WO2007050040A1

WO2007050040A1 - Immobilization unit and device for isolation of nucleic acid molecules

Info

Publication number: WO2007050040A1
Application number: PCT/SG2005/000374
Authority: WO
Inventors: Hongmiao Ji; Levent Yobas; Yu Chen; Wing Cheong Hui; Chew Kiat Heng; Tit Meng Lim
Original assignee: Agency For Science, Technology And Research; National University Of Singapore
Priority date: 2005-10-28
Filing date: 2005-10-28
Publication date: 2007-05-03
Also published as: WO2007050040A8

Abstract

An immobilization unit (820) for the detection of dexoyribonucleic acid (DNA) and ribonucleic acid (RNA) molecules capable of immobilizing nucleic molecules, said immobilization unit comprising a channel of a given depth, width and length and having at least one surface. The surface is adapted to immobilize nucleic molecules under suitable conditions. The channel has at least one straight portion and at least one meandering portion. The at least one meandering portion is essentially u-shaped and the at least one straight portion is an arm of the u-shaped meandering portion.

Description

Immobilization unit and device for isolation of nucleic acid molecules

TECHNICAL FIELD

[001] The present invention relates to immobilization units that can be used for isolating, purifying and extracting nucleic acid molecules such as deoxyribonucleic acid (DNA) and/or ribonucleic acids (RNA). The invention also relates a device for isolating, purifying and extracting ribonucleic acids (RNA) in which such an immobilization unit incorporated. The invention also relates to a corresponding method of purifying and extracting ribonucleic acids by means of an immobilization unit and device as described herein.

BACKGROUND TO THE INVENTION

[002] Recent epidemics such as Severe Acute Respiratory Syndrome (SARS) or the imminent threat from influenza viruses such as the bird flu virus, for example, highlight the importance of having an automated sample preparation for virus and pathogen detection. Methods such as immunofluorescence assays (IFA) or enzyme-linked immunosorbent assays (ELISA) are often used in clinical settings. These tests, however, may fail to detect the viral RNA at the early stage due to low viral load or pre-seroconversion in a sample. Lack of indicative clinical symptoms at the early stage can also contribute to the disease progress going undetected. Further challenges facing the immunoassays include relatively long incubation times, and false positives due to cross-reactivities.

[003] As an alternative to using assays, molecular techniques involving detection of viral RNA in the sample could lead to early diagnosis of the disease before it becomes symptomatic. This would be beneficial during epidemics and pandemics to isolate and quarantine the confirmed cases earlier and hence contain the further spreading of the disease.

[004] Detection of viral nucleic acid, however, demands tedious laboratory procedures to prepare the sample and dealing with many repetitive steps of pipetting and centrifugations. Extensive handling of potentially infectious samples poses serious risk to the laboratory staff and environment. Detection of extracellular viruses from blood usually requires the separation of plasma or serum containing virus particles from other cellular components, such as red blood cells (RBC), for example. This is because hemoglobin from RBC is known to inhibit nucleic acid amplification while nucleic acids in white blood cells (WBC) can contribute to background noise during the detection phase. By sample preparation, the steps of detecting, identifying, isolating and purifying are meant.

[005] Typically, plasma is obtained from whole blood by a centrifugation step. However, doing so requires the inclusion of the centrifugation step followed by the transference of the plasma from the centrifuge to the detecting device. Accordingly, the transference step may cause the sample or the user to be susceptible to possible contamination especially in the case where viral RNA is being detected.

[006] A portable system which is automated and self-contained would analyze the suspected samples in the field without the need for transporting them to the laboratories. Sample preparation in such portable system could leverage from microfluidics to speed up biochemical reactions and reduce cost per test by saving reagents.

[007] The Biosensor Focus Interest Group (BFIG) in Singapore, following the outbreaks of viral diseases in Asia (e.g. SARS and Avian flu), has developed such a system. The purpose of the system is to minimize user's contact with the infected sample through miniaturization and automation. The majority of viral infections can be confirmed through serological immunoassays in which the patient's blood sample is screened against either specific viral antigens or antibodies mounted by the host against the viral pathogen.

[008] hi addition, U.S. Patent 5,234,809 describes a process for isolating nucleic acid. The process requires a user to mixing the initial test sample with a chaotropic substance and a nucleic acid binding solid phase. Subsequently, the solid phase is separated from the nucleic acid bound thereto from and the process concludes with a washing phase of the solid phase nucleic acid complexes. However, the process described in U.S. Patent 5,234,809 requires several individual steps to be carried out thereby increasing the risk of contamination to the test sample or to the user, should the test sample contain virulent strands of viral RNA.

[009] A nucleic acid purification chip and process of isolating nucleic acids, for example, wherein mixing and the detection reaction do not require human interference, is disclosed in International Patent Application WO 2005/066343 and in Kim at el., Proc. of the IEEE Conference on MEMS 2002, Las Vegas, 15, 2002, pages 133 - 136.

[0010] Despite these more recent developments, there is a still need for a device that is capable of isolating, detecting and purifying a sample of viral RNA from a test sample such as blood, for example. There is also a need for such a device that is both cost- effective and easy to fabricate.

SUMMARY OF THE INVENTION

[0011] Accordingly, an aspect of the invention provides for an immobilization unit for the isolation of nucleic acid molecules such as deoxyribonucleic acid (DNA) and/or of ribonucleic acid (RNA) molecules capable of immobilizing DNA and/or RNA molecules. The immobilization unit comprises (or consists of) a channel of a given depth, width and length and having at least one surface, which is adapted to immobilize DNA and/or RNA molecules under suitable conditions, wherein the channel has at least one straight portion and at least one meandering portion, wherein the at least one meandering portion is essentially u-shaped and the at least one straight portion is an arm of the u-shaped meandering portion.

[0012] Another aspect according to the invention provides an immobilization unit for the isolation of nucleic acid molecules such as deoxyribonucleic acid (DNA) and/or ribonucleic acid (RNA) molecules capable of immobilizing such nucleic molecules, said immobilization unit comprising a channel of a given depth, width and length having at least one surface, said surface being adapted to immobilize nucleic acid molecules under suitable conditions, wherein the channel is essentially formed to route any fluid flowing therein, in a substantially spiral flow pattern.

[0013] Another aspect of the invention relates to a device for the isolation of nucleic acid molecules comprising an immobilization unit and a corresponding method of detecting anucleic aicd molecule, said method comprising contacting a liquid sample suspected to contain nucleic acid with the immobilization unit as mentioned above, hi one such embodiment, the device of the invention is a device for isolating viral RNA. [0014] In this regard, an immobilization unit, a device incorporating said immobilization unit and a method of use thereof, as defined in the appended claims, provides a DNA or RNA detection device that is cost effective and is capable of reproducing accurate results. The various embodiments of the present invention are described below.

DETAILED DESCRIPTION OF THE INVENTION

[0015] The immobilization unit, as mentioned above, for the isolation of deoxyribonucleic acid (DNA) and/or ribonucleic acid (RNA) molecules capable of immobilizing DNA or RNA molecules includes a channel of a given depth, width and length. The channel has at least one surface that is adapted to immobilize ribonucleic acid molecules under suitable conditions. The channel itself includes at least one straight portion and at least one meandering portion. The at least one meandering portion is essentially u-shaped and the at least one straight portion is an arm of the u-shaped meandering portion.

[0016] The essentially u-shaped meandering portion is not strictly restricted to being u- shaped. The meandering portion may be, for example, also v-shaped or have its bends at right angles instead of smooth curves. Accordingly, the term "u-shaped" essentially refers to a portion of the channel of the immobilization unit that can direct or vary the direction of flow of a fluid sample by from about 45 degrees to about 180 degrees, thus including, for example also about 90 degrees. In case of a meandering portion that is shaped such that is provides for a u-turn of about 180 degrees, the direct of flow of a fluid to be analyzed is essentially reversed. The various alternative embodiments with regard to the shape of the meandering portion will be further discussed in the illustrations below.

[0017] In the immobilization unit the straight portion forms an arm of the meandering portion. The arm formed by the straight portion may either be at the begώning of the end of the meandering portion. As an illustrative example, in the u-shaped meandering portion, two straight portions bound the curved portion on either side. However, in one embodiment, the curved portion may be bounded only on one side by a straight portion. Accordingly, in all embodiments described herein, the arm of any of the meandering portions is to be taken as being formed by a straight portion. [0018] In this regard, straight portion is taken to mean that the said portion is substantially straight (with respect to the direction of the fluid flow) and does not cause any significant or substantial variation in fluid flow direction as said fluid travel along the channel. Accordingly, a straight portion may not change the overall direction of flow of a fluid sample while traveling through that straight portion or only to a minor extent, for example up to 10, or 20 degrees. The edges of the straight portion may not be perfectly parallel to each other, but can also be substantially parallel to each other and thus may narrow or widen along the length of the straight portion. In addition, the edges may not be straight but also, for example, be corrugated or serrated, as long as they overall still retain a high degree of parallelism with respect to each other. In one embodiment, the straight portion may also split into a plurality (two or more) sub-channels and converge back into a single channel prior to entering the meandering portion of the immobilization unit.

[0019] In an alternative embodiment, the (channel of the) immobilization unit may include a plurality of meandering portions. In such an embodiment, each of the at least one straight portion couples a pair of meandering portions to one another. As an exemplary embodiment, the immobilization unit may include at least two meandering portions. Ih this exemplary embodiment, at least one straight portion couples one end of the first meandering portion to another end of the second meandering portion.

[0020] Apart from having a plurality of meandering portions, the immobilization unit may also include a plurality of straight portions (for example, but by no means limited to, 3, 4, 5, 6 or 7), such that each straight portion couples two meandering portions to each other, as described previously.

[0021] The at least one surface of the channel of the immobilization unit comprises or consists of a material that is adapted to provide for or enhance (reversible) binding affinity witii DNA or RNA molecules. The material of the immobilization unit may have inherent binding affinity towards nucleic acid molecules, including ribonucleic acids, under the conditions chosen for isolating and purifying such molecules. For example, nucleic acid molecules such as DNA or RNA bind to silica in high salt concentration of chaotropic salts such as guanidine hydrochloride or perchlorate and elute in low salt conditions (see for example, Melzak et al., "Driving forces for DNA absorption to silica in perchlorate solutions, J. Colloid and Interface Science, Vol. 181, pp. 635-644,1996, Kim at el., Proc. of the IEEE Conference on MEMS 2002, supra, or WO 2005/066343). Accordingly, at least one surface of the channel or also the entire channel of title immobilization unit may comprise or be made of a silica material such a glass, for example, normal glass or photosensitive glass as described in Kim et al, Proc. of the IEEE Conference on MEMS 5 2002, supra. The at least one surface of the channel may also comprise silica beads, or a (ribo)nucleic acid binding material such as silanes, polylysine, tethered antibodies or poly T DNA molecules. In another embodiment, the at least surface of the channel that has affinity to (ribo)nucleic acid molecules may be obtained from silicon as follows (see also WO 2005/066343). As a first method, a bare silicon wafer (surface) can be oxidized by 0 thermal oxidation to a suitable thickness of for example 0,5 μm. Alternatively, the thermal oxide treatment can be followed or combined with a treatment with a solution of hydrogen peroxide/sulfuric acid ("Piranha", comprising a 3:1 mixing ratio of cone. H₂SO₄: 30% H₂O₂). Another suitable approach for rendering a silicon surface able to bind nucleic acids such as DNA and RNA is a thermal oxide treatment used in combination with subsequent plasma etching. The plasma etching may comprise the use of a tetrafluormethane (CF₄), triflourmethane (CHF₃) or (O₂) atmosphere or an atmosphere comprising CF₄ and/or CHF₃ together with oxygen (O₂). The surface having affinity to nucleic acid molecules can also be obtained from plasma enhanced chemical vapour deposition (PEVCD) of silane based silicon oxides. Such processes generate a surface that is very suitable for nucleic acid binding and, if wanted, also elution. Also, using such kind of surface, there are no further process steps required for the modification of a surface of a silicon chip, if the immobilization unit is part of a silicon chip that is used for nucleic acid isolation and subsequent detection, for example. It is noted in this conjunction that a plasma treatment step can be carried out during the wafer front side nitride stripping process which is a usual step in a fabrication of such a chip anyway (see in this regard WO 2005/066343, for example). Irrespective of the method used to provide the surface with the ability to bind nucleic acid molecules, in a final step, the surface treatment can comprise contacting the surface with distilled or deionized water. If the immobilization unit is made from a semiconductor substrate/chip, this treatment (washing) with distilled water is carried out after the substrate (into which the channel of the immobilization, and optionally also other functional unit of a device as described herein, have been formed) has been bonded to a suitable cover. [0022] The channel of the immobilization unit may be of any depth and width that are suitable for the intended use of the immobilization unit. Thus, the depth and width may differ if the immobilization unit is used in/as a microfluidic system or on a conventional macroscale. If the immobilization unit is to be used as microfluidic device on its own or is part of a microfluidic chip, the depth of the channel is in one exemplary embodiment approximately between about 50 to about 500 micrometers. Similar, if the immobilization unit is a microfluidic device or integrated into a microfluidic chip, the width of the channel is approximately between about 50 to about 500 micrometers. As a general guideline, the dimensions of the channel should, irrespective of the context in which the immobilization is used, such that the width to depth ratio of the channel should be approximately between about 0.1 to about 10. Generally, in order to provide a binding channel of suitable length for detecting DNA or RNA, the length traversed by a fluid in the channel of the immobilization unit can approximately be between about 10,000 to about 100,000 micrometers.

[0023] The width and the depth of the channel can vary along the length of the channel. In this regard, the channel may also narrow or widen to increase or decrease the flow rate respectively. Irrespective of the width of the channel, the depth may also vary along the length of the channel. If used for microfluidic applications, it is useful that the overall dimensions of the channel maintain within in the above-mentioned ratio between the width and depth.

[0024] In a further embodiment of the invention, the channel of the immobilization unit may be essentially formed such that it routes a fluid flowing therein in a substantially spiral flow pattern. Such an immobilization unit may comprise one or more essentially spirally formed channels. It may, for example, have the shape of a micromixer that is described for mixing of fluids in the International patent applications WO 2004/108261 and WO 2005/066343. In other embodiments, the immobilization unit may have the shape as the mixer that is described below, in which the channel is formed as two spirals connected to each other, wherein the inlet channel of the immobilization unit spirals inwards to a central point in a clockwise direction, and then enters an outlet channel which spirals in an anticlockwise manner circumferentially outwards. [0025] Again, as with the above-mentioned embodiments, at least one surface of such an embodiment of the immobilization unit comprises or consists of a material that is adapted to provide for or enhance the binding affinity with DNA or RNA molecules. The material of this immobilization unit may have inherent binding affinity towards nucleic acid molecules, including ribonucleic acids, under the conditions chosen for isolating and purifying such molecules.

[0026] Also in embodiments of the immobilization wherein the channel provides for an essentially spiral fluid pattern, the immobilization unit may be of any depth and width that are suitable for the intended use of the immobilization unit. In some embodiments, for example, but not limited to the one where the immobilization unit is part of a microfluidic chip, the dimensions of the depth and width lie within the range of about 50 to about 500 micrometers respectively. Essentially, the same width to depth ratio is typically maintained. That is a width to depth ratio approximately between about 0.1 to about 10. As above, the length traversed by a fluid in the channel of the immobilization unit may approximately be, but is not limited to, between about 10,000 to about 100,000 micrometers. The width and depth of the channel that formed to route any fluid flowing therein in a substantially spiral flow pattern may not be constant by can vary. In other words, the dimensions along the channel may vary and may not be uniform throughout.

[0027] A further aspect of the present invention is a device for the isolation, purification and extraction of deoxyribonucleic acid (DNA) and/or ribonucleic acid (RNA) molecules. The device is essentially a DNA or RNA purification or isolation chip having at least one of the immobilization units as described above. In addition to the immobilization unit, the DNA or RNA purification chip may further includes a microfluidic mixing chamber and a nano filter. The DNA or RNA purification chip, which includes the immobilization unit, ήano filter and microfluidic mixer, can be formed on a single monolithic substrate. The immobilization unit of the invention may accordingly be integrated into a nucleic acid purification or detection chip as described in WO 2005/066343 or Kim et al, Proc. of the IEEE Conference on MEMS 2002, supra. For instance, in the device described in WO 2005/066343 the immobilization unit of the invention may replace the region termed binder in that patent application. The device of the invention can be fabricated as a monolithic device starting from a silicon chip using standard methods such as DRIE etch that are well known to the person of average skill in the art and that are described in WO 2005/066343 or US Patent 6,379,929, for example.

[0028] The microfluidic mixer in a device for the isolation or purification and extraction of deoxyribonucleic acid (DNA) and/or ribonucleic acid (RNA) molecules is adapted to mix at least two fluids in a substantially spiral manner. The inlet channel of the microfluidic mixer spirals inwards to a central point in a clockwise direction, for example and then enters an outlet channel which spirals in an anti-clockwise manner circumferentially outwards till the fluid being mixed exits the mixer.

[0029] In one embodiment of a device for detection/purification and/or extraction of the present invention, a novel nano filter is provided. This nano filter according to the present invention includes an inlet and an outlet. This nano filter further comprises a plurality of periodical pillars arranged between said inlet and outlet to form a channel of a given depth and width, each of said pillars being separated by a gap from each adjacent pillar. The inlet, outlet and periodical pillars can be formed on a silicon wafer and covered by a suitable covering material. The covering material can comprise or be made of any biocompatible material. Examples of such materials include, but are not limited to, glass, silicon, or a polymeric material. The glass can be any conventional glass slip, or a Pyrex wafer, for example. The polymeric material can be formed as a polymeric sheet (plastic sheet) or foil, for example. One example of suitable biocompatible polymeric materials are thermoplastics. Examples of thermoplastics include, but are not limited to, polycarbonate, poly(meth)acrylate, polyoxymethylen, polyamide, polybutylenterephthalat, or polyphenylenether. Another suitable class of polymeric materials from which the cover (sheet or substrate) can be made are polymeric silicones. Such a polymeric silicone can for example be polydimethylsiloxane (PDMS), polydiethylsiloxane and polydipropylsiloxane. The covering material (or cover) can be bonded to the silicon wafer by any suitable bonding method. Examples of suitable bonding methods include room temperature bonding, bonding at elevated temperature or anodic bonding, to name only a few.

[0030] The gaps of the nano filter of the present invention separating the periodical pillars are, in one exemplary embodiment, approximately about 0.8 to about 1.6 micrometers wide, and the depth and width of the channel of the nano filter is between approximately

65 and approximately 195 micrometers, respectively. In this embodiment the gap between the pillars is basically constructed such that the periodical pillars are adapted to allow for blood plasma to pass through the gaps and to prevent cellular material such as white blood cells, red blood cells or platelets from entering a mixing or detection region of a (ribo)nucleic acid purification or detection device.

[0031] In one embodiment the immobilization unit or nucleic acid purification device of the invention that includes an immobilization unit as described here is manufactured using a semiconductor substrate such as a silicon or gallium arsenide wafer. A respective method comprises in a first step providing a semiconductor wafer having a frontside and a backside. In a second step material from the frontside of the wafer is removed in such a way that at least the channel of the immobilization unit is formed. If the device comprises a nano filter as described above, material is also removed in such a manner that a plurality of pillars is formed, wherein the pillars are separated from each other by a gap and the pillars forming a channel. If a mixer is to be included in the device, the corresponding channel is also formed in this step. The same applies to the channels that fluidly connect the functional units to each other. This removal of material can be done using standard lithography processing that is known to the person of average skill in the art. In a further step an oxide material is formed on at least one side of the wafer and material from the backside of the wafer is removed, thus forming backside holes. Thereafter a covering material such as a plastic sheet or a glass wafer is bonded on top of immobilization unit or the device.

[0032] Another aspect of the invention is a method of isolating DNA and/or RNA molecules, said method comprising contacting a liquid sample suspected to contain DNA and/or RNA with an immobilization unit as described above. In one embodiment, the nucleic acid to be isolated is viral RNA.

[0033] The following drawings illustrate various exemplary embodiments of the present invention. However, it should be noted that the present invention is not limited to the exemplary embodiments illustrated in the following figures.

BRIEF DESCRIPTION OF THE DRAWINGS [0034] Figures Ia - Id illustrate various exemplary embodiments of a minimal element of an immobilization unit comprising a meandering portion with an arm and Fig.le, Fig.lf and Fig.lg illustrate exemplary embodiments of a straight portion of a immobilization unit;

[0035] Figure 2 shows an embodiment of an immobilization unit having an arrangement of a plurality of the minimal elements as shown in Figure Ic;

[0036] Figure 3 shows an immobilization unit having an arrangement of a plurality of rrώώnal elements shown in Figure Ia;

[0037] Figure 4 shows an immobilization unit having a channel of substantially spiral shape;

[0038] Figure 5 is an immobilization unit according to Figure 2;

[0039] Figure 6 is another embodiment of an immobilization unit;

[0040] Figure 7 shows anano filter of a device of the invention;

[0041] Figure 8 shows a device of the invention including a nano filter of Figure 7, a micromixer and an immobilization unit of Figure 2;

[0042] Figure 9 shows a system of the invention including reagents and elution apparatus.

[0043] Figure 10 shows several embodiments of a nano filter of the invention, wherein Figure 10a shows a diagram of a microfilter chip in: (a) plane view with inset showing close-up of channel defined by pillars and: (b) cross section profile; Figure 10b shows plasma separation from whole blood (undiluted): (a) plasma escaping (arrows) through narrow slits between pillars (b) close-up view of red blood cells inside the channel, and Figure 10c show nano-filters (microfilter #1, #2 and #3) being fabricated on a silicon chip in design configurations mainly differing in chip size and shape of the meander-type channel; [0044] Figure 11 shows an agarose gel electrophoresis experiment for the filtering effect of a nano-filter of the invention using Reverse Transcriptase-Polymerase Chain Reaction RT-PCR products of Cymbidium Mosaic Virus RNA that are separated by agarose gel electrophoresis, wherein lane 1 to 5 show dilution-series of standards, lanes 6 and 7 show plasma from spiked blood prepared by centrifuge, and lane 9 and 10 show plasma from spiked blood prepared by the microfilter shown in Figure 9c.

[0045] Figure 12 shows an exemplary process of fabrication a device of the invention as shown in Figure 8; and

[0046] Figure 13 shows a flow diagram of a method of extracting and immobilizing viral RNA using a device of Figure 9;

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

[0047] Figures Ia — Ic illustrate various exemplary embodiments of a minimal element of the invention comprising a meandering portion with an arm (or straight portion) 118. In Figure Ia, the meandering portion essentially comprises of two sections. The first section 102 is approximately at a right angle to the arm 118 and the second section 104 is approximately at a right angle to the first section 102, so resulting in an essentially u- shaped form. The design of the meandering portion of Figure Ia is also such that the direction of flow of a fluid therein is turned by about 180 degrees.

[0048] Figure Ib shows another embodiment of the minimal element of the invention. In said embodiment, two u-shaped meandering portions (a first and a second meandering portion, 112 and 114) are linked together to comprise the minimal element of the invention. Essentially, a non-corresponding arm of the first u-shaped meandering portion

112 links to an arm of the second meandering portion 114. hi other words, in the illustrated embodiment of Figure Ib, the left arm of the first meandering portion 112 would always connect with the left arm of the second meandering portion 114 in the orientation and arrangement shown.

[0049] In this embodiment the direction of flow of the fluid within the minimal element is reversed twice resulting in the direction of flow of the fluid exiting the niinimal element, via the second meandering portion 114, to be the same as the direction of flow of the fluid entering the minimal element, via the first meandering portion 112.

[0050] Figure Ic is another embodiment of the invention and is similar to the embodiment illustrated in Figure Ia. The meandering portion 120 is similar to that shown in Figure Ib (112 or 114). As in Figure Ia, the meandering portion 120 connects with the arm (or straight portion) 118. Again, as in Figure Ia, the direction of flow of a fluid therein is turned by about 180 degrees.

[0051] Figure Id illustrates another embodiment of the minimal element of the invention. The minimal element of Figure Id comprises two arms 106 and 108 (or straight portions). The arm 106 is connected to the arm 108, resulting in an essentially u-shaped form. The angle between the arms 106 and 108 may be acute or obtuse. Subsequently, the arm 108 may be connected to another arm 110 should there be a need to redirect the flow of the fluid.

[0052] Fig. Ie shows a straight portion 108 the edges of which narrow along the length of the straight portion. Fig.lf shows a straight portion 108 the edges of which are corrugated and Fig.lg shows a the straight portion 108 that it split into three sub-channels which converge back into a single channel prior to entering the meandering portion of the immobilization unit.

[0053] Figure 2 shows an embodiment of an immobilization unit having an arrangement of a plurality of the minimal elements shown in Figure Ic. Meandering portions 210 are arranged to connect with arms 220. Each meandering portion 210 is connected to two arms 220. However, the first and last arms 220, through which the fluid enters and leaves respectively, have only one end connected to a meandering portion. The corresponding end of the first and last arms 220 may be attached to other apparatus or devices.

[0054] Figure 3 shows an embodiment of the invention having an arrangement of a plurality of minimal elements shown in Figure Ia. Meandering portions 310 are arranged to connect with arms 320. Each meandering portion 210 is connected to two arms 320. However, the first and last arms 320, through which the fluid enters and leaves respectively, have only one end connected to a meandering portion. The corresponding end of the first and last arms 320 may be attached to other apparatus or devices.

[0055] Figure 4 shows an embodiment of the invention having a channel of substantially spiral shape. The channel essentially comprises of a plurality (two or more) of meandering portions that are arranged to result in the directional flow of the fluid to be substantially spiral. The arms through which the fluid enters and leaves respectively may be attached to other apparatus or devices.

[0056] Figure 5 is an embodiment of an immobilization unit according to Figure 2. This embodiment includes an inlet 550 and an outlet 520 through which a fluid containing viral RNA molecules may enter and exit. The meandering portions 530, as previously described with respect to Figure 2, are connected to arms 510.

[0057] Figure 6 is another embodiment of the invention which is similar to that shown on Figure 5. In this embodiment, an inlet 630 and an outlet 640 provide for the entry and exit of a sample fluid. Meandering portions 620, which are similar to those described in connection to Figure Ia, and are in connection with arms 610, allow for the direction of flow of the fluid to be changed accordingly.

[0058] Figure 7 shows a nano filter of a device according to the invention. The inlet 716 and outlet 718 are linked by a plurality of basic units, as shown in Figure 2. In this embodiment, channels are defined by the walls of periodical pillars 710 and gaps 712 to realize the filtration. The periodical pillars 710 have a dimension of 20 x 30 μm separated by the gap 712 of size 20 x 0.8 μm. In an exemplary embodiment, the gap 712 between the adjacent pillars 710, which determines the filtration size, is nominally 1.6μm. The channel width is 195 μm. The microstructures (periodic pillars and gaps) of the nano filter are covered by a Pyrex glass at the front side and all of the ports (inlet 716 and outlet 718) are linked to the environment through the etched holes.

[0059] Red blood cells, white blood cells and platelets are blocked based on their sizes by the narrow gaps 712 between the periodical pillars 710. When blood sample containing the various cells and plasma are pumped into the reservoir from the inlet 716, the plasma goes through the small gaps 712 into the big chamber 714 as the pumped sample moves forward through the channel and eventually, through the outlet 718.

[0060] Figure 8 shows a device of the invention including the nano filter 824 of Figure 7, a micromixer 822 and the immobilization unit 820 of Figure 2. The nano filter 824 connects to the micromixer 822, which in turn connects to the immobilization unit 820.

[0061] Figure 9 shows a system of the invention including reagents 926 and elution apparatus 935. It consists of one inlet 920 to pump in the initial sample from the reverse side of the wafer, one outlet 938 to collect the residues while the other two outputs 912 to collect the samples with plasma inside for further study. The rounded rectangle 910 represents a silicon wafer whilst the rectangles formed of dashed lines 940 are the anodic bonded glass wafer to cover the two reservoirs and all of the micro-channels.

[0062] Another aspect of the invention is a method of detecting a RNA molecule, said method comprising contacting a liquid sample suspected to contain viral RNA with an immobilization unit as described above.

EXAMPLES Example 1: Separation of viral particles using a "filter chip" from whole blood

[0063] As a test vehicle, particles of orchid plant virus (harmless to human) are employed to spike blood samples. Specifically, Cymbidium Mosaic Virus (CyMV), a type of orchid virus elongated in shape, is used.

[0064] Most commercial kits relying on the solid-phase extraction of viral RNA utilize porous columns and demand a cell-free source material (e.g. serum or plasma). This is mainly to simplify the daunting task of nucleic acid extraction. Unlike the genomic nucleic acids, the viral nucleic acid in the blood is quite scarce during the onset of diseases. Once the blood cells get lysed, sub-cellular components are released and might compete with viral nucleic acids to bind to the column. Moreover, the cell debris could also clog up the pores in the column, causing the nucleic acids getting trapped. Separating cells, however, requires in traditional approaches a centrifugal spinning of the source material. However, a centrifuge is not amenable to miniaturization on chip. For this purpose, in a device (chip) of the invention the separation method is the filtration of the blood cells based on the size exclusion principle. Most viral particles are sub-micron in size whereas blood cells measure at least several micrometers.

[0065] A microfilter of the invention made on the basis of a silicon chip is diagrammatically shown in Fig.10. The chip contains a chamber etched about 65-μm deep into silicon by deep reactive ion etching and capped with a glass wafer by anodic bonding. Plasma can be collected through anisotropically-etched backside holes in silicon located at two diagonal corners. At the other corners, backside holes allow blood to flow in and out of the chip through a meander type channel defined by silicon pillars. As blood flows inside the channel, plasma can escape through narrow slits between pillars due to combined action of capillary forces and pressure gradient. The nominal gap between the pillars is about 1.6μm wide, which can retain most blood cells but allows passage of virus particles. The microfilter chips have been fabricated in three design configurations mainly differing in chip size and shape of the meander-type channel (Table 1).

[0066] Table 1 : Microfilter chip designs and their efficiencies

[0067] Fig. 10b shows on-chip collection of plasma escaping through the slits between pillars as the anticoagulant-treated whole blood flows through the meander-type channel. Anticoagulant-treated blood was pumped through the chips at lOμl/min and at different dilutions of phosphate buffered saline (PBS) solution. RBC counts in the blood pumped in (RBC_bIood) and the plasma collected (RBC_plasma) were obtained by a hemocytometer. Table I shows volume of the collected plasma samples and percent efficiency of each microfilter chip (% EF ) as calculated by:

[0068] As shown, chips based on any of the three designs had higher than 99% efficiency for the Blood:PBS ratio of 25:75. The efficiency deteriorated with an increase in the blood:PBS ratio but stayed above 90% for all three microfilter chips. Further, experiments were conducted to test whether the plasma filtered by the microfilter chips can be used for detection of virus particles in blood. Anticoagulant-treated whole blood at a volume of 140μl was spiked with virus (Cymbidium Mosaic Virus) suspension in water at a volume of 70μl and concentration of 0.26μg/μl. Approximately, 180μl of the spiked blood was pumped through microfilter #1 at lOμl/min. The plasma filtrate was used for extraction of viral RNA via a commercial kit and amplified by RT-PCR. The amplified products were separated by agarose gel electrophoresis and ethidium bromide-stained products were visualized on a UV transilluminator. As can be seen in Fig.l 1, viral RNA from the plasma filtered by microfilter chip #1 could be amplified, demonstrating a successful substitute for the conventional centrifugation step.

Example 2: VIRAL RNA EXTRACTION

[0069] An RNA purification device (chip) as shown in Fig.9 comprising a nano filter, a micromixer and an immobilization unit was fabricated using title process flow for the v- RNA chip fabrication as shown in the following Table 2 and also in Fig.12.

[0070] As starting point of the RNA chip fabrication process flow, an 8-inch p-type (100) silicon wafer is provided, a cross-section of which is shown in Table 2 and Fig.12. hi a first process step (Step 1), a masking oxide layer (mask) is formed on one side of the wafer (in the following referred to as the frontside of the wafer), and the mask is then patterned, hi another process step (Step 2), the Si wafer is etched from the frontside by deep Si etching (e.g. deep reactive ion etching) using the patterned mask. The deep etching is carried out in such a way that the channel of the immobilization unit, the channel of the micro mixer and a plurality of pillars for the nano-filter are formed and that these units are in fluid connection with each other. Each of the pillars is separated by a gap from each adjacent pillar, and the pillars form a channel of given depth and width. In another process step (Step 3), the masking oxide layer is removed (stripping oxide). In a further process step (Step 4), an oxide material (e.g. silicon oxide) is grown on the frontside of the wafer and on the opposite side (in the following referred to as the backside) of the wafer, e.g. by thermal oxidation. In addition, a nitride material (e.g. silicon nitride) is deposited on both sides. In another process step (Step 5), a mask is formed on the backside of the wafer and the silicon wafer is etched from the backside by anisotropic wet etching, for example with KOH, from the backside using the mask, so forming backside holes. In another process step (Step 6), the oxide material and the nitride material on the backside, and also the nitride material on the frontside are removed by stripping. The stripping includes an etching with a plasma that comprises an atmosphere of CF₄ or CHF₃ together with oxygen (O₂). In a final process step (Step 7), the chip is capped with a cover such as a glass wafer. The bonding can be carried out thermally or by anodic bonding, wherein room temperature bonding is preferred. Thus an RNA chip is obtained, comprising a nano filter, a mixer and an immobilization unit. After the bonding the surface of the immobilization unit or the one of a device that includes the immobilization unit can be treated/washed with deionized water.

Table 2: Process flow for fabrication of a RNA chip

[0071] The nanofilter was designed to separate out plasma containing virus particles from blood cells with a main goal to increase the percentage ratio of the virus particles that can pass through the filter while retaining most of the red and white blood cells. The virus particles being sub-micrometer in size can easily pass through nano-slits of the filter while the blood cells being several micrometer in size, can not.

[0072] The micromixer was used to mix two reagents such as plasma containing viral particles and lysis buffer. The macroscopic mixers use stirring parts to create turbulence in the liquids to be mixed. However, with the micro-mixer used in a nucleic acid purification device of the invention, the Reynolds number is very low for turbulence to take place. In the micromixer, the mixing is done mainly by diffusion. A spiral design is used here to increase the contact area between the two reagents flowing side by side and hence facilitate their mixing.

[0073] The RNA immobilization unit is designed as a chamber that provides large surface area for nucleic acids (e.g. RNA) to get bound in the presence of high salt concentration. The surface of the immobilization unit and rest of the chip has received a plasma treatment with an atmosphere comprising triflourmethane (CHF₃) and oxygen (O₂) to facilitate reversible binding of nucleic acids.

[0074] The purification/extraction device was designed for the following experimental conditions: The input into the purification device is whole blood spiked with viral particles (10OuI - ImI). The output is viral RNA elution (<lml) that can be amplified and detected by nucleic acid sequence based amplification (NASBA) and reverse transcriptase polymerase chain reaction (RT-PCR).

[0075] In order to investigate the viral RNA extraction on the chip and as the same time to avoid filtration and save tune, the extraction of viral RNA was performed without separating virus particles from other blood cells. Instead, whole blood containing CymMV particles was mixed with lysis buffer before being injected into the chip. Since the sample lysis begins outside the chip, the entire exposed area of the chip surface can be used to immobilize RNA. This immobilization is not exclusive to a particular (viral) RNA but to all nucleic acids that interact with the chip surface. Selectivity is achieved in the subsequent step of NASBA amplification by including primers for target RNA of interest. The NASBA results confirmed the presence of the CymWTV RNA in the elutions collected from the chip.

[0076] The design flow chart using this set-up is shown in Fig. 13. The chip used in the present example contained a fluid volume of about 12ul.The first step is to pump the blood in through the inlet of the nano-filter. The design of the nanofilter allows the blood to flow easily flown through the nano-filter without trapping any air bubble. The blood flowing through the nano-filter is collected at the nano-filter outlet. This collected blood includes most of the red and white blood cells as they cannot pass through the nano-filter. However, the viral particles, although hardly visible even under the optical microscope, can escape from the nano-filter along with the plasma and travel to the immobilization unit by passing through the micromixer. Simultaneously, the lysis buffer is also pumped in to break open protein coatings around the virus particles and thereby release their RNA. The viral RNA gets bound to the surface of the immobilization unit in the presence of high salt (for example, lysis buffer with a 6 molar guanidine hydrochloride) and can be eluted out later in a low ionic strength buffer such as TE buffer (10 mM Tris-HCl, lmm EDTA) or deionized. The design of the immobilization unit of the invention is less susceptible to the trapping of air bubbles. Before the low-salt elution, the chip was washed with a high-salt solution to make sure the debris removed.

Table 3 Experimental results (Summary)

[0077] It is to be noted that the present invention is by no means limited to the above- mentioned illustrated embodiments alone. The illustrated embodiments merely serve as exemplary embodiments to facilitate the understanding of and to better illustrate the working principles behind the present invention.

Claims

CLAIMSWhat is claimed is:

1. An immobilization unit for the isolation of nucleic acid molecules capable of immobilizing nucleic acid molecules, said immobilization unit comprising: a channel of a given depth, width and length and having at least one surface, which is adapted to immobilize nucleic molecules under suitable conditions, wherein the channel has at least one straight portion and at least one meandering portion, wherein the at least one meandering portion is essentially u- shaped and the at least one straight portion is an arm of the u-shaped meandering portion.

2. The immobilization unit of claim 1, wherein the nucleic acid molecules to be immobilized are either deoxyribonucleic acid (DNA) and/or ribonucleic acid (RNA) molecules.

3. The immobilization unit of claim 1 or 2, comprising a plurality of meandering portions.

4. The immobilization unit of claim 3 , wherein each of the at least one straight portion couples a pair of meandering portions to each other.

5. The immobilization unit according to any of the preceding claims comprising at least two meandering portions.

6. The immobilization unit of claims 5, wherein the at least one straight portion couples one meandering portion to the other.

7. The immobilization unit according to any of the preceding claims comprising two straight portions, wherein each straight portion couples two meandering portions to each other.

8. The immobilization unit according to any of the preceding claims, wherein the at least one surface of the channel is adapted to provide for or enhance binding affinity with DNA and/or RNA molecules.

9. The immobilization unit according to claim 8, wherein the at least one surface comprises silica or silicon that has undergone a plasma treatment.

10. The immobilization unit according to any of the preceding claims, wherein the depth of the channel is approximately between about 50 — about 500 micrometers.

11. The immobilization unit according to any of the preceding claims, wherein the width of the channel is approximately between about 50 — about 500 micrometers.

12. The immobilization unit according to any of the preceding claims, wherein the width to depth ratio of the channel is approximately between about 0.1 - about 10.

13. The immobilization unit according to any of the preceding claims, wherein the length traversed by a fluid in the channel of the immobilization unit is approximately between about 10,000 - about 100,000 micrometers.

14. The immobilization unit according to any of the preceding claims, wherein the width and the depth of the channel varies.

15. An immobilization unit for the isolation of nucleic acid molecules capable of immobilizing nucleic acid molecules, said immobilization unit comprising: a channel of a given depth, width and length having at least one surface, said surface being adapted to immobilize nucleic acid molecules under suitable conditions, wherein the channel is essentially formed to route any fluid flowing therein, in a substantially spiral flow pattern.

16. The immobilization unit of claim 15, wherein the nucleic acid molecules to be immobilized are either deoxyribonucleic acid (DNA) and/or ribonucleic acid

(RNA) molecules.

17. The immobilization unit of claim 16, wherein the at least one surface of the channel is adapted to provide for or enhance binding affinity with DNA or RNA molecules.

18. The immobilization unit according to claim 17, wherein the at least one surface comprises silica or silicon that has undergone a plasma treatment.

19. The immobilization unit of any of claims 15 to 18, wherein the depth of the channel is approximately between 50 - 500 micrometers.

20. The immobilization unit of any of claims 15 to 19, wherein the width of the channel is approximately between 50 — 500 micrometers.

21. The immobilization unit of any of claims 15 to 20, wherein the width to depth ratio of the channel is approximately between about 0.1 to about 10.

22. The immobilization unit of any of claims 15 to 21, wherein the length traversed by a fluid in the channel of the immobilization unit is approximately between 10,000 - 100,000 micrometers.

23. The immobilization unit of any of claims 15 to 22, wherein the width and depth of the channel along said channel that is essentially formed to route any fluid flowing therein, in a substantially spiral flow pattern varies.

24. A device for the isolation of ribonucleic acid (RNA) molecules comprising an immobilization unit as defined in any of the claims 1 to 23.

25. The device of claim 23 further comprising a microfluidic mixing chamber and a nano filter, wherein said microfluidic mixer is adapted to mix at least two fluids in a substantially spiral manner.

26. The device of claims 24 or 25, wherein the immobilization unit, nano filter and microfluidic mixer are formed on a monolithic substrate.

27. The device of claim 24 or 25, wherein the nano filter comprises: an inlet and an outlet, a plurality of periodical pillars arranged between said inlet and outlet to form a channel of a given depth and width, each of said pillars being separated by a gap from each adjacent pillar, wherein the inlet, outlet and periodical pillars are formed on a silicon wafer and covered by a biocompatible covering material

28. The device of claim 27, wherein the biocompatible covering material comprises glass, silicon, or a polymeric material.

29. The device of claim 28, wherein the covering material is bonded to the silicon wafer by bonding.

30. The device of claim of 29 wherein bonding of the covering material to the silicon wafer occurred by thermal bonding, bonding at room temperature or anodic bonding.

31. The device of any of claims 27 to 30, wherein the gaps separating the periodical pillars are approximately 0.8 - 1.6 micrometers.

32. The device of claims 29, wherein the periodical pillars are adapted to allow for plasma to pass through the gaps.

33. The device of any of claims 27 to 33, wherein the depth and width of the channel is approximately 65 and 195 micrometers respectively.

34. The device of any of claims 24 to 33, wherein the device is a microfmidic device.

35. A method of isolation of a nucleic acid molecule, said method comprising: contacting a liquid sample suspected to contain DNA or RNA with an immobilization unit as defined in any of claims 1 to 23.