential long-term geochemical consequences. At the base of the mantle, water can react with core iron to produce a super-oxidized and hydrated phase, FeO₂H_x, with the potential to profoundly alter the mantle and even the surface and atmosphere redox state, but only if enough water can reach such depths [1].Current estimates for bulk mantle water content based on the average H₂O/Ce ratio of oceanic basalts from melt inclusions and the most un-degassed basalts, coupled with mass balance constraints for Ce, indicate a fraction under one ocean mass [2], a robust estimate as long as the basalts sampled at the surface tap all mantle reservoirs. The mantle likely contains some primordial water but given that the post-accretion Earth was very hot, water has low solubility and readily degasses from magma at low pressures, and its solubility in crystallizing liquidus minerals is also very low, the mantle just after accretion may have been relatively dry. Thus, it is plausible that most or even all of the water in the current mantle is ‘recycled’, added primarily by subduction of hydrated lithospheric plates. If transport of water to the core–mantle boundary is an important geological process with planet-scale implications, then surface water incorporated into subducting slabs and transported to the core–mantle boundary may be a requirement.Water is added to the basaltic oceanic crust and peridotitic mantle in lithospheric plates (hereafter, slab crust and slab mantle, respectively) at mid-ocean ridges, at transform faults, and in bending faults formed at the outer rise prior to subduction [3]. Estimates vary but about 1 × 10¹² kg of water is currently subducted each year into the mantle [4], and at this rate roughly 2–3 ocean masses could have been added to the mantle since subduction began. However, much of this water is returned to the surface through hydrous magmatism at convergent margins, which itself is a response to slab dehydration in an initial, and large, release of water. Meta-basalt and meta-sediments comprising the slab crust lose their water very efficiently beneath the volcanic front because most slab crust geotherms cross mineral dehydration or melting reactions at depths of less than 150 km, and even if some water remains stored in minerals like lawsonite in cooler slabs, nearly complete dehydration is expected by ∼300 km [5].Peridotitic slab mantle may have much greater potential to deliver water deeper into the interior. As shown in Fig. 1a, an initial pulse of dehydration of slab mantle occurs at depths less than ∼200 km in warmer slabs, controlled primarily by breakdown of chlorite and antigorite when slab-therms cross a deep ‘trough’, sometimes referred to as a ‘choke point’, along the dehydration curve (Fig. 1a) [6]. But the slab mantle in cooler subduction zones can skirt beneath the dehydration reactions, and antigorite can transform directly to the hydrated alphabet silicate phases (Phases A, E, superhydrous B, D), delivering perhaps as much as 5 wt% water in locally hydrated regions (e.g. deep faults and fractures in the lithosphere) to transition zone depths [6]. Estimates based on mineral phase relations in the slab crust and the slab mantle coupled with subduction zone thermal models suggest that as much as 30% of subducted water may have been transported past the sub-volcanic dehydration front and into the deeper mantle [4], although this depends on the depth and extent of deep hydration of the slab mantle, which is poorly constrained. Coincidentally, this also amounts to about one ocean mass if water subduction rates have been roughly constant since subduction began, a figure tantalizingly close to the estimated mantle water content based on geochemical arguments [2]. But what is the likely fate of water in the slab mantle in the transition zone and beyond?Open in a separate windowFigure 1.(a) Schematic phase relations in meta-peridotite modified after [6,10,12]. Slab geotherms are after those in [4]. Cold slabs may transport as much as 5 wt% water past ‘choke point 1’ in locally hydrated regions of the slab mantle, whereas slab mantle is dehydrated in warmer slabs. Colder slab mantle that can transport water into the transition zone will undergo dehydration at ‘choke point 2’. How much water can be transported deeper into the mantle and potentially to the core depends on the dynamics of fluid/melt segregation in this region. (b) Schematic showing dehydration in the slab mantle at choke point 2. Migration of fluids within slab mantle will result in water dissolving into bridgmanite and other nominally anhydrous phases with a bulk storage capacity of ∼0.1 wt%, potentially accommodating much or all of the released water. Migration of fluids out of the slab into ambient mantle would also hydrate bridgmanite and other phases and result in net fluid loss from the slab. Conversely, migration of hydrous fluids into the crust could result in extensive hydration of meta-basalt with water accommodated first in nominally anhydrous phases like bridgmanite, Ca-perovskite and NAL phase, but especially in dense SiO₂ phases (stishovite and CaCl₂-type) that can host at least 3 wt% water (∼0.6 wt% in bulk crust).Lithospheric slabs are expected to slow down and deform in the transition zone due to the interplay among the many factors affecting buoyancy and plate rheology, potentially trapping slabs before they descend into the lower mantle [7]. If colder, water-bearing slabs heat up by as little as a few hundred degrees in the transition zone, hydrous phases in the slab mantle will break down to wadsleyite or ringwoodite-bearing assemblages, and a hydrous fluid (Fig. 1a). Wadselyite and ringwoodite can themselves accommodate significant amounts of water and so hydrated portions of the slab mantle would retain ∼1 wt% water. A hydrous ringwoodite inclusion in a sublithospheric diamond with ∼1.5 wt% H₂O may provide direct evidence for this process [8].But no matter if slabs heat up or not in the transition zone, as they penetrate into the lower mantle phase D, superhydrous phase B or ringwoodite in the slab mantle will dehydrate at ∼700–800 km due to another deep trough, or second ‘choke point’, transforming into an assemblage of nominally anhydrous minerals dominated by bridgmanite (∼75 wt%) with, relatively, a much lower bulk water storage capacity (< ∼0.1 wt%) [9] (Fig. 1a). Water released from the slab mantle should lead to melting at the top of the lower mantle [10], and indeed, low shear-wave velocity anomalies at ∼700–800 km below North America may be capturing such dehydration melting in real time [11].The fate of the hydrous fluids/melts released from the slab in the deep transition zone and shallow lower mantle determines how much water slabs can carry deeper into the lower mantle. Presumably water is released from regions of the slab mantle where it was originally deposited, like the fractures and faults that formed in the slab near the surface [3]. If hydrous melts can migrate into surrounding water-undersaturated peridotite within the slab, then water should dissolve into bridgmanite and coexisting nominally anhydrous phases (Ca-perovskite and ferropericlase) until they are saturated (Fig. 1b). And because bridgmanite (water capacity ∼0.1 wt%) dominates the phase assemblage, the slab mantle can potentially accommodate much or all of the released water depending on details of how the hydrous fluids migrate, react and disperse. If released water is simply re-dissolved into the slab mantle in this way then it could be transported deeper into the mantle mainly in bridgmanite, possibly to the core–mantle boundary. Water solubility in bridgmanite throughout the mantle pressure-temperature range is not known, so whether water would partially exsolve as the slab moves deeper stabilizing a melt or another hydrous phase, or remains stable in bridgmanite as a dispersed, minor component, remains to be discovered.Another possibility is that the hydrous fluids/melts produced at the second choke point in the slab mantle at ∼700 km migrate out of the slab mantle, perhaps along the pre-existing fractures and faults where bridgmanite-rich mantle should already be saturated, and into either oceanic crust or ambient mantle (Fig. 1b). If the hydrous melts move into ambient mantle, water would be consumed by water-undersaturated bridgmanite, leading to net loss of water from the slab to the upper part of the lower mantle, perhaps severely diminishing the slab’s capacity to transport water to the deeper mantle and core. But what if the water released from slab mantle migrates into the subducting, previously dehydrated, slab crust?Although slab crust is expected to be largely dehydrated in the upper mantle, changes in its mineralogy at higher pressures gives it the potential to host and carry significant quantities of water to the core–mantle boundary. Studies have identified a number of hydrous phases with CaCl₂-type structures, including δ-AlOOH, ϵ-FeOOH and MgSiO₂(OH)₂ (phase H), that can potentially stabilize in the slab crust in the transition zone or lower mantle. Indeed, these phases likely form extensive solid solutions such that an iron-bearing, alumina-rich, δ-H solid solution should stabilize at ∼50 GPa in the slab crust [12], but only after the nominally anhydrous phases in the crust, (aluminous bridgmanite, stishovite, Ca-perovskite and NAL phase) saturate in water. Once formed, the δ-H solid solution in the slab crust may remain stable all the way to the core mantle boundary if the slab temperature remains well below the mantle geotherm otherwise a hydrous melt may form instead [12] (Fig. 1a). But phase δ-H solid solution and the other potential hydrated oxide phases, intriguing as they are as potential hosts for water, may not be the likely primary host for water in slab crust. Recent studies suggest a new potential host for water—stishovite and post-stishovite dense SiO₂ phases [13,14].SiO₂ minerals make up about a fifth of the slab crust by weight in the transition zone and lower mantle [15] and recent experiments indicate that the dense SiO₂ phases, stishovite (rutile structure—very similar to CaCl₂ structure) and CaCl₂-type SiO₂, structures that are akin to phase H and other hydrated oxides, can host at least 3 wt% water, which is much more than previously considered. More importantly, these dense SiO₂ phases apparently remain stable and hydrated even at temperatures as high as the lower mantle geotherm, unlike other hydrous phases [13,14]. And as a major mineral in the slab crust, SiO₂ phases would have to saturate with water first before other hydrous phases, like δ-H solid solution, would stabilize. If the hydrous melts released from the slab mantle in the transition zone or lower mantle migrate into slab crust the water would dissolve into the undersaturated dense SiO₂ phase (Fig. 1b). Thus, hydrated dense SiO₂ phases are possibly the best candidate hosts for water transport in slab crust all the way to the core mantle boundary due to their high water storage capacity, high modal abundance and high-pressure-temperature stability.Once a slab makes it to the core–mantle boundary region, water held in the slab crust or the slab mantle may be released due to the high geothermal gradient. Heating of slabs at the core–mantle boundary, where temperatures may exceed 3000°C, may ultimately dehydrate SiO₂ phases in the slab crust or bridgmanite (or δ-H) in the slab mantle, with released water initiating melting in the mantle and/or reaction with the core to form hydrated iron metal and super oxides, phases that may potentially explain ultra-low seismic velocities in this region [1,10]. How much water can be released in this region from subducted lithosphere remains a question that is hard to quantify and depends on dynamic processes of dehydration and rehydration in the shallower mantle, specifically at the two ‘choke points’ in the slab mantle, processes that are as yet poorly understood. What is clear is that subducting slabs have the capacity to carry surface water all the way to the core in a number of phases, and possibly in a phase that has previously seemed quite unlikely, dense SiO₂.  相似文献   

Modulating patterns of two-phase flow with electric fields     

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 = μ_aU_a/σ, and electric Bond number, Bo_e = E²(εD/σ). Ca and Bo_e describe the ratio of viscous force to interfacial tension force and the ratio of electric-field force to interfacial tension force, respectively. Here, μ_a (1 mPa s), σ (8.5 mN/m), ε (7.1 × 10⁻¹⁰ F/m), E, U_a, 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 Bo_e, 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 Bo_e 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 Bo_e 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   

Further improved results on stability and dissipativity analysis of static impulsive neural networks with interval time-varying delays     

R. Manivannan  R. Samidurai  Quanxin Zhu 《Journal of The Franklin Institute》2017,354(14):6312-6340

  相似文献   

A high-throughput cellulase screening system based on droplet microfluidics     

Raluca Ostafe  Radivoje Prodanovic  W. Lloyd Ung  David A. Weitz  Rainer Fischer 《Biomicrofluidics》2014,8(4)

A new ultra-high-throughput screening assay for the detection of cellulase activity was developed based on microfluidic sorting. Cellulase activity is detected using a series of coupled enzymes leading to the formation of a fluorescent product that can be detected on a chip. Using this method, we have achieved up to 300-fold enrichments of the active population of cells and greater than 90% purity after just one sorting round. In addition, we proved that we can sort the cellulase-expressing cells from mixtures containing less than 1% active cells.Cellulases are important enzymes with numerous applications across multiple industries, including biofuel, pulp, paper, textile and laundry, food, feed, brewing, and agriculture.¹ Most cellulases have low activity and stability, so improving these properties would have substantial impact on numerous industrial processes.Enzymatic properties can be improved by protein engineering² but the limiting step is the screening process. Classical screening uses microtiter plates (MTPs), where each well contains cells expressing a single type of mutant enzyme. However, this type of screening is the bottleneck in directed evolution, because a maximum number of 10⁵ clones can be screened over the course of weeks or even months³ and large quantities of reagents and consumables are needed. High-throughput screening methods based on either fluorescence activated cell sorting (FACS)^4–7 or microfluidic devices⁸ increase the number of clones that can be screened and reduce the amount of consumables required. Here, we demonstrate the use of a high-throughput screening system for cellulases by combining lab-on-chip sorting devices with an emulsion-based fluorescent assay previously developed for use in flow cytometry.⁵Water–in-oil emulsions are needed to maintain the connection between genotype and phenotype by compartmentalizing individual cells expressing a mutant enzyme together with the components of the fluorescence assay corresponding to the enzyme activity.⁷ For FACS, double emulsions (water-in-oil-in-water) are required because the instrument''s mobile phase is an aqueous solution. Such double emulsions can be produced by stirring or agitation,^9,10 but the resulting emulsions are polydisperse and multiple water droplets may be scattered within a single oil droplet. In addition, large droplets tend to produce more fluorescence because there are more substrate molecules available for conversion into the fluorescent product. The emulsions are produced in bulk, so each droplet will be detected at a different time point from the start of the reaction. This means that increased fluorescence may result because an enzyme has worked on the substrate for a longer amount of time, and the fluorescence of the droplet may plateau before sorting as the enzyme consumes all the available substrate. Cell loading is difficult to control because the average number of cells per droplet scales with droplet volume. Also, if several inner droplets, containing cells with different activities, are encapsulated within the same outer droplet, false positives may occur upon sorting. Consequently, it is impossible to differentiate fluorescence changes due to enzyme activity from those due to other effects using polydisperse double emulsions in FACS, but it is possible to achieve plus/minus screening,⁴ separating cells with activity from those without.Droplet microfluidics overcomes many of the drawbacks of high-throughput enzyme sorting with FACS. Both the size and composition of the droplets can be tuned precisely. Furthermore, once the enzyme is mixed with the substrate, the incubation time can be controlled and all compartments will have the same conditions in terms of concentration and total number of substrate molecules. Although cell loading is still subject to Poisson statistics, the probability for cells to be loaded into a given droplet is the same and can be adjusted by tuning the input cell density. These characteristics make the microfluidic method more sensitive, flexible, and quantitative at detecting changes in enzyme activity than the FACS-based sorting of double emulsions.Here, we report a method in which droplet microfluidics is used to sort libraries containing different percentages of cells expressing cellulase activity and demonstrate enrichment of the cells expressing active cellulases. The entire process is summarized in Figure ​Figure11.Open in a separate windowFIG. 1.General overview of cellulase screening using droplet microfluidics. In the emulsification device, suspensions of yeast surface displayed libraries are co-flowed with the substrate solution at equal flow rates to a drop-forming junction where they mix. A stream of perfluorinated oil then breaks the aqueous mixture into monodisperse water-in-oil emulsions. Within each droplet, the cellulase reaction starts after compartmentalization and the fluorescent product is formed by a coupled enzymatic cascade in droplets containing cells that express the active enzyme. After a fixed incubation time, the emulsion droplets are re-injected into a microfluidic sorting device, where they are analyzed and sorted based on their fluorescence.To detect cellulase activity, we designed an assay that uses a chain of coupled enzymatic reactions to yield fluorescence corresponding to cellulase activity without needing artificial substrates (which may lead to confounding effects, such as improved binding of the enzyme specifically to the artificial compound but not the natural substrate). In this method, cellulase hydrolyzes cellulose, its natural substrate, into monosaccharides and oligosaccharides that are further detected by the enzymatic cascade⁵ (Figure ​(Figure11).Based on previous FACS experiments, no difference in activity can be detected between the positive and the negative droplets before 2 h incubation time.⁵ Based on these observations, we expected the cells to require more than 2 h of incubation in droplets for the reaction to develop.Emulsions were formed using a co-flow flow-focusing Polydimethylsiloxane device prepared by soft lithography as previously described⁸ and using fluorocarbon oil containing 1% (v/v) Krytox-PEG-Krytox detergent synthesized as reported in an earlier study.^11,14 The solutions, one containing library cells (S. cerevisiae YPH500 cells, Agilent Technologies, Santa Clara, USA) and the other with the substrate,¹⁴ were mixed at the same flow rate, giving a one-to-one mixing ratio. The library cells were a defined mixture of cells transformed with cel5A pESC-Trp (positive cells) or empty pESC-Trp (negative cells). The two solutions therefore mixed just prior to encapsulation, minimizing the chance that fluorescent products would enter neighboring droplets. The substrate solution contained carboxymethyl cellulose (CMC), which has a high viscosity. To prevent fluctuations in the flow of substrate during the emulsification process, we optimized the flow rate and the concentration of CMC and found that a CMC concentration of 0.33% (w/v) produced monodisperse emulsions.We discovered that the HOx required for the enzymatic cascade causes droplet coalescence. HOx alone was sufficient to cause the observed change in droplet stability because droplets containing only hexose oxidase in buffer exhibited the same amount of coalescence as those containing the full set of assay components. We hypothesized that the enzyme might be surface active, disturbing the emulsion interface, but emulsions of an inactivated form of the enzyme were stable (Figure 2(a)). One possible explanation is that active HOx may interact with the detergent through the active site. Adding bovine serum albumin (BSA), which is known to have a stabilizing effect,¹² to the mixture improved droplet stability (Figure 2(a)). Emulsions of the assay mixture with BSA were stable for more than 1 day at room temperature.Open in a separate windowFIG. 2.(a) Transmission light micrographs of water-in-perfluorinated-oil emulsions produced using the microfluidic emulsification devices after 2 h incubation at room temperature. The emulsions contain 3 U/ml HOx either in its native form (left image), inactivated by heating at 99 °C for 20 min (middle image), or supplemented with 1 mg/ml BSA (right image). (b) Images of the results of the agar plate Congo Red cellulase assay before and after sorting, with the percentage of positive colonies indicated. The cells expressing cellulase activity show clear hallos.The time required for the cellulase reaction to produce detectable quantities of fluorescent product was monitored using the droplet screening instrument. These devices proved to have a higher sensitivity than the FACS system because the optics are designed for the droplet size selected for the assay. We were able to detect cellulase activity just 20 min after the compartmentalization of cells. This shorter incubation time allowed us to couple the emulsification device directly to the droplet sorting device using a short piece of tubing. The rate of emulsion flow and the dimensions of the tube set the droplet incubation time.Using the optimized conditions, we used droplet microfluidics to sort cellulase-expressing cells from a set of reference libraries. The reference libraries were created by mixing different concentrations of positive S. cerevisiae YPH500 cells expressing Cel5A cellulase and negative S. cerevisiae YPH500 cells transformed with the pESC-Trp empty vector. The mixed populations were emulsified together with the assay components in water-in-perfluorinated-oil emulsions and incubated at room temperature for 20 min. The gated population was sorted and the cells were spread on yeast nitrogen base casaminoacids (YNB CAA) Glu agar plates. An aliquot of the reference library was also plated on agar plates prior to sorting. Approximately, 100 cells before and after sorting were transferred to YNB CAA CMC Gal/Raf induction plates, and the Congo red assay¹³ was used to detect cells expressing cellulase. In this assay, colonies of positive cells developed transparent halos around them.¹⁴ The results before and after sorting are presented in Figure 2(b).We enriched cellulase-expressing cells from a pool of negative cells, regardless of the starting concentration of positive cells. We were able to isolate the cellulase-expressing cells even when starting from a low percentage of active cells (0.1%). We obtained high enrichment factors of up to 300 when starting from low concentrations of positive cells, and we were able to sort to a purity of greater than 90%. These results exceed those obtained by comparable experiments using FACS.⁵In conclusion, we developed a high-throughput screening system for cellulase activity based on droplet microfluidics. We optimized the emulsification conditions to produce highly stable and monodisperse droplets. The low dispersity of the emulsion enables the sensitive, tunable, and quantitative detection of cellulase activity. In addition, we substantially reduced the reaction time needed for the development of a fluorescent product from 2 h to 20 min. As a result, we sorted reference libraries of cellulases with various ratios of positive to negative cells, and regardless of the starting population of positive cells we were always able to enrich the active population to a higher purity than that obtained by FACS.  相似文献   

Preface to Special Topic: Papers from the 2009 Conference on Advances in Microfluidics and Nanofluidics, The Hong Kong University of Science & Technology, Hong Kong, 2009     

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   

Flow manipulation and cell immobilization for biochemical applications using thermally responsive fluids     

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   

Enhancing conjugation rate of antibodies to carboxylates: Numerical modeling of conjugation kinetics in microfluidic channels and characterization of chemical over-exposure in conventional protocols by quartz crystal microbalance     

Sasan Asiaei  Brendan Smith  Patricia Nieva 《Biomicrofluidics》2015,9(6)

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