Abstract:
A microfluidic device and method for enzymatic processing of ultra-long macromolecules is accomplished using a microfluidic device a reaction chamber with a first manifold, a second manifold, and a plurality of reaction channels. Each reaction channel extends from the first manifold to the second manifold. First inlet and outlet channels fill the reaction channels via the manifolds with one or more macromolecule containers suspended in a first carrier fluid. The first inlet and outlet channels are configured such that a flow is guided through the reaction channels, and an enzymatic reagent is fed to the reaction chamber essentially without displacing the macromolecule containers trapped in the reaction channels. The second set of inlets and outlets are configured such that a flow established from the second inlet to the second outlet is guided through at least one of the manifolds and bypasses the reaction channels.

Description:
TECHNICAL FIELD  
       [0001]    The invention relates to a microfluidic device for enzymatic processing of macromolecules, more particularly for preparation of isolated single macromolecules for subsequent processing. In a further aspect, a method is provided for preparing isolated single macromolecules for subsequent processing. The invention is suited for preparing and processing ultra-long macromolecules, i.e. macromolecules with a length of about 1 million base pairs and more. An example for such macromolecules is DNA. 
         [0002]    According to a further aspect, the present invention relates to systems, apparatus, kits, algorithms and methods for handling, preparing analysing and characterizing (in any order) biological samples. The invention also describes uses of the invention, particularly in relation to nucleic acid sequencing technologies. 
       BACKGROUND  
       [0003]    In the recent decades, micro- and nanofluidic devices and methods have been developed for integrating, miniaturising and automating numerous laboratory tasks. Furthermore, due to the characteristic length scales involved, analysis tasks not previously available have been made possible. 
         [0004]    Considerable efforts have been directed to providing a reliable, rapid and affordable analysis of very long macromolecules, such as single DNA molecules, including amplification and/or sequencing steps. However, the maximum fragment lengths analysed by existing methods, are typically limited to about 35-1000 base pairs as compared to the length of bacterial DNA of about 1-10 million base pairs, and at least 50 million base pairs of a complete human DNA molecule. 
         [0005]    A recent article on “Single molecule linear analysis of DNA in nano-channel labelled with sequence specific fluorescent probes”, published in 2010 in Nucleic Acids Research by S. K. Das et al., discloses a nanofluidic method of analysing DNA molecules. The DNA molecules analysed have all a length less than 200 kilo base pairs. A further article on nanofluidic analysis of DNA by Reisner and co-workers, published in Proceedings of the National Academy of Sciences of the USA, vol. 107, p. 13294, 2010, discloses a method for analysing DNA applicable to long DNA molecules, where “long” refers to a length of about 100 kilo base pairs. 
         [0006]    It is one of the merits of the present invention to recognise, that the main factor limiting the length of the fragments analysed in a nanofluidic system lies in the sample preparation and transfer steps. As a principal reason for this limitation, the fragility of isolated DNA molecules or similar long macromolecules, due to shearing forces acting on the DNA, has been identified. According to the present invention, the sample preparation and transfer steps are thus identified to be critical for increasing the fragment length that can be analysed in micro- and nanofluidic systems, and eventually being able to process, e.g. for sequencing or amplification, a complete isolated DNA molecule or similar long macromolecule. 
         [0007]    A chromosome prior to replication comprises a single length of DNA. The ability to visualize the DNA from each chromosome, from one end to the other, would enable the native long-range organization of the genome and its variation between homologous chromosomes and between individuals to be investigated. Entropic confinement in nano-channels/grooves, as demonstrated for bacteriophage genomes (&lt;200 Kbp length), forces DNA into an extended conformation co-linear with the information encoded therein. However, to linearize whole large genomes (e.g. Human), direct from source without cloning, two problems must be considered: Firstly, during extraction or loading into a device, genomic DNA can become fragmented due to shear forces, and secondly, DNA tends to form folded, globular states in solution rather than the extended conformation. Although methods for mapping sequence motifs and patterns (Neely et al Chem. Sci. 2010; Xiao et al Nucleic Acids Res. 35: e16 2007; Reisner et al PNAS107(30):13294-9) on linearized DNA have been developed, new approaches are needed to handle, if not whole chromosomal lengths of human DNA, then portions of chromosomes that are large enough to span the haplotype blocks and much of the structural variation found in large diploid genomes. 
         [0008]    Moreover genome analysis methods with minimal sample preparation are needed. Direct single molecule analysis of genomic DNA can achieve this; recently a whole genome has been sequenced using single molecule technology (Pushkarov et al, Nature Biotechnology 27: 847). Even so, in current methods, DNA extraction is done off-chip and the DNA handling (e.g. pipetting) leads to reduction in size of the genome fragments due to fragmentation by shearing. 
         [0009]    There is a pronounced need for single molecule analysis of long macromolecules. For example, there are an estimated 200 cell types in the human body. However, all cells within a seemingly homogeneous population of a given a cell type are not necessarily alike. Stochastic expression at the gene and protein level is well documented. Stochastic effects lead to widely differing responses to stimuli: fast, slow, extreme or subdued. Ensemble analysis of cell populations masks the variation that is clearly evident when individual cells from a population are analysed. 
         [0010]    There is substantial heterogeneity between cells in a tumour biopsy, including differences in chromosome number (aneuploidy), mutational profiles, methylation profiles and expression at the RNA and protein level. Analysis of single cells within tumours is important for understanding tumour pathology and is expected to contribute to cancer diagnosis, staging, and prognosis. Biopsies may contain on the order of 10,000 cells. Systematic, high throughput and preferably automatable analysis is therefore needed to address the population cell by cell. 
         [0011]    In addition single cell analysis is important for genetic diagnosis, particularly for pre-implantation genetic analysis, which in the future may require analysis of more than one or a few genes, as the scientific community makes increasingly more connections between genotype and phenotype. 
         [0012]    In many cases sample material is limiting, for example from archived material or for the analysis of fetal material in a mothers circulating blood or shed tumour cells or metastatic cells in circulating blood. In these cases better methods are needed for analysis of single or a few cells or a small amount of material. In the case of analysis of material in circulating blood the task may be compared to finding a needle in a haystack because the target material is a small fraction of a complex sample. 
         [0013]    The genome and its epigenetic modifications can be analysed by modern genomic methods, the most comprehensive approach being complete genome sequencing. However, despite the emergence of technologies that have increased throughput and spectacularly lowered sequencing cost, a number of bottlenecks remain that serve as barriers to the effective translation of genomic knowledge. Although much attention has been given to throughput/cost of the sequencing process itself, the same cannot be said of preparation of the sample for sequencing. A first bottleneck is that sequencing technologies require days of upfront sample preparation. A second bottleneck is that upfront sample processing is further increased when goal is to sequence selected parts of the genome. A third bottleneck arises because all the existing technologies produce short sequence reads and thus genome assembly relies on comparing reads to the reference genome. But since the reference sequence is a composite of several genomes, such comparisons do not reveal the phenotypically significant structural variation that exists between individual genomes (rearrangements, copy number, translocations, inversions). 
         [0014]    As mentioned above, it is one of the merits of the present invention to recognise, that the main factor limiting the length of the fragments analysed in a micro- and/or nanofluidic system lies in the sample preparation and transfer steps. 
         [0015]    With this insight in mind, the object of the present invention is providing an improved technique for preparing long macromolecules for subsequent processing in a micro- and/or nanofluidic device overcoming the problems of the prior art or at least providing an alternative. 
       DISCLOSURE OF THE INVENTION  
       [0016]    According to a first aspect of the invention, a microfluidic device for enzymatic processing of macromolecules comprises a reaction chamber with a first manifold, a second manifold, and a plurality of reaction channels, each reaction channel extending from the first manifold to the second manifold. The device further comprises first inlet and outlet channels for filling the reaction channels via the manifolds with one or more macromolecule containers suspended in a first carrier fluid, wherein the first inlet and outlet channels are configured such that a flow established from the first set of inlets to the first set of outlets is guided through the reaction channels. The device further comprises second inlet and outlet channels for feeding an enzymatic reagent to the reaction chamber essentially without displacing the macromolecule containers trapped in the reaction channels, wherein the second set of inlets and outlets are configured such that a flow established from the second inlet to the second outlet is guided through at least one of the manifolds and bypasses the reaction channels. 
         [0017]    The microfluidic device is filled through an input port with a sample solution containing macromolecule containers. The input port is via the one or more first inlet channels connected to the first manifold, which via the reaction channels (“isolation zones”) in the reaction chamber (“trap area”) is in fluid communication with the second manifold, which in turn via the first outlet channel is connected to an outlet port. The reaction channels are filled with the sample solution and a flow of sample solution is established from the inlet port to the outlet port. The flow of sample solution thus carries at least one macromolecule container into at least one of the reaction channels. When an isolated macromolecule is identified to be present in one of the reaction channels, the flow of sample solution is stopped and a flow of enzymatic reagent is established from the one or more second inlet channels via the manifolds to the one or more second outlet channels. 
         [0018]    The second inlet and outlet channels are configured for feeding the enzymatic reagent to the reaction chamber without displacing the macromolecules trapped in the reaction channels. Displacement of the trapped macromolecules is avoided by carefully balancing the pressure in the first and second manifolds, i.e. the pressure applied on either end of each of the reaction channels. The reaction channels thus form a stagnant volume of the flow and only the sample solution in the manifolds is replaced by the enzymatic reagent. The enzymatic reagent diffuses from the manifolds into the reaction channels, and thus also into the reaction channel comprising the identified isolated macromolecule container. The enzymatic reagent interacts with the isolated macromolecule container, thereby producing an intact isolated macromolecule in the reaction channel. The isolated macromolecule may then by fluid handling be retrieved from the reaction channel and extracted from the reaction chamber in order to transfer the macromolecule to its destination for single molecule analysis or any other single molecule processing in one or more subsequent stages. The second inlet and outlet channels may also be used for flushing buffer solutions and/or feeding any reagents, to be applied to the macromolecule container and/or to the released macromolecule. 
         [0019]    Preferably, the second inlet and/or outlet channels are made shallower than the microchannels in the reaction chamber. Thereby, flow of sample fluid into the reagent channels is avoided or at least kept at a minimum. 
         [0020]    An important advantage of the device according to the invention is that sample macromolecules are provided to the input of the device in a macromolecule container. The macromolecule container protects the macromolecule from mechanical shearing during sample preparation and handling of the macromolecules, e.g. by pipetting. The macromolecule container thus acts as a “carrier” for the macromolecule. A macromolecule container may be formed by encapsulation of the macromolecule, by complexing the macromolecule with a protein scaffold or by complexing the macromolecule with polycations. A DNA molecule may, for example be encapsulated by a cell wall, in a nucleus, or carried by cell extract, preferably a metaphase chromosome. Preferably, DNA molecules are provided to the microfluidic device in a solution containing chromosomes acting as a container for the DNA molecules. In the case where cells are loaded into the device, metaphase chromosomes are obtained from the cell on-chip before releasing DNA from the chromosomes. Where cell extract is loaded, DNA can be released directly from the chromosomes. Alternatively a macromolecule container may be formed by complexing the macromolecule with a protein scaffold or in some cases with polycations, such as spermine or spermidine. 
         [0021]    The macromolecules are released from their container on-chip where shearing forces are minimal and/or are controlled. Releasing DNA from metaphase chromosome comprises the addition of one or more enzymatic reagents. This may include the addition of proteases. The preferred protease is Proteinase K. Other protease may also be useful (e.g. Trypsin). It may also include the addition of topoisomerases before or after adding the protease. Where topoisomerase is added after protease care is taken to kill the activity of the protease and/or to remove the protease. Preferably, the proteases used are appropriate for digesting at least histones or protamine. Releasing DNA may comprise the creation of a substantially naked DNA molecule, essentially devoid of proteins. However, in some cases it may be desirable to retain binding of one or more class of proteins to the DNA. Therefore, releasing DNA may alternatively comprise the creation of chromatin fibres to which proteins are still associated. 
         [0022]    Once released, the isolated macromolecule may be manipulated by means of on-chip fluid handling and transferred to subsequent processing/analysis stages. Thereby the mechanical forces exerted on the released macromolecule may be controlled to a level so as to avoid unintended breaking of the macromolecule. The device according to the invention may thus prepare isolated macromolecules with a length that by far exceeds the length of macromolecules prepared by known techniques. 
         [0023]    The invention thus provides a device, system and method for releasing macromolecules, such as biomolecules, on chip from small amounts of sample material, single cells, nuclei or chromosomes in a manner that keeps the macromolecules substantially intact. In particular, in an embodiment applied to genomic DNA, megabase lengths of DNA can be kept intact. Advantageously for this embodiment, following releasing of the DNA, the DNA is linearly elongated and displayed for detection. In a further embodiment the chip design allows reagents to be flushed over the DNA and allows features of interest to be labelled and then mapped. Events along the span of the DNA region being imaged can be followed in real-time. The invention provides an unprecedentedly long-range view of the genome, which encompasses the haplotype blocks as well as the structural organization of the genome. The long-range view will facilitate the de novo identification of a significant amount of previously characterized and uncharacterised copy number/structural variation. Furthermore, the mapping can be used to barcode individual genome fragments, which enables parts of the genome, bearing specific map patterns to be selected. After analysis, individual megabase length molecules can be transported to an output port of the chip, from where they can be further processed. For example, the DNA can be isothermally amplified in- or off-chip. The amplified DNA can then be collected from the output port and subjected to further molecular analysis including sequencing by any available method. Also a DNA fragment sent to the outlet port can be a component in the assembly of artificial chromosomes or used for synthesis of complex macromolecules. 
         [0024]    Advantageously, the method of the invention may be used at the front-end of sequencing pipelines and significantly enhances the quality and throughput of DNA sequencing. In one embodiment of the invention a micro-/nano-fluidic device processes a population of individual cells in a high-throughput manner: releasing and purifying DNA from each cell and preparing the DNA through to the final steps for sequencing. Moreover, the sample preparation is done in a highly innovative way which has the double side-benefit that the long-range haplotype map of the genome can be obtained and specific parts of the genome can be selected for sequencing. It is the downstream choice of the investigator whether to collect data separately from single cells or whether to amalgamate the data from the population of cells. If the latter, the investigator still benefits because less sample material is required. 
         [0025]    As well as applications in the research environment the microfluidic or micro/nanofluidic devices of this invention can be used as part of point-of-care systems for medical testing as well as devices for monitoring samples in the field or in various industries (e.g. water-treatment, food processing). 
         [0026]    In a preferred embodiment of a device according to the invention, a single pair of second inlet and outlet channels is provided and the reaction chamber is essentially symmetric with respect to a mirror axis connecting said second inlet and second outlet, a longitudinal axis of the reaction channels being oriented essentially transverse to the mirror axis. 
         [0027]    The single pair of second inlet and outlet channels has a single second inlet and a single second outlet. The second inlet channel is via an inlet branch symmetrically connected to both the first and the second manifolds. The second outlet channel is via an outlet branch symmetrically connected to both the first and the second manifolds. 
         [0028]    The second inlet and the second outlet are thus configured to supply enzymatic reagent symmetrically to the manifolds on either end of the reaction channels, thereby maintaining substantially equal pressure, equal flow rate, and thereby for each reaction channel a balanced reagent concentration on either end. 
         [0029]    Alternatively, an embodiment of a device according to the invention may comprise a mirror-symmetric trap area, wherein the reaction channels extend essentially transverse to the mirror axis from a first manifold to a second manifold, and wherein each of the manifolds is provided with an inlet at a first end of the manifold and an outlet at a second end opposite to the first end. In this embodiment, the reaction channels of the trap area may be loaded with a sample fluid containing macromolecule containers by establishing a diagonal flow through the reaction chamber, i.e. from an inlet of one of the manifolds (first/second manifold) via the reaction channels to an outlet of a different manifold (second/first manifold). Once the presence of at least one target macromolecule container has been determined, i.e. the trapping of a macromolecule container in a reaction channel, the filling flow is stopped and a secondary flow of flushing agents/buffers and/or reagents may be established. Any flushing agents/buffers and/or reagents are provided by a symmetric flow bypassing the reaction channels, i.e. where a flow is driven through each of the manifolds from the respective inlet to the respective outlet without passing through a reaction channel. The pressure in the manifolds is balanced with respect to each other, such that at the same pressure is present at either end of a given reaction channel, thereby avoiding any flow/displacement of the trapped target macromolecule in the reaction channel. The reagent required for releasing the macromolecule from its container is supplied via diffusion from the manifold into the reaction channel. 
         [0030]    In a further embodiment of a device according to the invention, the device is provided with a viewport in the region of the reaction channels, the view port allowing for the visual detection of a trapped macromolecule container in at least one of the reaction channels. The visual detection is typically performed by monitoring one or more of the reaction channels during filling of the channels. 
         [0031]    The term “visual” comprises any form of optical observation, and in particular any microscopic imaging technique and may, advantageously be combined with a machine vision system comprising recognition modules providing target detection signals representing the presence of a target macromolecule/macromolecule container. A machine vision system may also provide further signals responsive to the visual monitoring of any process steps performed on the target following detection, including releasing the macromolecule from its container, labelling, fragmenting, de-/re-naturation and/or transferring of the released macromolecule. Alternatively, target detection signals and any further monitoring signals may be provided by other means, such as integrated or external electrical and/or magnetic sensors. Any detection and/or further monitoring signals may be used as input to a control unit for controlling the processing in an auto-mated manner. 
         [0032]    In a further embodiment of a device according to the invention, the total effective cross-sectional area for flow through the reaction chamber in the region of the reaction channels is enlarged as compared to the first inlet channel by a ratio of at least 2:1, alternatively at least 5:1, or alternatively at least 10:1. For a given throughput this reduces the flow velocity of the sample fluid, thereby facilitating the detection of a target macromolecule. 
         [0033]    In a further embodiment of a device according to the invention, the flow resistance of reaction channels is decreased with increasing distance from the first inlet channel. When establishing a filling flow passing through the reaction channels, a pressure drop occurs along the manifolds, wherein the pressure decreases for increasing distance from the inlet channel. Consequently different driving conditions for the flow through the reaction channels may occur. To counter this effect, and to equalize the flow through the reaction channels with respect to each other, the resistance of the reaction channels is decreased, advantageously by increasing the width of the reaction channels with increasing distance from the first inlet channel. Advantageously, the distance is increased according to a linear relationship. Equalizing the filling flow has the advantage that it facilitates detection of target macromolecule containers in any of the reaction channels. 
         [0034]    In a further embodiment of a device according to the invention, the device only comprises passive microfluidic components. An important advantage of the device according to the invention is that it does not require any active fluidic components on chip for trapping the macromolecule container, and for releasing and retrieving the isolated macromolecule. Any flow driving and control components, such as pumps, valves or the like can thus be provided external to the device. The device only comprising passive microfluidic components can thus be provided e.g. as a chip interacting/interfacing with an analysis apparatus providing such infrastructure, whereas the chip is produced cheaply, e.g. as a disposable consumable. 
         [0035]    In a further embodiment of a device according to the invention, the reaction chamber has a rectangular layout, the rectangle having a first edge, a second edge parallel thereto, and a third and a fourth edge essentially perpendicular to the first and second edges, the first and second manifold extending along the first and second edge, respectively, the reaction channels extending from the first to the second manifold in a direction essentially parallel to the third and fourth edge, the first inlet and outlet being arranged at diagonally opposing corners of the rectangle, and the second inlet and outlet being arranged at the third and fourth edge respectively, wherein both the second inlet and the second outlet are in symmetric fluid communication with both the first and the second manifold through edge channels extending parallel to the reaction channels along the third and fourth edge. 
         [0036]    This configuration allows in a simple manner for establishing a diagonal filling flow, i.e. a flow passing from a first inlet via a first manifold, through essentially all reaction channels, and via a second manifold to an outlet channel. 
         [0037]    According to a broader aspect of this embodiment, the first inlet and outlet channels are arranged at opposing edges of the rectangular reaction chamber. Preferably, at least a first inlet channel is provided at the first edge and at least a first outlet channel is provided at the second edge opposite to the first edge. Advantageously, the axis connecting the first inlet and outlet channels is not a symmetry axis of the reaction chamber. The asymmetric arrangement has the advantage of promoting distribution of the injected sample fluid over the reaction channels. 
         [0038]    Preferably, the filling flow is injected from the first inlet into the reaction chamber at an angle that is inclined with respect to the direction of the flow barriers defining the reaction channels. Injecting the filling flow at an angle with respect to the flow barriers/reaction channels further promotes distribution of the sample fluid over the reaction channels. 
         [0039]    Advantageously said angle of injection is selected from the range between 10 to 90 degrees, alternatively from the range between 30 to 60 degrees, preferably about 45 degrees. For reasons of disambiguation only angles between 0 and 90 degrees are recited. Angles in excess of 90 degrees are mapped back to said range between 0 and 90 degrees by always measuring the smallest angle between the direction of injection and the direction of the flow barriers defining the reaction channel walls. 
         [0040]    The single pair of second inlet and outlet channels is in symmetric fluid communication with the first and second manifolds. This means that by injecting a fluid through the second inlet channel, a symmetric pressure distribution is established in the reaction chamber, and a balanced pressure level is established on either end of each of the reaction channels. While the pressure may drop in the manifolds along the direction of flow, the device is configured such that the pressure drop in one of the manifolds is a mirror of the pressure drop in the other manifold with respect to the symmetry axis of the reaction chamber. 
         [0041]    Operation of the device is particularly simple and reliable, because it only requires simple actuation of external valves and/or flow driving components, yet keeping sample fluids and reagent fluids well separated. Correct establishing of the appropriate type of flow for sample fluids (diagonal flow) and for reagent fluids (symmetric flow bypassing the reaction channels) is taken care off by the geometric lay-out of the device. 
         [0042]    In a further embodiment of a device according to the invention, the device further comprises an extraction channel connected to one of the manifolds. 
         [0043]    Reaction products/fractioned components of the macromolecule may be collected and extracted from the reaction chamber through a suitable extraction channel connected to one of the manifolds, and either directly transferred to a subsequent processing stage or retrieved from an extraction port. 
         [0044]    Preferably, the extraction channel is a wide, but shallow channel. In order to stretch out the macromolecule in the extraction channel, the height/depth of the extraction channel should be in the same length scale as the persistence length of the macromolecule, e.g. the characteristic length of the macromolecule representing the flexibility of the macromolecule. For double stranded DNA, this length is about 64 nm depending on salt concentration in the carrier fluid. A preferred range for the height/depth of the extraction channel is between 50 nm and 100 nm. The width of the extraction channel should be larger than this characteristic length so that the macromolecule can rearrange in order to resolve folds or similar disarrangements. 
         [0045]    In order to facilitate high volume production of the device, defining the lateral dimensions of the device including the extraction channel should be compatible with microscale pattern transfer techniques, i.e. patterning techniques for reliably producing lateral feature widths of about 1 μm and above, such as UV-lithography, microinjection moulding or any other high volume micro-fabrication technique. In order to achieve a satisfactory yield in a laboratory scale production, the extraction channel width is typically larger than 4 μm. Furthermore, an extraction channel nanoslit with a micro-scale width is advantageous for avoiding excessive pressure build-up in the device and the associated fluidic system under operation. Therefore, the width of the extraction channel is preferably also adapted according to fluidic design considerations for a given type of application. In order to shunt a macromolecule into and through the shallow extraction channel, it is desirable to achieve a reasonable flow rate through the extraction channel, thereby avoiding excessive pressure build-up inside the device. Increasing the width of the channel is a mean to limit the maximum pressure to achieve the desired flow rate as high pressure can jeopardize the sealing of the device or the microfluidic handling system, such as pumps, valves, fluidic connectors, and the like. 
         [0046]    As mentioned above, the device according to the invention may be integrated into an automated system for enzymatic processing and analysis of macromolecules. The automated system may comprise automated fluid handling for handling sample and reagent fluids. Any active components of the automated fluid handling system may be arranged external to the microfluidic device and interface with the microfluidic device through any known type of fluid connection technology. 
         [0047]    A system for enzymatic processing of macromolecules comprising a device according to the invention may further comprise a machine vision system for the automated detection of a trapped macromolecule container in at least one of the reaction channels. A suitable machine vision system includes appropriate optics for imaging the macromolecule on an electronic image sensor using any known microscopic technique. 
         [0048]    Furthermore, a system using the microfluidic device according to the present invention may comprise a nanofluidic processing/analysis portion, such as a sequencing portion or an amplification stage, arranged in direct extension of the extraction channel. 
         [0049]    According to a further aspect of the invention, a method for preparing isolated macromolecules is provided. The method has the same advantages has mentioned above with reference to a microfluidic device for enzymatic processing of macromolecules. 
         [0050]    A method for preparing isolated macromolecules and using a microfluidic device according to any of the above-mentioned embodiments comprises the steps of 
         [0051]    a) at an input port connected to the first inlet, providing a first fluid containing macromolecule containers, 
         [0052]    b) establishing a flow of the first fluid from the first inlet, via the first manifold, through the reaction channels, and via the second manifold to the first outlet, 
         [0053]    c) stopping the flow, when a macromolecule container is detected in one of the reaction channels, thereby trapping the macromolecule container in said reaction channel, 
         [0054]    d) at an input port connected to the second inlet, providing a second fluid containing an enzymatic reagent, 
         [0055]    e) in at least one of the manifolds, replacing the first fluid by the second fluid by establishing a flow from the second inlet to the second outlet, wherein the flow of the second fluid essentially by-passes the reaction channels, and 
         [0056]    f) allowing the enzymatic reagent to diffuse from the at least one manifold into the reaction channels to perform an enzymatic reaction releasing an isolated macromolecule from its container. 
         [0057]    In a further embodiment of a method according to the invention, the macromolecule container is a metaphase chromosome and the macromolecule is a DNA-molecule. 
         [0058]    In a further embodiment of a method according to the invention, a pre-defined level of enzyme concentration is maintained in the at least one manifold by maintaining in the at least one manifold a continuous flow of reagent with a pre-defined enzyme concentration. 
         [0059]    In a further embodiment of a method according to the invention, in step c), trapping of the macromolecule container in one of the reaction channels is visually detected by an operator and/or by means of a machine vision system. 
         [0060]    In a further embodiment of a method according to the invention, in step e) the first fluid is replaced by the second fluid essentially simultaneously in both manifolds, wherein at least for the reaction channel comprising the trapped macromolecule container, the hydrostatic pressures on either end of said reaction channel are balanced during said replacement. 
         [0061]    In a further embodiment of a method according to the invention, the method further comprises the step of 
         [0062]    g) transferring the isolated macromolecule via an extraction channel to a subsequent processing step, such as sequencing and/or amplification. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS  
         [0063]    In the following, the invention is described in detail with reference to schematic drawings, where like numerals refer to like parts. The drawings show on 
           [0064]      FIG. 1  an embodiment of a microfluidic device according to the invention, 
           [0065]      FIG. 2  a detail of the embodiment of the device shown in  FIG. 1 , 
           [0066]      FIG. 3  cross-sectional view of the reaction chamber along line III-III in  FIG. 2 , 
           [0067]      FIG. 4  cross-sectional view of the device along line IV-IV in  FIG. 2 , 
           [0068]      FIG. 5  detail of the reaction chamber region of an alternative embodiment of a microfluidic device according to the invention in (a) a first operational state, and (b) in a second operational state, 
           [0069]      FIG. 6   a - 6   d  different operational states for a device according to another embodiment of the invention, 
           [0070]      FIG. 7  a sequence of micrographs showing the filling of the manifolds with an enzymatic reagent and the diffusion thereof into the reaction channels, 
           [0071]      FIG. 8  micrographs showing digestion of chromosomes in the reaction chamber, 
           [0072]      FIG. 9  micrographs showing A) a released DNA molecule being shunted to the extraction port, and B) and C) details of the released DNA molecule in the extraction port, and 
           [0073]      FIG. 10  a further embodiment of a device according to the invention. 
       
    
    
     DETAILED DESCRIPTION OF THE INVENTION  
       [0074]      FIG. 1  shows schematically a microfluidic device  100  according to one embodiment of the invention. At the centre, the device  100  comprises a reaction chamber  101  (trap area) comprising reaction channels  102  defined by flow barriers  103 . A fluid sample may be loaded at a sample input port  112  and transferred to the reaction chamber  101  via a first inlet channel  110 . The sample fluid may leave the reaction chamber  101  via a first outlet channel  111  and is discharged at a sample output port  113 . Reagents may be provided in the form of a reagent fluid at a reagent input port  122  and are injected into to the reaction chamber  101  via a second inlet channel  120 . A flow of reagent fluid may leave the reaction chamber  101  via a second outlet channel  121  and is discharged through a reagent output port  123 . Processed samples may be retrieved through an extraction channel  130  and output at an extraction port  131 . 
         [0075]      FIG. 2  shows schematically a detail of the reaction chamber portion  201  of a microfluidic device  100  according to one embodiment of the invention. Notably, the reaction chamber  201  has a square lay-out with a first edge  206 , a second edge  207  parallel thereto, and a third edge  208  and a fourth edge  209  essentially perpendicular to the first and second edges  206 ,  207 . The reaction chamber  201  comprises a first manifold  204  and a second manifold  205  extending along the first edge  206  and the second edge  207 , respectively. The reaction chamber  201  further comprises reaction channels  202  defined by flow barriers  203  and extending from the first manifold  204  to the second manifold  205  in a direction essentially parallel to the third and fourth edge  208 ,  209 . A first inlet  210  and outlet  211  are arranged at diagonally opposing corners of the square, wherein the principal axis of the first inlet channel  210  and the second outlet channel  213  are oriented at an angle of about 45 degrees with respect to the flow barriers  203  and thus with respect to the reaction channels  202 . This arrangement of reaction chamber layout and sample fluid inlet/outlet configuration provides for an improved distribution of the sample fluid flow throughout the reaction chamber  201 , thereby facilitating an even filling of the reaction channels  202 . 
         [0076]    A second inlet  220  and outlet  221  are arranged at the third and fourth edge  208 ,  209 , respectively. The reaction chamber is essentially symmetric with respect to a mirror axis M connecting the second inlet  220  and the second outlet  221 , and a longitudinal axis of the reaction channels  202  is oriented perpendicular to the mirror axis M. Both the second inlet  220  and the second outlet  221  are in symmetric fluid communication with both the first and the second manifold  204 ,  205  through edge channels  298 ,  299  extending parallel to the reaction channels  202  along the third and fourth edge  208 ,  209 . 
         [0077]    The reaction channels  202  thus act as zones for isolation of a macromolecule container (e.g. chromosome, nucleus, or cell) from which a macromolecule/polymer (e.g. Nuclear Acid) can be released. The released macromolecule can then be passed to an extraction channel  230  from where it may be retrieved for subsequent processing, for example through an extraction port  231 . 
         [0078]      FIG. 3  shows schematically a cross-sectional view of the reaction chamber along line III-III in  FIG. 2 . The microfluidic device  300  comprises a micro-structured first part  341  with access holes  342 ,  343  for access to the sample input and output ports  312 ,  313 , respectively. A cover part  340  is bonded to the first part  341  so as to define closed microfluidic channels. The cross-section passes from the sample inlet port  312  through the first inlet channel  310 , via the first manifold  304 , through reaction channels  302  defined by flow barriers  303 , via the second manifold  305 , and through the first outlet channel  311  to the sample output port  313 . 
         [0079]      FIG. 4  shows schematically a cross-sectional view of the device along line IV-IV in  FIG. 2 . As in mentioned above with respect to  FIG. 3 , a micro-structured first part  441  defines together with a cover part  440  the fluid channels of the device. The cross-section illustrates how a released macromolecule  452  is transferred from a coiled state in the second manifold  405  to a linearized state in the extraction channel  430  and further to a recoiled state in an extraction port  431 . Note, that the first part  431  in lateral directions (i.e. in the plane of  FIG. 2 ) may be patterned with micron-scale resolution techniques whereas in a vertical direction (i.e. in the plane of the cross-sections of  FIG. 3  or  FIG. 4 ) shallow milling may be performed to achieve nano-scale structures in some regions, and micron-scale milling may be performed to obtain deeper channels in other regions. In particular, the extraction channel  430  may have a depth c of 100 nm, whereas the depth of the manifold  405  is for example 10 μm. Also the length a of the extraction channel is chosen depending on the application, and is for example 450 μm, but may also be several millimetres or even a few centimetres. 
         [0080]      FIG. 5  shows schematically a detail of the reaction chamber region of an alternative embodiment of a microfluidic device according to the invention. Like the embodiment of  FIG. 2 , the reaction chamber  501  has a square layout with a first manifold  504  and a second manifold  505 , both manifolds  504 ,  505  extending in a first direction along parallel edges of the square. Reaction channels  502  are defined by flow barriers  503  extending perpendicular thereto from the first manifold  504  to the second manifold  505 . s  502 . A shallow extraction channel  530  is centrally connected to the second manifold  505 . 
         [0081]    The embodiment of  FIG. 5  differs from the embodiment of  FIG. 2  in the arrangement of fluid connection channels  510 ,  511 ,  514 ,  515  connecting the reaction chamber to fluidic interface ports (not shown) of the device. The four fluid connection channels  510 ,  511 ,  514 ,  515  extend outwardly from the four corners of the rectangle, one connection channel from each corner, at an angle of between 30 and 60 degrees, preferably about 45 degrees with respect to the direction of the reaction channel. As shown in the schematic drawing, the connection channels  510 ,  511 ,  514 ,  515  have essentially the same dimensions and are arranged mirror symmetrically at least with respect to a centre axis M. 
         [0082]      FIG. 5   a  shows a first operational state, where a diagonal flow is established for filling the reaction chamber  501  with a sample fluid. Due to the symmetry of the device, any of the connection channels  510 ,  511 ,  514 ,  515  may be chosen as a first inlet channel for injecting the sample fluid (here  510 ). A diagonal flow is established by using the connection channel at the diagonally opposing corner as the first outlet channel (here  515 ), while keeping the two other connection channels blocked/closed (here  511 ,  514 ). 
         [0083]      FIG. 5   b  shows a second operational state, where parallel reagent flows are established in both manifolds  504 ,  505  by simultaneously injecting reagent into the first manifold  504  and the second manifold  505  through connection channels at the same edge and discharging the reagent through corresponding connection channel at the opposite edge. In the operational state shown in  FIG. 5   b , reagent is injected from connection channel  510  acting as a second inlet channel, passes through the first manifold  504 , and is discharged through connection channel  511  acting as a second outlet channel. Symmetrically thereto, reagent is simultaneously injected from connection channel  514  acting as a second inlet channel, passes through the second manifold  505 , and is discharged through connection channel  515  acting as a second outlet channel. The flow and pressure in both manifolds  504 ,  505  may be controlled independently, and is adjusted to bypass the reagent channels  502  such that the macromolecule containers are fluidically immobilised in the stagnant volume of the reaction channels  502 . Typically in practice, this is done by balancing the pressure in the two manifolds  504 ,  505  so as to achieve substantially equal pressures on either end of each of the reaction channels  502 . 
         [0084]    Referring to  FIG. 10 , a microfluidic device  1000  according to a further embodiment may comprise a first component for trapping/isolating macromolecule containers and releasing a single macromolecule therefrom by means of an enzymatic reaction in a reaction chamber  1001 . Advantageously, the first component may essentially correspond to the above-mentioned embodiments, e.g. as described with reference to  FIG. 1-4 . The released macromolecule may be shunted to an extraction channel  1030 , and passed to a subsequent second component of the device  1000 , arranged in direct extension of the extraction channel  1030 . The second component comprises a first nanoslit  1030   a,  a second nanoslit,  1030   b,  and a third nanoslit  1030   c,  which together with the nanoslit of the extraction channel  1030  form a cross with a longitudinal axis defined by two channels  1030  and  1030   a,  and a transverse axis perpendicular thereto by the two other channels  1030   b,    1030   c.  The channels  1030   a - c  may be accessed through fluidic interface ports  1031   a - c.  The nanoslits  1030 , and  1030   a - c  have typically the same height of up to a few hundred nanometres, typically about 100 nm, depending on the actual application. The width of the channels may be between a few micrometres up to a couple of hundred micrometres, typically about 50 μm, compatible with state-of-the-art microscale pattern transfer techniques. 
         [0085]    The longitudinal channels  1030 ,  1030   a  of the cross in the second component may be used to linearize, stretch, observe/sequence, label or otherwise analyse/process the single macromolecule produced in the first component. The transverse channels  1030   b,    1030   c  may be used for fluidic manipulation of the macromolecule, and/or for providing additional reagents as required by the processes performed in the second component. The combination of the first and the second component in the integrated device  1000  of  FIG. 10  has the advantage that the analysis/processing steps in the second component benefit from the ultra-long macromolecules that are produced in the first component. 
       EXAMPLE  
       [0086]    Referring to  FIGS. 6   a - 6   d,    7 ,  8  and  9 , in the following, an example is given for the design, fabrication and use of a device for handling and releasing DNA from metaphase chromosomes.  FIG. 6  gives a schematic view of the device used in the example and its operation.  FIGS. 7-9  show micrographs visualising different aspects of the operation of the device. The device design of the example aims to immobilize a single metaphase chromosome in an isolation zone through which reagents can be exchanged by diffusion enabling proteins to be digested. The DNA thus extracted can then be shunted out of the isolation zone into a nanoslit for stretching. 
         [0087]      FIG. 6  shows schematically a sequence of operational states for a device according to another embodiment of the invention. The device of  FIG. 6  has, apart from the number of reaction channels  602 , the same configuration of the reaction chamber region, and in particular of the first inlet/outlet channels  610 ,  611 , the second inlet/outlet channels  620 ,  621 , and the extraction channel  630 . The sequence shows in  FIG. 6   a  loading of a sample  650  containing metaphase chromosomes  651 . The sample  650  is loaded at an input port  612 , which is connected to the reaction chamber  601  via first inlet channel  610 . Referring to  FIG. 6   b , the reaction chamber  601  is filled with the sample  650  by establishing a diagonal sample flow through the reaction chamber  601 : a sample injection flow  660  through first inlet channel  610  transfers the sample fluid  650  to a first manifold flow  661 . The first manifold flow  661  branches into a number of reaction channel flows  662 . After passing the reaction channels  602 , the reaction channel flows  662  are collected by a second manifold flow  663 , which is leaves the reaction chamber in a sample discharge flow  664 . Single chromosomes  651  carried by the reaction channel flows  662  may be observed by optical microscopy, and the sample flow may be stopped when the presence of an isolated target chromosome  651  is determined.  FIG. 6   c  shows how protease is introduced in the reaction chamber  601  without displacing the chromosome  651  of interest by establishing a pressure balanced parallel flow through the manifolds  604 ,  605 . A reagent injection flow  670  carrying protease splits essentially symmetrically into a first manifold flow  671  and a second manifold flow  672 , and recombines again before leaving the reaction chamber  601  in a reagent discharge flow  674 . The manifolds flows  671 ,  672  essentially by-pass the reaction channels  602 . The protease enters the reaction channels  602  by diffusion  672  to act on the immobilised target chromosome  651  in order to release a DNA molecule  652 . In  FIG. 6   d , the released DNA  652  is retrieved from the reaction channel  602  and shunted to the extraction port  630  by applying appropriate shunting pressures  680 ,  681 ,  682 ,  683  through the connection channels  610 ,  611 ,  620 , and  621 , respectively. The released DNA  652  is stretched through a 100 nm high, 450 μm long and 50 μm wide nanoslit forming the extraction channel  630 . 
         [0088]    The device was designed, with the aid of finite element simulations (COMSOL, USA), to have a series of isolation zones to slow down the chromosomes in the trap area while maintaining a high flow rate through the device. The parallel isolation zones increased in area with increasing distance (3000 μm 2 , 6000 μm 2 , 9000 μm 2 , etc.) from the sample entry point, in order to obtain a homogeneous flow rate into each of the zones during the introduction of the sample. This was to ensure that all chromosomes entering isolation zones were moving at the same horizontal speed in order to facilitate selection of individual chromosomes from the parade of chromosomes and cell debris flowing through the device. 
         [0089]    The device was fabricated using UV lithography and reactive ion etching of a silicon substrate. Briefly, a 500 nm dry thermal oxide was grown on a silicon wafer. The protease inlet slit and the slit for DNA stretching were defined by UV masking and deep reactive ion etching in the oxide at the depth of 500 nm and 100 nm, respectively. The 50 μm wide microfluidic channels connecting the inlet ports and the 400×400 μm trap area were defined using a third UV lithography step and were etched in silicon at a depth of 10 μm. A thermal oxide was grown in order to later allow fusion bonding. Inlet holes were made by powder blasting from the backside of the device which was finally sealed by fusion bonding to a 500 μm thick borofloat glass wafer. 
         [0090]      FIG. 7  visualizes the filling of the manifolds with an enzymatic reagent and the diffusion thereof into the reaction channels. The protease reagent was introduced from the second inlet port (located at the top edge of the frames) with flow occurring perpendicular to the reaction channels acting as isolation zones for the isolation of individual chromosomes. In this configuration there was no flow into the isolation zones; reagent exchange with the stagnant volume inside the isolation zones occurred by diffusion only. We used streptavidin labelled with Cy3 to visualize the diffusion of the reagent into the isolation zones to verify device operation before chromosome isolation and protease digestion was conducted. Observation of the introduction and spread of the Cy3 fluorescent marker into the isolation zones validated the device design and indicated that the reagent is able to spread quite well throughout the isolation zones by time, 300 s. The sequence of micrographs was taken with a time-lapse of 30 s between frames and shows the increasing fluorescence in the reaction chamber (“trap area”) due to the diffusion of stretavidin-Cy3 as it is injected at 0.6 nL min −1 . The diffusion constant is 60×10 −12  m 2  s −1 . 
         [0091]    After experimental verification of the device design, the sample and reagent exchange process was applied to a sample containing metaphase chromosomes. The chromosomes were isolated from Jurkat cells (DSMZ, Germany: ACC282) in a polyamine buffer as described by Cram et al. (L. S. Cram, C. S. Bell and J. J. Fawcett, Methods Cell Sci., 2002, 24, 27-35) with some modifications. Briefly, the Jurkat cells were grown at 37° C. in a 5% CO 2  atmosphere. At exponential growth, they were arrested in metaphase with colcemide at 0.06 μg mL −1  for 12-16 hours. The cells were collected at 200 g for 10 minutes and re-suspended in a swelling buffer (55 mM NaNO 3 , 55 mM CH 3 COONa, 55 mM KCl, 0.5 mM spermidine, 0.2 mM spermine) at approximately 10 6  cells per mL and incubated for 45 minutes at 37° C. The swollen cells were collected at 200 g for 10 minutes and re-suspended in an ice-cold isolation buffer (15 mM Tris-HCl, 2 mM EDTA, 80 mM KCl, 20 mM NaCl, 0.5 mM EGTA, 0.5 mM spermidine, 0.2 mM spermine, 0.12% digitonin, and 7 mM mercaptoethanol) at approximately 8×10 6  cells per mL. The cells were lysed by vigorous vortex for 30 s. The chromosome content was estimated to be in the order of 10 7  cells per mL. 
         [0092]    The device was mounted on a holder interfacing the inlet holes of the device with pressured air allowing movement of the solution inside the device as described elsewhere (W. Reisner, N. B. Larsen, H. Flyvbjerg, J. O. Tegenfeldt and A. Kristensen, Proc. Natl. Acad. Sci. U.S.A., 2009, 106, 79-84). Fluorescence imaging was performed using an inverted microscope (Nikon Eclipse TE2000, Japan) equipped with a 60×/1.00 water immersion objective and an EMCCD camera (Photometrics Cascade II512, USA). The temperature inside the device was controlled by a cartridge heater held in contact with the backside of the silicon device. Inlet holes were loaded with 30 mL of solution unless otherwise mentioned. 
         [0093]    Prior to receiving the chromosomes  651 , the device was flushed by 1% sodium dodecyl sulfate, buffer solution (0.5_TBE, 3% b-mercaptoethanol (BME) and 0.5% Triton X-100) and BSA at 1 mg mL −1  for 10 minutes. A sample  650  with 1000-2000 chromosomes  651  were added to the diagonal sample inlet port  612 ; the depth of the microfluidic structure allowed the cell extract to be flushed quickly through the isolation zones formed by the reaction channels  602  while watching for the appearance of chromosomes  651  that could be isolated. A single chromosome  651  was trapped in a reaction channel  602  of the device. 
         [0094]    Simultaneously the temperature was adjusted to 37° C. and a 100 μg mL −1  solution of protease K (1 mM of YOYO-1 is added to the protease K solution for staining the DNA strands while cut free from the chromatin in the vicinity of the bright chromosome body) was introduced. The device enabled a high flow rate of 0.6 nL min −1  allowing the protease to diffuse quickly into the stagnant volume within the isolation zone  602 . 
         [0095]    Moreover, a continuous flow  670 ,  671 ,  672 ,  673 ,  674  through the device ensured that after 4 minutes the protease concentration around the isolated chromosome  651  was maintained above 50 μg mL −1  and that the digestion products were washed away from the isolation zone through diffusion. 
         [0096]    The series of micrographs in  FIG. 8  (A) shows the digestion of a single metaphase chromosome  651  with protease at 37° C. Subsequent frames are taken with a time-lapse of 5 minutes. As proteolysis took place, the chromosome  651  swelled and self-aligned in the plane of the device allowing reliable and reproducible fluorescence time-lapse imaging. Although no visible change of the chromosome  651  was observed after t=25 minutes, digestion was allowed to proceed for one hour as recommended by protocols for digestion in bulk solution (J. Sambrook, E. F. Fritsch and T. Maniatis, Molecular Cloning: a Laboratory Manual, Cold Spring Harbor Laboratory, 1989).  FIG. 8  (B) shows different individually isolated chromosomes after 40 minutes digestion. Even after a digestion treatment that should be sufficient to remove all proteins, sister chromatids could still be clearly identified and chromosomes of different sizes and with different centromere positions could be seen. Moreover heterogeneity in the chromatin folding morphology could be observed at the micrometre scale. 
         [0097]    The chromosomal DNA  652  could be easily manipulated by using the sample inlet/outlet microchannels  610 ,  611  and the reagent inlet/outlet slits  620 ,  621  as a bi-directional flow system inside the reaction chamber  601 . This enabled the chromosomal DNA  652  to be moved in front of the 100 nm high nanoslit forming the extraction channel  630  and then forced in. Although, the bi-directional flow in the reaction chamber  601  would enable DNA  652  extracted from chromosomes  651  trapped in different isolation zones  602  to each be individually manipulated and moved toward the extraction channel  630 , in the present example a dilute solution of chromosomes  651  was used and only one chromosome  651  at a time was processed. 
         [0098]    After completion of the digestion, DNA  652  released from an individual chromosome  651  is passed through a 100 nm high nanoslit forming the extraction channel  630 .  FIG. 9A  shows a micrograph of a released DNA molecule  652  being shunted to the extraction channel  630 ; The post-digestion chromosomal DNA  652  was observed as a densely packed core composed of separated loops. The chromosomal DNA  652  was highly pliable: the DNA  652  stretched by increasing the flow through the nanoslit  630  and recoiled when the flow was stopped. 
         [0099]      FIG. 9B  and  FIG. 9C  show micrographs with details of the released DNA molecule  652  in the extraction channel  630 .  FIG. 9B  is a close-up of loops of DNA emanating from the core package of the chromosomal DNA.  FIG. 9C  is a close-up of the linear DNA strand emerging from the DNA package. A longer separate strand stretched across the whole length of the 450 μm long nanoslit and out into a microchannel ( FIG. 4C ). This corresponded to a minimal length of about 1.3 Mbp (1.3 million bases) of fully elongated DNA. Such separated DNA strands were also visible around the chromosomal DNA before the introduction to the nanoslit. 
       LIST OF REFERENCE NUMBERS  
       [0100]    Throughout the application, like numerals refer to like parts, wherein x is to be replaced by the numbers 1, 2, 3 . . . 10 as appropriate. 
         [0101]    x 00  device 
         [0102]    x 01  reaction chamber 
         [0103]    x 02  reaction channel 
         [0104]    x 03  flow barrier 
         [0105]    x 04 , x 05  first and second manifold 
         [0106]    x 06 -x 09  first, second, third and fourth edges 
         [0107]    x 10 , x 11  inlet/outlet channel 
         [0108]    x 12 , x 13  connection ports 
         [0109]    x 14 , x 15  inlet outlet channel 
         [0110]    x 20 , x 21  inlet/outlet channel 
         [0111]    x 22 , x 23  connection ports 
         [0112]    x 30  extraction channel 
         [0113]    x 31  connection port 
         [0114]    x 40 , x 41  device parts 
         [0115]    x 42 , x 43  connection holes 
         [0116]    x 50  sample 
         [0117]    x 51  macromolecule container 
         [0118]    x 52  macromolecule 
         [0119]    x 60 -x 64  sample flow 
         [0120]    x 70 , x 71  reagent flow 
         [0121]    x 72  reagent diffusion 
         [0122]    x 73 , x 74  reagent flow 
         [0123]    x 80 -x 83  fluidic manipulation 
         [0124]    x 98 , x 99  edge channels 
         [0125]      1030   a - c  nanoslits 
         [0126]      1031   a - c  connection ports 
         [0127]    M symmetry axis