Synergism between particle-based multiplexing and microfluidics technologies may bring diagnostics closer to the patient
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In the field of medical diagnostics there is a growing need for inexpensive, accurate, and quick high-throughput assays. On the one hand, recent progress in microfluidics technologies is expected to strongly support the development of miniaturized analytical devices, which will speed up (bio)analytical assays. On the other hand, a higher throughput can be obtained by the simultaneous screening of one sample for multiple targets (multiplexing) by means of encoded particle-based assays. Multiplexing at the macro level is now common in research labs and is expected to become part of clinical diagnostics. This review aims to debate on the “added value” we can expect from (bio)analysis with particles in microfluidic devices. Technologies to (a) decode, (b) analyze, and (c) manipulate the particles are described. Special emphasis is placed on the challenges of integrating currently existing detection platforms for encoded microparticles into microdevices and on promising microtechnologies that could be used to down-scale the detection units in order to obtain compact miniaturized particle-based multiplexing platforms.
KeywordsBioassays Biochips/high-throughpout screening Microfluidics/microfabrication Encoded particles Multiplexing Integrated systems
Many automated systems have been introduced in the field of medical diagnostics to enable more rapid and efficient data collection from the incredible amount of samples that hospitals deal with daily. However, such automated equipment is mostly not suitable for use in small diagnostic and research laboratories and for decentralized point-of-care testing, as they require highly qualified personnel, are often not portable and/or are too expensive. Hence, there is an increasing need for (a) accurate, (b) quick, (c) miniaturized, and (d) cheap innovative tools which should bring medical diagnostics closer to the patient.
There is no doubt that the recent progress in microfluidics technologies will strongly support the development of miniaturized analytical devices . Microfluidics involves the manipulation, transport and analysis of fluids in micrometer-sized channels. A “liquid microspace” has characteristic features which differ from the properties of a “liquid bulk”: high interface-to-volume ratio, small heat capacity and, especially, short diffusion distances. The latter is a useful property in analysis, because the time a molecule needs to diffuse from point a to point b is proportional to the square of the distance between a and b ; While it takes several hours to overcome 1 cm, it only takes tens of seconds to overcome 100 μm.
The microfluidic concept has already evolved into promising analytical “lab-on-a-chip” (LOC) tools . The LOC concept, or the “micro total analysis system” (μTAS) as it is today commonly referred to, was proposed in the early 1990s by Manz et al. . Since that time, the field has bloomed and branched off into many areas with different applications, such as single molecule analysis , single cell processing and analysis , biological and chemical analysis [7, 8, 9, 10, 11, 12, 13, 14], point of care testing [15, 16], clinical and forensic analysis , molecular and medical diagnostics [18, 19, 20, 21], combinatorial chemistry  and drug discovery . The fact that LOC systems are compact, which allows the automation of complex tasks, makes them very attractive .
A higher throughput in (bio)analysis can be obtained (a) by the parallel screening of multiple samples for one target, (b) by the simultaneous screening of one sample for multiple targets (multiplexing), or (c) by a combination of both, as recently reviewed by Situma et al. . In microfluidics, a higher throughput is currently obtained by the parallel screening of a number of samples in a number of channels in one device. Sato et al.  fabricated a device with branching multichannels that allows four samples to be processed simultaneously. The assay time for four samples was 50 minutes, instead of 35 min for one sample in a single-channel assay. Another way to realize higher throughput analysis in microfluidic devices is by multiplexing, i.e., the simultaneous detection of multiple analytes in a sample present in one channel. Kartalov et al. reported a multi-antigen microfluidic fluorescence immunoassay which measures up to five analytes for each of ten samples in a 100-chamber polydimethylsiloxane (PDMS) microchip . Multiplexing at the “macro” level is now common in research labs and is expected to become part of clinical diagnostics [28, 29, 30]. Both “planar arrays” (often called “microarrays”) and “suspension arrays” (particle-based arrays) have been developed for multiplexing purposes.
Because microarrays allow (ultra)high density analysis of samples, they have become standard tools for gene expression analysis . Multiplexing necessitates an encoding scheme for molecular identification; the code allows the capture probe bound at a particular position on the array to be identified, and so it is also possible to know which analyte is analyzed. Whereas planar arrays strictly rely on spatial positional encoding, particle-based arrays have used a great number of encoding schemes that can be classified as optical, graphical, electronic or physical [32, 33]. Particle-based arrays benefit from (a) “near-solution” kinetics, which means that the kinetics between a molecule bound to the surface of a particle and a free molecule equals those between two free molecules, (b) lower instrument-related costs, (c) higher sample throughput, and (d) good quality control by batch synthesis [34, 35]. When compared with microarrays, particle-based arrays offer a more flexible choice of the “probe set;” the detection of extra targets only implies the addition of extra microparticles to the sample, while a new microarray has to be made in the case of microarray-assaying. Particle-based arrays are especially favorable compared to microarrays when a modest rather than a very high number of targets must be analyzed simultaneously. This feature may explain the recent exponential increase in particle-based applications at the “macro” level .
Currently, multiplexing at the microlevel is mainly done by combining flat surface microarrays with microchannels. Delehanty and Ligler  used noncontact microarray printing to immobilize biotinylated capture antibodies at discrete locations on an avidin-coated microscope slide and processed the samples with a six-channel flow module. Assays were completed in 15 min. The group of Delamarche combined concepts of micromosaïc immunoassays and microfluidic networks to detect C-reactive protein (CRP) and other cardiac markers [37, 38]. By using 20 μm × 10 μm channels (5 mm in length), CRP was detected in ten minutes in only one microliter of human plasma down to concentrations of 30 ng/ml. So far only a few examples have been reported of multiplexing by particles in microfluidic devices . One of the problems is that the implementation of the detection systems currently used to analyze particles in macro-assays into microfluidic devices is not straightforward.
This review discusses the “added value” we can expect from (bio)analysis with particles in microfluidic devices. Technologies to (a) decode, (b) analyze and (c) manipulate the particles are described. Also, an interdisciplinary effort is made to overview possibilities for the integration of different processes, like decoding and sorting of encoded particles.
Strategies to decode particles
Clearly, to achieve multiplexing with particles in microfluidic devices, one should be able to decode the positive particles. In macro assays with encoded particles, three decoding platforms are generally used: flow (cyto)meter platforms, optical reading platforms, and fibre optic platforms. Although some well-written reviews have been published on this matter [32, 33, 40, 41], the next section provides a brief overview.
Flow (cyto)meter platform
A totally different approach is the use of light-powered 100 μm × 250 μm × 250 μm microtransponders, called electronic radio frequency microchips. A serial number is stored electronically and allows the probe which is attached to the surface of a transponder to be identified. Such electronically encoded microcarriers are also analyzed by high-speed flow (cyto)meters modified to detect radiofrequencies .
A flow (cyto)meter can rapidly process optically/ physically/electronically encoded particles, making it a popular reading platform for multiplexing. However, it has also several disadvantages, including: (i) its lack of portability, as flow meters are bulky; (ii) the cost (especially when multiple lasers and detectors are needed), and; (iii) the potential interference between the fluorescence from the fluorophores which make up the code and the fluorescence generated at the surface of the particles in the case of a positive reaction.
(Fluorescence) microscope platform
Optical fiber platform
Multifunctionality of particles in microfluidic devices
Particles in microfluidic-based assays may have different functionalities—a particularly attactive and powerful trait—as outlined below.
Particles offer a huge analytical surface
Clearly, when compared with flat supports, three-dimensional particles offer a huge surface which should improve the (bio)chemical reaction rates. A decade ago, Zammatteo et al. demonstrated faster nucleic acid hybridization kinetics when DNA probes were coated onto the surfaces of 4.5 μm particles instead of onto the surfaces of the wells of microtiterplates . Because the microparticles continuously move in the surrounding fluid (a very dynamic process), the reactions at their surface follow “near solution” kinetics. (Spherical) microparticles also have a high surface-to-volume ratio, which enables reactions to be performed in smaller volumes without needing to resort to a smaller reaction surface. This again leads to a smaller diffusion distance and a shorter analysis time. Interestingly, even in microfluidic devices, hybridization kinetics are faster if the probes are coupled to the surface of particles instead of the walls of the microchannels, as recently shown by Kim et al. . The authors showed that the analysis of 2 μl volumes of samples took on the order of a few minutes, with flow rates of some hundreds of nanoliters per second. Not just hybridization reactions but also protein–protein reactions take advantage of the size effect of the liquid microspace. Sato et al. demonstrated that the reaction time between antibodies and antigens coupled to the surfaces of 45-μm polystyrene particles in a microfluidic device is 1/90 of the time needed in a conventional microtiterplate . The overall analysis time was shortened from 24 h to less than 1 h, and troublesome operations could be substantially limited. It is worth mentioning that new technologies are in progress which offer high flexibility for the surface coating of microparticles, which will further broaden their molecular applications [34, 67].
Particles allow mixing
Another advantage is that particles allow mixing, which is important for (bio)chemical reactions. Because of the dimensions, the Reynolds number of fluid flows in microfluidic devices is extremely small (usually less than 1). This means that the flow profile is laminar and that molecular transport only occurs by diffusion, which is relatively time-consuming despite the rather small dimensions involved in the assay. The lack of turbulence makes mixing in microdevices a very challenging issue. Liu et al. have shown that oscillating the sample within a microchip accelerates the hybridization of nucleic acids to their probes spotted on the bottom of the channel . A factor of five improvement the signal was achieved with sample oscillation after 15 minutes of hybridization. Similar conclusions on hybridization kinetics using conventional microarrays were made by Pappaert et al.; they showed that a shear-driven flow reduces the analysis time (from 16 hours down to 30 minutes) . The effect of sample oscillation could be improved even further by using microparticles which are continuously moved around in the sample to cause a local turbulent flow. This has been demonstrated by Seong et al. , who studied how enzymes that are immobilized on microparticles convert their substrates. Herrmann et al. recently described a microfluidic ELISA (enzyme-linked immunosorbent assay) reaction at the surfaces of microparticles, using about 106 paramagnetic particles 1 μm in diameter trapped in a reaction chamber with dimensions of 6 mm × 2 mm × 50 μm, and showed that mixing through the application of an external magnetic field enhances the reaction speed .
Particles allow sorting
This is an important feature, as particles allow the (a) enrichment of molecules of interest from complex samples, and (b) the separation of cells, viral particles and bacteria from a large population . Technical aspects related to sorting of samples with particles in microfluidics will be considered in the next section.
Particles are very practical
Particles are also of interest from a practical point of view. For example, it is much easier to handle (detect, trap, transport) microparticles than single molecules in microfluidic systems. Also, it is much easier to modify the surfaces of microparticles than to modify the walls of a microchannel in a chip. The surfaces of microparticles can easily be modified off-chip. Adding extra probes to an existing microfluidic-based assay can be achieved by simply adding microparticles bearing the probes; it is not necessary to produce a new device. By adding more or fewer microparticles it is also straightforward to change the total capture surface (related to the number of probe molecules) in the assay (note that the total capture surface of a flat array in a microwell or microarray is constant). This can result in higher signals from particles than from flat arrays, for instance for enzyme/substrate reactions .
(Encoded) particles allow multiplexing
This is highly important in situations where the amount of sample is very limited, such as in the analysis of blood from newborns, tumor tissue from biopsies, etc. Additionally, multiplexing allows more efficient and therefore less expensive use of reagents, and because the different targets are screened simultaneously they experience equal conditions at each step of the assay procedure. The integration of microparticles into microdevices for multiplexing is still in its early stage and only a few publications have demonstrated this synergism [39, 65, 83]. Technical challenges related to this integration process will be overviewed in another section.
Particle trapping and sorting in microfluidics
Propulsion of fluids in microdevices
Microfluidics involves the transport, manipulation and analysis of fluids or substances in fluids in micrometer-sized channels. Flow in microfluidics can be generated (a) mechanically (by pressure), (b) electrokinetically (electroosmotic flow; EOF), (c) by capillary forces or (d) by centrifugal forces. The type of propulsion force used is highly dependent on the application, the requested flow rate and the material composition of the microchannel.
Propulsion by capillary forces, which are driven by local heating of the fluid and are due to the high heat-exchange rate in the microchannel, is still at an early stage of development and might not be compatible with (bio)chemical assaying in microfluidics, as the high temperature can have a negative effect on the assay.
Centrifugal fluidic platforms (called “lab-on-a-disc”) were recently reviewed by Madou et al. . Due to the rotational speed, it is possible to have identical flow rates, to load identical volumes, and to have identical incubation times in parallel assay capillaries. Therefore, they have great potential for parallel screening .
Manipulation of particles in microdevices
To perform (bio)chemical reactions on a set of particles in microfluidic devices and to take full advantage of their multifunctionality (mixing, sorting, multiplexing, etc.), they usually have to be trapped into a constrained volume inside the chip while samples and reagents are flushed through the device. Sometimes more “selective” methods are needed which aim to isolate and manipulate individual particles. For example, an excellent review on the manipulation of single cells in microfluidic devices has recently been published by Toner and Irimia . Some of the methods described herein are applicable to microparticles as well. However, as certain microparticles have unique properties, complementary techniques for separating and sorting microparticles in microchannels exist too.
Magnetic microparticles can also be immobilized and trapped in microdevices by means of magnetic forces exerted by an external rare-earth magnet [81, 92]. The relatively large size of such an external magnet may, however, complicate the precise handling of the microparticles. This issue has recently been solved by using microfabricated 3-D magnetic devices positioned in a continuous flow-through microfluidic chamber (10 mm × 5 mm × 0.1 mm) . Magnetic particles between 1 and 5 μm in diameter were trapped at flow rates on the order of 10–100 μl/min. Another original concept is the manipulation of groups of magnetic particles, as described by Rida and Gijs . The local rotational motion of the particles in a microfluidic flow, generated by an external local alternating magnetic field, enhances the interaction between the particles and the liquid: 95% mixing efficiency was achieved over a mixing length of 400 μm at flow rates on the order of 5 mm/s. However, the accurate manipulation (and separation) of individual particles by magnetic forces remains a challenge. Note that the presence of magnetic material in/on the microparticles is sometimes a limitation because it often renders them opaque.
Electrically polarizable microparticles can be manipulated by dielectrophoresis (DEP) . When such microparticles are subjected to an alternating electric field, a dipole moment is induced in the particles. In a nonuniform electrical field, the polarized particles experience a dielectrophoretic force which may move them to regions of high or low electrical field. The motion depends on the particle polarizability compared to the suspending medium. The magnitude and direction of the dielectrophoretic force on a particle also depends on its dielectric properties, so that a heterogeneous mixture of microparticles in a continuous flow can be spatially separated to produce a more homogeneous population in an appropriate electrical field.
The separation of microparticles by DEP in a microdevice (“DEP migration”) has been demonstrated by several research groups. Kentsch et al. developed a particle-based assay for the detection of viruses in serum . Kralj et al. simulated the flow behavior of spherical particles in a DEP-based device and verified the model for sorting differently sized particles using DEP experimentally . The separation efficiency can be improved by combining DEP with other physical forces (’DEP retention”). Microparticles mechanically driven through a microdevice by pressure-based flow fields can be separated by a dielectrophoretic force perpendicular to the flow, because the particles acquire different velocities due to the parabolic flow profile, depending on their dielectric characteristics. This is an example of what is called field-flow fractionation (FFF). In FFF particles move in a flow and become separated by an external force perpendicular to the flow. Particles with different properties attain different positions relative to the chamber wall due to a number of possible forces: diffusive, hydrodynamic, gravitational (sedimentational), electrophoretic, dielectric and other forces, or a combination thereof [98, 99].
To obtain a more precise (single particle) trapping, DEP and optical tweezing have been combined by Arai et al. They describe a device in which DEP and laser trapping forces are used to selectively isolate one single microbe from of a huge population in a microdevice in less than 20 s . Laser trapping was used to trap the microbe of interest, while DEP forces were applied to exclude other objects around the target microbe. Reichle et al. combined DEP and optical tweezing (OT) for receptor–ligand interactions on single cells in microdevices . Ligands were coupled to particles 4.1 μm in diameter which were brought into contact with the cell (receptors) by OT. The latter one was held in a DEP cage.
Integration of decoding and detection platforms
Despite the popularity of conventional flow (cyto)meters for multiplexing, it is unfortunately not straightforward to combine them with microfluidic chips; after carrying out the (bio)chemical reactions in the chip (the recipient), the particles must be transferred to the flow cytometer (which is also the case when an optical fiber platform is used), which is not desirable. Also, the flow cytometers currently available are relatively expensive, cumbersome (difficult to handle because of their size and weight), and need trained personnel.
(Fluorescent) microscope reading platforms, which allow both the (fluorescence) analysis of the (bio)chemical reaction at the particle’s surface as well as decoding, by simply placing the particle containing microfluidic device under a microscope, are of high interest. Nevertheless, some requirements need to be fulfilled for such purposes. First, the part of the microfluidic device where decoding and detection of the particles occurs should be optically transparent and compatible with the microscope optics. Besides thin glass, other materials like poly(dimethylsiloxane) (PDMS) can be used to this end [115, 116, 117]. Second, the movement of the particles must be negligible during image acquisition in the case of graphically encoded particles to avoid blurring the code. This can be accomplished by the trapping techniques described above. If the microparticles are located close to each other, parallel detection of multiple particles should be possible. The number of particles detected simultaneously will depend on the trapping system, the dimensions of the detection chamber, the field of view and the size of the particles. Thirdly, suitable dimensions should be selected for the particle detection chamber since the encoded particles have to be arranged in a monolayer. For example, Yuen et al. developed a microdevice in which glass microbarcodes can be arranged next to each other by means of centrifugal forces . The device consists of a central 1-mm-high reservoir surrounded by a 35-μm-high sorting region (less than twice the height of the 20 μm × 20 μm × 100 μm microbarcodes). The outside of the sorting region was connected to a network of sixty 20-μm-wide microchannels (equal to the width of the microbarcodes). After loading a suspension of the microbarcodes into the central reservoir, a monolayer of microbarcodes was formed in the sorting region by spinning the device. The microchannels stopped the microbarcodes from passing through, but acted as a drain for the liquid. The group of Ducree arranged particles in a monolayer within a disk-based detection chamber, which allowed parallel read-out of multiplexed particle-based immunoassays [39, 119].
As mentioned above, apart from microscope reading systems, other types of currently available reading instruments are not easily compatible with microfluidic devices. Microtechnology research into integrating electronics, optics, and detectors in microfluidic devices is currently ongoing. The next section overviews recent advances in this field.
Micro flow (cyto)meter
Advances in flow (cyto)metric analysis of cells and particles in microfluidic devices have recently been described [120, 121]. Meanwhile, the first commercial microfabricated flow (cyto)meter has also become commercially available (2100 Bioanalyzer, Agilent Technologies). Similar to conventional flow meters, micro flow meters require precise fabrication to obtain optimal fluid flows in which particles are hydrodynamically focused into a single-file stream. The cost and complexity of fabricating fluidic components, traditionally made of glass, can be reduced by using inexpensive polymers like PDMS or SU-8 [122, 123]. Although sheath liquid-based hydrodynamic focusing serves as a standard technology in both conventional and micro flow (cyto)meters, it requires a large volume of sheath liquid to process a very small amount of sample (up to 1 L for 1 mL of sample), preventing further reductions in the size and volume of the whole system. It also needs continuous pumping of sheath liquid at high flow rates to generate a thin sample stream. Alternatively, ambient air can be used .
Light-emitting diodes and detectors
In the previous section it was explained that high-throughput screening of encoded microcarriers using existing optical reading instruments is possible “on a chip,” while the optical components, such as the light source, sensors, lenses and waveguides, remain “off the chip.” Researchers have also taken on-chip high-throughput screening systems of encoded microcarriers one step further by integrating optics on the chip. The group of deMello reported thin-film polymer (polyfluorene-based) light-emitting diodes (LEDs) and thin-film organic photodiodes used as integrated excitation sources and detectors, respectively [132, 133]. Since the LED is a very small, low-power, inexpensive device, it can be integrated into microfluidics as a disposable light source. Recently, the same group made progress in the fabrication of disposable high-quality monolithically integrated optical filters . Chabynic et al. reported the integration of an optical fiber and a fluorescence detector based on a microavalanche photodiode (μ-APD) into a microfluidic device fabricated in PDMS . No transfer optics were necessary, because the pixel size of the μ-APD matched the dimensions of the channels and the μ-APD was incorporated in close proximity to the microchannel. However, in this system there was a lot of light loss because focusing of the LED light was not possible on the optical fiber (100 μm diameter) that coupled light into the microdevice, resulting in ineffective illumination and insensitive analyses. This can be circumvented, as reported by Miyaki et al., by placing the light-emitting face of the LED close to the microchannel by incorporating it into a chip fabricated through in situ polymerization. In this case, the detection sensitivity was comparable to that of laser-induced fluorescence . Seo and Lee have reported work on a disposable integrated device with self-aligned planar microlenses for bioanalytical systems, which has LEDs as excitation sources and photodiodes as detectors . The lenses enable increased detection sensitivity and reduced time for optical alignment.
Optical imaging fibers
Multiple articles describe the implementation of optical imaging fibers into microfluidics [117, 135]. However, the implementation of optical fiber arrays is still under development. The group of Walt recently developed the first microfluidic platform equipped with an optical imaging fiber microarray capable of detecting DNA at the attomolar level. The use of a microfluidic platform enabled faster DNA hybridizations, lower sample volumes and 100-fold more sensitive detection when compared with a static platform (where the fiber is submerged in the target DNA sample and hybridization occurs by diffusion only); the minimal detectable concentration with the microfluidic platform was equal to 10 aM after 15 minutes of hybridization of a 50 μl target DNA sample at flow rates of 1 μl/min, compared with 1 pM detection with the static platform after 30 min of hybridization of a 200 μl target DNA sample . As is the case for the microscope reading platforms, future developments in order to incorporate multiplexed microparticle arrays, optics, fluidic channels and a detection unit are necessary before a portable system becomes reality.
Conclusions and future perspectives
This review focused on miniaturized multiplexing using encoded microparticles. The combination of microfluidic technologies with encoded microparticle arrays is a very promising lab-on-a-chip tool, due to the remarkable characteristics of both technologies, which complete each other. A special emphasis was placed on the challenges of integrating current detection platforms for encoded microparticles into microdevices. The flow cytometer is currently a very popular detection platform for medium-throughput particle-based “macroscopic” multiplexing assays. When comparing the opportunities of conventional decoding instruments for miniaturized multiplexing, it seems that microscope reading platforms have a clear advantage over other platforms, because the microparticles can remain on the chip for decoding as long as the microdevice is optically transparent. Recent research shows that optical fibers may be usable “on-chip” too. However, the fiber (carrying the encoded particles) will therefore have to be inserted into a microchip in a sealed way without liquid leakage.
Work in the field of microtechnologies aimed at down-scaling the decoding unit to the microlevel, which circumvents the need to use conventional instruments for miniaturized multiplexing, is underway. Considerable cost savings can potentially be realized by integrating the optics, electronics, and detection instruments on-chip, in close proximity to the microchannels carrying the multiplexed microparticle arrays. Although this field is still in its infancy, it will probably result in new fundamental concepts for the decoding of miniaturized multiplexed microparticle assays with the same throughput as existing conventional decoding instruments, which will eventually replace the latter ones. Promising examples are the first generation of micro flow cytometers, integrated light-emitting diodes and detectors, and so on.
Which type of detection system will become popular for multiplexing in microfluidics devices will not only depend on parameters associated with the decoding system, such as portability, costs and ease of use, but also on, for instance, the level of multiplexing that can be achieved. The latter depends on the way in which the microparticles are encoded, and is mainly defined by the intended application of the microparticle array (genotyping, protein analysis, gene expression analysis, etc.). Advances in encoding technologies of microcarriers are expected to result in new multiplexing platforms and will therefore certainly influence the future choice of detection/decoding system.
Finally, the goal of those integrated lab-on-a-chip tools is point-of-care assessment. Therefore, the real challenge will come from the coupling of the decoding modules under investigation in this study in an appropriate way on one chip to other advanced modules used for other sub-tasks, such as blood processing, extraction of DNA, RNA or proteins, and so on. In order to bring diagnostics closer to the patient, future requirements will also involve progress in non-hardware tools, like data acquisition, data management, etc. Research into each of these fields holds much promise, and we can probably expect to see the first prototypes of multiplexed particle-based LOC tools within the next decade, assuming that new nano-engineering technologies are rapidly accepted.
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