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Ligation-based molecular tools for lab-on-a-chip devices
New Biotechnology Volume 25, Number 1 June 2008
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Research Paper
Ligation-based molecular tools for lab-on-a-chip devices Jonas Melin, Jonas Jarvius, Chatarina Larsson, Ola So¨derberg, Ulf Landegren and Mats Nilsson Department of Genetics and Pathology, Rudbeck Laboratory, University of Uppsala, SE 75185 Uppsala, Sweden
Abstract
Molecular diagnostics can offer early detection of disease, improved diagnostic accuracy, and qualified follow-up. Moreover, the use of microfluidic devices can in principle render these analyses quickly and user-friendly, placing them within the reach of the general practitioner and maybe even in households. However, the progress launching such devices has been limited so far. We propose that an important limiting factor has been the difficulty of establishing molecular assays suitable for microfabricated formats. The assays should be capable of monitoring a wide range of molecules, including genomic DNA, RNA and proteins with secondary modifications and interaction partners, and they must exhibit excellent sensitivity and specificity. We discuss these problems and describe a series of molecular tools that may present new opportunities for lab-on-a-chip devices at the point-of-care. Production of microfabricated lab-on-a-chip devices The lab-on-a-chip concept promises rapid, inexpensive diagnostics in the context of a personalized medicine. Properties like rapid reaction kinetics and response times, minimal reagent consumption, convenient control over parallel and sequential reactions steps, and the easy availability of, for example, on-board optics all represent alluring features of this approach, but only modest progress has been seen so far in exploiting microfluidic devices in routine diagnostics. For reasons discussed below, these analytic devices so far remain largely confined to the substantially smaller research market, and even there success has been limited. The pioneering microdevices for chemical and biochemical analysis produced in the 1980s and early 1990s, respectively, were typically fabricated in silicon or glass, using methods adapted from the semiconductor industry. Photolithography permits accurate, micrometer resolution transfer of a pattern to a substrate. The important heritage from integrated electronics is still obvious in current technologies, though several polymer materials have now joined silicon as materials used for the fabrication of microstructures. The long cycle times and the sophisticated equipment Corresponding author: Landegren, U. ([email protected])
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required for silicon and glass microfabrication prompted the development of more convenient fabrication methods. In the mid-1990s polymer microreplication techniques became a frequently employed fabrication method. In academia silicone rubber materials such as poly(dimethyl) siloxane (PDMS) have become the standard material for microfluidic chip prototyping [1], and for small-scale fabrication. This material is easy to replicate by molding, and it is optically transparent between 240 and 1100 nm [2]. PDMS microstructures are also uncomplicated to seal to form closed channels and reservoirs, by irreversible bonding to lids made of, for example, PDMS, glass or silicon [1]. For large volume production PDMS has yet to prove its usefulness, however, because of the relatively long curing times. An alternative fabrication method is on the basis of injectionmolding of thermoplastic material. Most disposable lab materials used in biochemical analyses are produced using this method, since the technique offers an extremely high-throughput and a very low cost per produced unit. We have developed a platform for microfluidic chip fabrication that combines the virtues of high-throughput compact disc injection molding and elastomer technology [3]. The fabrication scheme involves rapid injection molding of thermoplastics, followed by silica deposition and
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New Biotechnology Volume 25, Number 1 June 2008
covalent attachment of an unstructured PDMS lid (Figure 1). This technique significantly reduces the time for chip production compared to standard PDMS molding technology. The sealing process via silica deposition preserves microstructure topology
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and does not introduce materials with unfavorable fluorescence properties. It also allows for observation of the microchannel with high magnification optics since the thickness of the PDMS film can be chosen to match the working distance of an immersion objective (<200 mm).
Besides the cost of production of most current microfabricated devices, the slow adoption of microfluidic devices in diagnostics may also stem partly from the challenge of dealing with highly complex biological samples such as serum and lysed cells in these devices. For example, human DNA analyses for the purposes of genotyping common variants or searching for recent mutations requires identification of variable nucleotides that occur as approximately 1 part per 13 billion in the 2 strands of each copy of the chromosomes of the diploid human genome. Similarly, analysis of RNA transcripts, while less complex than genomic DNA, requires copy number differences of five or more orders of magnitude to be negotiated in individual cells, and even more in mixtures of different cells. For proteins, finally, the detection of abundant ones presents little difficulty, but future analyses probably will require detection also of extremely low concentrations of proteins released to the bloodstream from diseased tissues anywhere in the body, as well as distinguishing protein variants subjected to processing steps, secondary modifications, and interacting with one another in specific diagnostic constellations. Current microfluidic diagnostic devices frequently employ miniaturized versions of molecular assay procedures previously applied in standard microtiter wells and the like. However, to take full advantage of the strength of the lab-on-a-chip concept it may be necessary to rethink also the molecular assay mechanisms. A case in point is the recent method for parallel DNA sequencing of millions of clonally amplified DNA sequences, immobilized on beads using water-in-oil-emulsion PCR [4,5], or on a planar surface using anchored PCR primers to generate confined amplification products (http://www.illumina.com/). These molecular clones are then sequenced in parallel in microfluidic devices using different sequencing by synthesis chemistries [4,6,7] (http://www.454.com/), or sequencing by ligation [5] (http://www.appliedbiosystems.com/). The purpose of this review is to describe a class of molecular probing schemes our lab has been involved in developing, and that may present advantages for future miniaturized test platforms. Before embarking on a description of these molecular tools and their possible roles in microfluidic assays, it is appropriate to present a short background on molecular probing techniques.
A brief history of molecular probing
FIGURE 1
Manufacturing process for the thermoplastic-PDMS microfluidic platform. From top to bottom, microchannels are produced using rapid compact disc injection molding, followed by dicing of individual chips. The chip surface is next coated with a thin layer of silica by sputtering or electron beam evaporation. Closed channels are formed by bonding a preformed unstructured lid PDMS or a PDMS-thermoplastic composite lid to the polymer chip. The bonding takes place at atmospheric pressure after oxidization of the chip and lid surface.
Techniques such as the parallel DNA sequencing methods mentioned above, and analogously, mass spectrometry of the protein composition of samples, allow the user to screen through and identify all components occurring at a sufficiently high concentration in a sample. Molecular probing schemes involving affinity reagents, in contrast, enable focused analyses of molecules of known diagnostic interest. Unfortunately, affinity reagents such as DNA hybridization probes and antibodies typically cannot be trusted to show exclusive affinity for their intended target molecules. Even if this affinity is higher than that for any other molecules, sufficient www.elsevier.com/locate/nbt
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Molecular assays in microfabricated devices
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numbers of non-target molecules frequently compromise detection specificity and preclude detection of rare species. Antibody-based protein assays became popular with the publication of the radioimmune assay by Rosalind Yalow in the late 1950s [8], winning her a Nobel Prize. Shortly thereafter, during the early 1960s several groups, including those of Paul Doty and Sol Spiegelman [9,10], realized that the complementarity of strands of nucleic acids renders these suitable for molecular probing of specific nucleic acids. Many versions of these types of affinity-based protein and nucleic acid assays saw the light of day during the 1960s and 1970s, but a particularly important next step was taken with the development in 1967 of the so-called sandwich immune assay [11]. In this format each protein molecule must be recognized twice. First by being captured to a solid support via an immobilized antibody, and then after washes, by a second antibody binding the immobilized target protein and carrying a suitable detectable moiety to report the detection reaction after renewed washes. The requirement for dual recognition greatly improved detection specificity, and this approach has remained the dominant format for affinity-based protein assays up till the present. The corresponding approach for nucleic acid detection via sandwich hybridization reactions was first established during the second half of the 1980s [12]. Also other means have been used to improve detection specificity, for example, by combining size separation with affinity probing. In this manner the eponymous Southern blot [13], for the first time allowed convenient analyses of human single copy genes, and even single nucleotide variants via their effect on recognition by restriction enzymes. As a consequence it thereby became possible to establish the first genetic linkage maps of man, and to search for the location of inherited lesions in monogenic disease. The analogous method for identifying proteins by gel separation and affinity probing with antibodies was coined by the Western blot, published by Renart et al. [14] in 1979, and has remained popular ever since for accurate detection of proteins. The next revolutionary development of DNA analyses came in 1985 with the publication of the PCR method [15], where the specific detection of target DNA sequences by two oligonucleotide primers, aided by a DNA polymerase, results in the exponential accumulation of target sequences to levels where remaining unamplified bystander sequences can be safely ignored. Until recently, nothing like the sensitivity and specificity of PCR has been available for protein analysis, largely precluding detection of very low copy numbers or modified variants of specific proteins in complex samples.
Ligase-mediated detection of target molecules For a number of years we and other groups have been developing a family of DNA ligation-based methods for detection of an everincreasing range of biomolecules. The DNA ligation approach offers several attractive features, including the fact that affinitybased recognition reactions result in the formation of covalent and thus very stable links connecting detection probes. Moreover, it is possible to target ligated probes but not unreacted ones for DNA amplification reactions, greatly enhancing detection sensitivity. Finally, the amplifiable probes can be designed to include specific messages written in DNA code, to identify the detected target molecule upon subsequent decoding by hybridization or other forms of DNA sequence analysis. The following two ligase-based probing techniques are of particular interest in our work (Figure 2). 44
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Padlock probe ligation Linear oligonucleotides with target-complementary regions at both ends can be designed such that the two ends will be brought in juxtaposition when the probe encounters a target DNA or RNA sequence [16,17]. The act of joining the ends by ligation gives rise to DNA circles that are wound around their target molecules because of the helical probe-target duplex; hence the name padlock probes (Figure 2A). We have established that the requirement for dual hybridization and recognition by a ligase jointly provide
FIGURE 2
Ligation-based assays and amplified single-molecule detection. (A) Padlock probe ligation. The target-complementary regions at both ends of the padlock probe (red) are designed such that the two ends will be brought in juxtaposition when the probe encounters a target DNA or RNA sequence (blue). The ligation, catalyzed by a DNA ligase (yellow), results in a closed DNA circle for each recognized target molecule. (B) Proximity probe ligation. Upon coincident binding of two antibodies to a target protein molecule the attached oligonucleotide strands come into close proximity. Two connector oligonucleotides (red and blue) are added to form a circular structure guided by the oligonucleotide strands attached to the antibodies (green), resulting in the formation of a covalently closed DNA circle by ligation. (C) Amplified single-molecule detection through RCA. A padlock probe is circularized through enzymatic ligation upon identification of a target DNA sequence, resulting in the creation of a circular DNA molecule with nanometer dimensions (red and green). The circular DNA molecule is copied using rolling circle amplification (RCA), generating a large randomly coiled DNA strand (blue and green). This rolling circle product (RCP) is composed of the complement of the circle repeated several hundred times and, collapses into an amorphous blob of DNA owing to Brownian motions affecting the long flexible DNA chain. A short fluorescence-labeled oligonucleotide (red) is hybridized to the repeated sequence of the RCP resulting in a confined cluster of hundreds of fluorophores. Each fluorophore cluster is visible as a bright object with a diameter slightly less than 1 mm, when observed by fluorescence microscopy.
sufficient specificity to identify specific sequences in complex genomes such as that of man [18]. Moreover, because of the substrate requirement by the ligase, single-nucleotide mismatches at the ligation junction can be easily discriminated for genotyping purposes [19,20]. Several tens of thousands of such probing reactions can be performed in parallel in a DNA sample, followed by identification of the reacted padlock probes or a variant thereof called Molecular Inversion Probes (MIPs) [21–23]. This class of probes holds advantages for miniaturized DNA analyses, and the type of probes described below extends the range of detectable molecules to proteins and other macromolecules.
Proximity probe ligation These probes represent pairs of antibodies or similar affinity reagents equipped with short DNA strands [24,25]. Upon pairwise binding to the same target protein molecule the attached DNA strands are brought into proximity so that the high local concentration of one end about the other, typically in the micromolar range, allows the DNA strands to be joined efficiently by DNA ligation following introduction of a connector oligonucleotide for the two ends to hybridize to (Figure 2B). The ligated DNA strands can be amplified to desired levels using methods like PCR. By judicious assay design and using high-affinity antibodies the presence of even very few target molecules results in more ligation products than those observed in the absence of target, permitting detection of very low levels of proteins [26]. The assay mechanism can be applied using solid phase-bound probes or in homogenous phase with no need for washes [24,27]. By redesigning assays to depend on the simultaneous binding by three affinity probes with attached DNA strands rather than two, as little as hundreds of protein molecules can be detected, which is far below detection limits in current sandwich immunoassays [28]. It is also possible to interrogate proteins with respect to their binding to specific DNA sequences [29]. The two probing methods described above, padlock and proximity probing, have features that should render them suitable for efficient and highly specific on-chip probing in microfluidic devices. Moreover, it is possible to directly connect the probing reactions to an amplification step, greatly extending sensitivity, even to the level of single-molecule detection and analysis. Before describing this, we will first discuss opportunities for single molecule detection.
Detection of single molecules Most current biochemical analyses are geared to detecting millions or billions of molecules, while more weakly expressed molecules or molecular heterogeneity among target molecules often goes undetected. The introduction of single-molecule detection (SMD) techniques has revolutionized the study of biomolecular dynamics, allowing for direct observation of, for example, enzymatic mechanisms of purified molecules both in real-time and in situ [30,31]. However, SMD so far has had a limited role for analyzing complex mixtures of molecules in biological samples, and Dittrich and Manz recently described the combination of microfluidics and single-molecule detection as the Holy Grail in miniaturized analysis [32]. In addition to presenting the ultimate limit of detection, single-molecule detection makes highly precise quantifications possible. An inherent problem of any assay aiming to detect single molecules in biological samples is the difficulty of distinguishing
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among many crossreactive target molecules, some of which may be present at concentrations orders of magnitude higher than the investigated one, as well as nonspecifically bound detection reagents. Opportunities for SMD are thus limited by the specificity of detection. One possible approach to achieve sufficient detection specificity is to directly couple specific molecular recognition reactions to localized amplification reactions. An early approach involved running numerous PCRs on aliquots of a diluted sample [33]. The development of integrated microfluidic systems has allowed for microfabricated compartments for PCR [34] and such digital array chips that contain 9000 compartments are now commercially available. One drawback of using predefined compartments is that most of the compartments have to be empty to ensure that very few compartments contain more than one molecule. Millions of confined PCRs can be run in the aqueous compartments that form in a water-in-oil emulsion, and the PCR product can be trapped on single beads by anchoring one of the PCR primers to the bead [35,36]. The oil emulsion approach has been used in quantification assays, using flow cytometry to detect molecules bound to the beads, and it is used to prepare large numbers of DNA templates for parallel sequencing approaches [4,5]. PCRs have also been performed in a gel matrix, limiting diffusion [37,38] to assay both DNA and RNA targets [39,40]. An elegant way to generate a confined amplification product is to circularize the amplification target and use rolling circle amplification (RCA) instead of PCR (Figure 2C). During RCA the circular DNA molecule is copied by a DNA polymerase, generating a long DNA strand composed of tandem repeats of complements of the circular DNA strand. The rolling circle products (RCPs) form individual random-coil DNA molecules and thus no physical barriers are needed to confine the amplification products from individual molecules in one spot. The RCA principle was first applied for biotechnological purposes by Fire [41] and Kool [42], and later the discrete nature of the amplification products was established by Lizardi et al., studying RCPs linked to a solid support [43]. Individual RCPs generated from preformed DNA circles have also been used to score and quantify single nucleotide polymorphisms [44,45]. The RCA technique is particularly well suited for amplifying circular DNA strands that have arisen due to target detection by padlock or proximity probes.
Rolling-circle amplified padlock and proximity ligation assays We have used RCA to visualize specific molecular recognition events by nanometer-size padlock and proximity probes that give rise to micrometer-sized, intensely fluorescent bundles of singlestranded DNA, each composed of hundreds of tandem repeated copies of the reacted probes. This process results in one RCP for each recognized target, representing the discrete nature of the molecular population (Figure 2C). RCA-mediated detection of reacted padlock probes was demonstrated first using bulk average readout for DNA quantification [43,46–48]. Later RCA was applied in situ to amplify the detection signals of specifically reacted padlock probes, allowing for in situ genotyping of individual mitochondrial DNA molecules in cells [49], for the first time enabling robust detection of single-nucleotide polymorphisms in single DNA molecules in situ (Figure 3A). www.elsevier.com/locate/nbt
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In a related approach, proximity ligation has been combined with RCA to visualize the subcellular localization of proteins and interacting pairs of proteins at the single molecule level, for the first time permitting detection of individual endogenous protein– protein interaction events in cells and tissues [50] (Figure 3B)
Research Paper FIGURE 3
Single-molecule detection in situ using padlock probes and proximity probes coupled to RCA. (A) In situ genotyping of human mitochondrial DNA. Cells harboring both normal and mutant mitochondrial DNA, a state referred to as heteroplasmy, were cultured and fixed on a glass microscopy slide. The only difference between the two mitochondrial DNA variants is the A3243G single base-pair mutation. A pair of padlock probes was hybridized, ligated, rollingcircle amplified, and finally stained with red and green fluorescence-labeled detection probes. Green and red fluorescence indicate detection of mutant and wild-type mitochondrial DNA molecules, respectively. Cell nuclei were stained blue using DAPI. (B) Detection of protein interactions in situ using proximity ligation. The proto-oncogene c-myc is one of the most commonly dysregulated genes in cancer. Upon binding of c-Myc to its partner Max the protein complex can bind to DNA and promote transcription of genes containing a Myc-recognition motif (E-box). Interactions between c-Myc and Max were detected by proximity ligation (red dots) in cultured U2OS cells, counterstained with FITC labeled anti-actin antibodies (green) and Hoechst (blue), to visualize the cytoplasm and nucleus, respectively. As expected the c-Myc/Max heterodimers are present in cell nuclei.
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FIGURE 4
The homogenous amplified single-molecule detection set-up. Brightly fluorescent micrometer-sized RCPs are formed as a consequence of specific molecular probing reactions (Figure 2). The RCPs are pumped through the thermoplastic-PDMS analysis chip (Figure 1) and detected using a confocal microscope operated in line scanning mode, focused at the center of the channel. The recorded image (x-time stack) is binarized through thresholding to allow for digital counting and classification of individual RCPs.
(http://www.olink.com/). The method has also recently been applied to investigate tyrosine phosphorylation of stimulated growth factor receptors in cells in vitro and in samples of patient tissues [51]. The combination of highly specific probing of biomolecules with padlock and proximity probes, immediately followed by localized amplification by RCA, is promising for use in microfabricated diagnostic devices. We have illustrated this potential by establishing a homogenous method for amplified single-molecule detection of circularized padlock probes via RCA, followed by the enumeration of individual amplified molecules being pumped through a microfluidic channel with fluorescent detection as described below [52].
Homogenous rolling circle product quantification In contrast to the methods described previously for localized amplification of single molecules [4,5,35–40], RCA of reacted padlock probes can be performed in the homogenous phase with no need for a solid support. This is possible since the entire amplification product is one large, bundled-up DNA molecule [53]. For biomolecular quantification assays this permits a simple protocol, only involving serial additions of reagents and temperature variation before microfluidic quantification. The fluorescence-labeled RCPs are pumped through a microfluidic device and counted as individual objects via fluorescence imaging. The microfluidic device is fabricated by the process describe above [3] (Figure 1). These microfluidic devices thus combine ease of production with excellent optical properties, suitable for counting fluorescent objects.
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The homogenous amplified single molecule detection technique exhibits similar specificity and sensitivity of detection as realtime PCR, but it is characterized by better quantitative precision and a greater potential for multiplexing by fluorescence detection (Figure 4). As the spectral profile of each RCP is analyzed separately the multiplexing capability can be extended beyond the number of label colors by combinatorial labeling. The precision of counting RCPs depends on how accurately the investigated volume is measured. If confocal volume definition is used in the microfluidic readout, the precision is limited only by Poisson sampling statistics, providing a highly specific and precise molecule counter [54]. In conclusion, we have discussed the preparation of lab-on-achip devices and we have illustrated the gradual development of increasingly specific methods to probe nucleic acids and proteins. Assays with single-molecule sensitivity are within reach, and can be applied in miniaturized formats. Accordingly, novel probe designs in combination with microfluidic devices promise to reach the speed, accuracy and user friendliness required for implementation at the point-of-care and for environmental monitoring.
Acknowledgements The work in our group is supported by grants from the EU 6th Framework Programme (MolTools, Enlight, and COMICS), from the Swedish research councils for Medicine and for Natural Sciences and Technology, the Knut and Alice Wallenberg Foundation, the Beijer Foundation, VINNOVA/SSF, Uppsala-Bio, and the Swedish Defense Nanotechnology program.
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Ligation-based molecular tools for lab-on-a-chip devices Jonas Melin, Jonas Jarvius, Chatarina Larsson, Ola So¨derberg, Ulf Landegren and Mats Nilsson Department of Genetics and Pathology, Rudbeck Laboratory, University of Uppsala, SE 75185 Uppsala, Sweden
Abstract
Molecular diagnostics can offer early detection of disease, improved diagnostic accuracy, and qualified follow-up. Moreover, the use of microfluidic devices can in principle render these analyses quickly and user-friendly, placing them within the reach of the general practitioner and maybe even in households. However, the progress launching such devices has been limited so far. We propose that an important limiting factor has been the difficulty of establishing molecular assays suitable for microfabricated formats. The assays should be capable of monitoring a wide range of molecules, including genomic DNA, RNA and proteins with secondary modifications and interaction partners, and they must exhibit excellent sensitivity and specificity. We discuss these problems and describe a series of molecular tools that may present new opportunities for lab-on-a-chip devices at the point-of-care. Production of microfabricated lab-on-a-chip devices The lab-on-a-chip concept promises rapid, inexpensive diagnostics in the context of a personalized medicine. Properties like rapid reaction kinetics and response times, minimal reagent consumption, convenient control over parallel and sequential reactions steps, and the easy availability of, for example, on-board optics all represent alluring features of this approach, but only modest progress has been seen so far in exploiting microfluidic devices in routine diagnostics. For reasons discussed below, these analytic devices so far remain largely confined to the substantially smaller research market, and even there success has been limited. The pioneering microdevices for chemical and biochemical analysis produced in the 1980s and early 1990s, respectively, were typically fabricated in silicon or glass, using methods adapted from the semiconductor industry. Photolithography permits accurate, micrometer resolution transfer of a pattern to a substrate. The important heritage from integrated electronics is still obvious in current technologies, though several polymer materials have now joined silicon as materials used for the fabrication of microstructures. The long cycle times and the sophisticated equipment Corresponding author: Landegren, U. ([email protected])
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required for silicon and glass microfabrication prompted the development of more convenient fabrication methods. In the mid-1990s polymer microreplication techniques became a frequently employed fabrication method. In academia silicone rubber materials such as poly(dimethyl) siloxane (PDMS) have become the standard material for microfluidic chip prototyping [1], and for small-scale fabrication. This material is easy to replicate by molding, and it is optically transparent between 240 and 1100 nm [2]. PDMS microstructures are also uncomplicated to seal to form closed channels and reservoirs, by irreversible bonding to lids made of, for example, PDMS, glass or silicon [1]. For large volume production PDMS has yet to prove its usefulness, however, because of the relatively long curing times. An alternative fabrication method is on the basis of injectionmolding of thermoplastic material. Most disposable lab materials used in biochemical analyses are produced using this method, since the technique offers an extremely high-throughput and a very low cost per produced unit. We have developed a platform for microfluidic chip fabrication that combines the virtues of high-throughput compact disc injection molding and elastomer technology [3]. The fabrication scheme involves rapid injection molding of thermoplastics, followed by silica deposition and
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covalent attachment of an unstructured PDMS lid (Figure 1). This technique significantly reduces the time for chip production compared to standard PDMS molding technology. The sealing process via silica deposition preserves microstructure topology
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and does not introduce materials with unfavorable fluorescence properties. It also allows for observation of the microchannel with high magnification optics since the thickness of the PDMS film can be chosen to match the working distance of an immersion objective (<200 mm).
Besides the cost of production of most current microfabricated devices, the slow adoption of microfluidic devices in diagnostics may also stem partly from the challenge of dealing with highly complex biological samples such as serum and lysed cells in these devices. For example, human DNA analyses for the purposes of genotyping common variants or searching for recent mutations requires identification of variable nucleotides that occur as approximately 1 part per 13 billion in the 2 strands of each copy of the chromosomes of the diploid human genome. Similarly, analysis of RNA transcripts, while less complex than genomic DNA, requires copy number differences of five or more orders of magnitude to be negotiated in individual cells, and even more in mixtures of different cells. For proteins, finally, the detection of abundant ones presents little difficulty, but future analyses probably will require detection also of extremely low concentrations of proteins released to the bloodstream from diseased tissues anywhere in the body, as well as distinguishing protein variants subjected to processing steps, secondary modifications, and interacting with one another in specific diagnostic constellations. Current microfluidic diagnostic devices frequently employ miniaturized versions of molecular assay procedures previously applied in standard microtiter wells and the like. However, to take full advantage of the strength of the lab-on-a-chip concept it may be necessary to rethink also the molecular assay mechanisms. A case in point is the recent method for parallel DNA sequencing of millions of clonally amplified DNA sequences, immobilized on beads using water-in-oil-emulsion PCR [4,5], or on a planar surface using anchored PCR primers to generate confined amplification products (http://www.illumina.com/). These molecular clones are then sequenced in parallel in microfluidic devices using different sequencing by synthesis chemistries [4,6,7] (http://www.454.com/), or sequencing by ligation [5] (http://www.appliedbiosystems.com/). The purpose of this review is to describe a class of molecular probing schemes our lab has been involved in developing, and that may present advantages for future miniaturized test platforms. Before embarking on a description of these molecular tools and their possible roles in microfluidic assays, it is appropriate to present a short background on molecular probing techniques.
A brief history of molecular probing
FIGURE 1
Manufacturing process for the thermoplastic-PDMS microfluidic platform. From top to bottom, microchannels are produced using rapid compact disc injection molding, followed by dicing of individual chips. The chip surface is next coated with a thin layer of silica by sputtering or electron beam evaporation. Closed channels are formed by bonding a preformed unstructured lid PDMS or a PDMS-thermoplastic composite lid to the polymer chip. The bonding takes place at atmospheric pressure after oxidization of the chip and lid surface.
Techniques such as the parallel DNA sequencing methods mentioned above, and analogously, mass spectrometry of the protein composition of samples, allow the user to screen through and identify all components occurring at a sufficiently high concentration in a sample. Molecular probing schemes involving affinity reagents, in contrast, enable focused analyses of molecules of known diagnostic interest. Unfortunately, affinity reagents such as DNA hybridization probes and antibodies typically cannot be trusted to show exclusive affinity for their intended target molecules. Even if this affinity is higher than that for any other molecules, sufficient www.elsevier.com/locate/nbt
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Molecular assays in microfabricated devices
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numbers of non-target molecules frequently compromise detection specificity and preclude detection of rare species. Antibody-based protein assays became popular with the publication of the radioimmune assay by Rosalind Yalow in the late 1950s [8], winning her a Nobel Prize. Shortly thereafter, during the early 1960s several groups, including those of Paul Doty and Sol Spiegelman [9,10], realized that the complementarity of strands of nucleic acids renders these suitable for molecular probing of specific nucleic acids. Many versions of these types of affinity-based protein and nucleic acid assays saw the light of day during the 1960s and 1970s, but a particularly important next step was taken with the development in 1967 of the so-called sandwich immune assay [11]. In this format each protein molecule must be recognized twice. First by being captured to a solid support via an immobilized antibody, and then after washes, by a second antibody binding the immobilized target protein and carrying a suitable detectable moiety to report the detection reaction after renewed washes. The requirement for dual recognition greatly improved detection specificity, and this approach has remained the dominant format for affinity-based protein assays up till the present. The corresponding approach for nucleic acid detection via sandwich hybridization reactions was first established during the second half of the 1980s [12]. Also other means have been used to improve detection specificity, for example, by combining size separation with affinity probing. In this manner the eponymous Southern blot [13], for the first time allowed convenient analyses of human single copy genes, and even single nucleotide variants via their effect on recognition by restriction enzymes. As a consequence it thereby became possible to establish the first genetic linkage maps of man, and to search for the location of inherited lesions in monogenic disease. The analogous method for identifying proteins by gel separation and affinity probing with antibodies was coined by the Western blot, published by Renart et al. [14] in 1979, and has remained popular ever since for accurate detection of proteins. The next revolutionary development of DNA analyses came in 1985 with the publication of the PCR method [15], where the specific detection of target DNA sequences by two oligonucleotide primers, aided by a DNA polymerase, results in the exponential accumulation of target sequences to levels where remaining unamplified bystander sequences can be safely ignored. Until recently, nothing like the sensitivity and specificity of PCR has been available for protein analysis, largely precluding detection of very low copy numbers or modified variants of specific proteins in complex samples.
Ligase-mediated detection of target molecules For a number of years we and other groups have been developing a family of DNA ligation-based methods for detection of an everincreasing range of biomolecules. The DNA ligation approach offers several attractive features, including the fact that affinitybased recognition reactions result in the formation of covalent and thus very stable links connecting detection probes. Moreover, it is possible to target ligated probes but not unreacted ones for DNA amplification reactions, greatly enhancing detection sensitivity. Finally, the amplifiable probes can be designed to include specific messages written in DNA code, to identify the detected target molecule upon subsequent decoding by hybridization or other forms of DNA sequence analysis. The following two ligase-based probing techniques are of particular interest in our work (Figure 2). 44
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Padlock probe ligation Linear oligonucleotides with target-complementary regions at both ends can be designed such that the two ends will be brought in juxtaposition when the probe encounters a target DNA or RNA sequence [16,17]. The act of joining the ends by ligation gives rise to DNA circles that are wound around their target molecules because of the helical probe-target duplex; hence the name padlock probes (Figure 2A). We have established that the requirement for dual hybridization and recognition by a ligase jointly provide
FIGURE 2
Ligation-based assays and amplified single-molecule detection. (A) Padlock probe ligation. The target-complementary regions at both ends of the padlock probe (red) are designed such that the two ends will be brought in juxtaposition when the probe encounters a target DNA or RNA sequence (blue). The ligation, catalyzed by a DNA ligase (yellow), results in a closed DNA circle for each recognized target molecule. (B) Proximity probe ligation. Upon coincident binding of two antibodies to a target protein molecule the attached oligonucleotide strands come into close proximity. Two connector oligonucleotides (red and blue) are added to form a circular structure guided by the oligonucleotide strands attached to the antibodies (green), resulting in the formation of a covalently closed DNA circle by ligation. (C) Amplified single-molecule detection through RCA. A padlock probe is circularized through enzymatic ligation upon identification of a target DNA sequence, resulting in the creation of a circular DNA molecule with nanometer dimensions (red and green). The circular DNA molecule is copied using rolling circle amplification (RCA), generating a large randomly coiled DNA strand (blue and green). This rolling circle product (RCP) is composed of the complement of the circle repeated several hundred times and, collapses into an amorphous blob of DNA owing to Brownian motions affecting the long flexible DNA chain. A short fluorescence-labeled oligonucleotide (red) is hybridized to the repeated sequence of the RCP resulting in a confined cluster of hundreds of fluorophores. Each fluorophore cluster is visible as a bright object with a diameter slightly less than 1 mm, when observed by fluorescence microscopy.
sufficient specificity to identify specific sequences in complex genomes such as that of man [18]. Moreover, because of the substrate requirement by the ligase, single-nucleotide mismatches at the ligation junction can be easily discriminated for genotyping purposes [19,20]. Several tens of thousands of such probing reactions can be performed in parallel in a DNA sample, followed by identification of the reacted padlock probes or a variant thereof called Molecular Inversion Probes (MIPs) [21–23]. This class of probes holds advantages for miniaturized DNA analyses, and the type of probes described below extends the range of detectable molecules to proteins and other macromolecules.
Proximity probe ligation These probes represent pairs of antibodies or similar affinity reagents equipped with short DNA strands [24,25]. Upon pairwise binding to the same target protein molecule the attached DNA strands are brought into proximity so that the high local concentration of one end about the other, typically in the micromolar range, allows the DNA strands to be joined efficiently by DNA ligation following introduction of a connector oligonucleotide for the two ends to hybridize to (Figure 2B). The ligated DNA strands can be amplified to desired levels using methods like PCR. By judicious assay design and using high-affinity antibodies the presence of even very few target molecules results in more ligation products than those observed in the absence of target, permitting detection of very low levels of proteins [26]. The assay mechanism can be applied using solid phase-bound probes or in homogenous phase with no need for washes [24,27]. By redesigning assays to depend on the simultaneous binding by three affinity probes with attached DNA strands rather than two, as little as hundreds of protein molecules can be detected, which is far below detection limits in current sandwich immunoassays [28]. It is also possible to interrogate proteins with respect to their binding to specific DNA sequences [29]. The two probing methods described above, padlock and proximity probing, have features that should render them suitable for efficient and highly specific on-chip probing in microfluidic devices. Moreover, it is possible to directly connect the probing reactions to an amplification step, greatly extending sensitivity, even to the level of single-molecule detection and analysis. Before describing this, we will first discuss opportunities for single molecule detection.
Detection of single molecules Most current biochemical analyses are geared to detecting millions or billions of molecules, while more weakly expressed molecules or molecular heterogeneity among target molecules often goes undetected. The introduction of single-molecule detection (SMD) techniques has revolutionized the study of biomolecular dynamics, allowing for direct observation of, for example, enzymatic mechanisms of purified molecules both in real-time and in situ [30,31]. However, SMD so far has had a limited role for analyzing complex mixtures of molecules in biological samples, and Dittrich and Manz recently described the combination of microfluidics and single-molecule detection as the Holy Grail in miniaturized analysis [32]. In addition to presenting the ultimate limit of detection, single-molecule detection makes highly precise quantifications possible. An inherent problem of any assay aiming to detect single molecules in biological samples is the difficulty of distinguishing
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among many crossreactive target molecules, some of which may be present at concentrations orders of magnitude higher than the investigated one, as well as nonspecifically bound detection reagents. Opportunities for SMD are thus limited by the specificity of detection. One possible approach to achieve sufficient detection specificity is to directly couple specific molecular recognition reactions to localized amplification reactions. An early approach involved running numerous PCRs on aliquots of a diluted sample [33]. The development of integrated microfluidic systems has allowed for microfabricated compartments for PCR [34] and such digital array chips that contain 9000 compartments are now commercially available. One drawback of using predefined compartments is that most of the compartments have to be empty to ensure that very few compartments contain more than one molecule. Millions of confined PCRs can be run in the aqueous compartments that form in a water-in-oil emulsion, and the PCR product can be trapped on single beads by anchoring one of the PCR primers to the bead [35,36]. The oil emulsion approach has been used in quantification assays, using flow cytometry to detect molecules bound to the beads, and it is used to prepare large numbers of DNA templates for parallel sequencing approaches [4,5]. PCRs have also been performed in a gel matrix, limiting diffusion [37,38] to assay both DNA and RNA targets [39,40]. An elegant way to generate a confined amplification product is to circularize the amplification target and use rolling circle amplification (RCA) instead of PCR (Figure 2C). During RCA the circular DNA molecule is copied by a DNA polymerase, generating a long DNA strand composed of tandem repeats of complements of the circular DNA strand. The rolling circle products (RCPs) form individual random-coil DNA molecules and thus no physical barriers are needed to confine the amplification products from individual molecules in one spot. The RCA principle was first applied for biotechnological purposes by Fire [41] and Kool [42], and later the discrete nature of the amplification products was established by Lizardi et al., studying RCPs linked to a solid support [43]. Individual RCPs generated from preformed DNA circles have also been used to score and quantify single nucleotide polymorphisms [44,45]. The RCA technique is particularly well suited for amplifying circular DNA strands that have arisen due to target detection by padlock or proximity probes.
Rolling-circle amplified padlock and proximity ligation assays We have used RCA to visualize specific molecular recognition events by nanometer-size padlock and proximity probes that give rise to micrometer-sized, intensely fluorescent bundles of singlestranded DNA, each composed of hundreds of tandem repeated copies of the reacted probes. This process results in one RCP for each recognized target, representing the discrete nature of the molecular population (Figure 2C). RCA-mediated detection of reacted padlock probes was demonstrated first using bulk average readout for DNA quantification [43,46–48]. Later RCA was applied in situ to amplify the detection signals of specifically reacted padlock probes, allowing for in situ genotyping of individual mitochondrial DNA molecules in cells [49], for the first time enabling robust detection of single-nucleotide polymorphisms in single DNA molecules in situ (Figure 3A). www.elsevier.com/locate/nbt
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In a related approach, proximity ligation has been combined with RCA to visualize the subcellular localization of proteins and interacting pairs of proteins at the single molecule level, for the first time permitting detection of individual endogenous protein– protein interaction events in cells and tissues [50] (Figure 3B)
Research Paper FIGURE 3
Single-molecule detection in situ using padlock probes and proximity probes coupled to RCA. (A) In situ genotyping of human mitochondrial DNA. Cells harboring both normal and mutant mitochondrial DNA, a state referred to as heteroplasmy, were cultured and fixed on a glass microscopy slide. The only difference between the two mitochondrial DNA variants is the A3243G single base-pair mutation. A pair of padlock probes was hybridized, ligated, rollingcircle amplified, and finally stained with red and green fluorescence-labeled detection probes. Green and red fluorescence indicate detection of mutant and wild-type mitochondrial DNA molecules, respectively. Cell nuclei were stained blue using DAPI. (B) Detection of protein interactions in situ using proximity ligation. The proto-oncogene c-myc is one of the most commonly dysregulated genes in cancer. Upon binding of c-Myc to its partner Max the protein complex can bind to DNA and promote transcription of genes containing a Myc-recognition motif (E-box). Interactions between c-Myc and Max were detected by proximity ligation (red dots) in cultured U2OS cells, counterstained with FITC labeled anti-actin antibodies (green) and Hoechst (blue), to visualize the cytoplasm and nucleus, respectively. As expected the c-Myc/Max heterodimers are present in cell nuclei.
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FIGURE 4
The homogenous amplified single-molecule detection set-up. Brightly fluorescent micrometer-sized RCPs are formed as a consequence of specific molecular probing reactions (Figure 2). The RCPs are pumped through the thermoplastic-PDMS analysis chip (Figure 1) and detected using a confocal microscope operated in line scanning mode, focused at the center of the channel. The recorded image (x-time stack) is binarized through thresholding to allow for digital counting and classification of individual RCPs.
(http://www.olink.com/). The method has also recently been applied to investigate tyrosine phosphorylation of stimulated growth factor receptors in cells in vitro and in samples of patient tissues [51]. The combination of highly specific probing of biomolecules with padlock and proximity probes, immediately followed by localized amplification by RCA, is promising for use in microfabricated diagnostic devices. We have illustrated this potential by establishing a homogenous method for amplified single-molecule detection of circularized padlock probes via RCA, followed by the enumeration of individual amplified molecules being pumped through a microfluidic channel with fluorescent detection as described below [52].
Homogenous rolling circle product quantification In contrast to the methods described previously for localized amplification of single molecules [4,5,35–40], RCA of reacted padlock probes can be performed in the homogenous phase with no need for a solid support. This is possible since the entire amplification product is one large, bundled-up DNA molecule [53]. For biomolecular quantification assays this permits a simple protocol, only involving serial additions of reagents and temperature variation before microfluidic quantification. The fluorescence-labeled RCPs are pumped through a microfluidic device and counted as individual objects via fluorescence imaging. The microfluidic device is fabricated by the process describe above [3] (Figure 1). These microfluidic devices thus combine ease of production with excellent optical properties, suitable for counting fluorescent objects.
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The homogenous amplified single molecule detection technique exhibits similar specificity and sensitivity of detection as realtime PCR, but it is characterized by better quantitative precision and a greater potential for multiplexing by fluorescence detection (Figure 4). As the spectral profile of each RCP is analyzed separately the multiplexing capability can be extended beyond the number of label colors by combinatorial labeling. The precision of counting RCPs depends on how accurately the investigated volume is measured. If confocal volume definition is used in the microfluidic readout, the precision is limited only by Poisson sampling statistics, providing a highly specific and precise molecule counter [54]. In conclusion, we have discussed the preparation of lab-on-achip devices and we have illustrated the gradual development of increasingly specific methods to probe nucleic acids and proteins. Assays with single-molecule sensitivity are within reach, and can be applied in miniaturized formats. Accordingly, novel probe designs in combination with microfluidic devices promise to reach the speed, accuracy and user friendliness required for implementation at the point-of-care and for environmental monitoring.
Acknowledgements The work in our group is supported by grants from the EU 6th Framework Programme (MolTools, Enlight, and COMICS), from the Swedish research councils for Medicine and for Natural Sciences and Technology, the Knut and Alice Wallenberg Foundation, the Beijer Foundation, VINNOVA/SSF, Uppsala-Bio, and the Swedish Defense Nanotechnology program.
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