WO2009005476A1

WO2009005476A1 - Capillary sample separation apparatus

Info

Publication number: WO2009005476A1
Application number: PCT/SG2008/000235
Authority: WO
Inventors: Nam Trung Nguyen; Yien Chian Kwok; Yi Sun
Original assignee: Nanyang Technological University
Priority date: 2007-07-03
Filing date: 2008-07-02
Publication date: 2009-01-08

Abstract

The invention provides a capillary sample separation apparatus and uses thereof. The capillary sample separation apparatus comprises a monolithic solid, which comprises a first and a second lateral wall, arranged in opposing relationship to each other. The monolithic solid comprises a plurality of parallel capillary microtubes, one end of each being arranged in the first lateral wall and one end being arranged in the second lateral wall of the monolithic solid. One end of each capillary microtube is in fluid communication with a common sample loading port, the other end is in fluid communication with a common reservoir.

Description

CAPILLARY SAMPLE SEPARATION APPARATUS CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application makes reference to and claims the benefit of priority of an application for a "MicroChannel Bundle for Separation of Charged Compounds" filed on July 03, 2007 with the United States Patent and Trademark Office, and there duly assigned serial number 60/947,923. The contents of said application filed on My 03, 2007 is incorporated herein by reference for all purposes, including an incorporation of any element or part of the description, claims or drawings not contained herein and referred to in Rule 20.5(a) of the PCT, pursuant to Rule 4.18 of the PCT.

FIELD OF THE INVENTION

[0002] The present invention relates to a capillary sample separation apparatus. Provided is also the use of the capillary sample separation apparatus in separating a sample.

BACKGROUND OF THE INVENTION

[0003] The advent of micro-chemical analysis systems has led to a growing interest in microfabricated fluidic systems with length scales in the range of one to a hundred micrometers. Further advances allow making fluidic systems with length scale in the range of several ten nanometers to one micrometer. Such miniaturization promises realization of assays with low reagent volumes and costs. It permits scaling at the micrometer range, coupled with a potential or path for implementing multiplexed, arrayed assays of small size that may be used in laboratories and point-of-care medical devices. These are commonly known as lab-on-chip ("LOC") or micro total analytical systems (μTASs). [0004] In the emerging field of electrophoresis devices, sample separation can inter alia be realized in a channel or a capillary. Capillary electrophoresis (CE) usually involves high applied voltage. Since the concept of electrophoresis was introduced, it has been known that Joule heat generated as a result of the electric current passing through the electrophoretic buffer can adversely affect the quality of the separation. Due to the presence of electrical potential gradient and electrical current in electrokinetic flow, Joule heating is an inevitable phenomenon which leads to an increase in the overall temperature and to temperature gradients in the transverse and longitudinal directions inside the capillaries. Excess temperature elevations may cause bubble formation, denaturation of biological samples, and even breakdown of the chip systems. Large temperature gradients cause band broadening and dispersion that leads to inefficient and low quality separation. It was assumed that the use of microchannels would solve the problems associated with Joule heating, as the large channel surface-to-volume ratio (SVR) was expected to reduce the Joule heat and to increase the cooling rate through the channel wall. However, many research works in the past showed that Joule heating still affects electro- phoretic separation in microchannels. Comparing an applied field of 475 V/cm to 240 V/cm, Swinney et al. (Electrophoresis (2002) 23, 613-620) observed a 7 % reduction in separation efficiency for tris-boric acid buffer with the higher voltage. Chen & Chen (Electrophoresis (2000) 21, 165-170) reported that plate number in the separation of DNA fragments started to decrease when the electric field strength was above 300 V/cm.

[0005] Accordingly, there remains a need for devices that allow for an electrophoretic separation of samples at higher speed than conventional devices - with a comparably high resolution. Thus, it is an object of the present invention to provide an apparatus or device for sample separation that reduces the above discussed disadvantages occurring in electrophoretic sample separation.

SUMMARY OF THE INVENTION

[0006] According to a first aspect, the invention provides a capillary sample separation apparatus. The capillary sample separation apparatus includes a monolithic solid. The monolithic solid includes a first lateral wall and a second lateral wall. The first lateral wall of the monolithic solid is arranged in opposing relationship with the second lateral wall of the monolithic solid. Further, the monolithic solid includes a plurality of at least substantially parallel capillary microtubes. Each capillary microtube has a first end with a first aperture and a second end with a second aperture. The first end of each capillary microtube is arranged in the first lateral wall of the monolithic solid. The second end of each capillary microtube is arranged in the second lateral wall of the monolithic solid. Thus all microtubes of the plurality of capillary microtubes are fluidly coupled. The first aperture of each capillary microtube is in fluid communication with a common sample loading port. The second aperture of each capillary microtube is in fluid communication with a common reservoir. [0007] According to a further aspect, the invention provides a method of forming a capillary sample separation apparatus. The method includes providing a microfluidic device. The method also includes forming on the microfluidic device a sample loading port and a reservoir. The method further includes arranging on the microfluidic device a monolithic solid. The monolithic solid includes a first lateral wall and a second lateral wall. The first lateral wall of the monolithic solid is arranged in opposing relationship with the second lateral wall of the monolithic solid. Further, the monolithic solid includes a plurality of at least substantially parallel capillary microtubes. Each capillary microtube has a first end with a first aperture and a second end with a second aperture. The first end of each capillary microtube is arranged in the first lateral wall of the monolithic solid. The second end of each capillary microtube is arranged in the second lateral wall of the monolithic solid. Thus all microtubes of the plurality of capillary microtubes are fluidly coupled. By forming on the microfluidic device the sample loading port and the reservoir and arranging on the microfluidic device the monolithic solid the method the first apertures of the capillary microtubes are brought in fluid communication with the sample loading port. Further, the second apertures of the capillary microtubes are thereby brought in fluid communication with the reservoir.

[0008] According to a further aspect, the invention relates to the use of a monolithic solid to expose a single sample to capillary electrophoresis. The monolithic solid includes a first lateral wall and a second lateral wall. The first lateral wall of the monolithic solid is arranged in opposing relationship with the second lateral wall of the monolithic solid. Further, the monolithic solid includes a plurality of at least substantially parallel capillary microtubes. Each capillary microtube has a first end with a first aperture and a second end with a second aperture. The first end of each capillary microtube is arranged in the first lateral wall of the monolithic solid. The second end of each capillary microtube is arranged in the second lateral wall of the monolithic solid. Thus all microtubes of the plurality of capillary microtubes are fluidly coupled.

[0009] In a related aspect the invention relates to the use of a monolithic solid in the analysis of a single sample by capillary separation. The monolithic solid includes a first lateral wall and a second lateral wall. The first lateral wall of the monolithic solid is arranged in opposing relationship with the second lateral wall of the monolithic solid. Further, the monolithic solid includes a plurality of at least substantially parallel capillary microtubes. Each capillary microtube has a first end with a first aperture and a second end with a second aperture. The first end of each capillary microtube is arranged in the first lateral wall of the monolithic solid. The second end of each capillary microtube is arranged in the second lateral wall of the monolithic solid. Thus all microtubes of the plurality of capillary microtubes are fluidly coupled. In the present use of the monolithic solid the sample is allowed to enter the capillary microtubes that are included in the monolithic solid via the first apertures of the capillary microtubes. The sample is also allowed to migrate along the lengths of the capillary microtubes to the second apertures of the capillary microtubes. In the present use of the monolithic solid the sample is allowed to enter the capillary microtubes and to migrate along the lengths thereof in the absence of an additional pressure gradient.

[0010] According to yet a further aspect the invention provides a method of subjecting a sample to capillary separation. The method includes introducing the sample into a sample loading port of a capillary sample separation apparatus. The capillary sample separation apparatus includes a monolithic solid. The monolithic solid includes a first lateral wall and a second lateral wall. The first lateral wall of the monolithic solid is arranged in opposing relationship with the second lateral wall of the monolithic solid. Further, the monolithic solid includes a plurality of at least substantially parallel capillary microtubes. Each capillary microtube has a first end with a first aperture and a second end with a second aperture. The first end of each capillary microtube is arranged in the first lateral wall of the monolithic solid. The second end of each capillary microtube is arranged in the second lateral wall of the monolithic solid. Thus all microtubes of the plurality of capillary microtubes are fluidly coupled. The first aperture of each capillary microtube that is included in the monolithic solid is in fluid communication with the sample loading port. The second aperture of each capillary microtube is in fluid communication with a common reservoir. The present method of the invention further includes allowing the sample to enter the capillary microtubes that are included in the monolithic solid via the first apertures of the capillary microtubes. The present method of the invention further includes allowing the sample to migrate along the lengths of the capillary microtubes to the second apertures thereof. In the present method the sample is allowed to enter the capillary microtubes and to migrate along the lengths thereof in the absence of an additional pressure gradient.

[0011] In a related aspect the invention provides a method of subjecting a sample to capillary electrophoresis. The method includes introducing the sample into a sample loading port of a capillary sample separation apparatus. The capillary sample separation apparatus includes a monolithic solid. The monolithic solid includes a first lateral wall and a second lateral wall. The first lateral wall of the monolithic solid is arranged in opposing relationship with the second lateral wall of the monolithic solid. Further, the monolithic solid includes a plurality of at least substantially parallel capillary microtubes. Each capillary microtube has a first end with a first aperture and a second end with a second aperture. The first end of each capillary microtube is arranged in the first lateral wall of the monolithic solid. The second end of each capillary microtube is arranged in the second lateral wall of the monolithic solid. Thus all microtubes of the plurality of capillary microtubes are fluidly coupled. The first aperture of each capillary microtube that is included in the monolithic solid is in fluid communication with the sample loading port. The second aperture of each capillary microtube is in fluid communication with a common reservoir. The present method of the invention further includes applying an electric field along the lengths of the capillary microtubes.

BRIEF DESCRIPTION OF THE DRAWINGS

[0012] The invention will be better understood with reference to the detailed description when considered in conjunction with the non-limiting examples and the accompanying drawings.

[0013] Figure IA and IB depict scanning electron microscopy images of the cross- section of the bundle of capillary microtubes. Figure IB is an enlargement of the central part of the image of Fig. IA. Figure 1C depicts the layout of a microfluidic device in the form of a microchip with a monolithic solid that includes a bundle of capillary microtubes (1 : monolithic solid with plurality of capillary microchannels; 2: substrate; 3: sample loading port; 4: cathode; 5: anode; 6: waste).

[0014] Figure 2 depicts a monolithic solid (22) as included in a capillary sample separation apparatus of the invention. The monolithic solid (22) includes capillary microtubes (21) and may be arranged in such a way that the apertures of the capillary microtubes are in fluid communication with an electrode.

[0015] Figure 3 shows an Ohm's plots for the capillary sample separation apparatus of the invention and the glass microchannel. Triplicate readings were taken after 10 sec for each point. For the glass microchip (D) the Ohm's plot deviates from linearity at electric field larger than 700 V/cm. For the capillary sample separation apparatus according to the invention (o), the deviation from linearity sets in at electric field higher than 1100 V/cm.

[0016] Figure 4 depicts electropherograms of a mixture of 100 nM fluorescein and 200 nM Rhodamine 123 in 0. IxTBE on (A) a capillary sample separation apparatus of the invention and (B) glass microchannel under 700 V/cm; (C) capillary sample separation apparatus and (D) glass microchannel under 850 V/cm; (E) capillary sample separation apparatus and (F) glass microchannel under 1000 V/cm. Triplicate experiments were conducted for each experiment (a.u. = arbitrary units).

[0017] Figure 5 shows the flow profile of a DNA fragment in a photonic crystal fiber (PCF) during separation. Fluorescent images were taken at the detection point 1 cm from the buffer waste reservoir. [0018] Figure 6 depicts electropherograms of 5μg/mL ΦX174-Hae III dsDNA digest in 80 mM MES/40mM TRIS buffer with 1.5% hydroxypropylcelϊulose on (A) a capillary sample separation apparatus of the invention and (B) a glass microchannel under 500 V/cm with a separation length of 6 cm. The results were repeated three times. The standard deviations are less than 0.5 % and 5 % for migration time and half peak width, respectively.

[0019] Figure 7 depicts examples of polymeric capillary nanotubes for nanofluidics that may be used in the manufacture of a capillary sample separation apparatus of the invention. A: atomic force microscope image of planar nanochannels. B: Fluorescent solution in an array of nanochannels. C: Capillary filling of nanochanels. D: Capillary filling characteristics (position of advancing meniscus) of liquids with different ion concentrations (DI = deionized water, substantially free of NaCl). E: Velocity of the advancing meniscus as a function of time (DI = deionized water).

[0020] Figure 8 is a schematic representation of exemplary embodiment of an arrangement of a plurality of bundles of capillary microtubes in two (A) and three (B) dimensions.

[0021] Figure 9 is a schematic representation of the monolithic integration of a plurality of capillary capillary microtubes. A: fabrication of a two-dimensional arrangement of a plurality of open channels on a thin substrate layer; B: lamination of multiple layers to form a three-dimensional arrangement, defining a capillary bundle; C: bonding improvement such as annealing.

[0022] Figure 10 depicts examples of the fabrication of planar capillary nanotubes in silicon/glass technology. A: Bulk micromachining: nanochannels (I), microchannels and access holes (II) are etched in the substrate (7), then closed by a second substrate (8) (III), thereby forming tubes. B: Spacer technique: a thin layer is deposited and patterned (I)₅ microchannels and access holes (II) are etched in the substrate (7), then closed by a second substrate (8) to form tubes (III). C: Sacrificial technique: a thin layer is deposited and patterned (I), a structural layer is deposited and patterned (II), then the sacrificial layer is etched away (III).

[0023] Figure 11 depicts examples of techniques to pattern polymers. (A) Nanoimprint lithography (NIL): a thermoplastic resist (13) is spin-coated onto a substrate (14), the plastic is heated above its glass temperature Tg (I), a template (12), fabricated using silicon technologies) is pressed against it, and (II) the template (12) is released and the residual layer etched using an oxygen plasma. (B) Step-and-flash imprint lithography (SFIL): a photoresist (13) is deposited onto a substrate (14), exposed to UV while the template (12) is pressed against it (I), and the template is released and the residual layer etched using an oxygen plasma (II). (C) Reversal imprint: a liquid polymer or prepolymer (15) is spin-coated onto the template (12), then transferred on a substrate (14) and hardened to a hardened polymer (16) (I), (c) and the template is released and the residual layer etched. Figure HD depicts a further technique based on photolithography, including the final generation of nanotubes. Using a mold (19), fabricated on a four- inch silicon wafer by standard photolithography and reactive ion etching, a polymer waver (17) is embossed (I), and the mold released (II). Subsequently, a second polymer wafer is bonded to the first sheet (III). [0024] Figure 12 illustrates the differences in the electric potential and the ionic concentration in a microtube (A) and in a nanotube (B). In a microtube, the electrical double layer is much smaller than in conventional dimensions. The potential is neutral in most of the tube. In a nanochannel, the Debye length is not negligible compared to the typical dimensions, leading to an excess of counterions in the electrolyte. [0025] Figure 13 is a schematic representation of the technique for further reducing the cross-sectional size of a plurality of capillary microtubes (21) in a monolithic solid by thermal stretching. A: original form of a corresponding device and the cross-section of the monolithic solid; B: device and cross-section of the monolithic solid under axial stress and heat.

DETAILED DESCRIPTION OF THE INVENTION [0026] Throughout this specification a reference to micro is to be taken as including a reference to nano and vice versa.

[0027] Unless expressly stated or implicitly referred to otherwise, throughout this specification a reference to a microchannel is to be taken as including a reference to a hollow tube, a capillary or an enclosed channel (e.g. a duct) with cross-section dimensions on the order of micrometers or nanometers, thus including the ranges referred to in the art as the sub- micrometer range and the nanometer range. Thus the term "channel" is generally used herein interchangeably with the term "capillary microtube".

[0028] Throughout this specification a reference to a width of a capillary microtube is to be taken to refer to a dimension in a plane that is perpendicular to the length of the capillary microtube. Generally, the term "width" includes a cross-sectional width, aperture size, including bore size, lumen size and vice versa. It further includes a diameter, where applicable.

The maximal with corresponds to the maximal diameter where the cross-section of a capillary microtube or aperture is of ovoid or circular shape. Where reference is made to a circular cross-section, the terms "width" and "diameter" can be used interchangeably.

[0029] In one aspect the present invention relates to a sample separation apparatus. The apparatus can be used in the separation of samples, for instance for carrying out chemical and

5 biochemical analysis. Thereby the composition of the sample may for instance be analysed, such as determining how many components or how many main components are included in a sample. The device of the invention may also be used in combination with other techniques established in the art for assessing or determining, which components or which main components are included in a sample. Any sample may be analysed using the apparatus of the

10 invention as long as it can be dissolved in a liquid such as water. Thus, the sample can originate from a large variety of sources. As an illustrative example, the sample may be provided in form of an aqueous solution.

[0030] Such a sample may for instance be of biological origin, e.g. derived from plant material and animal tissue (e.g. insects, fish, birds, cats, livestock, domesticated animals and

15 human beings), as well as blood, urine, sperm, stool samples obtained from such animals. Biological tissue of not only living animals, but also of animal carcasses or human cadavers can be analysed, for example, to carry out post mortem tissue biopsy or for identification purposes, for instance. In other embodiments, samples may be water that is obtained from nonliving sources such as from the sea, lakes, reservoirs, or industrial water to determine the

20 presence of a targeted bacteria, pollutant, element or compound. Further embodiments include, but are not limited to, dissolved liquids or suspensions of solids.

[0031] Accordingly, any of the following samples selected from, but not limited to, the group consisting of a soil sample, an air sample, an environmental sample, a cell culture sample, a bone marrow sample, a rainfall sample, a fallout sample, a sewage sample, a ground

25 water sample, an abrasion sample, an archaeological sample, a food sample, a blood sample, a serum sample, a plasma sample, an urine sample, a stool sample, a semen sample, a lymphatic fluid sample, a cerebrospinal fluid sample, a nasopharyngeal wash sample, a sputum sample, a mouth swab sample, a throat swab sample, a nasal swab sample, a bronchoalveolar lavage sample, a bronchial secretion sample, a milk sample, an amniotic fluid sample, a biopsy

30. sample, a cancer sample, a tumour sample, a tissue sample, a cell sample, a cell culture sample, a cell lysate sample, a virus culture sample, a nail sample, a hair sample, a skin sample, a forensic sample, an infection sample, a nosocomial infection sample, a production sample, a drug preparation sample, a biological molecule production sample, a protein preparation sample, a lipid preparation sample, a carbohydrate preparation sample, a space sample, an extraterrestrial sample or any combination thereof may be processed in a method of the invention. Where desired, a respective sample may have been pre-processed to any degree. As an illustrative example, a tissue sample may have been digested, homogenised or centrifuged prior to being used with the device of the present invention. Accordingly, the capillary sample separation apparatus of the present invention can be used in a large variety of areas such as the field of life sciences, including medical diagnostic or forensic purposes - e.g. the typing of single nucleotide polymorphisms - as well as e.g. analysing the molecular distribution of synthetic polymers or the analysis of molecular changes induced by restoration techniques or the cleaning of paintings.

[0032] The sample may furthermore have been prepared in the form of a fluid, such as a solution. Examples include, but are not limited to, a solution or a slurry of a nucleotide, a polynucleotide, a nucleic acid, a peptide, a polypeptide, an amino acid, a protein, a synthetic polymer, a biochemical composition, an organic chemical composition, an inorganic chemical composition, a metal, a lipid, a carbohydrate or of any combinations thereof. Further examples include, but are not limited to, a suspension of a cell, a virus, a microorganism, a pathogen or any combinations thereof. It is understood that a sample may furthermore include any combination of the aforementioned examples. In some embodiments the sample may include additional components such as detergents that assist in, effect or provide dissolving the sample components to be analysed. As an illustrative example, such sample components may generally include or be, without being limited to, one or more nucleic acid molecules, oligonucleotides, saccharides (e.g. oligosaccharides or polysaccharides), lipids, proteins or peptides. Such components may be of e.g. human or animal origin, including mammalian origin, for example a human or mouse sample, including an extract, e.g. of total mRNA or of proteins of a subcellular organelle.

[0033] The term "nucleic acid molecule" as used herein refers to any nucleic acid in any possible configuration, such as single stranded, double stranded or a combination thereof. Nucleic acids include for instance DNA molecules, RNA molecules, analogues of the DNA or RNA generated using nucleotide analogues or using nucleic acid chemistry, locked nucleic acid molecules (LNA)₅ and protein nucleic acids molecules (PNA). LNA has a modified RNA backbone with a methylene bridge between C4' and O2', providing the respective molecule with a higher duplex stability and nuclease resistance. DNA or RNA may be of genomic or synthetic origin. A respective nucleic acid may furthermore contain non-natural nucleotide analogues and/or be linked to an affinity tag or a label.

[0034] Many nucleotide analogues are known and can be used in nucleic acids used in the methods of the invention. A nucleotide analogue is a nucleotide containing a modification at for instance the base, sugar, or phosphate moieties. As an illustrative example, a substitution of 2'-OH residues of siRNA with 2'F, 2'0-Me or 2'H residues is known to improve the in vivo stability of the respective RNA. Modifications at the base moiety include natural and synthetic modifications of A, C, G, and T/U, different purine or pyrimidine bases, such as uracil-5-yl, hypoxanthin-9-yl, and 2-aminoadenin-9-yl, as well as non-purine or non-pyrimidine nucleotide bases. Other nucleotide analogues serve as universal bases. Universal bases include 3-nitro- pyrrole and 5-nitroindole. Universal bases are able to form a base pair with any other base. Base modifications often can be combined with for example a sugar modification, such as for instance 2'-O-methoxyethyl, e.g. to achieve unique properties such as increased duplex stability.

[0035] If desired, further matter may be added to the respective sample, for example dissolved or suspended therein. As an illustrative example an aqueous solution may include one or more buffer compounds. Numerous buffer compounds are used in the art and may be used to carry out the various processes described herein. Examples of buffers include, but are not limited to, solutions of salts of phosphate, carbonate, succinate, carbonate, citrate, acetate, formate, barbiturate, oxalate, lactate, phthalate, maleate, cacodylate, borate, N-(2-acetamido)- 2-amino-ethanesulfonate (also called (ACES), N-(2-hydroxyethyl)-piperazine-N'-2-ethanesul- fonic acid (also called HEPES), 4-(2-hydroxyethyl)-l-piperazine-ρroρanesulfonic acid (also called HEPPS), piρerazine-l,4-bis(2-ethanesulfonic acid) (also called PIPES)₃ (2-[Tris(hydro- xymethyl)-methylamino]-l-ethansulfonic acid (also called TES), 2-cyclohexylamino-ethansul- fonic acid (also called CHES) and N-(2-acetamido)-iminodiacetate (also called ADA). Any counter ion may be used in these salts; ammonium, sodium, and potassium may serve as illustrative examples. Further examples of buffers include, but are not limited to, triethanol- amine, diethanolamine, ethylamine, triethylamine, glycine, glycylglycine, histidine, tris(hy- droxymethyl)aminomethane (also called TRIS), bis-(2-hydroxyethyl)-imino-tris(hydroxy- methyl)methane (also called BIS-TRIS), and N-[Tris(hydroxymethyl)-methyl]-glycine (also called TRICINE), to name a few. The buffers may be aqueous solutions of such buffer compounds or solutions in a suitable polar organic solvent. One or more respective solutions may be used to accommodate the suspected biological analyte molecule as well as other matter used, throughout an entire method of the present invention. Further examples of matter that may be added, include salts or chelating compounds. As yet a further illustrative example, nuclease inhibitors may need to be added in order to maintain a nucleic acid molecule in an intact state.

[0036] The apparatus of the present invention is based on a monolithic solid. The solid may include or be of any solid material as long as the material is of a stability and stiffness that provides the solid with the capability of containing a plurality of capillary tubes. Further, the monolithic solid is generally of such rigidity that the arrangement, shape and integrity of capillary microchannels included therein remain at least essentially intact during the operation of the selected separation technique, e.g. electrophoresis, including isoelectric focusing or field-amplified sample stacking (for an introduction into capillary isoelectric focusing see e.g. Silvertand, L.H.H., et al., J. Chromatography A (2008), doi:10.1016/j.chroma.2008.05.057 and references cited therein). In addition, the monolithic solid is in typical embodiments of such rigidity that both its overall shape and integrity, and the arrangement, shape and integrity of capillary microchannels included therein remain at least essentially intact during the operation of the selected separation technique. In some embodiments the rigidity of the monolithic solid further allows for the shape and integrity of both the monolithic solid and the microchannels to remain at least essentially intact during assembly and/or storage. The solid may for instance include a metal, a metalloid, ceramics, a metal oxide, a metalloid oxide, oxide ceramics, a polymer and composites thereof. A respective polymer may for example be plastic (such as thermoplastics) or an elastomer (such as PDMS or elastic silicone rubber). Examples of suitable metalloids include, but are not limited to silicon, boron, germanium, antimony and composites thereof. Examples of suitable metals include, but are not limited to iron (e.g. steel), aluminium, gold, silver, chromium, tin, copper, titanium, zinc, aluminium, lead and composites thereof. A respective oxide of any of these metalloids and metals may be used as a metalloid oxide or metal oxide respectively. As an illustrative example, the surface may be of quartz or glass. As a further illustrative example, a silicon oxide or germanium oxide surface may be obtained by etching a silicon substrate or germanium substrate, respectively, with piranha solution, i.e. a mixture of sulphuric acid and hydrogen peroxide solution at a molar ratio of 7:3. Examples of ceramics include, but are not limited to, silicate ceramics, oxide ceramics, carbide ceramics or nitride ceramics. [0037] The apparatus of the present invention includes a plurality of capillaries. These capillaries are typically arranged as a bundle. Previously a chip that includes a bundle of capillaries and that can be used for affinity detection/analytical purposes has been described in US patent application 2002/0086325. Further, a cartridge that contains a plurality of packed and nominally aligned solid polymer fibers has been disclosed in US patent application US 2006/0032816. The apparatus according to the present invention includes a plurality of capillary microtubes (including nanotubes) with dimensions in terms of their width in the micrometer range, the sub-micrometer range and/or the nanometer range. The capillary tubes may for instance be of a width of 5 μm or below, such as 1 μm and below, hi some embodiments the capillary microtubes have a width from about 1 nm to about 500 nm, such as about 5 nm to about 200 nm, about 5 nm to about 100 nm, about 10 nm to about 200 nm, about 10 nm to about 100 nm, or about 5 nm to about 80 nm. At least some of the capillary microtubes may be of at least substantially the same width, hi some embodiments some capillary microtubes of the plurality of microtubes differ in their width.

[0038] The capillary microtubes may be of any desired length. The length of the microtubes may for instance be selected in the micrometer range, the submillimeter range, the millimeter range, the sub centimeter range, the centimeter range, or above. The microtubes may for instance be of a length of about 0.1 cm, 0.2 cm, 0.5 cm, 0.7 cm, 0.9 com or above or about 1 cm or above, such as in the range from about 1 mm to about 100 cm, such as about 10 mm to about 100 cm, about 10 mm to about 10 cm or about 0.5 cm to about 10 cm.

[0039] The capillary microtubes of the apparatus of the invention are included in the monolithic solid (supra). They may possess any internal surface characteristics, as long as they allow for the separation of a selected sample included therein. Where for instance an aqueous liquid sample is provided, internal surfaces of the capillary microtubes may thus be rendered water- attracting, typically polar and hydrophilic, or water-repellent, typically non-polar and hydrophobic. Furthermore, different internal areas of capillary microtubes may provide different surface characteristics. Thus, some areas on the capillary microtubes, such as walls or wall- portions, may be rendered polar, while others areas may be rendered non-polar. The internal surface of tube walls may have subjected to a treatment that leads to an alteration of the respective surface characteristics that lasts long enough for a subsequent separation of a liquid sample to be affected. Typically, this treatment does not affect the composition of a sample contacting the respective surface area, hi some embodiments the treatment does not affect the composition of any fluid, including any liquid that contacts the respective surface area. [0040] A treatment that may be carried out to alter surface characteristics may include various means, such as mechanical, thermal, electrical or chemical means. A method that is commonly used in the art is a treatment with chemicals having different levels of affinity for the fluid sample. As an example, the surface of plastic materials can be rendered hydrophilic via treatment with dilute hydrochloric acid or dilute nitric acid. As another example, a polydimethylsiloxane (PDMS) surface can be rendered hydrophilic by an oxidation with oxygen or air plasma. Alternatively, the surface properties of any hydrophobic surface can be rendered more hydrophilic by coating with a hydrophilic polymer or by treatment with surfactants. Examples of a chemical surface treatment include, but are not limited to exposure to hexamethyldisilazane, trimethylchlorosilane, dimethyldichlorosilane, propyltrichlorosilane, tetraethoxysilane, glycidoxypropyltrimethoxy silane, 3-aminopropyltriethoxysilane, 2-(3,4- epoxy cyclohexyl)ethyltrimethoxysilane, 3-(2,3-epoxy propoxyl)propyltrimethoxysilane, polydimethylsiloxane (PDMS), γ-(3,4-epoxycyclohexyl)ethyltrimethoxysilane, poly (methyl meth- acrylate), a polymethacrylate co-polymer, urethane, polyurethane, fluoropolyacrylate, poly- (methoxy polyethylene glycol methacrylate), poly(dimethyl acrylamide), poly[N-(2-hydroxy- propyl)methacrylamide] (PHPMA), a-phosphorylcholine-o-(N,N-diethyldithiocarbamyl)unde- cyl oligoDMAAm-oligo-STblock co-oligomer (see Matsuda, T et al., Biomaterials (2003), 24, 24, 4517-4527), poly(3,4-epoxy-l-butene), 3,4-epoxy-cyclohexylmethylmethacrylate, 2,2-bis- [4-(2,3-epoxy propoxy) phenyl] propane, 3,4-epoxy-cyclohexylmethylacrylate, (3',4'-epoxy- cyclohexylmethyl)-3 ,4-epoxycyclohexyl carboxylate, di-(3 ,4-epoxycyclohexylmethyl)adipate, bisphenol A (2,2-bis-(p-(2,3-epoxy propoxy) phenyl) propane) or 2,3-epoxy-l-propanol.

[0041] The capillary microtubes present in the apparatus of the invention are free of branches and loops. Thus any one of the capillary microtubes has two ends, each end having an aperture, such as an opening. These two apertures are typically the only apertures included in the capillary microtubes. Further, each capillary microtube stretches in at least substantially one main direction. The capillary microtubes may be bent at a maximal angle that is below 90 °, such as below about 50 °, below about 40 °, below about 30 °, below about 20 ° or below about 10°. In typical embodiments the capillary microtubes are at least substantially straight. They are in some embodiments at least substantially linear, hi typical embodiments each capillary microtube is further free of kinks, dents and constrictions. In some embodiments the maximal width of the cross-section of at least some capillary microtubes remains at least substantially constant. In some embodiments the maximal width of the cross-section of each capillary microtube remains at least substantially constant. Likewise, in some embodiments the minimal width of the cross-section of at least some capillary microtubes remains at least substantially constant. In some embodiments the minimal width of the cross-section of each capillary microtube remains at least substantially constant, hi some embodiments both the maximal and the minimal width of the cross-section of at least some, of most or of at least substantially all capillary microtubes remains at least substantially constant. [0042] In the apparatus of the invention these capillary microtubes (henceforth also generally referred to as "the tubes" or "the channels") are arranged in an at least substantially parallel manner. Further, they may be arranged at a certain distance from each other. The distance between some or all individual microtubes may differ or be at least substantially similar or identical. The distance between individual microtubes may be selected in the range from about 1 nm to about 10 mm, such as about 5 nm to about 2 mm. about 1 ran to about 1 mm, about 5 nm to about 1 mm, about 10 nm to about 1 mm, about 50 nm to about 1 mm, about 50 nm to about 1 mm or about 100 nm to about 0.5 mm. Generally, the capillary microtubes are arranged in a two-dimensional or three-dimentional array (in the following also addressed as a "microchannel bundle"). Thus, the plurality of capillary microtubes may for instance be arranged in a row. In one embodiment three capillary microtubes are arranged at an at least substantially equal distance to each other. Thereby they define an at least substantially equilateral triangle when viewed along the length of the capillary microtubes. hi some embodiments four capillary microtubes are arranged at an at least substantially equal distance, defining a square, when viewed along the length of the capillary microtubes. Some, most or all capillary microtubes may be arranged at an at least substantially equal distance to each other, thereby defining an at least substantially regular polyhedron when viewed along the length of the capillary microtubes. The capillary microtubes can be positioned in an inter-digitated manner to improve the visibility of the individual microchannel, especially in applications requiring optical detection.

[0043] Separation of biomolecules can be realized effectively inside a plurality of capillary microtubes such as a bundle of capillary microtubes. Such a plurality of capillary microtubes may for example have numerous applications in electrophoretic device platforms, e.g. the emerging field of capillary electrophoresis devices, and may outperform conventional single-channel microfluidic networks. Compared to the single microtubes or nanotubes used in all current electrophoretic devices, the use of a plurality of microchannels offers unique properties that significantly improve separation and results. Without being bound by theory it is believed that the arrangement of the capillary microtubes in a monolithic solid provides an advantageous heat conduction, thereby minimizing the negative effect of Joule heat on the resolution obtainable in sample separation. As further explained below, the present inventors have further found that an apparatus of the invention can also be used in other separation techniques, such as for a chromatographic separation of a sample. Throughout this specification a reference to electrophoretic devices is to be taken as including portable lab-on- chip devices, conventional benchtop capillary electrophoretic devices and other instruments designed for the separation of charged molecules and vice versa.

[0044] The monolithic solid has a first lateral wall and a second lateral wall. These two walls are arranged in an opposing relationship, facing away from each and generally facing opposite directions. The plurality of at least substantially parallel capillary microtubes is arranged in the monolithic solid in such a way that the first ends of the microtubes are located in the first of the two walls and the second ends are located in the second wall. Thus the two apertures included in the two ends of each microtube are generally likewise facing opposite directions. In the apparatus of the invention the monolithic solid is further arranged in such a way that a plurality of the capillary microtubes included in the monolithic solid are in fluid communication with the ambience of the monolithic solid. In typical embodiments all capillary microtubes are accessible once the monolithic solid is arranged in the apparatus of the invention. Thus all microtubes included in the monolithic solid are typically in fluid communication with the ambience thereof. Thereby the microtubes are fluidly coupled, typically all first ends of the microtubes being fluidly coupled and all second ends of the microtubes being fluidly coupled.

[0045] The ambience of the monolithic solid includes geometric elements of or integrated into the apparatus of the invention, such as a reservoir, a channel or a chamber. One such geometric element that may be included in the apparatus of the invention is a sample loading port. The sample loading port may be a chamber defined by a circumferential wall and a base, into which the sample to be separated is to be filled. In the apparatus of the invention each accessible first aperture, typically each first aperture, of a microtube included in the monolithic solid is in fluid communication with this common sample loading port. Another such geometric element included in the apparatus of the invention is a reservoir. This reservoir may be of any desired dimensions and geometry. In embodiments where the reservoir is included in the apparatus of the invention the size of the reservoir is generally limited by the dimensions of the apparatus, m some embodiments the reservoir is of a dimension or volume that allows some or all of the second apertures of the monolithic solid to be in fluid communication with each other. In some embodiments the reservoir is of dimensions that allow for the accommodation of an electrode such as an anode. [0046] Each accessible second aperture, typically each second aperture, of a microtube included in the monolithic solid is in fluid communication with this common sample loading port. In some embodiments only one of the sample loading port and the reservoir is included in the apparatus, while the other of these two elements situated in a location that differs from the apparatus of the invention. In some embodiments neither the sample loading port nor the reservoir are included in the apparatus. Ih any case, the sample loading port is in fluid communication with, typically fluidly connected to, each accessible first aperture of the plurality of capillaries included in the monolithic solid. Further, in any case is the reservoir in fluid communication with, typically fluidly connected to, each accessible second aperture of the plurality of capillaries.

[0047] The monolithic solid may be of any desired size and shape as long as it has a first and a second lateral wall arranged in at least substantially opposing relationship as described above, which are arranged at a distance from each other that allows for the capillary microtubes to be included in the monolithic solid with a length desired for a selected separation. The capillary microtubes can then be allowed to span the distance between the first and the second wall inside the monolithic solid. The monolithic solid may for instance be of rounded shape, e.g. ovoid or circular shape or it may be of tubular shape. It may also include quoins, corners, flanges or brinks in any desired number and for instance be of cuboid or cube shape. [0048] As explained above, in some embodiments the capillary microtubes included in the monolithic solid are at least substantially straight. The length of such elongate microtubes thus defines an axis of the at least substantially parallel plurality of microtubes. In some embodiments the first and/or the second lateral wall of the monolithic solid are orientated to be at least substantially perpendicular (also used interchangeably with the term 'orthogonal') to this axis. In this manner, the capillary microtubes may all be of about the same length, i.e. provide about the same distance between the first and the second aperture in each microtube. Further, in such an embodiment the plane of each aperture, included in a lateral wall of the monolithic solid, is at least substantially perpendicular to the axis defined by the length of the corresponding microtube. By the term 'substantially perpendicular', it is meant that the angle between the plane of the opposing lateral walls of the monolithic solid may be arranged not exactly at 90° to the axis defined by the lengths of the plurality of microtubes. The angle may deviate from 90°, as long as the lengths of the capillary microtubes are of a homogeneity that is sufficient to achieve a desired sample separation. In embodiments where the first and the second lateral wall of the monolithic solid are at least substantially plain and at least substantially parallel to each other, the lengths of the microtubes may be similar or even at least substantially identical, even where the above defined axis is not perpendicular to the plane defined by the first and the second lateral walls. In such embodiments the planes of the tube apertures is however not perpendicular to the above defined axis. Depending on the complexity of the sample used, an apparatus with such a monolithic solid may well be suited for a separation to be carried out.

[0049] The cross-section of the capillary microtubes can have any shape, which can mainly be determined by the way the microtubes are fabricated as well as by the materials used for such a fabrication. The cross-section may for instance be ovoid, circular, triangular, rectangular, square or of the shape of any polyhedron. An overview on the formation of micro- and nanotubes has for instance been given by Abgrall et al. (Analytical Chemistry (2008) 80, 7, 2326-2341, incorporated herein by reference in its entirety). In some embodiments at least an substantial portion of at least some of the capillary microtubes have a cross-section of a shape selected from the group of ovoid shape, at least substantially circular shape and the shape of a polyhedron. A substantial portion is understood to refer to a portion that amounts to more than a marginal portion of a respective capillary, such as a portion that amounts to at least 10 %, at least 20 %, at least 30 %, at least 40 %, at least 50 %, at least 60 %, at least 70 %, at least 80 %, at least 85 % at least 90 %, at least 95 % or about 100% of the entire inner surface of the respective channel. A substantial portion of the capillary tube may also amount to at least 10 %, at least 20 %, at least 30 %, at least 40 %, at least 50 %, at least 60 %, at least 70 %, at least 80 %, at least 85 % at least 90 %, at least 95 % or about 100% of the entire length of the respective channel. The cross-section of some or all capillary microtubes may also vary in shape along the length of the capillary microtube to any extent as long as the microtubes are still capable of achieving a desired separation of a specific sample.

[0050] Bonding of two substrates, one (or both) of which may have been patterned by means of etching, including the use of a sacrificial layer or a thin spacer layer (see Fig. 10), or by means of photolithography, imprint lithography, scanning beam lithography or scanning probe lithography (see also Fig. 11) may for example be used. A combination of thermal bonding and hot-embossing of a polymeric substrate such as polymethylmethacrylate (cf. Fig. HD) may also be used, as disclosed by Abgrall et al. (Lab on a Chip (2007) 7, 520-522, incorporated herein by reference in its entirety). Using the latter technique nanotubes with rectangular cross-section of a depth of 50 nm and a depth to width ratio from 0.008 to 0.05 have been obtained, i.e. channels with a significant difference in their maximal and minimal width due to a low aspect ratio (ibid).

[0051] The plurality of capillaries may be implemented into the monolithic solid by any desired technique. These techniques may be classified into monolithic integration and hybrid integration (see Fig. 9). Monolithic integration of a two-dimensional microchannel bundle may be implemented by any microtechnology used for the fabrication of conventional electrophoretic devices. Monolithic integration of a three-dimensional microchannel bundle can be implemented with the technologies depicted in Figure 8 and Figure 9.

[0052] The fabrication of a three-dimensional microtube bundle starts with the fabrication of a two-dimensional arrangement of microtubes using for instance any micromachining technique including but not limited to etching or hot embossing in a thin film of materials including but not limited to polymers, glass or silicon. Subsequently, the layers may be laminated and bonded to form the bundles. Bonding techniques including but not limited to solvent-assisted bonding, anodic bonding and thermal fusion bonding can be used for this purpose. This technique also allows the fabrication of larger reservoirs and microchannels for delivery purpose (cf. Fig. 9).

[0053] If the size of the microtube is limited by the fabrication technology, smaller microtubes can be achieved by thermal stretching of the monolithic solid. For example, the bundle can be first fabricated with the technique described in Figure 9. Subsequently, the entire bundle can be heated to reach the plastic region of the material (e. g. above the glass temperature). Under axial stress, the cross-section of the capillary bundle and its microtubes are reduced, as shown in Figure 13. This technique of manipulating PCFs and other suitable monolithic devices could also be adopted to replace the capillaries of single-lumen-capillary systems. As a result, the PCFs have the potential to be adopted for e.g. all current and future capillary electrophoresis applications. The advantage of the described methods for microchannel bundle fabrication using PCF is the ability to create bore sizes of extremely narrow width within the bundle. Previous techniques are limited in their ability to reliably and reproducibly create microchannel bundles containing individual microchannel bore sizes of such small size. [0054] An off-the-shelf monolithic solid with a plurality of microtubes, such as a photonic crystal fibre (PCF), can be integrated with a conventionally micro-machined microfluidic device system (e.g. an electrophoretic device) by hybrid integration to form an apparatus of the invention. The monolithic solid may for instance be cut into small pieces that can then be placed in their positions on the microfluidic device system. The monolithic solid may then be embedded in the respective device by thermal bonding or adhesive bonding. Figure 1C schematically indicates the implementation of a monolithic solid with a plurality of microtubes into a microfluidic device that may be used for separation based on capillary electrophoresis. The same device can be implemented monolithically using the technique depicted in Figure 8.

[0055] In some embodiments the apparatus of the invention further includes an electrode. The term "electrode" as used herein is employed in its conventional sense, thereby referring to an object that is capable of serving as an electric conductor, through which an electrical current or voltage may be brought into and/or out of a medium in contact with the electrode. Typically an electrode is one of at least two terminals of an electrically conducting medium. In some embodiments the apparatus of the invention includes two electrodes, an anode and a cathode. The electrodes of a respective electrode pair are arranged at a distance from one another. Li embodiments where two electrodes are provided, the two electrodes may for instance be separated by a distance in which the monolithic solid is arranged. In some embodiments the two electrodes are at least substantially parallel. The one or more electrodes included in the apparatus of the invention may be of any desired dimension and shape. They may for example have the shape of a flat, arched, concave or convex slab, hi some embodiments they may have the shape of a ring (for an example see Green, BJ, & Hudson, J.L., Phys. Rev. E (2001), 63, 026214; see also Fig. 4A). In some embodiments interdigital electrodes are provided, which typically include a digitlike or fingerlike pattern of parallel in- plane electrodes (see Mamishev, A.V., Proc. IEEE (2004), 92, 5, 808-845, or Matsue, T., Trends Anal. Chem. (1993), 12, 3, 100 - 108 for examples). Li some embodiments an array of electrodes may be provided. If desired, one or more floating electrodes may be used, hi some embodiments the electrodes that are provided are of similar size, for example of identical size.

[0056] The distance between the two or more electrodes may be of any dimension. Where the one or more electrodes are to be used for electrophoretic separation of a sample, they should be able to provide an electric field of sufficient strength to the respective region can be determined in the method of the present invention, hi some embodiments the apparatus of the invention may include more than two electrodes. In such embodiments the distance at which the electrodes are arranged may in some embodiments be identical between each of the respective electrodes. In other such embodiments the distance at which the electrodes are arranged maybe identical between some of the respective electrodes, hi yet other embodiments where more than two electrodes are provided, each distance at which two electrodes are arranged may be different from another distance at which two electrodes are arranged.

[0057] For the electrode that is included in the apparatus of the invention a zone can be defined in which an electric field of the electrodes is effective. The first apertures of the capillary microtubes are arranged within this zone. Typically the plurality of capillary microtubes is arranged within this zone in its entirety, such that the entire length of each microtube can be exposed to an electrical field of the electrode. In some embodiments an electrode that is included in the apparatus of the invention, is in fluid communication with the first apertures or with the second apertures of the capillary microtubes. As noted above, in some embodiments the apparatus of the invention includes a pair of electrodes. In such embodiments the first apertures of the capillary microtubes may be arranged within the zone where an electric field of the first electrode is effective and the second apertures of the capillary microtubes may be arranged within the zone where an electric field of the second electrode is effective. In some embodiments the pair of electrodes is arranged at a distance that permits an electric field of the pair of electrodes to be formed. In such embodiments the plurality of capillary microtubes is generally arranged in the zone where an electric field of the pair of electrodes is effective, for instance in between the pair of electrodes. For instance the first electrode may face the first apertures and the second electrode may face the second apertures of the capillary microtubes. Additionally, the first electrode may be in fluid communication with the first apertures and the second electrode may be in fluid communication with the second apertures of the capillary microtubes. As an illustrative example, the pair of electrodes may include an anode and a cathode. The first lateral wall of the monolithic solid that includes the first apertures of the capillaries may be opposing the anode and the second lateral wall of the monolithic solid may be opposing the cathode. [0058] In some embodiments the monolithic solid is located on or in vicinity to a semiconductor based transistor or conductively connected thereto. As an example, the monolithic solid may be or be included in the surface of a gate electrode of a field effect transistor (FET). In some embodiments the immobilisation unit is located in the zone where the electric field of the field effect transistor is effective. The monolithic solid may also be located in the zone where the electric field a floating gate electrode of a field effect transistor is effective. A power supply unit, which may be any power supply unit, e.g. as commonly used in the art, may be included in the apparatus for applying a voltage to any electrode(s) included in the apparatus.

[0059] Thus in some embodiments separation of matter in a sample (e.g. proteins, nucleic acids, ions etc.) is achieved via electrophoresis. An introduction into the separation of a number of biological samples by means of electrophoresis using a single capillary tube has been given e.g. by Lloyd et al. (J. Chromatography B (2008) 866, 154-166). It is understood that the same underlying principles apply for a plurality of capillary microtubes as used in the apparatus and method of the present invention. Using the apparatus of the invention, the sample is introduced into 'the sample loading port. Depending on the electrophoretic properties of the analyte, the sample will be separated along the length of the capillary microtubes under a specified electric field. Its components may be separated largely based on the velocity of the charged component in the electric field. Where desired, this can then be detected with a detector. For example, DNA within a given sample could be labelled with an intercalating agent such as ethidium bromide and bands of DNA of a given size detected under exposure to UV light. Depending on the molecule of interest and the detection method used, the signal can provide qualitative and quantitative information of the sample contents. In previous separation systems, reduction of the bore size of a capillary tube for increased heat dissipation was limited by the minimum bore size required to maintain accurate detection of the sample. The increased surface area of the plurality of capillary microtubes described here allows for detection from most or all of the microchannels concurrently, leading to a stronger signal than that of a dot array or a simple microchannel. This overcomes the limitation of the bore size of previous systems.

[0060] The introduction of a sample may also be achieved or assisted by other active means such as electrokinetic pumping or using external pumps and valves. However, capillary action is generally sufficient to load the sample. In particular at the nanoscale, capillary pressure can locally reach very high values. Nevertheless the apparatus of the invention may include means for filling the plurality of capillary microtubes with the sample and/or a separation medium. Illustrative examples of such means are a pump and a geometric element, which may be or include a microcapillary channel, with the element being included in or on a substrate in or on which the monolithic solid is located. Further, a coordination of different geometric and/or surface characteristics of elements, such as connecting channels leading from the sample loading port to the first apertures of the microtubes, may assist in loading the sample.

[0061] The monolithic solid of the invention is thought to assist in dissipating heat generated during electrophoresis in the capillary microtubes. Further, in typical embodiments of the invention the width of the cross-section of the capillary microtubes is small enough to lead to a reduced or even to an at least substantially diminished Joule heat effect. The amount of heat generated in an electrical system can be calculated by

where J is the heat generated (Joules) as a function of time t, when a potential drop is applied across a microtube of resistance, R, with area of cross-section A and length L. The smaller the tube cross-section, the larger the electrical resistance. Consequently, less electrical current and less heat is generated for a given voltage. In addition, the heat can be dissipated more quickly from a microtube with a smaller cross-sectional area. This is because a decrease in cross- section leads to an increase in the SVR of the microtube, which offers better heat dissipation. The equation for the SVR of a microchannel with a unit length is defined as

S Perimeter of crosssection r_sv = — = - ^J - (2)

V Area _of _cr oss section

For example, the SVR of a capillary with 4 μm id is 25 times larger than that of a 100 μm id capillary. However, due to the limits of current fabrication techniques and detection devices, the typical dimensions of conventional capillary electrophoresis microtubes are in the range of 50 μm to 200 μm. Accordingly, embodiments of the apparatus of the invention with a monolithic solid that includes microtubes with smaller cross-sectional areas and larger surface areas arranged in the form of a bundle achieve enhanced heat dissipation from each microtube. In some embodiments the width of the cross-section of the capillary microtubes is small enough to prevent Joule heat from at least substantially affecting sample separation at a selected electric field. As depicted in Fig. 3, an apparatus of the invention can for instance permit electrophoresis to be carried out at at least 100 V/cm. Thus an apparatus according to the present invention can be used for applications involving higher electric field strengths than possible with single conventional microcapillary systems. [0062] While an apparatus according to the present invention advantageously dissipates joule heat energy, when incorporated into an electrophoretic device, it may also be integrated with cooling elements including additional heat sinks or electronic cooling devices

[0063] Furthermore, a bundle of microchannels overcomes difficulties encountered with single microchannel systems in regards to sample detection. One of the major problems in electrophoretic device systems is the limited sensitivity of detection due to the small sample volume and the limited analyte concentration in a sample. The relation between the sample volume V and the sensor efficiency η_s (0 < η_s < 1) is given by [Nguyen, N.T., & Wereley, S. T., Fundamentals and Applications of Microfluidics, 2^nd edition, Artech House, 2006]:

V = - (3)

where A₁ is analyte concentration and N_A is the Avogadro number. Equation (3) shows that at a given analyte concentration, the sample volume is determined by the sensor efficiency. A microchannel bundle offers a large surface area thus a stronger signal for fluorescence-based detection systems, thus increasing the sensor efficiency η_s . Thus, the total sample volume V can be reduced with the use of microchannel bundles, allowing for smaller microchannel bore sizes to be used to achieve greater heat dissipation. [0064] With regard to the selection of the width of the microtubes it is further noted that electrokinetic differences between channels widths of micrscale and nanoscale can be exploited (Abgrall et al, 2008, supra). Most surfaces submerged in an aqueous solution gain a net charge density which may originate from chemical reactions (e.g., protonation or deprotonation), adsorption, or defects in a crystalline structure. Surface charges can also stem from an external electrical potential. In the liquid, these charges are shielded by a layer of adsorbed ions, the Stern layer, and a mobile layer, the diffuse layer. In a microtube, the screening length of the electrical double layer is small compared to the typical tube dimensions and, without an external field, the electric potential is neutral almost everywhere in the tube but at the liquid/solid interface (Fig. 12A). As depicted in Fig. 12B, the situation is different in a nanochannel. As the dimensions are reduced (and/or the ionic concentration is decreased), the electrical double layer occupies a nonnegligible fraction of the tube and the quantity of surface charges becomes comparable to the quantity of charges in the bulk electrolyte. Because of the electroneutrality requirement, the ratio of counterions to co-ions in the tube is becoming larger and larger, and the electric potential is not neutral anymore. These phenomena are at the origin of the Donnan or co-ion exclusion effect well known in semipermeable membrane technologies. They also explain the higher conductivity observed at low salt concentration in nanochannels, the influence of the surface treatments on it, and other charge-selective effects.

[0065] In some embodiments at least a portion of the plurality of capillary microtubes may be filled with a stationary phase, also termed matrix, for instance in the form of a polymer - whether linear or cross-linked such as a gel - for electrophoresis. Examples of a respective stationary phase include, but are not limited to agarose, polyacrylamide, polyacrylamide/bis- acrylamide copolymer, polyvinylpyrrolidone and a cellulose material such as hydroxypropyl cellulose. As an example, the capillary microtubes may be partially filled with a matrix such as a Pluronic® polymer, e.g. Pluronic® F 127 (Sedlakova, P., & Svobodova, I.M., J Chromatography B (2006) 839, 112-117). As a further example, a linear homopolymer, also termed "non-gel sieving matrix" such as linear polyacrylamide, poly(N,N-dimethylacrylamide) poly-(ethylene- oxide), polyvinylpyrrolidone, cellulose or an other polymer or copolymer or a mixture thereof may be present in the entire capillary microtubes or a part thereof. In some embodiments a polymer, including a linear and a cross-linked polymer, optionally an emulsifier, and a plurality of nanoparticles may be included in the capillary microtubes as e.g. disclosed by Zhou et al. (Electrophoresis (2007) 28, 1072-1080; see also Shiddiky, M.J.A., & Shim, Y-B., Anal. Chem. (2007) 79, 3724-3733). In some embodiments the microtubes are filled with a liquid medium that is replaced by the sample upon loading. As an illustrative example the capillary microtubes may be filled with an aqueous buffer solution that corresponds to or is identical with a buffer solution in which the sample is included. In some embodiments the capillary microtubes are void of any liquid and filled with a gas such as air before use.

[0066] Accordingly, the present invention also relates to the use of a monolithic solid as defined above to expose a single sample to capillary electrophoresis. Thus in the use of the present invention just one sample is separated in a plurality of capillary microtubes that are included in a monolithic solid. The monolithic solid may in some embodiments be arranged in such a way that the first aperture of each capillary microtube is in fluid communication with a common sample loading port. The monolithic solid may in some embodiments be arranged in such a way that the second aperture of each capillary microtube is in fluid communication with a common reservoir. In some embodiments the first aperture of each capillary microtube is in fluid communication with a sample loading port and the second aperture of each capillary microtube is in fluid communication with a reservoir. In one embodiment the monolithic solid is arranged on or included in a capillary sample separation apparatus as defined above. [0067] The increased surface-area-to-volume ratio (SVR) offered by the PCF (but not restricted to PCF, so long as there are many narrow microchannels near to one another) allows for the immobilisation, e.g. covalent bonding, coordinative bonding or non-covalent bonding, of matter in the capillary microtubes of the monolithic solid. An example of such matter is a binding partner molecule or moiety that is capable of interacting with certain matter that may be included in the sample, thereby reducing its mobility. Such a binding partner molecule or moiety has an affinity to one or more molecules or ions or classes of molecules or ions, which may be suspected to be included in the sample. In some embodiments the binding partner molecule or moiety, in the following also termed "binding partner", is capable of forming a complex with such a molecule. The binding partner may therefore be selected according to the sample. Examples of a binding partner include, but are not limited to, a nucleic acid molecule, an oligonucleotide, a protein, an oligopeptide, a polysaccharide, an oligosaccharide, a synthetic polymer, a drug candidate molecule, a drug molecule, a drug metabolite, a metal ion, and a vitamin. As an illustrative example, the binding partner may be nucleic acid binding polypeptide. In some embodiments the binding partner may for example be a receptor molecule for a biological analyte molecule suspected to be present in a sample. In such embodiments the receptor molecule and the biological analyte molecule define a specific binding pair.

[0068] Three illustrative examples of suitable binding partner molecule or moiety are biotin, dinitrophenol or digoxigenin. Where the analyte molecule is a protein, a polypeptide, or a peptide, further examples of a binding partner include, but are not limited to, a streptavidin binding tag such as the STREP-TAGS® described in US patent application US 2003/0083474, US patent 5,506,121 or 6,103,493, an immunoglobulin domain, maltose-binding protein, glutathione-S-transferase (GST), calmodulin binding peptide (CBP), FLAG-peptide (e.g. of the sequence Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys-Gly), the T7 epitope (Ala-Ser-Met-Thr- Gly-Gly-Gln-Gln-Met-Gly), maltose binding protein (MBP), the HSV epitope of the sequence Gln-Pro-Glu-Leu-Ala-Pro-Glu-Asp-Pro-Glu-Asp of herpes simplex virus glycoprotein D, the Vesicular Stomatitis Virus Glycoprotein (VSV-G) epitope of the sequence Tyr-Thr-Asp-Ile- Glu-Met-Asn-Arg-Leu-Gly-Lys, the hemagglutinin (HA) epitope of the sequence Tyr-Pro- Tyr-Asp-Val-Pro-Asp-Tyr-Ala and the "myc" epitope of the transcription factor c-myc of the sequence Glu-Gln-Lys-Leu-Ile-Ser-Glu-Glu- Asp-Leu. Where the analyte molecule is a nucleic acid, a polynucleotide or an oligonucleotide, a binding partner may furthermore be an oligonucleotide. Such an oligonucleotide tag may for instance be used to hybridize to an immobilised oligonucleotide with a complementary sequence (see below). A respective binding partner may be located within or attached to any other molecule.

[0069] A further example of a binding partner is an immunoglobulin, a fragment thereof or a proteinaceous binding molecule with immunoglobulin-like functions. Examples of (recombinant) immunoglobulin fragments are F_ab fragments, F_v fragments, single-chain F_v fragments (scFv), diabodies, triabodies (Iliades, P., et al., FEBS Lett (1997) 409, 437-441), decabodies (Stone, E., et al., Journal of Immunological Methods (2007) 318, 88-94) and other domain antibodies (Holt, L.J., et al., Trends Biotechnol. (2003), 21, 11, 484-490). An example of a proteinaceous binding molecule with immunoglobulin-like functions is a mutein based on a polypeptide of the lipocalin family (WO 03/029462, Beste et al., Proc. Natl. Acad. ScL USA (1999) 96, 1898-1903). Lipocalins, such as the bilin binding protein, the human neutrophil gelatinase-associated lipocalin, human Apolipoprotein D or glycodelin, posses natural ligand- binding sites that can be modified so that they bind to selected small protein regions known as haptens. Examples of other proteinaceous binding molecules are the so-called glubodies (see e.g. international patent application WO 96/23879 or Napolitano, E. W., et al., Chemistry & Biology (1996) 3, 5, 359-367), proteins based on the ankyrin scaffold (Mosavi, L.K., et al., Protein Science (2004) 13, 6, 1435-1448) or crystalline scaffold (e.g. internation patent application WO 01/04144) the proteins described in Skerra, J. MoI. Recognit. (2000) 13, 167- 187, AdNectins, tetranectins and avimers. Avimers contain so called A-domains that occur as strings of multiple domains in several cell surface receptors (Silverman, J., et al., Nature Biotechnology (2005) 23, 1556-1561). Adnectins, derived from a domain of human fϊbronectin, contain three loops that can be engineered for immunoglobulin-like binding to targets (Gill, D.S. & Damle, N.K., Current Opinion in Biotechnology (2006) 17, 653-658). Tetranectins, derived from the respective human homotrimeric protein, likewise contain loop regions in a C-type lectin domain that can be engineered for desired binding (ibid.). Peptoids, which can act as protein ligands, are oligo(N-alkyl) glycines that differ from peptides in that the side chain is connected to the amide nitrogen rather than the α carbon atom. Peptoids are typically resistant to proteases and other modifying enzymes and can have a much higher cell permeability than peptides (see e.g. Kwon, Y.-U., and Kodadek, T., J. Am. Chem. Soc. (2007) 129, 1508-1509). If desired, a modifying agent may be used that further increases the affinity of the respective binding partner for any or a certain form, class etc. of analyte molecules.

[0070] As an illustrative example, the binding partner may be a metal ion bound by a respective metal chelator, such as ethylenediamine, ethylenediaminetetraacetic acid (EDTA), ethylene glycol tetraacetic acid (EGTA), diethylenetriaminepentaacetic acid (DTPA), N₅N- bis(carboxymethyl)glycine (also called nitrilotriacetic acid, NTA), l,2-bis(o-aminophenoxy)- ethane-N,N,N',N'-tetraacetic acid (BAPTA), 2,3-dimercapto-l-propanol (dimmercaprol), por- phine or heme. A respective metal ion may define a receptor molecule for a peptide of a defined sequence, which may also be included in a protein. In line with the standard method of immobilised metal affinity chromatography used in the art, for example an oligohistidine tag of a respective peptide or protein is capable of forming a complex with copper (Cu²⁺), nickel (Ni²⁺), cobalt (Co²⁺), or zinc (Zn²⁺) ions, which can for instance be presented by means of the chelator nitrilotriacetic acid (NTA).

[0071] The binding partner, for example a nucleic acid binding partner, used in the method according to the present invention, may be of any suitable length, hi some embodiments the binding partner is a nucleic acid molecule with a nucleic acid sequence of a length of about

7 to about 30 bp, for example a length of about 9 to about 25 bp, such as a length of about 10 to about 20 bp.

[0072] In some embodiments the binding partner is a PNA molecule. As indicated above, a PNA molecule is a nucleic acid molecule in which the backbone is a pseudopeptide rather than a sugar. Accordingly, PNA generally has a charge neutral backbone, in contrast to DNA or RNA. Nevertheless, PNA is capable of hybridising at least complementary and substantially complementary nucleic acid strands, just as e.g. DNA or RNA (to which PNA is considered a structural mimic).

[0073] Li the capillary microtubes of the monolithic solid any typical chromatographic stationary phase may also be immobilised. Examples include, but are not limited to, aliphatic amine groups, aliphatic acyl groups, aliphatic silane groups such as octadecylsilane chains, to perform open-channel chromatography or electrochromatography. Because the analytes to be separated have to diffuse to the walls to interact with the stationary phase, the diffusion distances, i.e. capillary dimensions, generally need to be small. The capillary diameter, typically less than 5 μm, is reduced considerably for improved interaction between the separated analytes and stationary phase. This avoids the use of micrometer-sized porous particles to pack a 50 - 75 μm diameter microtube, as this greatly increases the fluidic resistance, and high-performance pumps are hence needed. Detection problems that may be associated with narrow microtubes are addressed, by collecting signals from a bundle of microtubes, as arranged in the invention described here.

[0074] Any other dynamic or permanent immobilisation of matter known in the art for microchip devices such as microchip electrophoresis devices may be applied to any inner surface area of the capillary microchannels of the apparatus of the invention. The capillary microtubes of the monolithic solid may for example be modified by the functional groups, moieties, radiation (e.g. UV), grafting and other physical and/or chemical processes reviewed by Muck & Svatos (Talanta (2007) 74, 333-341).

[0075] As mentioned above, capillary action provides a means of avoiding or reducing the dependency on peripheral macro scale support infrastructures through reducing the dependency on external driving forces as for instance electrical currents, mechanical forces, pressure changes, or temperature differences. It is therefore no surprise that they have been explored extensively to control and/or direct the flow of fluid (see e.g. US patent application

03/0138941). Capillary forces result from surface affinities between matters and depend on material properties such as their surface chemistry, surface morphology and structure. The reduced structure scale of microdevices increases any effects of surface forces/tension and capillary actions. There is hence a potential to use such forces to deliver and enclose fluid in designated cavities for subsequent applications such as conduction of reactions under changing pressures and temperatures. Although surface tension is able to drive fluid flow without external forces, designing a system that relies completely on capillary forces for the indicated applications is a challenging task.

[0076] The present invention also relates to the use of a monolithic solid as defined above in the analysis of a single sample by capillary separation. The single sample is allowed to enter the capillary microtubes that are included in the monolithic solid via the first apertures thereof. As explained above, in typical embodiments capillary action is sufficient to allow the sample entering the microtubes. Likewise, the sample is typically capable of migrating along the lengths of the capillary microtubes to the second apertures thereof, in the absence of an additional pressure gradient. Surface tension at the advancing meniscus generally provides a sufficient force to cause the sample to enter the capillary microtubes, usually by flowing.

[0077] Without being bound by theory it is assumed that separation observed by the inventors occurs via interactions with the internal walls of the microtubes, via electrostatic charges or other surface charges, via interactions with binding partner molecules or via other non-covalent interactions such as dipole-dipole interactions. Non-covalent short- to long-range macroscopic scale interactions between different surfaces such as the surface of a molecule and the surface of a capillary tube include, but are not limited to, Lifshiz-van der Waals attractions, electrical double layer repulsion and electron-acceptor/electron-donor interactions (for an overview see e.g. van Oss, C.J., J. MoI Recognit. (2003) 16, 177-190). Contrary to standard chromatography techniques, which involve the flow of a sample in a mobile phase through a stationary phase, this use - as well as a corresponding method - of the invention does not require a continuous flow of a mobile phase.

[0078] In this regard the present inventors have found that nanochannels fabricated in a polymer such as polymethylmethacrylate (supra) are sensitive to surface charge. Ion transport in nanochannels is determined by the surface charge. This property can thus be exploited for sample separation. Using a monolithic solid that includes or consists of a polymer and in which the capillary microtubes are formed a sample can be separated according to the net charge of its components without applying an electric field.

[0079] In some embodiments the use and a corresponding method of the invention includes introducing the sample into a sample loading port of a capillary sample separation apparatus as defined above.

[0080] In some embodiments the capillary sample separation apparatus of the invention includes a plurality of monolithic solids. Each monolithic solid may include a plurality of capillary microtubes. Each monolithic solid may for instance be a photonic crystal fiber. In one such embodiment the sample separation apparatus may include one common sample loading port. The first apertures of the capillary microtubes included in each monolithic solid may be in fluid communication with this sample loading port. Such an arrangement may further increase the capacity and sensitivity of the apparatus in separation and detection. In one such embodiment the sample separation apparatus may include a plurality of sample loading ports. In such an embodiment the first ends of the capillary microtubes of each monolithic solid may be in fluid communication with one of the plurality of sample loading ports. The number of sample loading ports may match the number of monolithic solids included in the apparatus. In one such embodiment the apertures of the first ends of the microtubes of each monolithic solid are in fluid communication with a different sample loading port. In such embodiments the apparatus of the invention may be used to separate, for instance simultaneously or at independent points in time, a plurality of samples. Each monolithic solid may then be dedicated to the separation of an individual sample. Accordingly the methods and uses of the invention, as described above, may include separation of a plurality of samples. It should be noted that while previously described devices use n microtubes to separate n samples (n being an integer), the invention described here uses n pluralities of microtube bundles - with each plurality being included in a monolithic solid - for the separation of n samples. [0081] There is also potential for devices integrating the concepts described here to be connected in series with other analyte manipulating devices. For example, amplification of DNA samples by polymerase chain reaction (PCR) could be conducted on a thermal cycler designed to automatically feed amplified samples into an apparatus containing a plurality of microchannel bundles to electrophoretically separate the amplified samples. In this case, each sample would be fed to a separate bundle of microtubes and separation of each sample detected appropriately in parallel.

[0082] The monolithic integration of the capillary microtubes shown in Figure 8 is schematically depicted in Figure 9. Microtubes with heights of less than 100 nm can be micromachined in a PMMA substrate [Abgrall P., et al., Lab on a Chip (2007) 7, 520-522]. In contrast to the previously published technique [ibid.], a thin layer of polymeric material is first deposited on a carrier wafer. After machining the two-dimensional microchannel array on this polymeric layer, the whole layer is transferred to the device wafer. The layer is released from the carrier wafer by using sacrificial techniques or simply peeling it off. This layer transfer process, referred to here as lamination, can be repeated with an unlimited number of times to construct a three-dimensonal microchannel array. Access holes and large microtubes can be integrated during this lamination process. Further reducing the size of the microtube bundle can be realised by thermal stretching as depicted in Figure 13.

[0083] The invention is further illustrated by the following non limiting examples.

EXEMPLARY EMBODIMENTS OF THE INVENTION

[0084] In the present example electrophoretic separation was performed in the form of capillary electrophoresis in a photonic crystal fiber (PCF) which consists of a bundle of narrow microtubes, each microtube with a diameter of 3.7 μm. Microtubes with such small cross- section can sustain high electric field up to 1000 kV/cm and give better separations due to enhanced heat dissipation and smaller temperature differences across the cross-section of microtube. Furthermore, as the fluorescence signals are collected from all the microtubes within a bundle, the detection volume of this device is similar to that of a normal microchip.

[0085] PCF is originally made of silica glass with a microstructure of hollow channels that run along the cladding, confining light to the core [Chillcce, E. F., Cordeiro, C. M. B., Barbosa, L. C, Cruz, C. H. Brito, J. Non-Crystalline Solids (2006) 352, 3423-3428]. The scanning electron microscopy (SEM) images of the cross-section of the microtube bundle are shown in Figures IA and IB. The PCF (ESF-12-01, Blaze Photonics Limited, UK) had an outer diameter of 125 μm. There were 54 microtubes with a tube diameter of 3.7 μm each, giving a total cross-section area of 580.3 μm². The layout of the chip is presented in Figure IC A simple cross structure was engraved in polymethyl methacrylate (PMMA) substrate by CO2 laser ablation [Sun, Y., Kwok, Y. C, Nguyen, N. Ti, J. Micromech. Microeng. 2006, 16, 1681-1688]. It consisted of a 100 μm wide and 30 μm deep T-shaped injector for electrokinetic sample injection and a 200 μm wide and 150 μm deep straight channel right after the T-shaped injector for the insertion of PCF. Access holes were also drilled to form reservoirs. To guarantee that the sample could only flow through the bundle of microtubes, before the insertion, a few drops of polydimethylsiloxane (PDMS) (Best Chemicals, Singapore) was dripped along the straight channel. PDMS is a viscous liquid at room temperature so that it conforms well to the shape of the microtube. After a 0.5 cm long PCF was inserted, the entire substrate was baked at 60 ⁰C on a hotplate for 30 minutes, and the solidified PDMS completely blocked the straight PMMA microtube. To form a closed microfluidic apparatus, the substrate was then covered by a layer of adhesive tape (Adhesives Research Inc., Glen Rock, PA). The performance of the microtube bundle was compared to a commercially available glass microchip (X8050, Micronit Microfluidic BV, The Netherlands). The microtube was 50 μm wide and 20 μm deep. Due to the nature of isotropic etching, the cross-sectional area of the glass microtube was approximately 600 μm², comparable to that of the sum of 54 capillary microtubes of the microtube bundle. Using the current monitoring technique, the electro-osmotic mobility of the microtube bundle and the glass microchip were measured to be 4.46 x 10^~4 cm²/V-s and 4.52 x 10"⁴ cm²/V-s, respectively, which were also very similar.

[0086] To examine the flow in separation tubes, both the capillary bundle and the glass microchip were loaded with 1 μM fluorescein (Invitrogen, CA, USA) in 0.16 xTris-borate- EDTA (TBE; Sigma Chemical, MO, USA). The two insets in Fig. 3 are fluorescent images taken near the T-shaped injector region by an inverted microscope (BX51, Olympus, Japan). For the glass microchip, the whole tube was filled with fluorescein solution. While for the capillary bundle, fluorescein was well confined in the capillaries and there was no leakage to the PMMA microtube, indicating the microtube was completely sealed by PDMS and electrophoretic separation will be independently carried out in each narrow capillary.

[0087] The capability to dissipate the Joule heat was investigated by monitoring the electrical current as a function of Hie applied field strength. Conductivity measurements are temperature dependent. The degree to which temperature affects conductivity can be calculated using the following formula: σ_τ = σ_Tcal ( + α( T - T_cal )) (4) where σ^ is conductivity at any temperature T in ⁰C, σ_jcai is conductivity at calibration temperature T_cai in ⁰C, α is the temperature coefficient of the solution at T_cai in ⁰C. Here, 0. IxTBE buffer was used as running buffer and electrophoresis was performed by applying electric fields from 100 V/cm to 1500 V/cm in 100 V/cm increments. Ohm's plots for both chips are summarized in Figure 3. The current in the glass microchip is generally higher than that in microtube bundle, indicating a higher temperature inside the lumen of glass microtube as electric conductivity of solution would increase with temperature. For the glass microchip, the Ohm's plot deviates from linearity at electric field larger than 700 V/cm. For the microtube bundle, the deviation from linearity sets in at an electric field larger than 1100 V/cm. This difference demonstrated that by introducing a monolithic solid with a plurality of capillary microtubes, the effective range of separation electric field strength is greatly increased. As mentioned earlier, Joule heating is dissipated via the tube surface. From Eq. (1) above, the SVR of the bundle of capillary tubes is 1.1 μm"¹, around eight times larger than that of glass microchip which is 0.15 μrrr¹. Therefore, the monolithic solid with the bundle capillary microtubes can dissipate Joule heat more effectively and thus be able to sustain much higher electric field strengths.

[0088] CE separation of a mixture of 100 nM fluorescein and 200 nM Rhodamine 123 (Invitrogen Ltd, California, USA) was carried out with an inverted confocal microscope (TCS SP2, Leica, Germany) as the detection apparatus [Chillcce, E. F., et al., J. Non-Crystalline Solids (2006) 352, 3423-3428]. The voltages for the four reservoirs were generated by a high voltage power supply (MCP 468, CE Resources, Singapore). Figures 4A to 4F are the electropherograms obtained at various electric field strengths, i.e., 700, 850 and 1000 V/cm, with the glass microtube and the microtube bundle. The corresponding electrophoretic data are summarized in Table 1. As the glass microchip can only sustain an electric field strength of 600 V/cm, Joule heat generated at high electric field caused severe sample peak dispersion and band broadening, which greatly reduced separation efficiency and analysis resolution. The adverse effect of Joule heating on the separation efficiency is more pronounced at higher electric field, e.g. 1000 V/cm. Under the same electric field, the apparatus of the invention with a capillary microtube bundle always provided better performance. Compared to the glass microchip, the improvement in resolution is 20%, 40% and 60% for 700 V/cm, 850 V/cm and 1000 V/cm, respectively. This is due to the higher efficiency of heat dissipation in the microtube bundle. Thus, the microtube bundle can be used at very high electric fields to achieve fast separation with high resolution.

[0089] In a further example CE separation of the 11 -fragment ΦX174-Hae III dsDNA (Invitrogen Ltd, California, USA) was carried out using both the capillary microtube bundle and the glass microchip with an inverted confocal microscope (TCS SP2, Leica, Germany) as the detection apparatus [Sun, Y., Kwok, Y. C, Nguyen, N. T., J. Micromech. Microeng. 2006, 16, 1681-1688]. The voltages for the four reservoirs were generated by a high voltage power supply (MCP 468, CE Resources, Singapore). 1.5% hydroxypropylcellulose (HPC) in 80 mM MES/40 mM TRIS buffer was prepared as the separation medium. For DNA separation, an electric field of 500 V/cm was applied across the buffer and the buffer waste reservoirs.

[0090] The flow profile of DNA sample in PCF during separation was investigated by taking fluorescent images at the point 1 cm from the buffer waste reservoir. Figure 5 shows that DNA fragments of the same size moved at the same speed in all the microtubes, and there was no variation in migration velocities which would otherwise lead to broader bands and reduced resolution. In the capillary bundle, the DNA sample was well confined in the capillary tubes and there was no leakage to the PMMA microchannel, indicating that the PCF was completely sealed by PDMS and electrophoretic separation was independently carried out in each narrow capillary.

[0091] Figures 6A and 6B show the electropherograms obtained in the glass microchip and the capillary bundle chip, respectively. As seen from the results, for the glass microchip, fragments 271/281 as well as 1078/1353 were unable to be separated. Moreover, migration times for each fragment were relatively shorter than those in the chip with the monolithic solid that included a capillary bundle. This provided evidence of inefficient heat dissipation in the glass microchip as higher temperature would result in a corresponding decrease in the viscosity of the HPC matrix and thus faster migration velocities of the DNA fragments. The lower viscosity directly led to deformed sieve size and reduced resolving power of the sieving matrix. Since the quality of DNA separation largely depends on the matrix, the decreased viscosity of separation medium caused by Joule heat adversely affected separation efficiency and analysis resolution in the glass microchip. In contrast thereto, under the same electric field strength, a remarkable improvement in resolution was obtained using the chip with the monolithic solid that included a capillary bundle. This could be attributed to the relatively larger SVR of the plurality of capillary microtubes and the better controlled Joule heating and temperature gradients, consequently, viscosity as well as resolving power of sieving matrix were retained at higher voltage. The enhanced resolution obtained using the plurality of capillary microtubes would facilitate its wide applicability for DNA separation in various fields.

[0092] A CE microchip with a microchannel bundle for high performance electrophoretic separation was demonstrated. PCFs including 54 narrow capillary tubes were used as the separation column. Compared to a normal glass microchip with a similar cross-section area, the capillary bundle had a much larger SVR. Because of the efficient heat dissipation, the microchip could sustain an electric filed strength as high as 1000 V/cm and improved separation results were attained. As the PCF is commercially available and the fabrication process to encapsulate PCF to PMMA substrate is straightforward, this simple and novel concept could be widely applied to chip-based CE for enhanced performance.

[0093] The listing or discussion of a previously published document in this specification should not necessarily be taken as an acknowledgement that the document^' is part of the state of the art or is common general knowledge.

[0094] The invention illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms "comprising", "including," containing", etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by exemplary embodiments and optional features, modification and variation of the inventions embodied therein herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention.

[0095] The invention has been described broadly and genetically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

[0096] Other embodiments are within the following claims. In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.

Claims

ClaimsWhat is claimed is:

1. A capillary sample separation apparatus comprising a monolithic solid, wherein the monolithic solid comprises a first lateral wall and a second lateral wall, the first lateral wall of the monolithic solid being arranged in opposing relationship with the second lateral wall thereof, wherein the monolithic solid comprises a plurality of at least substantially parallel capillary microtubes, each capillary microtube having a first end with a first aperture and a second end with a second aperture, the first end being arranged in the first lateral wall of the monolithic solid and the second end being arranged in the second lateral wall of the monolithic solid, such that all microtubes of the plurality of capillary microtubes are fluidly coupled, and wherein the first aperture of each capillary microtube is in fluid communication with a common sample loading port, and the second aperture of each capillary microtube is in fluid communication with a common reservoir.

2. The capillary sample separation apparatus of claim 1, wherein the sample loading port and the reservoir are comprised in the capillary sample separation apparatus.

3. The capillary sample separation apparatus of claims 1 or 2, further comprising an electrode, wherein the first aperture of each capillary microtube comprised in the monolithic solid is in fluid communication with the electrode.

4. The capillary sample separation apparatus of claims 1 or 2, further comprising an electrode, wherein the second aperture of each capillary microtube comprised in the monolithic solid is in fluid communication with the electrode.

5. The capillary sample separation apparatus of claims 3 or 4, further comprising a power supply unit for applying a voltage to the electrode.

6. The capillary sample separation apparatus of any one of claims 3 - 5, comprising a pair of electrodes, wherein the electrodes are arranged at a distance from one another, and wherein the monolithic solid is arranged in between the pair of electrodes.

7. The capillary sample separation apparatus of claim 6, wherein the pair of electrodes comprises an anode and a cathode, and wherein the first lateral wall of the monolithic solid is opposing the anode and the second lateral wall of the monolithic solid is opposing the cathode.

8. The capillary sample separation apparatus of any one of claims 1 -7, wherein each capillary microtube comprised in the monolithic solid has one single inlet, the single inlet being comprised in the first end of the capillary microtube.

9. The capillary sample separation apparatus of any one of claims 1 -8, wherein each capillary microtube comprised in the monolithic solid has one single outlet, the single outlet being comprised in the second end of the capillary microtube.

10. The capillary sample separation apparatus of any one of claims 1 - 9, wherein the capillary microtubes comprised in the monolithic solid are of about the same lengths.

11. The capillary sample separation apparatus of any one of claims 1 - 10, wherein at least some of the capillary microtubes comprised in the monolithic solid are of cylindrical shape.

12. The capillary sample separation apparatus of any one of claims 1 - 11, wherein at least an substantial portion of at least some of the capillary microtubes have a cross-section of a shape selected from the group of ovoid shape, at least substantially circular shape and the shape of a polyhedron.

13. The capillary sample separation apparatus of any one of claims 1 - 12, wherein at least some of the capillary microtubes comprised in the monolithic solid have a maximal width of about 5 μm or below.

14. The capillary sample separation apparatus of claim 13, wherein the maximal width of the capillary microtubes is from about 5 nm to about 200 run.

15. The capillary sample separation apparatus of claim 14, wherein the maximal width of the capillary microtubes is from about 10 nm to about 100 nm.

16. The capillary sample separation apparatus of any one of claims 13 - 15, wherein the maximal width of the capillary microtubes is small enough to prevent Joule heating from at least substantially affecting sample separation at a selected electric field.

17. The capillary sample apparatus apparatus of any one of claims 1 - 16, wherein the maximal width of the capillary microtubes is small enough to allow capillary electrophoresis to be carried out at at least 1000 V /cm.

18. The capillary sample separation apparatus of any one of claims 1 - 17, wherein at least some of the capillary microtubes comprised in the monolithic solid have a length of about 0.1 cm, or about 0.5 cm, or about 1 cm, or above.

19. The capillary sample separation apparatus of any one of claims 1 - 18, wherein the capillaries in the monolithic solid are arranged at a distance from each other.

20. The capillary sample separation apparatus of claim 19, wherein the distance separating adjacent capillaries is selected in the range from about 1 nm to about 10 mm.

21. The capillary sample apparatus apparatus of claims 19 or 20, wherein the capillary microtubes in the monolithic solid are arranged at an at least substantially equal distance to each other, thereby defining a line or an at least substantially regular polygon.

22. The capillary sample separation apparatus of any one of claims 1 - 21, further comprising means for filling the plurality of capillary microtubes with a separation medium.

23. The capillary sample separation apparatus of claim 22, wherein the means for filling the plurality of capillary microtubes with a separation medium is one of a pump and a geometric element of a substrate in or on which the monolithic solid is comprised.

24. The capillary sample separation apparatus of claim 23, wherein the geometric element comprises a micro capillary channel.

25. The capillary sample separation apparatus of any one of claims 1 - 24, wherein the capillary sample separation apparatus comprises a microfluidic device and wherein the monolithic solid is arranged on or within the microfluidic device.

26. The capillary sample separation apparatus of claim 25, wherein the sample loading port and the reservoir are arranged in or on the microfluidic device.

27. The capillary sample separation apparatus of claims 25 or 26, wherein an electrode is arranged in or on the microfluidic device.

28. The capillary sample separation apparatus of any one of claims 1 - 27, wherein the monolithic solid comprises one of a metal, a metalloid, ceramics, a metal oxide, a metalloid oxide, oxide ceramics, carbon, a polymer or a combination thereof.

29. The capillary sample separation apparatus of any one of claims 1 -28, wherein the monolithic solid is defined by a photonic crystal fiber.

30. The capillary sample separation apparatus of any one of claims 1 - 29, comprising a plurality of monolithic solids, each monolithic solid comprising a plurality of capillary microtubes.

31. The capillary sample separation apparatus of claim 30, wherein each monolithic solid is defined by a photonic crystal fiber.

32. The capillary sample separation apparatus of claims 30 or 31, further comprising a plurality of sample loading ports, wherein the first ends of the capillary microtubes of each monolithic solid are in fluid communication with one of the plurality of sample loading ports.

33. The capillary sample separation apparatus of claim 32, comprising a number of sample loading ports matching the number of monolithic solids, wherein the apertures of the first ends of the microtubes of each monolithic solids are in fluid communication with a different sample loading port.

34. The use of a monolithic solid to expose a single sample to capillary electrophoresis, wherein the monolithic solid comprises a first lateral wall and a second lateral wall, the first lateral wall of the monolithic solid being arranged in opposing relationship with the second lateral wall, wherein the monolithic solid comprises a plurality of at least substantially parallel capillary microtubes, each capillary microtube having a first end with a first aperture and a second end with a second aperture, the first end being arranged in the first lateral wall of the monolithic solid and the second end being arranged in the second lateral wall of the monolithic solid, such that all microtubes of the plurality of capillary microtubes are fluidly coupled.

35. The use of claim 34, wherein the sample is selected from the group consisting of a soil sample, an air sample, an environmental sample, a cell culture sample, a bone marrow sample, a rainfall sample, a fallout sample, a space sample, an extraterrestrial sample, a sewage sample, a ground water sample, an abrasion sample, an archaeological sample, a food sample, a blood sample, a serum sample, a plasma sample, a urine sample, a stool sample, a semen sample, ,a lymphatic fluid sample, a cerebrospinal fluid sample, a naspharyngeal wash sample, a sputum sample, a mouth swab sample, a throat swab sample, a nasal swab sample, a bronchoalveolar lavage sample, a bronchial secretion sample, a milk sample, an amniotic fluid sample, a biopsy sample, a nail sample, a hair sample, a skin sample, a cancer sample, a tumour sample, a tissue sample, a cell sample, a cell lysate sample, a virus culture sample, a forensic sample, an infection sample, a nosocomial infection sample, a production sample, a drug preparation sample, a biological molecule production sample, a protein preparation sample, a lipid preparation sample, a carbohydrate preparation sample, a solution of a nucleotide, a solution of polynucleotide, a solution of a nucleic acid, a solution of a peptide, a solution of a polypeptide, a solution of an amino acid, a solution of a protein, a solution of a synthetic polymer, a solution of a biochemical composition, a solution of an organic chemical composition, a solution of an inorganic chemical composition, a solution of a lipid, a solution of a carbohydrate, a solution of a combinatory chemistry product, a solution of a drug candidate molecule, a solution of a drug molecule, a solution of a drug metabolite, a suspension of a cell, a suspension of a virus, a suspension of a microorganism, a suspension of a metal, a suspension of metal alloy, a solution of a metal ion, and any combination thereof.

36. The use of claims 34 or 35, wherein the monolithic solid is defined by a photonic crystal fiber.

37. The use of a monolithic solid in the analysis of a single sample by capillary separation, wherein the monolithic solid comprises a first lateral wall and a second lateral wall, the first lateral wall of the monolithic solid being arranged in opposing relationship with the second lateral wall, wherein the monolithic solid comprises a plurality of at least substantially parallel capillary microtubes, each capillary microtube having a first end with a first aperture and a second end with a second aperture, the first end being arranged in the first lateral wall of the monolithic solid and the second end being arranged in the second lateral wall of the monolithic solid, such that all microtubes of the plurality of capillary microtubes are fluidly coupled, wherein the single sample is allowed to enter the capillary microtubes comprised in the monolithic solid via the first apertures thereof, and to migrate along the lengths of the capillary microtubes to the second apertures thereof, in the absence of an additional pressure gradient.

38. The use of claim 37, wherein the sample comprises macromolecules or ions.

39. The use of claims 37 or 38, wherein the monolithic solid is defined by a photonic crystal fiber.

40. A method of forming a capillary sample separation apparatus, the method comprising providing a microfluidic device, forming thereon a sample loading port and a reservoir, and arranging thereon a monolithic solid, wherein the monolithic solid comprises a first lateral wall and a second lateral wall, the first lateral wall of the monolithic solid being arranged in opposing relationship with the second lateral wall, wherein the monolithic solid comprises a plurality of at least substantially parallel capillary microtubes, each capillary microtube having a first end with a first aperture and a second end with a second aperture, the first end being arranged in the first lateral wall of the monolithic solid and the second end being arranged in the second lateral wall of the monolithic solid, such that all microtubes of the plurality of capillary microtubes are fluidly coupled, and thereby bringing the first apertures of the capillary microtubes in fluid communication with the sample loading port and the second apertures of the capillary microtubes in fluid communication with the reservoir.

41. The method of claim 40, wherein applying the monolithic solid comprises monolithic or hybrid integration.

42. The method of claims 40 or 41, further comprising applying thermal stretching along the length of the capillary microtubes, thereby reducing the width of the capillary microtubes.

43. The method of any one of claims 40- 42, further comprising arranging on the microfluidic device an electrode, thereby bringing the first aperture of each capillary microtube in fluid communication with the electrode.

44. A method of subjecting a sample to capillary separation, comprising introducing the sample into a sample loading port of a capillary sample separation apparatus, wherein the capillary sample separation apparatus comprises a monolithic solid, the monolithic solid comprising a first lateral wall and a second lateral wall, the first lateral wall of the monolithic solid being arranged in opposing relationship with the second lateral wall, wherein the monolithic solid comprises a plurality of at least substantially parallel capillary microtubes, each capillary microtube having a first end with a first aperture and a second end with a second aperture, the first end being arranged in the first lateral wall of the monolithic solid and the second end being arranged in the second lateral wall of the monolithic solid, such that all microtubes of the plurality of capillary microtubes are fiuidly coupled, wherein the first aperture of each capillary microtube comprised in the monolithic solid is in fluid communication with the sample loading port, and the second aperture of each capillary microtube is in fluid communication with a common reservoir, and allowing the sample to enter the capillary microtubes comprised in the monolithic solid via the first apertures thereof, and to migrate along the lengths of the capillary microtubes to the second apertures thereof, without applying an additional pressure gradient.

45. The method of claim 44, wherein the monolithic solid is defined by a photonic crystal fiber.

46. The method of claims 44 or 45, wherein the sample comprises macromolecules and wherein the method is a method of analytically separating the macromolecules.

47. The method of any one of claims 44-46, wherein allowing the sample to enter the capillary microtubes and to migrate along the lengths thereof comprises applying an electrical field along the lengths of the capillary microtubes.

48. The method of claim 47, wherein the electrical field is applied by an electrode.

49. A method of subjecting a sample to capillary electrophoresis, comprising introducing the sample into a sample loading port of a capillary sample separation apparatus, wherein the capillary sample separation apparatus comprises a monolithic solid, the monolithic solid comprising a first lateral wall and a second lateral wall, the first lateral wall of the monolithic solid being arranged in opposing relationship with the second lateral wall, wherein the monolithic solid comprises a plurality of at least substantially parallel capillary microtubes, each capillary microtube having a first end with a first aperture and a second end with a second aperture, the first end being arranged in the first lateral wall of the monolithic solid and the second end being arranged in the second lateral wall of the monolithic solid, such that all microtubes of the plurality of capillary microtubes are fluidly coupled, wherein the first aperture of each capillary microtube comprised in the monolithic solid is in fluid communication with the sample loading port, and the second aperture of each capillary microtube is in fluid communication with a common reservoir, and applying an electrical field along the lengths of the capillary microtubes.

50. The method of claim 49, wherein the electrical field is applied by an electrode.

51. The method of claim 50, wherein the first or the second aperture of each capillary microtube comprised in the monolithic solid is in fluid communication with the electrode.

52. The method of one of claims 49 to 51, wherein the monolithic solid is defined by a photonic crystal fiber.

53. The method of any one of claims 49 - 52, wherein the sample comprises macromolecules and wherein the method is a method of analytically separating the macromolecules.

54. The method of claim 53, wherein the macromolecules are selected from the group consisting of polypeptides, oligonucleotides, polynucleotides, carbohydrates, lipids, and organic molecules.