WHEN MICROFLUIDIC DEVICES GO BAD How does fouling occur in microfluidic devices, and what can be done about it?
T Rajendrani Mukhopadhyay
he advantages of miniaturizing analytical instruments are well known. Only tiny sample and reagent volumes are needed, reaction times are quicker, tasks can be carried out in parallel to increase throughput, and so on. But miniaturization means that as instruments shrink, the surface-to-volume ratios in the devices increase. More surface is now available inside the device for fouling. Fouling occurs in various ways; one common problem is that components from samples and reagents
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stick to the surfaces in the device. The instrument’s parts become encrusted, clogged, and eventually useless. Paul Yager at the University of Washington says that fouling “is one of microfluidics’ dirty little secrets—literally dirty! It’s a problem for anything except electrophoresis of DNA.” Fouling is a daunting issue to tackle because every application for microfluidic devices comes with its own set of baggage. The nature of the sample, the materials from which the
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device is fabricated, and the type of flow applied to move samples can all contribute to the problem. Methods to prevent nonspecific adsorption to surfaces have been developed, and cleaning protocols exist for certain types of devices; however, the remedies tend to be specific and are usually found by trial and error. “It’s an incredibly complex problem, and there’s no magic bullet,” says Deborah Leckband of the University of Illinois at Urbana–Champaign. Researchers don’t always realize that fouling is occurring in their miniaturized instruments. “A lot of people don’t even know whether there’s a fouling problem. They never run the assay 200 times in the same channel,” says Anup Singh at the Sandia National Laboratories in Livermore, Calif. “If you only run the assay five times, then fouling doesn’t make the assay look that bad, and you can get away with it.” Some experts say that the way in which microfluidic devices are tested can attempt to circumvent fouling issues altogether. “Researchers often evaluate the detection limit of sensors using clean samples, where the sample only contains one to two contaminants in it, and that’s it,” says Darren Branch at the Sandia National Laboratories in Albuquerque, N.M. “But in the real world of field-portable microsensors, you don’t have the luxury to take [a] complex sample and clean it prior to testing.” With microfluidic devices, experts say that it’s more often the industrial labs, rather than the academic ones, that relentlessly pursue the problem of fouling. The issue is so multifaceted that unless the intention is to make a profit from a device, the motivation to map out the sources and solutions for fouling isn’t overwhelming. “[Fouling] is critical if you’re talking about making a real device that you want to sell to someone who wants it to work reliably. That’s where the strongest motivation is—to go [to] that extent and prove your device will survive long enough or over the variety of samples,” says Josh Molho at Caliper Life Sciences. “We have to look at [fouling] because if our devices don’t work reliably, then no one’s going to buy them!”
Sources of fouling The parameters in fouling are closely interconnected. Take, for example, a microfluidic device that is designed to carry out a diagnostic test on blood samples. Blood contains a whole host of molecules that exhibit a range of properties. Constituents of blood can be as minuscule as NaCl or as gargantuan as red blood cells. Molecules can be charged, neutral, or hydrophobic. They can even change their charge properties over time. A protein may be hydrophilic on the surface, but upon unfolding, it might expose a hydrophobic interior. All these properties come into play at the interface between the sample and the device. “For example, if you choose a material which is charged, then the molecules that have the opposite charge will tend to nonspecifically stick to the surface. That can create fouling of your channel surface. It will also mean the loss of molecules from your sample,” says Singh. But that’s not all. “Size is a problem, because if your channel has a constriction somewhere or is [made of] a porous material, then big things can get stuck and clog,” adds Singh. The big things could be the analytes or background species in the sam430 A
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ple. “Now [the clog] can interfere with the flow through your device, disrupting the microfluidic analysis, in addition to loss of macromolecular analytes, such as cells,” Singh points out. In addition, with blood samples, the nature of the substances sticking to the walls changes with time. This phenomenon, known as the Vroman effect (1), means that fouling is dynamic in nature. The effect arises from a combination of concentration, diffusion, and adsorption coefficients. When any material is placed in contact with blood, components with the highest diffusion coefficient will adsorb to the material’s surface first. However, eventually, a series of different components will stick in place of them. “You’ll have a sequence of different proteins on a polymer surface,” says Yager. “Watch over time—and time could be hours to days—[and] you’ll actually see a sequence of different things at the surface.” The simple adsorption of molecules to the device’s walls isn’t the only source of fouling. Biological reactions within the sample can be triggered as the sample travels through the device. “For example, [if ] you have platelets and you run them through a shear field by [a] mechanism, they become activated and sticky,” says George Whitesides at Harvard University. “That can be either a good thing, if you’re studying the biology of [platelet] activation, or a bad thing, if the platelets are sticking to the wall of your device.” For microfluidic assays that involve bacteria, he adds, “If the bugs you’re growing [in your device] start producing a biofilm, then the jelly layer they make can act as a fouling component.” Some experts say that filtration of samples to remove the offending parts is one way to prevent fouling. But others think the need for preliminary sample preparation steps for a micro-total analysis system (µTAS) is counterintuitive. “It means you’ve rehired your lab tech,” says Yager. Essentially, you are saying, “‘We have this really nifty microfluidic device. Now we need to hire you to clean the samples up to go into it.’ In my mind, this defeats the purpose of [µTAS] in the first place.” The design of the microfluidic device can either prevent or promote fouling. Tejal Desai at the University of California, San Francisco, explains that issues arise related to the number of turns in a channel or the angle of channels relative to each other. “As solutions flow, if there are regions that are static where things can pool, those can become areas where fouling occurs, much like in the body. When you have a plaque buildup within the arteries, it’s generally because there has been disruption of flow that causes some sort of buildup,” she says. Brian Kirby at Cornell University agrees that the microchannel design contributes to fouling. “[U]sually, simple geometries that are very easy to design on a CAD program and are very easy to conceptualize are typically not the best geometries for optimizing flow through microdevices,” he says. But there aren’t any hard-and-fast rules for which microchannel designs will foul. “It’s not something I can define very precisely. It depends a lot on what sort of technique you’re using,” he adds. Methods for moving samples around in a device can contribute to fouling or can be adversely affected by fouling. Singh explains that, for pressure-driven flow, if the channels clog even partially, higher and higher pressures will be required. On the
other hand, if the device is run at a constant pressure, the flow rate will decrease, the elution times will increase, and the device will yield irreproducible results. For cases in which capillary action or electroosmotic flow moves the fluid, fouling of the surfaces will also change the flow drastically, he notes. For flows that are driven by voltages, fouling can alter the surface charges and, thus, wreak havoc with the flow of fluid in the channels. Frances Ligler at the U.S. Naval Research Laboratory describes a time when she and her colleagues attempted to pump groundwater samples through a microfluidic device. “We tried the [sensors] in an electroosmotic flow,” she recounts. “Sometimes the flow went one way, and sometimes it completely reversed and went the other way!” Surface charge on the channel walls of a device is determined by the material used for fabrication. For practical applications of microfluidic devices, the two major categories of materials are glass and plastics. Within plastics, PMMA, PDMS, and polyimide are popular. Each type of material fouls in its own way. Glass is hydrophilic with a net negative charge, so substances with the opposite charge tend to stick to it. Plastics such as PDMS tend to be hydrophobic, so fouling can occur via hydrophobic interactions. Experts say one way to overcome fouling at the material–sample interface is to modify the surface of the material, a process known as passivation.
Passivating surfaces The ways to change a material’s surface properties to prevent nonspecific adsorption are numerous but not generic. “Most of the coatings are based on trial and error, your particular application, and the particular material or substrate that you’re using,” says Singh. “There is no one coating that will be a universal fix to all the problems.” For glass devices, one of the simplest options is to “pre-foul” the device, says Kirby. Researchers will often introduce bovine serum albumin or powdered skim milk into channels so that the surfaces that would normally attract analytes from the samples are now covered with a coat of inexpensive protein. However, this approach can have serious drawbacks, warns Yager. For example, even if proteins do not stick to a coated surface, small molecules may still be lost because they may adsorb to the coating. Another option is to change the surface chemically, though these modifications tend to be specific to a given material. Selfassembled monolayers (SAMs) based on alkane thiolates and polymeric coatings are popular approaches. SAMs that use siloxane chemistry can modify the hydrophilic character of glass. Gold–thiol chemistry-based SAMs, which are commonly applied to microarrays, are not practical in microfluidic devices because it’s hard to evenly coat a channel with the requisite gold layer. With PDMS, oxidation is a simple way to convert the hydrophobic surface into a hydrophilic one. However, some experts caution that the PDMS oxidation is only a temporary fix. A more permanent one is to oxidize the PDMS surface and then
attach a polymer coating via silane chemistry; this provides a longer-lasting hydrophilic layer. Getting the polymer coating onto a polymeric plastic can be tricky, according to Kirby, because the chemistry of these surfaces is not as well understood as that of glass. Polyacrylamide, polyethylene oxide, or polyethylene glycol (PEG) coatings are often used to inhibit nonspecific protein adsorption on both glass and plastic devices. Although these coatings are generally successful, they can cause secondary reactions.
Fouling “is one of microfluidics’ dirty little secrets—literally dirty! It’s a problem for anything except electrophoresis of DNA.” —Paul Yager For instance, Yager notes that PEG triggers platelet activation in blood samples. Uniformity and stability are two general problems associated with coatings. “The ability to coat very small channels becomes an issue, because if you use conventional macroscale approaches, you often don’t get uniform coatings in very small channels,” says Desai. “You’re relying on bulk diffusion to carry that solution. You may get more even coatings at the beginning of your channel, but then it tapers off towards the end.” Singh explains that it is difficult to achieve a monolayer with polymeric coatings. “You can get a monolayer in some areas; in others, you get more than a monolayer. In the worst cases, you ended up polymerizing the whole channel,” he says. Furthermore, no reliable method exists to confirm the uniformity of a coating inside a channel. Singh says researchers typically coat a flat surface, such as a microscope slide, that is made out of the same material as their microfluidic device; then, they characterize the coating. “They use those results to assume whatever they see on the flat surface is what is happening inside the channel,” he says. “It’s a leap of faith there that the chemistry works exactly the same way on a flat substrate [as in] a confined channel.” The stability of a coating over extended periods of time can also be a concern. According to Milan Mrksich at the University of Chicago, SAMs are generally thought to be stable up to 60–70 °C. They can start to desorb at 100 °C and are not stable to UV light. At room temperature, at which a lot of biological experiments are done, monolayers are stable in cell culture conditions for about a week. “If you’re doing microfluidics experiments or other kinds of diagnostic experiments that take less than an hour or on the order of hours, then the stability is not a concern,” he explains. “If there are experiments that require a surface to be in contact with tissue for a period of weeks, then they would not be stable.” When SAMs are applied to substrates, the covalent bond holding the monolayer to the surface is a siloxane bond. “That N O V E M B E R 1 , 2 0 0 5 / A N A LY T I C A L C H E M I S T R Y
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bond basically has the same properties as glass,” says Kirby. “By that, I mean it will resist any pH ranging from 3 to 7.5. As soon as you go to a basic pH, then the Some experts say that fouling is receiving its basic solution will break that bond,” he says. Technically, glass is somewhat soluble in a base of pH 8, he notes: due attention, while others contend that it’s Only a single monolayer may get stripped off. “But if your one monolayer is the one that includes the chemibeing pushed to the back to make room for cal modification, then that’s very important,” he adds. If a polymeric coating is only physisorbed onto a surmore easily solved problems. face and not covalently attached, it can be lost via interactions with the sample. For example, Leckband points out that biopolymers in a solution could competitively displace materials on the surface. “As a result, you would tend to considered adequate. But for clinical purposes, experts say that use high-molecular-weight polymers to coat the surface because disposable microfluidic devices will probably be the way to go. “Human health-care applications will require single-use [de[they are] less likely to be displaced by proteins or lower-molecvices],” says Whitesides. “You’re not going to be able to run ular-weight components in the solution.” Finally, a process known as “blooming” occurs with polymer- samples over and over through [the same channel] because of ic coatings. Many polymers have additives, such as antislip and problems with contamination from sample to sample.” Because antistatic agents, that modulate their properties. The polymers the devices will be single-use, they have to be fabricated on a may also contain low-molecular-weight oligomers as contami- large scale that is cost-effective. For this reason, many renants. These additives and oligomers have a tendency to migrate searchers expect that the instruments will be made of plastics. to the surface. So even if an effective surface treatment is developed, the final coating may be compromised by the migrating The future of fouling additives and oligomers that cover it up. Opinions differ on whether fouling in microfluidic devices is being adequately addressed at the moment. Some experts say that fouling is receiving its due attention, while others contend that Clean or dispose of the device? Several ways exist to approach fouling in the long run, according it’s being pushed to the back to make room for more easily to Branch. “One camp [says that] by producing devices at ex- solved problems, such as instrument designs and detection tremely low cost, even if they foul, they can be replaced by new schemes. It helps to remember that fouling is not a problem unique to devices.” He says the second camp acknowledges that fouling will even- microfluidics. “People have been trying to work on antifouling tually be a problem with a reusable or implantable device; this is coatings since the 1950s. There’s [a] huge literature on preventwhere the surface passivation techniques play a critical role. ing protein adsorption. It primarily focused on prosthetic deHowever, Branch warns that chemical passivation methods will vices. What we’re talking about with regards to microfluidic defail over time: “Even the smallest defect at the interface can lead vices is not so different. . . . You’re trying to change the surface to fouling, because you now have a difference in the surface chemistry to prevent protein adsorption,” says Leckband. Whitesides sees fouling as an area that needs interdisciplinary chemistry in those locations.” Branch says the third camp accepts that chemistry is only part collaboration, especially for clinical microfluidic devices. He says, of the answer. Their philosophy is that “active methods must be “If you ask me, the group that is needed consists of good fabriused in conjunction with surface chemistry in an effort to main- cation people, cell biologists, and physiologists sitting down totain a clean [device]. This involves potentially using surfactants gether to systematically pick the problem apart and really find or surface acoustic actuation methods to clean the fouled inter- out what’s going on. I think there’s an enormous amount of opportunity there that scientists are not really exploiting at this face when possible,” he says. Stephen Quake at Stanford University says his group is in the stage.” As microfluidics research continues to push the limits, having third camp. While he and his colleagues were developing a chip with six nanoscale bioreactors (2), they had trouble finding a suit- various strategies to prevent fouling will become more critical. able coating. “We were never able to find a coating that would last Desai says, “We’re dealing with such small volumes that it’s imfor a couple of weeks, which is how long we’d like to keep our portant” to think about fouling at every step of the development chemostat,” he says. “We found some that worked well in the process, from choosing the materials to designing the elements. short term but not in the long term.” Their devices became fouled by bacterial biofilms over extended periods of time. References “That’s why we developed an active antifouling system where we (1) Vroman, L.; et al. Blood 1980, 55, 156–159. would use the power of microfluidic plumbing to repeatedly clean (2) Balagaddé, F. K.; et al. Science 2005, 309, 137–140. the walls of the different segments of the chamber,” he explains. If a device is fabricated out of glass and used in a laboratory Rajendrani Mukhopadhyay is an associate editor of Analytical setting, cleaning routines, such as a soak in bleach, are generally Chemistry. 432 A
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