WO2014065758A1

WO2014065758A1 - A method of isolating nucleic acids in an aqueous sample using microfluidic device

Info

Publication number: WO2014065758A1
Application number: PCT/SG2013/000457
Authority: WO
Inventors: Haiqing Gong; Chun Chau SZE; Rui Zhang; Xudong ZENG
Original assignee: Star Array Pte Ltd
Priority date: 2012-10-25
Filing date: 2013-10-25
Publication date: 2014-05-01

Abstract

A method of isolating nucleic acids in an aqueous sample (201 ) using a microfluidic device is disclosed. The device has at least one well (1042) in fluid communication with an adjacent space (1062) external to the opening of the at least one well (1042), and the aqueous sample (201 ) comprises biological and/or chemical materials and/or impurities, and is held in the at least one well. The method comprises introducing a first fluid (203) substantially immiscible with the aqueous sample (201 ) into the adjacent space (1062) to partition impurities to the first fluid (203); and removing the first fluid (203) with the impurities leaving the nucleic acid in the aqueous sample (201 ) in the well (1042). Other related methods are also disclosed.

Description

A method of isolating nucleic acids in an aqueous sample using a

microfluidic device

Field of the invention

The present invention relates to a method of isolating nucleic acids in an aqueous sample using a microfluidic device.

Background of the invention

Bacterial pathogen infections pose great danger to human and public health alike in general, and such infections were reported to be the leading cause of death worldwide, especially for the young and the elderly. Therefore, rapid and sensitive identification and quantification of bacterial pathogens is of key importance in many fields including contaminant screening of food and drinking water supply, clinical diagnostics and bio- defense. The recent development of microfluidics platforms for DNA-based quantitative PCR (q-PCR) or RNA-based quantitative reverse transcription PCR (q-RT- PCR) analysis has significantly accelerated and enabled identification of bacterial contamination from a variety of sources. However, these microfluidic bacterial pathogen detection devices in clinical or environmental applications have limited capability because isolation of nucleic acids at micro-scales from a small number of bacteria is extremely difficult, especially for gram-positive bacteria with rigid cell walls.

Presently, solid phase nucleic acid extraction is the most widely adopted technique in microfluidic platform for on-chip bacterial nucleic acid preparation. Typically, bacterial cells are disrupted by chemicals combined with enzymatic lysis, thermal lysis, or mechanical lysis, and thereafter the lysate of which are then passed through selected solid phase for nucleic acid isolation and purification. One well documented solid phase extraction medium in microfluidic nucleic acid isolation module is micro-pillars coated with silica gel, which can preferentially absorb nucleic acids in high ionic strength buffer solutions. Nucleic acids can later be eluted out for downstream assays after unwanted impurities are washed away. However, compromised nucleic acids recovery was reported due to insufficient binding of nucleic acids to silica gel, loss during washing, or failure to be eluted because of irreversible bonds. As a result, concentrated samples containing thousands to millions of bacteria are required as a starting material, which is far greater than the desired detection sensitivity in many applications. Another popular solid phase extraction medium for nucleic acid purification in microfluidic platform uses functionalized magnetic beads, based on a proposed complexly structured chip processor with fifty-four integrated micromechanical valves to allow sequential flow of lysis buffer, wash buffer and elution buffer into and out of reaction chambers for nucleic acid recovery. DNA from as little as twenty-eight bacteria cells extracted using this device can be detected with off-chip q-PCR assay. But important issues such as initial sample volume, reproducibility, and sample carryover were however not reported.

It is therefore desirable to address some of the problems identified and/or to provide a choice that is useful in the art.

Summary of the invention

According to a 1^st aspect of the invention, there is provided a method of isolating nucleic acids in an aqueous sample using a microfluidic device, the device having at least one well in fluid communication with an adjacent space external to the opening of the at least one well, wherein the aqueous sample comprises biological and/or chemical materials and/or impurities, and is held in the at least one well. The method comprising introducing a first fluid substantially immiscible with the aqueous sample into the adjacent space to partition impurities to the first fluid; and removing the first fluid with the impurities leaving the nucleic acid in the aqueous sample in the at least one well.

The method may further comprise moving the first fluid in the adjacent space for a defined period, after the first fluid is introduced into the adjacent space. Also, the method may further comprise angularly arranging the microfluidic device for a defined period to partition the impurities to the first fluid, if the first fluid is denser than the aqueous sample, and/or if the size of the at least one well is able to hold the aqueous sample therewithin by surface tension. Specifically, angularly arranging the microfluidic device may include inverting the microfluidic device. Further, the method may further comprise drying the at least one well having the nucleic acids, after the first fluid is removed. Preferably, the method may further comprise sequentially introducing and removing different constituents of the first fluid into the adjacent space, after the first fluid is removed. It is appreciated that the biological and/or chemical materials may be selected from the group consisting of: primers, short nucleotides, and adaptors for nucleic acid amplification, reverse transcription, and next generation sequencing applications, cells, cell debris, tissues, plants, viruses, antibodies, proteins, enzymes, molecules, peptides, nucleic acids, polynucleotides, oligonucleotides, short fragments of genes or probes, reaction constituents, lysis buffer constituents, bacteria, protozoa, pathogens, fluorescent chemicals or molecules, crystals, liquid droplets, metal ions, and solid particles. Preferably, the biological and/or chemical materials may comprise solid, dried, partially dried, or liquid forms. On the other hand, the solid particles may include fluorescent particles, fluorescent dye chemicals, nanoparticles, glass beads, or magnetic beads. Also, the nucleic acids may include DNA, RNA, mRNA, microRNA, or cDNA. 16. The impurities may include proteins, DNA, RNA, unwanted chemicals, metal ions and salt.

Preferably, the first fluid may include a composition of phenol, and/or chloroform and/or isoamyl alcohol, and/or other chemicals to facilitate the partitioning of the impurities from the aqueous sample to the first fluid. And the composition of phenol, chloroform and isoamyl alcohol may be · in a volume ratio of approximately 25:24:1 , if the nucleic acids to be isolated are DNAs. Specifically, a pH value of the first fluid may be approximately 8.0. Alternatively, the composition of phenol, chloroform and isoamyl alcohol may be in a volume ratio of approximately 125:24:1 , if the nucleic acids to be isolated are RNAs. And a pH value of the first fluid may be approximately 4.6.

More preferably, the method may further comprise providing a cover to the at least one well to configure the adjacent space into a fluid channel, prior to introducing the first fluid into the adjacent space. And drying the at least one well may include using vacuum evaporation and/or heat drying and/or freeze drying. The defined period may be approximately 15 minutes. Also, circulating the first fluid may comprise arranging the first fluid to circulate with forward and reverse flows for a plurality of cycles, wherein each cycle lasts approximately 5 seconds. In particular, the first fluid may be circulated at a flow rate of 0.25 ml/min, 0.45 ml/min, or 0.65 ml/min for one cycle. Moreover, if the device is inverted, the device may be restored back to the position with the adjacent space above the at least one well after removing the first fluid, the method may further comprise introducing the aqueous sample into the at least one well, prior to introducing the first fluid into the adjacent space. Specifically, introducing the aqueous sample into the at least one well may comprise introducing biological and/or chemical substances together with an aqueous fluid. Optionally, introducing the aqueous sample into the at least one well may comprise introducing an aqueous fluid into the at least one well, which is pre-loaded with biological and/or chemical substances. The method may further preferably comprise introducing into the at least one well lysis buffers and/or physical means such as ultrasound, electric current, thermal stress via freeze thawing process cycles or rapid heating and cooling cycles, or solid beads grinding under agitation such as vortex and ultrasound to lyse the biological substances to release nucleic acids therefrom. Furthermore, the method may further comprise depositing the aqueous fluid into the at least one well; and introducing the biological and/or chemical materials into the at least one well using diffusion, body forces, electrophoretic forces, dielectrophoretic forces, flow-induced hydrodynamic forces, electric forces or magnetic forces, wherein the aqueous liquid and the biological and/or chemical materials together form the aqueous sample. The body forces may include gravity and centrifugal forces. Also specifically, the bottom of the at least one well may be arranged to be substantially porous to enable at least some particulate impurities to pass through, and a pore size of the porous bottom may be configured to be smaller than the size of the nucleic acids. In this respect, the bottom of the at least one well may include being formed using a Telfon filter paper or a Polypropylene track-etched filter paper.

Preferably, introducing the aqueous sample into the at least one well may include using manual or robotic pipette loading. The method may further comprise vacuuming the at least one well to remove air trapped therein. Also, the method may further comprise depositing the aqueous sample adjacent to the bottom of the at least one well using a pipette. Yet optionally, the method may further comprise depositing the aqueous sample substantially adjacent to the top of the at least one well; and centrifuging the microfluidic device to cause the aqueous sample to move to the bottom of the least one well. More preferably, wherein the at least one well comprise walls with hydrophilic surfaces and a porous base, the method may further comprise depositing the aqueous sample into the at least one well through capillary forces between the aqueous sample and the hydrophilic surfaces, wherein air and/or particulate impurities in the at least one well are expelled through the porous base while the aqueous sample moves into and is retained in the at least one well. Yet also, introducing the aqueous sample into the at least one well may include using microfluidic loading, wherein the adjacent space is configured as a fluid channel.

The method may further include depositing the aqueous sample into the at least one well using vacuum loading, centrifugal loading, pressure loading, or capillary loading. Yet additionally, the method may further comprise washing the at least one well with a cleaning fluid after the at least one well is dried to substantially remove residual first fluid. The cleaning fluid may comprise ethanol of a concentration between 40% to 100%. In particular, the concentration of the ethanol may approximately be 70%.

Moreover, the method may further comprise removing the cleaning fluid from the adjacent space after the at least one well is washed. And the method may further comprise drying the at least one well to substantially remove residual cleaning fluid after the at least one well is washed. Also, the method may further comprise vacuum evaporating the at least one well for approximately 5 minutes. Preferably, the at least one well or the cover may be formed of a material selecting from the group consisting of Polydimethylsiloxane (PDMS), Teflon, Polypropylene, glass and ceramics. The at least one well may have an edge length of approximately between 0.05 μιη to 10000 μηΊ . Preferably, the method may further comprise introducing a reaction mixture suitable for polymerase chain reaction (PCR), q-PCR, q-RT-PCR, isothermal amplification, reverse transcription, DNA amplification used for DNA sequencing, into the at least one well having the nucleic acids. Also, the method may further comprise introducing a layer of mineral oil into the adjacent space to seal the at least one well filled with the reaction mixture to enable PCR, RT-PCR, q-PCR, q- RT-PCR, isothermal amplification, reverse transcription, or DNA amplification used for DNA sequencing assays to be subsequently performed on the nucleic acids. And the method may alternatively further comprise removing the aqueous sample in the at least one well for storage and/or further analysis. More preferably, removing the nucleic acids may include using pipette aspiration, centrifugation of the nucleic acids into a desired collection device, or collecting the nucleic acids through an aperture configured at the bottom of the at least one well. Preferably, the method may further comprise introducing an aqueous buffer into the at least one well having the nucleic acids to re-suspend the dried nucleic acids; and removing the re-suspended dried nucleic acids for further analyses.

According to a 2^nd aspect of the invention, there is provided a method of isolating nucleic acids from an aqueous sample comprising: introducing the aqueous sample into at least one internal well at the base of a tube, introducing a first fluid substantially immiscible with the aqueous sample in the space of the tube external to the opening of the well to partition impurities into the first fluid, optionally agitating the first fluid to improve partitioning; removing the first fluid from tube leaving the nucleic acids in the internal well of the tube; and drying the tube.

Preferably, the method may further comprise disposing materials into the at least one internal well to enable biological assays being one of nucleic acid amplification, cell assay and assays involving a plurality of biological particles and chemical agents. The materials disposed in the at least one well may include primers and/or probes for nucleic acid amplification, or same or different primers and/or probes. Optionally, the method may further comprise removing the nucleic acids from the internal well for storage and/or further analysis. Also, the method may further comprise removing the nucleic acids from the internal well for further analysis, wherein the nucleic acids are partially or fully amplified in the internal well.

According to a 3^rd aspect of the invention, there is provided a method of isolating nucleic acids in an aqueous sample using a microfluidic device, the device having at least one well in fluid communication with an adjacent space external to the opening of the at least one well. The method comprise introducing a first fluid substantially immiscible with the aqueous sample into the adjacent space and the at least one well to partition impurities to the first fluid; introducing the aqueous sample as at least one droplet into the first fluid which subsequently settle into the at least one well, wherein the aqueous sample comprises biological and/or chemical materials and/or impurities; and removing the first fluid with the impurities leaving the nucleic acid in the aqueous sample in the at least one well. More preferably, the method may further comprise angularly arranging the microfluidic device for a defined period to partition the impurities to the first fluid, if the first fluid is denser than the aqueous sample, and/or if the size of the at least one well is able to hold the aqueous sample therewithin by surface tension. And- angularly arranging the microfluidic device may include inverting the microfluidic device. The method may further comprise drying the at least one well having the nucleic acids, after the first fluid is removed. Also, the size of the at least one drop may be arranged to be substantially equally to the size of an opening of the at least one well. Further, the at least one droplet may include a plurality of droplets, and the at least one well includes a plurality of wells, and wherein a number of the droplets generated is less than a number of the wells.

Yet preferably, each droplet may include different biological and/or chemical materials from the other droplets. The method may further comprise pre-loading each of the wells with a biological and/or chemical material to enable interaction with the different materials held in the droplets.

According to a 4^th aspect of the invention, there is provided a method of isolating nucleic acids in an aqueous sample using a microfluidic device, the device having at least one well in fluid communication with an adjacent space external to the opening of the at least one well. The method comprise introducing an aqueous fluid into the adjacent space to fill the at least one well; introducing biological and/or chemical materials into the aqueous fluid which subsequently settle into the at least one well, wherein the biological and/or chemical materials include the nucleic acids; removing the aqueous fluid from the adjacent space; introducing a first fluid substantially immiscible with the aqueous fluid into the adjacent space to partition impurities to the first fluid; and removing the first fluid with the impurities leaving the nucleic acid in the aqueous sample in the at least one well. Preferably, the method may further comprise angularly arranging the microfluidic device for a defined period to partition the impurities to the first fluid, if the first fluid is denser than the aqueous sample, and/or if the size of the at least one well is able to hold the aqueous sample therewithin by surface tension. Angularly arranging the microfluidic device may include inverting the microfluidic device. The method may further comprise drying the at least one well having the nucleic acids, after the first fluid is removed. The size of the biological and/or chemical materials may arranged to be substantially equally to the size of an opening of the at least one well. Also, the at least one well may include a plurality of wells, and wherein a number of the biological and/or chemical materials introduced is less than a number of the wells.

According to a 5^th aspect of the invention, there is provided a method of isolating nucleic acids in an aqueous sample using a microfluidic device, the device having at least one well in fluid communication with an adjacent space external to the opening of the at least one well. The method comprises introducing an aqueous fluid pre-mixed with biological and/or chemical materials into the adjacent space to fill the at least one well, wherein the biological and/or chemical materials include the nucleic acids; removing the aqueous fluid from the adjacent space after the biological and/or chemical materials have settled into the at least one well; introducing a first fluid substantially immiscible with the aqueous fluid into the adjacent space to partition impurities to the first fluid; and removing the first fluid with the impurities from the adjacent space leaving the nucleic acid in the aqueous sample in the at least one well. According to a 6^th aspect of the invention, there is provided a method of isolating nucleic acids in an aqueous sample using a microfluidic device, the device having at least one well in fluid communication with an adjacent space external to the opening of the at least one well. The method comprises forming a mixture of the aqueous sample as at least one droplet with a first fluid substantially immiscible with the aqueous sample; introducing the said mixture into the adjacent space and the at least one well to partition impurities to the first fluid, allowing at least one droplet to settle into the at least one well, wherein the aqueous sample comprise biological and/or chemical materials and/or impurities; and removing the first fluid with the impurities from the adjacent space leaving the nucleic acid in the aqueous sample in the at least one well.

It would be understood that features relating to one aspect of the invention may also be applicable to the other aspects of the invention. These and other aspects of the invention will be apparent from and elucidated with reference to the embodiments described hereinafter.

Brief Description of the Drawings

Embodiments of the invention are disclosed hereinafter with reference to the accompanying drawings, in which:

Figure 1 includes Figures 1a and 1 b, which respectively show a schematic view and a photographic view of a microfluidic device, according to a first embodiment of the invention;

Figure 2 includes Figures 2a to 2h, which show respective schematic steps of a method for isolating nucleic acids from other particles in an aqueous sample using the microfluidic device of Figure 1 ;

Figure 3 is a table showing selected primer pairs for q-PCR and q-RT-PCR assay; Figure 4 depicts a photographic view of a vacuum system setup developed by StarArray Pte Ltd of Singapore for loading a PCR reagent into wells of the microfluidic device of Figure 1 ;

Figure 5 includes Figure 5a to 5d, which show respective schematic steps of a method for loading the PCR reagent using the vacuum system setup of Figure 4;

Figure 6 includes Figures 6a and 6b, which respectively show a schematic view and a photographic view of a different design of the microfluidic device of Figure 1 , which is to be used for analysis of DNA or RNA from single bacterial cells;

Figure 7 shows a schematic top view of the miGrofluidic device of Figure 6, in which single bacterium is each isolated into the wells of the device;

Figure 8 includes Figures 8a and 8b, which depict corresponding results of recovery of fluorescence labelled BSA, DNA and RNA in aqueous phase at pH values of 8.0 and 4.6 respectively; Figure 9 includes Figures 9a to 9d, which depict corresponding results of Off-chip q- PCR (i.e. Figures 9a and 9b) and q-RT-PCR (i.e. Figures 9c and 9d) analysis of DNA and RNA isolated by chip based liquid phase and column based solid phase nucleic acid purification methods from P. aeruginosa (i.e. Figures 9a and 9c) and S. aureus (i.e. Figures 9b and 9d) ranging from 5000 CFU to 5 CFU;

Figure 10 includes Figures 10a to 10d, which depict corresponding results of On-chip q-PCR amplification of genomic DNA isolated from 5000 to 5 P.aeruginosa and S. aureus cell (i.e. Figures 10a and 10b) and q-RT-PCR amplification of RNA isolated from 5000 to 5 P.aeruginosa and S. aureus cell (i.e. Figures 10c and 10d);

Figure 11 includes Figures 11a to 11d, which depict corresponding On-chip melting curve analysis for PCR product of genomic DNA isolated from 5000 to 5 P.aeruginosa and S. aureus cell (i.e. Figures 11a and 11 b) and RNA isolated from 5000 to 5 P.aeruginosa and S. aureus cell (i.e. Figures 11c and 1 d) to test the purity of the amplified product;

Figure 12 includes Figures 12a to 12d, which depict corresponding photos of results of On-chip amplification of DNA isolated from single P. aeruginosa (i.e. Figure 12a) and S. aureus (i.e. Figure 12c) and On-chip q-RT-PCR amplification of RNA isolated from single P. aeruginosa (i.e. Figure 12b) and S. aureus (i.e. Figure 12d);

Figure 13 includes Figures 13a and 13b, which collectively depict a method for preloading the array of wells of the microfluidic device of Figure 1, according to a second embodiment;

Figure 14 includes Figures 14a to 14d, which collectively depict a method for preloading the array of wells of the microfluidic device of Figurel , according to a third embodiment;

Figure 15 includes Figures 15a and 15b, which collectively depict a method for preloading the array of wells of the microfluidic device of Figurel , according to a fourth embodiment;

Figure 16 includes Figures 16a to 16c, which collectively depict a method for preloading the array of wells of the microfluidic device of Figurel , according to a fifth embodiment;

Figure 17 shows a microfluidic device, based on a sixth embodiment- Figure 18 includes Figures 18a and 18b, which collectively depict a method for preloading the array of wells of the microfluidic device of the sixth embodiment in Figurel 7; Figure 19 shows a device, based on a seventh embodiment, in which the device has a test tube-like arrangement;

Figure 20 includes Figures 20a to 20c, which collectively depict a method of processing the purified nucleic acids isolated in the array of wells of the microfluidic device of Figure 1 , according to an eighth embodiment; and

Figure 21 shows an alternative embodiment of the wells of the microfluidic device of Figure 1.

Detailed description of the invention

1. Materials and Methods

1.1 Chip design and fabrication

Figure 1 includes Figures 1a and 1b, which respectively show a schematic view and a photographic view of a microfluidic device 100, according to a first embodiment of the invention. In this embodiment, the microfluidic device 100 is realised in the form of a chip, also termed as a liquid phase nucleic acid purification chip. It is also to be appreciated that the microfluidic device 100 may be designed for single-use (i.e. disposable) and/or multi-use applications. The structure of the microfluidic device 100 comprises five different layers from top to bottom. Starting from the bottom layer, a base 102 made of glass substrate is provided at the bottom of the microfluidic device 100, and upon which there is a well layer 104 including a plurality of (micro )wells 042 (hereinafter "array of wells") arranged in a central portion of the base 102. Each well 1042 is of a same size and generally cuboid-shaped, and is adapted to hold fluid and biological/chemical materials (in dried, partially dried, or liquid forms), for example primers, short nucleotides, and adaptors for nucleic acid amplification, reverse transcription, and next generation sequencing applications, cells, cell debris, viruses, antibodies, proteins, enzymes, molecules, peptides, nucleic acid molecules (e.g. DNA, RNA, mRNA, microRNA, cDNA etc), polynucleotides, oligonucleotides, short fragments of genes, probes etc., reaction constituents, bacteria, protozoa, pathogens, fluorescent chemicals/molecules, crystals, solid particles such as fluorescent particles, fluorescent dye chemicals, and other impurities (e.g. proteins, DNA (i.e. if RNA is to be isolated), RNA (i.e. if DNA is to be isolated), unwanted chemicals, metal ions and salt) etc. Each generally cuboid-shaped well 1042 is arranged to be equally spaced apart from immediate neighbouring wells 1042, and each well 1042 has an edge length of approximately between 0.05 μιη to 10000 μιτη (i.e. 10 mm). More specifically, in this instance, each well 1042 has a dimension of 1 mm x 1 mm x 1 mm (i.e. length x width x height) and is arranged to hold about 1 μΙ of fluid. It is to be appreciated that for illustration simplicity, only ten wells 1042 (arranged in a row) in the array of wells 110 are shown in Figures 1a and 1b, but however not to be construed as limiting in any manner. The well layer 104 is made of glass. In other words, the well bottom or well inner surfaces are preferably made of glass or coated with a glass layer. The advantage of this feature is that glass helps to hold DNA/RNA after the sample is dried, which enables the DNA/RNA in contact with the glass to consequently bind to the glass bottom. Then, when the wells 1042 are later washed with a cleaning fluid (containing ethanol), the DNA/RNA already bonded to the glass bottom is less likely to be flushed away (or diffused into the cleaning fluid and be removed).

As will b& appreciated, the term "well" 1042 has a standard meaning known in the art. Specifically, each well 1042 is a depression for holding a fluid sample and is formed by removing a part of a solid mass (e.g. using chemical/electrochemical etching or sculpting a depression out of a solid mass). The depression may also be formed using moulding or casting a curable liquid to produce a solid mass having the depression (e.g. using a pre-fabricated die to produce a complementary shape). Non-limiting examples of possible shapes for the well 042 include cylindrical, conical, pyramid-like, prism-like and truncated variants etc. The shape defining the well 1042 is arranged with an opening through which fluid can enter/exit the well 1042. It is apparent that the opening for the well 1042 can be rectangular (including square) or circular in shape.

On top of and adjacent to the well layer 104, there is an intermediate layer 106 comprising a headspace channel 1062 (but also known as a fluid channel) in fluid communication with the array of wells 1042, and a protector layer 108 overlays the intermediate layer 106 to seal the headspace channel 1062 and the array of wells 1042 from contamination and exposure to external environment. The headspace channel 1062 is arranged as an adjacent space to the array of wells 1042 and usefully provides phase partitioning using an organic phase, which will be elaborated below, and has a dimension of 20 mm x 3 mm x 1mm (i.e. length x width x height) in this instance. The organic phase is a mixture of phenol, chloroform, and isoamyl alcohol (Ambion, Life Technologies, USA), often abbreviated as PCI. The intermediate layer 106 and protector layer 108 are also made of Polydimethy!siloxane (PDMS). -^~ But it will be appreciated that each of the well layer 104, intermediate layer 106, and protector layer 108 may also be formed of other suitable materials that are substantially inert to the biological/chemical materials, samples or fluids which the microfluidic device 100 may come into contact with, and the materials include (for example) PDMS, Teflon, Polypropylene, plastics, glass, metal, ceramics and the like. Further, there is an inlet tubing 110a at one end of the headspace channel 1062, and an outlet tubing 110b at an opposing end of the headspace channel 1062 to enable introduction of fluids into the headspace channel 1062, the purpose of which will be more apparent below. In this instance, both the inlet and outlet tubings 110a, 110b each has an inner diameter of about 1.60 mm and an outer diameter of about 3.18 mm, and are made of suitable materials, for example silicon. Additional Telfon tubings 112 may be attached to each of the inlet and outlet tubings 110a, 110b to facilitate extension to syringe pumps (not shown) for coupling thereto. In particular, the inflow and outflow of the organic phase are directed by an inlet and an outlet Teflon tubing 112a, 112b, each having an inner diameter of about 1.32 mm and an outer diameter of about 1.93 mm. Both inlet and outlet Teflon tubings 1 2a, 112b are then connected to the headspace channel 1062 by detachable insertion to the respective inlet and outlet tubings 1 0a, 10b to prevent liquid leakage. The inlet Teflon tubing 112a is connected to a polypropylene syringe (not shown) to allow introduction of the organic phase into the headspace channel 1062, and a flow rate of the organic phase is controlled by a programmable syringe pump (not shown).

Finally, at the top of the microfluidic device 100 is an acrylic embracer 114, which serves to protect the remaining four underlying layers 102, 104, 106, 108. The acrylic embracer 114 is essentially a piece of acrylic substrate. It will be appreciated that all of the five layers 102, 104, 106, 108, 14 of the microfluidic device 100 are of similar shape and size, and more specifically of substantially flat rectangular-like shape. More specifically, the well layer 104, intermediate layer 106, protector layer 108 and acrylic embracer 114 are all formed to be substantially transparent to facilitate visibility when a sample is being worked on in the microfluidic device 100.

Regarding the formation of the array of wells 1042 in the well layer 104, the PDMS structure is patterned using a pulsed C0₂ laser, according to a method described in a previous work known to skilled persons. Briefly, Dow Corning Sylgard 184 PDMS polymer (i.e. as 10:1 parts A and B, in which part A comprises PDMS pre-polymer and part B comprises a catalyst for polymerization) is homogenously mixed and then subjected to vacuum for 30 min to remove any air bubbles in the PDMS mixture. A desired respective thickness of the well layer 104, intermediate layer 06, and protector layer 108 (all of which are formed of PDMS) is achieved by volume controlled casting of PDMS mixture on an elevated surface in an oven at around 80 ^°C for about three hours. The well layer 104 is then patterned using a commercial C0₂ laser cutting instrument (e.g. VersaLaser VLS 2.30 from Universal Laser System Inc). As afore described, the well layer 104 and the headspace channel 1062 are arranged to be sandwiched between the base 102 (which is a piece of glass substrate) and the protector layer 108 to form the array of wells 1042. Thereafter, the acrylic embracer 114 is placed on top of the protector layer 108 to complete the microfluidic device 00. But however, it is to be appreciated that both the protector layer 108 and acrylic embracer 114 are removably attached to the microfluidic device 100, and thus may be removed when necessary, for example to allow access to the array of wells 1042 if desired. In other words, the array of wells 1042 is exposed as such. Therefore, in that sense, the protector layer 108 and acrylic embracer 114 collectively function as a cover for the microfluidic device 100. It will also be apparent that once the protector layer 108 and acrylic embracer 114 are removed, headspace channel 1062 simply becomes an open space adjacent to the array of wells 1042. 1.2 pH dependant phase partitioning of protein, DNA and RNA in micro-chip

Figure 2, which includes Figures 2a to 2h, shows the respective schematic steps of a method 200 for isolating nucleic acids from other particles in an aqueous sample (also known as aqueous phase) using the microfluidic device 100 of Figure 1. The aqueous sample will be referred hereinafter to as the aqueous phase. In particular, at step 202a (see Figure 2a), an aqueous phase 201 containing biological/chemical materials are first deposited in the array of wells 1042. Specifically, in other embodiments, the biological/chemical materials may first be pre-loaded into the array of wells 1042, and an aqueous fluid is then thereafter added to form the aqueous phase 201. In particular, the biological and/or chemical materials (i.e. solid, dried, partially dried, or liquid forms) are selected from the group consisting of primers, short nucleotides, and adaptors for nucleic acid amplification, reverse transcription, and next generation sequencing applications, cells, cell debris, tissues, plants, viruses, antibodies, proteins, enzymes, molecules, peptides, nucleic acids (e.g. DNA, RNA, mRNA, microRNA, or cDNA), polynucleotides, oligonucleotides, short fragments of genes or probes, reaction constituents, lysis buffer constituents, bacteria, protozoa, pathogens, fluorescent chemicals or molecules, crystals, liquid droplets, metal ions, and solid particles (e.g. fluorescent particles, nanoparticles, glass beads and magnetic beads, or fluorescent dye chemicals). Examples of the aqueous phase 201 include, but not limited to, Bovine serum albumin (BSA) protein conjugated with fluorescein isothiocyanate (FITC) fluorescence dye (Sigma, Singapore), genomic DNA and total RNA purified from P. aeruginosa PA01 cells (ATCC, USA) are dissolved in either Tris-EDTA (TE) buffer (10 mM Tris base, 1mM EDTA, 0.1% Triton X-100, pH value equal to 8) or Sodium acetate- Acetic acid- EDTA (SAE) buffer (5 mM Sodium acetate, 5 mM Acetic acid, 1mM EDTA, 0.1% TritonX-100, pH value equal to 4.6) and the like. At this step 202a, the headspace channel 1062 is positioned above the array of wells 1042. In this instance, purely for illustration purposes, the biological/chemical materials used are DNA, RNA and proteins as the model macro-biomolecules analytes of bacteria lysate, and RNA is the type of nucleic acids to be isolated and purified using this method 200. The analytes mixed with an aqueous fluid at a concentration of 0.1 pg/μΙ (to form the aqueous phase 201 ) are introduced (e.g. pipette, centrifugation, or by microfluidic means like vacuum loading) into the array of wells 1042. It will be appreciated that the step 202a may also be carried out prior to execution of this method 200 of Figure 2, for example, the microfluidic device 100 with the array of wells 1042 filled with the biological/chemical materials can instead also be provided by another third party. At step 202b (i.e. see Figure 2b), an organic phase 203 is introduced and circulated into the headspace channel 1062, and the microfluidic device 100 is then angularly arranged at a desired suitable angle to the horizontal. Particularly, this step may be performed if the organic phase 203 is denser than the aqueous phase 201 , and/or if the size of the wells 1042 is able to hold the aqueous phase 201 therewithin by surface tension. In other words, the aqueous phase 201 is less dense than the organic phase 203 in this embodiment. It is thus to be appreciated that a suitable angle for angularly arranging the microfluidic device 100 is one that is still able to retain the aqueous phase 201 within the wells 1042. It is also highlighted that in this instance, angularly arranging the microfluidic device 100 means that the microfluidic device 100 is fully inverted. It is to be appreciated that the aqueous phase 201 and the organic phase 203 are substantially immiscible, and therefore will not be mixed together with the introduction of the organic phase 203. It is appreciated that "substantially" is used here because the phenol and other organic chemicals in the organic phase 203 may partially dissolve into the aqueous phase 201 under specific conditions. Depending on the type of nucleic acids desired to be isolated and extracted, a different volume ratio of the organic phase 203 is used. For example, an organic phase 203 with the phenol, chloroform, and isoamyl alcohol compositionally mixed in an approximate volume ratio of 25:24:1 (with a pH value equal to 8.0) is selected and used for DNA extraction, whereas a different approximate volume ratio of 125:24:1 (with a pH value equal to 4.6) is selected and utilised for RNA extraction. DNA or RNA extracted in this manner using method 200 is also respectively termed as On-chip DNA or On-chip RNA extraction. Since RNA extraction is of interest in this example, the latter mixture mentioned above is adopted. In addition, the organic phase 203 may also be rebalanced with TE buffer or SAE buffer prior to usage

The organic phase 203 is circulated within the headspace channel 1062 with continuous forward and reverse flows for a plurality of cycles. A flow rate is selected to be one from 0.25 ml/min, 0.45 ml/min, or 0.65 ml/min, and each cycle lasts for about 5 seconds, with a total time of about 15 minutes for the entire plurality of cycles to be completed. It is to be appreciated that in other instances, the total time for circulating the organic phase 203 may not be limited to 15 minutes. Instead, a length of time to circulate the organic phase 203 may be shorter or longer, depending on the time required to transfer the impurities to the organic phase 203. That in turn is dependent on the well size, a flow speed of the organic phase 203 in the headspace channel 062 (i.e. over the wells 1042), types of impurities being dealt with, types of organic phase 203 used, degree of sample purity required for subsequently sample analyses. In particular, the different flow rates also allow an efficiency of the phase partitioning to be evaluated. The microfluidic device 100 is inverted for a predetermined period of time, equal to the time for the plurality of cycles to be completed, to allow the unwanted impurities to drop down into the headspace channel 1062 and be removed by the circulating organic phase 203, as shown in step 202c (See Figure 2c). It is to be appreciated that the intention of inverting the microfluidic device 100 to allow the unwanted impurities to drop down into the headspace channel 1062 is to avoid the aqueous phase 201 rising from and moving out of the wells 1042, since it needs to be bear in mind that the aqueous phase 201 is less dense than the organic phase 203 in this instance. The impurities then move into the organic phase 201 due to the chemical properties of the impurities and the organic phase 203 (and not due to weight/gravity force acting on the impurities). Furthermore, inversion of the microfluidic device 100 may be required if the size of the wells 042 is sufficiently large (i.e. between a range of 0.1 mm to 10 mm). If the wells 1042 are however of a smaller size (i.e. between a range of 0.05 mm to mm), then inversion of the microfluidic device 100 may not be necessary, since surface tension from the well surface of the wells 1042 is able to retain the aqueous phase 201 within the wells 1042. But, of course, whether sufficient surface tension is generated to retain the aqueous phase 201 in the wells 104 depends on the type of aqueous phase 201 , type of well surface, type of the organic phase 203, and other relevant factors (as will be appreciated by skilled persons). At this step 202c, the inverted microfluidic device 100 is arranged with the headspace channel 1062 now positioned below the array of wells 1042. It is to be appreciated that this step 202 is optionally, and performed only if the organic phase 203 is denser in fluid property than the aqueous phase 201. Furthermore, the inversion of the microfluidic device 100 is carried out because the organic phase 203 is of greater fluid density than the aqueous phase 201. In this case, the unwanted impurities are the DNA and proteins, since RNA is to be extracted, as explained. This is also known as phase partitioning to remove unwanted impurities from the aqueous phase 201. Thus, as will be appreciated, by the time the entire plurality of cycles for circulating the organic phase 203 are completed, most of the unwanted impurities are substantially transferred from the aqueous phase 201 to the organic phase 203, while the RNA is retained in the aqueous phase 201 within the array of wells 1042. At step 202d (See Figure 2d), the organic phase 203 is then removed from the headspace channel 1062 and the microfluidic device 100 is then inverted again, such that the microfluidic device 100 is as per arranged in the state at step 202a. Then the array of wells 1042 is decontaminated by drying using vacuum evaporation, with the isolated RNA still in the array of wells 1042. It is however to be appreciated that the drying of the wells 1042 using vacuum evaporation in this step may be optional, since a user may remove the cover of the microfluidic device 100 now, or centrifuge the isolated RNA out of the wells 1042 for use subsequently.

From here onwards, steps 202e to 202h (of Figures 2e to 2h) are optional to the method 200 of Figure 2. At step 202e (see Figure 2e), a cleaning fluid 204 comprising a certain predetermined concentration of ethanol is circulated into the headspace channel 1062 to further decontaminate the array of wells 1042 of any residues of the organic phase 203 by repetitive washing of the array of wells 1042 using the cleaning fluid 204. In this instance, the concentration of the ethanol in the cleaning fluid is about 70%. But depending on the application, the concentration of ethanol in the cleaning fluid 204 may generally be between 40% to 100%. Thereafter the cleaning fluid 204 is removed from the headspace channel 1062, and the array of wells 1042 are further dried via vacuum evaporation to remove the cleaning fluid 204 at step 202f (see Figure 2f). Particularly, drying of the array of wells 1042 is generally necessary if it is desired to achieve high purity of the isolated nucleic acids. In this case, drying allows the cleaning fluid 204 to clean the residual organic phase 203 in the wells 1042. Also drying is desired if any assay needs to be performed, requiring a second fluid to be introduced into the wells 1042 (e.g. such as PCR reaction mixture, isothermal reaction mixture for next generation sequencing). In this case, the drying creates a space in the wells 1042 to allow the second fluid (e.g. reaction mixture) to enter the wells 1042 (flow in or dispense in). It is also important that the array of wells 1042 is not subjected to high temperatures (i.e. less than 70°C), if the nucleic acids isolated in the array of wells 1042 are RNA. Next at step 202g (i.e. see Figure 2g), a suitable PCR reagent (i.e. a reaction mixture) 206, depending on application (i.e. in this case a q-RT-PCR reaction mixture) is loaded into the array of wells 1042, and at step 202h (see Figure 2h), a layer of mineral oil 208 is introduction into the headspace channel 062 to seal the array of wells 042 carrying the isolated nucleic acids (RNA in this case) to enable On- chip q-RT-PCR assay to be subsequently performed. It is to be appreciated that the reaction mixture is suitable for polymerase chain reaction (PCR), q-PCR, qRT-PCR, isothermal amplification, reverse transcription, DNA amplification used for DNA sequencing. Thus, the layer of mineral oil 208 acts as a sealant. In other instances, q- PCR, PCR, RT-PCR, isothermal amplification, reverse transcription, or DNA amplification used for DNA sequencing assays if desired may also be performed, as will be appreciated by the person skilled in the art. As an example of an application of this method 200, fluorescence labelled BSA retained in the aqueous phase after the phase partitioning may be quantified using a fluorescence imager (e.g. StarCycler, Star Array Pte Ltd, Singapore), while the DNA and RNA rinsed out of the array of wells 1042 may be quantified using Picogreen DNA quantization kit and Ribogreen RNA quantification kit respectively (Invitrogen, USA) following the manufacturer's protocol.

1.3 Comparison of chip based liquid phase and column based solid phase DNA/RNA extraction from bacterial lysate

1.3.1 Preparation of bacterial nucleic acids by chip based liquid phase and column based solid phase extraction method In this section, to illustrate a specific application of the method 200 of Figure 2, extraction of DNA and RNA from bacterial lysate using the microfluidic device 100 by chip based liquid phase and column based solid phase purification technology is disclosed. Steps for preparation of the biological/chemical materials for the aqueous phase 201 are as follow below. Bacterial nucleic acids are released by enzymatic treatment followed by thermal lysis. Gram-negative bacteria P. aeruginosa PA01 (ATCC, USA) and Gram-positive bacteria S. aureus (ATCC 25923, ATCC, USA) are maintained in Luria broth (LB) containing 1% tryptone, 0.5% yeast extract, and 0.5% sodium chloride. Suspensions of P. aeruginosa and S. aureus colonies are then diluted in a series of 10 fold dilutions and treated with enzyme mixture containing 1 U/μΙ Ready-Lyse™ Lysozyme (Epicentre Biotechnologies, USA)-50 g/ml proteinase K (Qiagen, Germany) and 50 pg/ml lysostaphin (Sigma, USA)-50 pg/ml proteinase K respectively in TE buffer at 37^°C for about 5 minutes followed by thermal lysis at 85°C for about 5 minutes for DNA extraction. As for RNA extraction, enzymatic reaction was carried out in 1 mM EDTA (with a pH value equal to 7.0) with subsequent thermal lysis in SAE buffer at 85°C for 5 minutes. A bacterial concentration is determined by measuring the optical density at 660 nm (OD₆₆₀) and confirmed by CFU counting. 1 μΙ bacterial cell lysate of P. aeruginosa or S. aureus (forming the aqueous phase) from 5000 CFU to 5 CFU are pipetted into the array of wells 1042 of the microfluidic device 100. This corresponds to the step 202a of Figure 2a. Then corresponding organic phase 203 is introduced into the headspace channel 1062 to remove unwanted impurities by phase partitioning at a flow rate of 0.65ml/min with continuous forward and backward flow for 5 seconds as one cycle, and lasting for 15 minutes in total for completion of the entire series of cycles. These correspond to the steps 202b to 202c (of Figures 2b and 2c), after which the organic phase 203 is pumped out of the headspace channel 1062. At this step, the DNA and RNA of the bacterial lysate are now isolated in the array of wells 1042. Any residues of the organic phase 203 are further eliminated by vacuum evaporation of the array of wells 1042 for about 5 minutes, as per step 202d of Figure 2d. The cleaning fluid 204 with 70% ethanol is then infused in and pumped out of the microfluidic device 100 to wash away any residues of the organic phase 203, as per step 202e of Figure 2e. The array of wells 1042 is then air dried and further decontaminated under vacuum for 5 minutes to remove residues of the cleaning fluid 204, as per step 202f of Figure 2f. The DNA and RNA as isolated (and thus purified) in the array of wells 1042 are then rinsed out by repeated pipitting and quantified using On-chip q-PCR or q-RT-PCR analysis. 0457

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Alternatively, DNA and RNA from 1 μΙ bacterial lysate of each dilution may be purified using Qiagen DNeasy Blood & Tissue Kit and RNeasy Mini Kit (Qiagen, Germany) respectively following the manufacturer's protocol. It is to be highlighted that DNA eluted in TE buffer is first dialyzed against water using Slide-A-Lyzer Dialysis Cassettes, 10K MWCO (Fisher Scientific, USA) to eliminate TE interference for Off- chip Q-PCR assay and then vacuum dried in a PCR tube, while RNA recovered in distilled sterile water is vacuum dried in another PCR tube directly. The DNA and RNA of P. aeruginosa and S. aureus at 5*10⁸ CFU/ml may also be prepared using the Qiagen DNeasy Blood & Tissue Kit and RNeasy Mini Kit respectively. The purified DNA and RNA are diluted in distilled sterile water to provide theoretically equivalent quantities of nucleic acids for each bacterial dilution as reference control for 100% recovery. 3.2 Off-chip quantitative PCR analysis to compare nucleic acid recovery yield of chip based liquid phase and column based solid phase extraction method

Following from the above, the DNA and RNA purified by both chip based liquid phase and column based solid phased purification technology (described in section 1.3.1) are then quantified by Off-chip q-PCR and q-RT-PCR assay respectively to compare the nucleic acid recovery yield using RotorGene 3000 thermal cycler (Qiagen, Germany). One q-PCR reaction mixture includes 10μΙ of 10mM Tris-HCI (with a pH value equal to 9.0), 50 mM KCI, 0.1 % Triton X-100, 0.2 mM each of dATP, dCTP, dTTP and dGTP, 3 mM MgCI2, 0.2 μΜ each of forward and reverse primer, 0.2 U/μΙ of Platinum Taq DNA polymerase (Invitrogen, USA), 1 pg/μΙ of BSA, and 1 * Syber green (Invitrogen, USA). q-PCR is subsequently performed at initial denaturation temperature of 95 ^°C for about 5 minutes followed by 40 cycles of denaturation at 95 ^°C for 30 seconds, annealing at 60^°C for 30 seconds and extension at 72^°C for 30 seconds. q-RT-PCR assay is carried out in a volume of 10 μΙ with 3 mM MgS0₄ using Superscript III Platinum One-Step qRT-PCR Kit (Invitrogen, USA) following the manufacturer's protocol at an initial 50^°C for 20 minutes, then a denaturation temperature at 95°C for 2 minutes followed by 40 cycles of denaturation at 95°C for 15 seconds, annealing and extension at 60°C for around 30 seconds. Contamination of genomic DNA in purified RNA is examined by replacing Superscript III RT/Platinum ^® Taq Mix with 2 units of Platinum^® Taq DNA polymerase according to the manufacturer's protocol. Primers pairs for q-PCR and q- RT-PCR assay of two target genes of P. aeruginosa (16S rRNA, gene PA 0708) and two target genes of S. aureus (16S rRNA and ViK) are as shown in the table 300 of Figure 3.

1.4 On-chip real time PCR analysis

Figure 4 depicts a photographic view of a vacuum system setup 400 developed by StarArray Pte Ltd of Singapore for loading a PCR reagent (i.e. a reaction mixture) 502 into array of wells 042 of the microfluidic device 100 of Figure 1 (as per step 202g of Figure 2g), while Figure 5 includes Figure 5a to 5d, which show respective schematic steps for loading the PCR reagent 206 using the vacuum system setup 400 of Figure 4. For clarity and ease of explanation, a different reference numeral of 502 for the PCR reagent is used in this instance, rather than that of 206 as previously labelled in Figure 2 afore, but otherwise not to be construed as being different. Particularly, the vacuum system setup 400 is commercially termed as Universal WellArray loader by StarArray Pte Ltd of Singapore. Also, loading of the PCR reagent 502, which is facilitated using vacuum, is performed according to the manufacturer's protocol for on-chip quantitative PCR assay in the same array of wells 1042, where the nucleic acids are isolated to avoid nucleic acid loss due to liquid transfer, as well as to demonstrate feasibility of the microfluidic device 100 in providing an "all in one" solution for bacterial nucleic acids analysis. Briefly, vacuum system setup 400 mainly comprises a body portion and a base, in which the body portion is mounted at an angle (e.g. 45°) to the base to facilitate user operation. The microfluidic device 100 is detachably held in place (e.g. by latches) via an adjustable chip adaptor 402, which is arranged in the centre of the body portion of the vacuum system setup 400. The inlet Teflon tubing 112a of the microfluidic device 100 is then attached to a pipette tip of the vacuum system setup 400, which in turn connects to a reservoir 403 for the PCR reagent 502, and flow of the PCR reagent 502 into the headspace channel 1062 is controlled by an external mechanical pinch valve 404 located at the body portion. The reservoir 403 for the PCR reagent 502 is mounted to a standing support attached to the base of the vacuum system setup 400. 1 mM CaCI2 is supplemented in the PCR reagent 502 to remove the PCR inhibition induced by EDTA retained in the aqueous phase 201 after phase partitioning. Also, the outlet Teflon tubing 112b of the microfluidic device 100 is attached to a vacuum pump (not shown). To briefly recap, at this point, the array of wells 1042 already contains the isolated nucleic acids (e.g. RNA), as per step 202f of Figure 2f. Then (with reference to step 202g), to load the PGR reagent 502 into the array of wells 1042 of the microfluidic device 100, the vacuum pump is first powered on with the pinch valve 404 closed until the system internal air pressure is below 2 Bar, as per step 504a of Figure 5a. Afterwards, the pinch valve 404 is released to load the PCR reagent 502 held in the pipette tip into the array of wells 1042 by vacuum driven microfluidics, as per steps 504b and 504c of Figures 5b and 5c respectively. For this step only, the pinch valve 405 at the outlet tubing 112b can be optionally closed before the pinch valve 404 at the inlet Teflon tubing 112a opens. Any excess PCR reagent 502 is removed from the headspace channel 1062 as per step 504c. That is the vacuum is still maintained to remove the excess PCR reagent 502 from headspace channel 1062 and to isolate the array of wells 1042 at this step 504c. Then at step 504d of Figure 5d, the array of wells 1042 are overlaid by a layer of mineral oil 506 (acting as a sealant) which is introduced into the headspace channel 1062, followed by q-PCR or q-RT-PCR assay using a quantitative PCR machine, for example the StarCycler quantitative PCR machine (of Star Array Pte Ltd, Singapore) which is configured with multiple integrated functions such as thermo-cycling control, real time fluorescence imaging, on-line image processing and data analysis. For clarity of explanation, a different reference numeral of 506 for the layer of mineral oil is used in this instance, rather than that of 208 as per Figure 2 afore, but not to be construed as otherwise being different. It will also be appreciated that steps 504a-504d of Figures 5a-5d collectively correspond to steps 202g and 202h of Figures 2g and 2h, as afore described 1.5 High throughput nucleic acid isolation from single bacterium

To enhance and achieve high throughput analysis of DNA or RNA from single bacterial cells, the microfluidic device 100 of Figure 1 is re-designed into a chip 600 with a 2- dimension (2D) format with 900 number of wells arranged as an array of wells 602, and having an adjacent headspace channel 604 in fluid communication therewith. In particular, the array of wells 602 is arranged as a rectangular layout, but other different forms of layout may also be possible depending on specific applications. In this instance, the array of wells 602 is configured to be in a 30 wells by 30 wells arrangement (i.e. 30 x 30). Figure 6, which includes Figures 6a and 6b, show a schematic view and a photographic view of the 2D chip 600. It will be appreciated that the proposed 2D chip 600 is also a liquid phase nucleic acid purification chip, just like the microfluidic device 100 of Figure 1. Briefly, the 2D chip 600 is formed by sandwiching the array of wells 602 between a piece of glass substrate 606 (i.e. to be positioned at the base), and a transparent acrylic substrate 608 (i.e. to be positioned at the top). A PDMS protector layer 610 (also transparent) is attached to underneath of the acrylic substrate 608, and faces the array of wells 602. Two capillary channels are respectively arranged at any two opposing diagonal corners of the 2D chip 600 as inlet and outlet capillary channels 610a, 610b. In this instance, each well 602 has a dimensional of 0.5 mm χ 0.5 mm χ 0.5 mm (i.e. length x width x height) and configured to be able to hold fluid of approximate 125 nl. But it is to be appreciated that the 2D chip 600 is largely similar to the microfluidic device 100 of Figure 1 , and hence for brevity, further explanations of the similar components of the 2D chip 600 will not be repeated herein. Similarly, the method 200 of Figure 2 is applied here, but the specifics of each step of the method 200 are further detailed below. A process for loading a PCR reagent into the array of wells 602, depositing with single bacterial cells, is as per based on the described schematic steps of Figure 4. Specifically, an enzyme mixture of 1 U/μΙ Ready-Lyse™ Lysozyme - 50 pg/ml Proteinase K or 50 pg/ml lysostaphin - 50 pg/ml Proteinase K is first pre-dried in the array of wells 602 for On-chip lysis of P. aeruginosa and S. aureus respectively. Single bacterium is isolated into the individual wells 602 by loading P. aeruginosa and S. aureus of less than 0.3 CFU/well. According to Poisson statistics, a vast majority of the wells 602 may contain no more than a single bacterium in this condition, as shown in Figure 7. Figure 7 is a schematic top view of the 2D chip 600, showing single bacterium each being isolated in some of wells 602 in the array. For single bacterial DNA extraction, P. aeruginosa and S. aureus are diluted to less than 0.3 CFU/ well in TE buffer and loaded into the 2D chip 600 after which a low-viscosity PCR encapsulation reagent (Vapor-Lock, Qiagen, Germany) is infused into the headspace channel 604. The 2D chip 500 is then subjected to heat treatment at 37°C for about 5 minutes, and further at 85°C for around 5 minutes to lyse the captured bacteria. Following this, the Vapor-Lock reagent is from the headspace channel 604.

On the other hand, to isolate RNA from single bacterium, P. aeruginosa and S. aureus culture are first stabilized by adding 1/5 volume of ice cold phenol: ethanol (5:95) to minimize RNA degradation, and adjusted to desired concentration in 1 mM EDTA buffer (with a pH value equal to 7.0). Bacterial cells are then loaded into the 2D chip 600 with appropriate enzyme mixture pre-dried in the array of wells 602. The 2D chip 600 is then incubated at room temperature for about 5 minutes and then heated at 37°C until the liquid in the array of wells 602 has fully evaporated to avoid repeated usage of PCR encapsulation reagent. A buffer mixture of 5 mM sodium acetate and 5 mM acetic acid is then loaded into the 2D chip 600 to adjust the pH value to 4.6 for RNA recovery. The array of wells 602 are subsequently overlaid with the Vapor-Lock reagent (mentioned above) and heated at 85°C for 5 minutes to lyse the captured bacteria. DNA and RNA from single bacterial cell are prepared by chip based liquid phase extraction using an organic phase (i.e. PCI) with the pH values of 8.0 and 4.6 respectively, as afore described in step 202b of Figure 2b. Any residue organic phase in the array of wells 602 are decontaminated by vacuum evaporation and further washed twice with a cleaning fluid having 70% ethanol concentration. Thereafter, a PCR reagent with corresponding primers is loaded into the 2D chip 600, followed by q-PCR or q-RT-PCR analysis as desired.

2. Results and discussion

2.1. Phase partitioning of DNA, RNA, and protein

Briefly, during liquid phase extraction, components of bacterial cell lysate are selectively distributed into the organic phase or retained in the aqueous phase with mass transport occurring at the organic-aqueous interface. Hence maximising the contact area of the two immiscible phases is important for effective separation of nucleic acids from other cell components. This may conveniently be achieved using a vortex mixer to generate chaotic flow fields of the two phases in macrofluidic systems. However, in microfluidic modules, extensive mixing of two immiscible liquid phases cannot be easily accomplished, and so phase partitioning mainly depends on passive diffusion in stratified or droplet based flow of the two phases, as proposed in a related prior work. Nonetheless, in these scenarios, the organic phase that remains with the aqueous phase will interfere with downstream nucleic acid assays. As discussed above, the proposed microfluidic device 100 with the aqueous phase 201 isolated in the array of wells 1042, while the organic phase 203 is introduced into the headspace channel 1062, in fluid communication with the array of wells 1042, and circulated with repeated forward and reverse flows to achieve intensive mixing of the two different phases. The organic phase 203 is then pumped out of the headspace channel 1062 and further decontaminated by repeated washing and evaporating with the cleaning fluid 204 having 70% ethanol concentration, leaving the target molecule of interest dried in the array of wells 1042 for q-PCR or q-RT-PCR analysis to be performed.

.

Now with reference to Figure 8, the partitioning efficiency of three major types of macromolecules (i.e. DNA, RNA and protein) with the organic phase 203 set^' at different flow rates of 0 ml/min, 0.25 ml/min 0.45 ml/min, and 0.65 ml/min are examined and studied in three experiments. Specifically, Figure 8, which includes Figures 8a and 8b, depict corresponding results of recovery of fluorescence labelled BSA, DNA and RNA in the aqueous phase 201 at pH values of 8.0 (i.e. Figure 8a) and 4.6 (i.e. Figure 8b) respectively. It is to be noted that recovery of fluorescence BSA, DNA and RNA in the aqueous phase 201 are evaluated using fluorescence imaging, and via Picogreen and Ribogreen quantification assay respectively. More specifically, images of fluorescence labeled BSA retained in the aqueous phase 201 are shown beneath the corresponding BSA recovery bars with non-partitioned BSA in a far left image of those images. Each point represents the mean ± standard deviation error of the three replicated experiments. Fluorophore-conjugated BSA is selected to study the protein phase partitioning as previously proposed in a related work, while unmodified DNA and RNA purified from P. aeruginosa cells are chosen as the analytes to eliminate potential bias that may be introduced when using short synthetic DNA or RNA labeled with fluorescence dye because of their significant variations in molecular weights and structures as compared with natural nucleic acids. As shown in Figure 8, 92.9% protein and 93.2% RNA can be removed from the aqueous phase 201 at the organic phase 203 (with a pH value equal to 8.0) at the flow rate of 0.45 ml/min. However, considerably enhanced DNA partitioning (with more than 99.9% removal from the aqueous phase) with the organic phase 203 (with a pH value equal to 4.6) is only found at the flow rate of 0.65 ml/min. Further, 93.3% DNA and 94.2% RNA are recovered in the aqueous phase 201 when partitioned with the organic phase 203 at the pH value of 8.0 and 4.6 respectively at this flow rate of 0.65 ml/min, which is further adopted for nucleic acid isolation from bacterial lysate. It is also validated that washing the proposed microfluidic device 100 with the cleaning fluid 204 having 70% ethanol concentration do not result in significant sample loss, which is in consistency with the findings of a previously published report that nucleic acids can non-specifically bind to glass substrate without the interference of ethanol washing. 2.2 DN and RNA isolation from bacteria lysate

Figure 9, which includes Figures 9a to 9d, depicts corresponding results of Off-chip q- PCR (i.e. Figures 9a and 9b) and q-RT-PCR (i.e. Figures 9c and 9d) analysis of DNA and RNA isolated by chip based liquid phase and column based solid phase nucleic acid purification methods from P. aeruginosa (i.e. Figures 9a and 9c) and S. aureus (i.e. Figures 9b and 9d) ranging from 5000 CFU to 5 CFU. In particular, thermal lysis offered a simple and effective way^~ to disrupt bacterial membranes and have been validated for DNA and RNA isolation from both gram positive and gram negative bacteria. One major advantage to incorporate thermal lysis technique into microfluidic nucleic acid extraction devices is that the engineering complexity of the chip design can be significantly reduced. In this study, bacterial DNA and RNA are released by thermal lysis in TE buffer and SAE buffer respectively with lysozyme-proteinase K pre- treatment for P. aeruginosa and lysostaphin-proteinase K pre-treatment for S. aureus. As the enzyme activity of lysozme and lysostaphin is significantly weakened in acidic buffer, enzyme reaction for RNA extraction was carried out in 1 mM EDTA (with a pH value equal to 7.0) at 37°C for 5 minutes with subsequent thermal lysis in SAE buffer at 85°C for 5 minutes. No noteworthy DNA and RNA degradation are observed at this temperature as assessed by q-PCR or q-RT-PCR analysis (data are not shown). Nucleic acids are isolated from P. aeruginosa or S. aureus cell suspensions ranging from 5000 CFU to 5 CFU in 1 μΙ volume using both chip based liquid phase, and Qiagen column based solid phase column-based nucleic acid purification methods, and analyzed by q-PCR or q-RT-PCR assay. Threshold Ct values obtained are then compared with purified nucleic acids isolated from high cell density bacterial suspension (5*10⁸ CFU/ml) using Qiagen DNeasy/RNeasy DNA/RNA purification kit with appropriate dilutions referenced as 100% nucleic acid recovery. 16S rRNA is selected as the RNA target for q-RT-PCR because of their rich abundance in bacteria, so as to eliminate the factor of sensitivity limit of q-RT-PCR technology itself. Nucleic acid recovery is calculated (based on a previous related work) according to a standard curve constructed using the Ct values obtained with reference nucleic acid template of each bacterial dilution, and Ct values obtained with nucleic acids purified by chip based liquid phase or column based solid phase extraction method. As shown in Figure 9, recovery of nucleic acids prepared by Qiagen solid phase technology is significantly reduced when the input bacteria concentration decreased, with a limit of 50 CFU for DNA recovery, and around only 15-20% recovery for 5 CFU in RNA recovery. In contrast, 85%-120% nucleic acid recovery of all bacterial dilutions may be reached using the proposed microfluidic device 100, with the only exception of 70%-80% recovery in S. aureus RNA extraction probably because of insufficient removal of endogenous RNase during phase partitioning. It is highlighted that no considerable DNA contamination is observed in RNA prepared using the proposed method 200 shown in Figure 2. These findings are in accordance with previously published results that nucleic acid recovery yield is higher with the liquid phase phenol- chloroform extraction than with the column purification.

Next, Figure 10, which, includes Figures 10a to 10d, depicts corresponding results of On-chip q-PCR amplification of genomic DNA isolated from 5000 to 5 P. aeruginosa and S. aureus cell (i.e. Figures 10a and 10b) and q-RT-PCR amplification of RNA isolated from 5000 to 5 P.aeruginosa and S. aureus cell (i.e. Figures 10c and 10d). On the other hand, Figure 11 includes Figures 11a to 11d, which depict corresponding On-chip melting curve analysis for PCR product of genomic DNA isolated from 5000 to 5 P.aeruginosa and S. aureus cell (i.e. Figures 11a and 11b) and RNA isolated from 5000 to 5 P.aeruginosa and S. aureus cell (i.e. Figures 11c and 1d) to test the purity of the amplified product. In particular, the scenario set up to obtain the above results for on-chip q-PCR or q-RT-PCR assay with purified nucleic acid dried in the array of wells 1042 is as follows. The array of wells 1042 is washed twice with the cleaning fluid 204 having 70% ethanol concentration to remove phenol contamination before loading the PCR reagent 206 (corresponding to step 202e of Figure 2e). Although the array of wells 1042 are momentarily in contact with the PCR reagent 206, no substantial loss of nucleic acid sample, nor cross contamination is however observed. Nucleic acids isolated from 5000 down to 5 cells of P. aeruginosa and S. aureus bacteria may then be successfully amplified using On-chip q-PCR and/or q-RT-PCR. The On-chip q-PCR amplification and melting curves are shown in Figures 10 and 11 respectively. It is also to be highlighted that control experiments carrying no template are occasionally amplified in RNA based q-RT-PCR assay due to primer dimer formation. However, these control experiments emerged significantly later than the amplification with RNA isolated from bacteria of 5 CFU and may therefore be differentiated. In addition, the Tm values of these control experiments are different from that of the samples with templates, so positive PCR reaction with RNA from bacterial samples can still be recognized.

It is also appreciated that q-PCR assay may be applied to amplify target sequence of a single DNA molecule. However, quantification of RNA from single bacterium by q-RT- PCR assay has only recently been proven to be possible. In this recent work mentioned, researches show that single bacterial cell is selected by vision based aspiration and dispensing technique under the microscope followed by RNA extraction using column based nucleic acid purification technique. Nonetheless, only a limited number of single bacterium can be studied each time. Besides, RNA recovery rate and possible false positive signal generated by DNA contamination are not verified. To validate whether the present proposed method 200 of Figure 2 can reach single bacterium sensitivity as a possible way to study DNA and RNA heterogeneity in microbial populations, the microfluidic device 100 of Figure 1 is re-designed in a two dimensional format with 900 number of wells arranged as the array of wells 602, which is the design of the 2D chip 600 as afore described with reference to Figure 6. Enzyme mixture of lysozyme-proteinase K and lysostaphin- proteinase K are pre-dried in the array of wells 602 and re-suspended by loading P. aeruginosa and S. aureus cells of less than 0.3 CFU/well into the 2D chip 600 so that single bacterium can be captured and lysed for On-chip liquid phase nucleic acid purification. The corresponding results obtained are depicted in Figure 12, which includes Figures 12a to 12d, showing corresponding photos of results of On-chip amplification of DNA isolated from single P. aeruginosa (i.e. Figure 12a) and S. aureus (i.e. Figure 12c) and On-chip q-RT-PCR amplification of RNA isolated from single P. aeruginosa (i.e. Figure 12b) and S. aureus (i.e. Figure 12d). It is to be highlighted that the foregoing discussions for Figures 8-12 are in relation to the microfluidic device 100 of Figure 1.

It is to be highlighted that similar to nucleic acids, pre-dried enzyme in the array of wells 602 is retained in both activity and quantity during the following sample loading step to be described. P. aeruginosa and S. aureus RNA are stabilized by adding 1/5 volume of ice cold phenol: ethanol (5:95). Nucleic acids isolated from single bacterium are analyzed by On-chip q-PCR and q-RT-PCR analysis. Taqman q-RT-PCR is performed in replacement of Sybre green assay for single bacterium RNA analysis to eliminate the primer-dimer interference. The primers and probes are adopted based on references from previous related work. Bacteria cell densities are then confirmed by CFU counting on LB agar. The overall success rate for DNA and RNA isolated from single P. aeruginoa is determined to be around 92% and 85% respectively, compared with 87% and 71 % for DNA and RNA isolated from single S. aureus as calculated according to Poisson statistics. Specifically, the relatively lower success rate for RNA isolation from single bacterium may have resulted from insufficient cell lysis and incomplete removal of endogenous RNase. The absence of DNA contamination in RNA isolated from single bacterium is confirmed by no positive On-chip amplification when loading q-RT- PCR reaction mixture with Superscript III RT/Platinum^® Taq Mix replaced by 2 units of Platinum^® Taq DNA polymerase. The standard deviations of the Ct values obtained from q-PCR amplification with genomic DNA from single P. aeruginosa and S. aureus are 0.45 and 0.57 respectively and standard deviations of the Ct values obtained from q-PT-PCR amplification with total RNA from single P. aeruginosa and S. aureus being 0.62 and 0.68 respectively, indicating good reproducibility of the proposed method 200 of Figure 2.

From the foregoing, it will be appreciated that the proposed microfluidic device 100 of Figure 1 is able to selectively isolate DNA or RNA from a small number of bacterial cells (ranging from 5000 down to single bacterium) distributed in an array of wells of 1 μΙ and 125nl in sample volume and directly detected by quantitative PCR performed in the same array of wells, in which the nucleic acid is isolated. It is determined through experiments that 85%-120% nucleic acid recovery can be achieved from bacteria ranging from 5000 to 5 CFU in 1 μΙ sample volume using the proposed microfluidic platform, with the only exception of 70%-80% for S. aureus RNA recovery. This contrasted with the significantly compromised nucleic acid recovery using conventional Qiagen nucleic acid purification techniques which could only achieve 10-18% in DNA recovery for 50 bacteria and 15-20% in RNA recovery for 5 bacteria. Furthermore, DNA extracted from 5 bacteria using Qiagen nucleic acid purification technique cannot be detected by q-PCR assay. Also, successful high throughput RNA extraction from single bacterium with compatible on-chip quantitative reverse transcription PCR (q-RT- PCR) assay was achieved, using the microfluidic device 100 of Figure 1 re-configured as the 2D chip 600 of Figure 6 in a two dimensional format of 900 number of wells 602 arranged as the array of wells 602 to hold the sample volume of 125 nl/well, with single bacterium trapped in individual wells by loading at a cell density of less than 0.3 CFU/ml. False positive signals generated from DNA in q-RT-PCR assays may be effectively removed without the need for additional DNase treatment. 3. Other embodiments and variations

Further embodiments of the invention will be described hereinafter. For the sake of brevity, description of like elements, functionalities and operations that are common between the embodiments are not repeated; reference will instead be made to similar parts of the relevant embodiment(s).

It is highlighted that for all subsequent relevant embodiments discussed hereinafter, whenever Figure 2 is referenced, it is to be noted that the isolated nucleic acids residing in the wells 1042 (either at after step 202c where the organic phase 203 is removed but prior to drying the wells 1042 at step 202d, or subsequent to step 202d, or step 202f) are recovered from the wells 1042 for storage and subsequent analyses. Methods of recovery of the isolated nucleic acids from the wells 1042 include by pipette aspiration of the aqueous phase 201 from the wells 1042, centrifugation of the aqueous phase 201 from the wells 1042 into a set of collection tubes whose positions are arranged to match the those of the wells 1042 or into the headspace channel 1062 and thereafter removed from the headspace channel 1062, or by opening an aperture at the bottom of the associated wells for recovery through the aperture. It is to be appreciated that the term "recover" or "recovery" in this present context refers to removal of the isolated nucleic acids and/or aqueous phase 201 from the wells 042 to be collected into another device/vessel for storage or subsequent analyses of the isolated nucleic acids. Further, it is to be appreciated that after the isolated nucleic acids have been amplification as per Figure 2h, the aqueous phase 2012 may need to be recovered from the respective individual wells 042 to be sent for DNA sequencing of the isolated nucleic acids on a well-by-well basis, or for conducting gene expression study of the isolated nucleic acids on a well-by-well basis, especially for single cell analysis, in which each well 1042 may contain one or a few cells. Furthermore, it is to be appreciated that steps 202a to 202d of Figure 2 may also be repeated with the same or different organic phases, whereas steps 202e to 202f may be repeated with the same or different cleaning fluids. In addition, lysing of the nucleic acid-containing biological particles if they are deposited in the wells 1042, may be performed, if such a step is considered relevant to the associated embodiments. Also, at step 202h of Figure 2h, other types of polymer liquid may optionally be used as sealant fluids.

According to a second embodiment, Figure 13, which includes Figures 13a and 13b, collectively depicts a method for preloading the array of wells 1042 of the microfluidic device 100 of Figure! It is to be highlighted that this mentioned method only modifies the step 202a of Figure 2a, pertaining to the introduction of the aqueous phase 201 (having biological and/or chemical materials). That is, the remaining steps 202b-202h in the method 200 of Figure 2 are still performed as per afore described.

In this embodiment, the cover comprising the protector layer 108 and acrylic embracer 114 of the microfluidic device 100 is optionally removed prior to performing the step 202a (as modified) under this method in order to allow access to the array of wells 1042. Specifically, the array of wells 1042 (and the adjacent space thereto) of the microfluidic device 100 is first preloaded with the organic phase 203 and then droplets 1300 of the aqueous phase 201 are introduced (or alternatively, the aqueous droplets 1300 are pre-mixed into the organic phase 203 before loading into the adjacent space above the wells 1042), as shown in Figure 3a. Thereafter, the microfluidic device 100 is preferably disturbed by agitating the microfluidic device 100 to shake the droplets 1300 that might otherwise be resting on the walls between the wells 1042 into the wells 1042. In addition, centrifugation be further be needed to speed up the movement of the droplets 1300 into the wells 1042, and vortex be need to be applied to homogenize the droplet distribution in the organic phase 203 so that some droplets 300 enter into the wells 1042. The organic phase 203 can be removed from the adjacent space above the wells 1042 even if there are some droplets 1300 left over in the organic phase 203 after a predetermined period of time has lapsed to allow the droplets 1300 to settle into the array of wells 1042, as depicted in Figure 13b. The predetermined period of time required depends on the processes to move the particles into the wells 1042. It will also be appreciated that, by the end of the predetermined period of time, not all of the array of wells 1042 will be deposited with a droplet 1300 of the aqueous phase 201. Then, the protector layer 108 and acrylic embracer 14 of the microfluidic device 100 are both re-attached thereto, and the adjacent space above the array of wells 1042 consequently becomes the headspace channel 1062 of the microfluidic device 100. The organic phase is afterwards processed as per the steps 202b-202f in the method 200 of Figure 2 to start purification of the nucleic acids from the droplets 1300 of the aqueous phase 201. It is appreciated that the steps pertaining to recovery of the nucleic acids, as afore described, apply similarly in this instance.

Possible variations to some aspects of this method of Figure 13 include the following: 1. A size of the droplets 1300 is controlled, so that each well 1042 may only accommodate one single droplet 1300 (or alternatively a predetermined number of droplets 1300). It is to be appreciated that the controlling the droplet size is important for applications relating to single cell or dPCR analysis.

2. A number of the droplets 1300 may be controlled, so that the total droplet number is optionally smaller than the total number of wells 1042, in order to achieve the object of having only one or no droplet 300 in each well 1042. The droplets 1300 may include some or all constituents required for biological assays, including polymerase chain reactions (PCR), isothermal amplifications, DNA amplification for DNA sequencing, various cell assays, etc.

3. Each droplet 1300 may be dispensed to include different biological and/or chemical materials.

4. Each well 1042 may be pre-loaded with a reaction mixture and/or different oligonucleotides as primers for PCR, or other types of nucleic acid amplification or primer extensions such as those suitable for DNA sequencing, or various cell assays.

According to a third embodiment, Figure 14, which includes Figures 14a to 14d, collectively depicts another method for preloading the array of wells 1042 of the microfluidic device 100 of Figure! Similarly, this present method only modifies the step 202a of Figure 2a, pertaining to the introduction of the aqueous phase 201 (having biological and/or chemical materials), and the remaining steps 202b-202h in the method 200 of Figure 2 are in accordance to those described afore. Also, in this embodiment, the protector layer 108 and acrylic embracer 4 of the microfluidic device 100 are optionally removed prior to performing the step 202a (as modified) under this method in order to allow access to the array of wells 1042. Specifically, the array of wells 1042 of the microfluidic device 100 is preloaded with an aqueous buffer 1400 (e.g. distilled water) and thereafter, biological materials/particles 1402 (including nucleic acid, cells, and tissues) are then deposited into the aqueous buffer 1400, as shown in Figure 14a. For clarity, the aqueous buffer is simply an aqueous fluid. Then, the microfluidic device 100 is for a predetermined period of time to allow the biological materials/particles 1402 to settle into the array of wells 1042, as depicted in Figure 14b. The predetermined period of time required depends on the processes to move the particles into the wells 1042. It is to be appreciated that, by the end of the predetermined period of time, not all of the array of wells 1042 may be deposited with the biological materials/particles 1402. Next, as depicted in Figure 14c, the aqueous buffer 1400 residing above the array of wells 1042 is removed, so that only the aqueous buffer 1400 filled in the array of wells 1042 remains, together with the biological materials/particles 1402 that have already settled into the array of wells 1042. It will be appreciated that the aqueous buffer 1400 and biological materials/particles 1402 in the array of wells 1042 together thus form the aqueous phase 201. Subsequently, as shown in Figure 14d, the organic phase 203 is then added into the microfluidic device 100 (and with optional inversion of the device 100 if required) to fill the adjacent space above the array of wells 1042, and the protector layer 108 and acrylic embracer 114 of the microfluidic device 100 are also re-attached thereto to seal the array of wells 1042. Moreover, the adjacent space above the array of wells 1042 then becomes the headspace channel 1062 of the microfluidic device 100 with this re-attachment. The organic phase 203 is processed as per the steps 202b-202f in the method 200 of Figure 2 to start purification of the nucleic acids from the aqueous phase 201. Possible variations to some aspects of this method of Figure 14 include the following:

1. If the biological materials/particles 1402 to be analysed are cells or tissue particles, then a size of the well 1042 is configured to match the size of the biological materials/particles 1402, in order that one well 1042 may only accommodate one biological material/particle 1402 (or alternatively a pre-determined number of the biological materials/particles). It will be appreciated that this variation is important for performing single cell analysis.

2. A number of the biological materials/particles 1402 may be controlled so that the number of biological materials/particles 1402 is optionally smaller than a number of wells 1042 to achieve the object of having only one or no biological material/particle 1402 in each well 1042.

3. Also the biological materials/particles 1402 may alternatively be pre-mixed with the aqueous buffer 1400 before being loaded into the wells 1042, as opposed to what is described afore. Furthermore it is also to be appreciated that the various variations afore described for the second embodiment of Figure 13, apply to this third embodiment of Figure 14 mutatis mutandis, and so for sake of brevity, will not be repeated herein. According to a fourth embodiment, Figure 15, which includes Figures 15a. and 15b, collectively depicts a further method for preloading the array of wells 1042 of the microfluidic device 100 of Figured As per the second and third embodiments, this present method only modifies the step 202a of Figure 2a, and the remaining steps 202b-202h in the method 200 of Figure 2 are maintained as described afore. It is appreciated that the steps pertaining to recovery of the nucleic acids, as afore described, apply similarly in this instance. Of course, the protector layer 108 and acrylic embracer 114 of the microfluidic device 100 are optionally removed prior to performing the step 202a (as modified) under this method in order to allow access to the array of wells 1042. In Figure 15a, the array of wells 1042 of the microfluidic device 100 is preloaded with droplets 1500 of the aqueous phase 201. But it is also to be appreciated that not all of the array of wells 1042 will eventually be deposited with a droplet 1500 during this step. Then, in Figure 15b, the organic phase 203 is introduced into the microfluidic device 100 to fill the array of wells 1042 and the adjacent space thereabove. A further optional step that may be performed (after introducing the organic phase) is to de-gas the array of wells 1042 to remove any air trapped in the array of wells when introducing the organic phase 203. Then, the protector layer 108 and acrylic embracer 114 of the microfluidic device 100 are both re-attached thereto, and the adjacent space above the array of wells 1042 consequently becomes the headspace channel 1062 of the microfluidic device 100. The organic phase 203 is processed as per the steps 202b-202f in the method 200 of Figure 2 to start purification of the nucleic acids from the droplets 1500 of the aqueous phase 201.

According to a fifth embodiment, Figure 16, which includes Figures 16a, 16b and 16c, collectively depict an alternative method for preloading the array of wells 1042 of the microfluidic device 100 of Figure! As per the fourth embodiment, this present method only modifies the step 202a of Figure 2a, and the remaining steps 202b-202h in the method 200 of Figure 2 are maintained with no change, as described afore. But, it is highlighted that the sample recovery steps described earlier above are included. Needlessly to say, the protector layer 108 and acrylic embracer 114 of the microfluidic device 100 are also optionally removed prior to performing the step 202a (as modified) under this method in order to allow access to the array of wells 1042. It is also to be highlighted that the array of wells 1042 are in this instance configured with a porous bottom 1600 (e.g. using Teflon filter paper, a polypropylene/polycarbonate track etched filter paper, or micro-fabricated mesh). In Figure 16a, the array of wells 1042 of the microfluidic device 100 is preloaded with nucleic acid-containing biological particles (such as cells, viruses, protozoa, tissues, and plants) 1602 contained in the aqueous phase 201. But it is also to be appreciated that not all of the array of wells 1042 may be deposited with a biological particle 1602 during this step. It is also to be appreciated that the protector layer 108 and acrylic embracer 114 is not limited to the materials afore described, and will be apparent that many other suitable materials may also be used, as long as those materials are inert to the organic phase 203.

Figure 16b shows unwanted materials/chemicals in the aqueous phase 201 passing through the porous bottom 1600 under application of air or vacuum pressure differential, centrifugation, etc., whereas the biological particles 1602 are then retained within the array of wells 1042, thereby providing an additional means for nucleic acids purification, prior to the steps 202b-202f in the method 200 of Figure 2, including lysing of the biological particles 1602. Thus, it will be appreciated that the pore size of the porous bottom 1600 only allows air and the unwanted materials/chemicals to pass through (and also to enable breakup of the unwanted particles in the step shown in Figure 16a, such as lyse red blood cells and pass the lysate through the porous bottom 1600, while retain white blood cells within the wells 1042. Or alternatively, red blood cells and white blood cells may be lysed and the lysate is passed through the porous bottom 1600, while cancer cells are retained within the wells 1042), while still enable the desired nucleic acids in the biological particles 1602 (to be eventually isolated) to be retained in the array of wells 1042. In this instance, the pore size of the porous bottom 1600 may be configured to be between 0.2 pm to 15pm (if retention of cells is desired) or alternatively between 20 nm to 500 nm (if retention of viruses is desired). Subsequently, in Figure 16c, the organic phase 203 is introduced into the microfluidic device 100 to fill up the array of wells 042 and the adjacent space above them. It is further highlighted that the biological particles 1602 retained in the wells 1042 are lysed to release nucleic acids into the wells before adding the organic phase 203 into the microfluidic device 100. Then, the protector layer 108 and acrylic embracer 114 of the microfluidic device 100 are both re-attached thereto, and the adjacent space above the array of wells 1042 becomes the headspace channel 1062 of the microfluidic device 100. In order to start purification of the nucleic acids from the droplets 1500 of the aqueous phase 201 , processing of the organic phase 203 is as per the steps 202b-202f described in the method 200 of Figure 2. To clarify, it is to be appreciated that the aqueous phase 201 may pass through the porous bottom 1600 is pressure is applied. The organic phase 203 does not pass through the porous bottom 1600, if no pressure is applied and surface tension within the wells 1042 will hold the organic phase, or vice versa. The intention of passing the aqueous phase 201 through the porous bottom 1600 is to filter any unwanted impurities while retain required cells in the wells 1042, which may later be lysed to release DNA/RNA in the cells. Of course, the lysed cells do not pass the porous bottom 1600 since no pressure will be applied at that stage.

According to a sixth embodiment, Figure 17 shows a microfluidic device 1700, and Figure 18 (which includes Figures 18a and 18b) depicts a corresponding method for preloading an array of wells 702 of the microfluidic device 1700 in this embodiment. In particular, the microfluidic device 1700 of this embodiment is arranged with a longitudinal channel 1704 that is in fluid communication with each corresponding well 1702 at respective sections along the length of the longitudinal channel 1704. In addition, the microfluidic device 1700 in this instance has a cover 1706, which is simple adjacent to the longitudinal channel 1704, such that both the longitudinal channel 1704 and the array of wells 1702 are sealed off by the cover 1706, except at two opposing ends of the longitudinal channel 1704 which are configured as fluid inlet 1708a and outlet 1708b respectively. That is, the longitudinal channel 1202 replaces the headspace channel 1062 of the microfluidic device 100 of Figure 1. It will be appreciated that, in this instance, the longitudinal channel 1704 is also arranged adjacent to and above the wells 1702. More specifically, the longitudinal channel 1704 is arranged and formed in an appropriate winding configuration that enables fluid connection to all the wells 1702 of the microfluidic device 1700. It will be appreciated that other types of winding configuration or parallel channels are however also possible. Further, it is highlighted that the arrangement of the longitudinal channel 1704, as described, helps to further reduce sample wastage from the wells 1702, specifically during when introducing the aqueous sample 201 into the array of wells 1702. As per the various foregoing embodiments, this present method using the variant microfluidic device 1700 of Figure 17 only modifies the step 202a of Figure 2a, and the remaining steps 202b-202h in the method 200 of Figure 2 are maintained with no change, as described afore. Particularly, in Figure 18a, the wells 1702 are loaded with an aqueous buffer 1800 (e.g. distilled water) and biological materials/particles 1802 are then introduced into the wells 1702 by use of one of the following techniques: body forces (e.g. gravity or centrifugation forces), diffusion, flow induced hydrodynamic forces, electrophoretic forces, dielectrophoretic forces, electric forces, and magnetic forces. Then, the aqueous buffer 1800 is removed from the longitudinal channel 1704, once the biological materials/particles 1802 have settled into the wells 1702 (after a predetermined period has passed). It will be appreciated that the aqueous buffer 1800 and biological materials/particles 1802 in the wells 1702 together form the aqueous phase 201. Subsequently, in Figure 18b, the organic phase 203 is introduced into the longitudinal channel 1704, and is processed as per the steps 202b-202f in the method 200 of Figure 2 to start purification of the nucleic acids from the aqueous phase 201.

In a slight alternative variation to the microfluidic device 1700 of Figure 17, the array of wells 1702 may also be configured with a porous bottom (e.g. using Teflon filter paper or a polypropylene track etched filter paper), just like in the fifth embodiment. The porous bottom of each well 1702 enables the aqueous buffer 1800 to pass through to speed up the trapping of the biological materials/particles 1802 and further remove any unwanted chemicals in the aqueous buffer 1800 that may inhibit sample analysis in a later stage. It is to be appreciated that a pore size of the porous bottom is arranged to be smaller than the size of the nucleic acids desired to be isolated in the wells 1702, as will be apparent by now.

Figure 19 shows an alternative microfluidic device 1900, based on a seventh embodiment, in which the device 1900 has a test tube-like shape. Specifically, this device 1900 is particularly arranged to run Liquid Phase Extraction (LFE) therein, which refers to conventional phenol chloroform extraction of DNA/RNA. The device 1900 comprises an outer tube 1902 which has the same outer shape as a typical PCR or qPCR tube, an inner tube 1904, a removable tube cap 1906 for the outer tube 1902 for sealing purposes. The inner tube 1904 serves as a well for holding samples. The inner tube 1904 is positioned within and adjacent the bottom of the outer tube 1902, and is optionally smaller in size to the outer tube 1902, as will be appreciated. But it is also to be appreciated that for other embodiments, a planar array of the inner tubes 1904 may be arranged within the outer tube 1902. A bottom-most layer of material 1907 of the outer tube 902 that surrounds the inner tube 1904 is preferably arranged to be thermally conductive, and the inner tube 1904 is detachably attached to the outer tube 1902 by known suitable means. Both the inner and outer tubes 1904, . 902 may be made of any materials suitable to facilitate performance of PCR or real-time quantitative PCR or other types of nucleic acid amplification or primer extensions, as will be apparent to skilled persons. In addition, the materials to be adopted for forming the inner and outer tubes 1904, 1902, the bottom-most layer of material 1907 of the outer tube 1902, and the removable tube cap 1906 are preferably transparent and chemically compatible materials that are suitable to allow real-time PCR (or other types of nucleic acid amplification or primer extensions, or other applications requiring optical excitation and fluorescence detection of the sample inside the inner tube 1904) to enhance heat transfer during thermal cycling or heating/cooling during nucleic acid amplification.

To use the device 1900 for purifying nucleic acids, the removable tube cap 1906 is first removed (i.e. unplugged) from the outer tube 1902 to load an aqueous phase (i.e. a sample) 1908 into the inner tube 1904, and the outer tube 1902 is left open. Then, an organic phase 1910 is introduced into the outer tube 1902, and thereafter the device 1900 is optionally agitated to partition impurities from the aqueous phase 1908 to the organic phase 1910. It will be appreciated that the impurities move or diffuse into the organic phase 1910 due to the chemical nature of the organic phase 1910 and the impurities (not affected by the tube inversion), rather than by the gravity forces as a result of the tube inversion. It is also to be appreciated that the inner tube 1904 is initially positioned upright for loading of the aqueous phase 1908, and then tube inversion is carried out after the organic phase 1910 has covered the inner tube 1904 (holding the aqueous phase 1908 therein). But how fast the tube inversion is to be performed depends on the wells size, and the properties of the organic and aqueous phases 1910, 1908. Once the partitioning of the impurities is completed, the organic phase 1910 is then removed, the aqueous sample 1908 is optionally dried and the inner tube 1904 is optionally ethanol washed and dried, and a reaction mixture (not shown) for carrying out nucleic acid amplification or primer extensions is introduced into the inner tube 1904. A layer of oil 1912 may also optionally be loaded into the outer tube 1902 to cover the opening of the inner tube 1904. The outer tube 1902 is then closed off with the removable tube cap 1906 to carry out further nucleic acid amplification or primer extensions, as may be desired. It is however also to be appreciated that the nucleic acids may optionally be removed from the inner tube 1904 for further analysis, and in some instances, the nucleic acids may be partially or fully amplified in the inner tube 1904, before being removed. It is appreciated that the remaining volume of the outer tube 1902 above the layer of oil 1912 (or a similar sealant) is occupied by air, as will be apparent to skilled persons.

Figure 20 includes Figures 20a to 20c, which collectively depict a method of processing the purified nucleic acids isolated in the array of wells 042 of the microfluidic device 100 of Figure 1 , according to an eighth embodiment. In particular, different types of biological/chemical materials may be loaded into the wells 1042 for cell-cell, or cell- molecule interaction studies. That is, this embodiment enables biological assays using nucleic acid amplification, cell assay, or assays involving a plurality of biological particles and chemical agents. It is highlighted that this mentioned method only modifies the steps 202g and 202h of Figures 2g and 2h, pertaining to the introduction of the aqueous phase 201 (having biological and/or chemical materials). That is, the remaining steps 202a-202f in the method 200 of Figure 2 are still as per described afore. But of course not limited to the above stated, if desired, the step 202a may also be carried out using any of the methods afore described in the second to the sixth embodiments.

More specifically, as shown in Figures 20a to 20c, the configuration of the microfluidic device 100 remains the same as in Figure 1 , but differing slightly in steps for loading biological/chemical materials into the wells 1042 for interacting with the isolated nucleic acids. Primers are used as an example of the biological/chemical materials for illustration purpose in this case. The biological materials may include cells. As depicted in Figure 20a, the wells 1042 are preloaded with a first set of primers 2020a, 2020b, 2020c and filled with a fluid sample 5000. It will be appreciated that the fluid sample 5000 may be the organic phase 203 or a sample liquid containing sample droplets and biological particles. Thereafter, a portion of the fluid sample 5000 in each well 1042 is evaporated to create a space 2040 (on top of the respective primers 2020a, 2020b, 2020c) for loading a second set of primers 2060a, 2060b, 2060c, as shown in Figure 20b. Loading of the created spaces 2040 with the second set of primers 2060a, 2060b, 2060c and filling out the spaces 2040 with the fluid sample 5000 are as shown in Figure 20c. A sealant (e.g. mineral oil) may then be introduced to seal the wells 1042, like in afore embodiments. Subsequently, the first set of primers 2020a, 2020b, 2020c may then chemically/biologically interact with the second set of primers 2060a, 2060b, 2060c within the respective wells 1042. It will be appreciated that this eighth embodiment relates to loading of multiple samples of the biological/chemical materials, in contrast to loading of a single sample of the biological/chemical materials as afore described under the first embodiment. Thus, it will be appreciated by now that the materials disposed in the wells 1042 may include primers and/or probes for nucleic acid amplification, or same or different primers and/or probes.

Further, it is highlighted that the bottom 5002 of the wells 1042 in this instance may also be configured as a solid or porous layer. If the bottom 5002 is arranged as a solid layer, the fluid sample 5000 in the wells 1042 as per in Figure 20b is to be evaporated to create the space 2040 to allow the second set of primers 2060a, 2060b, 2060c to be loaded. However, if the bottom 5002 is instead a porous layer, a required portion of the fluid sample 5000 in the wells 1042 as per Figure 20b is first drained through the porous bottom 5002 to sufficiently create the space 2040 for subsequently allowing the second set of primers 2060a, 2060b, 2060c to be loaded. The characteristics of the porous bottom 5002 is as per described in the fifth embodiment, and will not be repeated for brevity sake.

In summary, purification of nucleic acids from small amount of bacteria in minute volume is important in many clinical and biological applications. For this, the proposed microfluidic platform (i.e. the microfluidic device 100 of Figure 1 ) for nucleic acid purification from a small number of bacteria based on a proposed liquid phase partitioning technique (i.e. the method 200 of Figure 2), using P. aeruginosa and S. aureus as model organisms, compatible for subsequent On-chip q-PCR or q-RT-PCR assay are disclosed. In particular, the microfluidic device 100 is configured to be able to selectively isolate DNA or RNA from a small number of bacterial cells (ranging from 5000 down to a single bacterium), distributed in the array of wells 1042, of 1 μΙ and 25 nl in sample volume and thereafter advantageously directly detected by quantitative PCR in the same array of wells 1042 in which the nucleic acids are isolated. Therefore, in this way, there is little or negligible loss of isolated nucleic acids, as will be appreciated. Also, no usage of solid phase is involved in the entire process. The performance of the microfluidic platform is evaluated using an aqueous phase containing protein, with DNA and RNA as the analytes. The aqueous phase is isolated in the array of wells 1042 of the microfluidic platform. And thereafter, an immiscible organic phase (i.e. PCI) is introduced in a headspace channel 1062 (formed of e.g. PDMS) which fluid communicates with the array of wells 1042. Continuous flow of organic phase increases the interfacial contact with the aqueous phase for sufficient partitioning of undesirable bio-molecules from the aqueous phase into the organic phase to achieve purification of nucleic acids in the array of wells 1042. It is to be appreciated that with the organic phase arranged to flow at an optimal flow rate, protein in the aqueous phase may then be effectively transferred from the aqueous phase to the organic phase, while DNA and RNA may thereafter be selectively recovered with minimal loss, in a pH-dependant manner. Residual organic phase in the array of wells 1042 is removed by repeated washing and vacuum evaporating with a cleaning fluid having 70% ethanol concentration. PCR reagent is then distributed into the micro-wells array by vacuum facilitated microfluidics for On-chip and/or Off-chip real time PCR amplification to avoid loss of nucleic acids due to liquid transfer. DNA, or RNA originating from 5 to 5000 bacteria, including both gram-positive (e.g. S. aurous) and gram-negative (e.g. P. aeruginsoa) bacteria in 1 Ι sample volume, may be selectively isolated depending on the arranged pH value of the organic phase and amplified with the nucleic acid recovery yield up to 10 folds higher than conventional column based nucleic acid extraction methods.

To further explore the capability of the proposed microfluidic platform for single bacterial cell analysis, the microfluidic device 100 of Figure 1 is modified and fabricated into a two dimensional format, being the 2D chip 600 of Figure 6 configured with 900 number of wells as the array of wells 602, in which a liquid holding volume of 125 nl/well. Single bacterium is isolated in the individual wells 602 probabilistically by loading of diluted sample bacteria with cell density of less than 0.3 CFU/well through vacuum facilitated microfluidics. Nucleic acids from single bacterium are then extracted using the proposed 2D chip 600 followed by On-chip q-PCR or q-RT-PCR assay. From experiments conducted, it is determined that successful high throughput RNA extraction from single bacterium with compatible on-chip quantitative PCR assay can be obtained. Specifically, it is found that 92% and 85% of DNA and RNA from single P. aeruginosa cell showed positive amplification, compared with 87% and 71% for DNA and RNA from single S. aureus cell. In all, the proposed microfluidic platform (of Figure 1 or Figure 6) and method 200 of Figure 2 therefore provide a valuable, but yet simple and effective solution for nucleic acid preparation with minimal loss and may be integrated for automated bacterial pathogen detection and high throughput transcriptional profiling assays (with minimum user exposure to the hazardous reagent), as well as for other critical applications such as in forensics, biodefence and etc.

In conventional liquid phase extraction (LFE), it works by dispersing the aqueous phase into a large number of droplets within the organic phase, and using vortex to perform partitioning of the impurities, It is then cumbersome to locate those desired aqueous droplets dispersed in the organic phase (by using centrifuge and highly skill dependent pipetting to collect those droplets). In contrast, for the proposed method 200 in Figure 2, the aqueous phase may be readily identifiable in fixed locations (i.e. in the wells). Another distinction over conventional methods is that the proposed method 200 allows a reaction mix to be added into the wells to be mixed with the aqueous phase to perform nucleic acid amplification and analysis in the same wells, thus minimising loss of nucleic acids during liquid transfer that will inevitably happen with all conventional methods. The described embodiments should not however be construed as limitative. For example, it will be appreciated that the step 202a of Figure 2a (relating to the introduction of the aqueous phase 201 ) may also be performed using manual and/or robotic pipette loading. More specifically, in all of the first to fourth variations to be discussed below, the protector layer 108 and acrylic embracer 114 (i.e. collectively the cover) are first removed from the microfluidic device 100 to expose the array of wells 1042. To clarify, the discussions with be with reference to the microfluidic device 100 of Figure 1. Biological and/or chemical materials (in liquid, solid or mixture forms) mixed with an aqueous buffer (to form the aqueous phase 201) are then subsequently dispensed into the exposed array of wells 1042 by contact or non-contact means of dispensing. The aqueous buffer may be distilled water, for example. It will be appreciated that in the variations, the aqueous phase 201 is dispensed in the form of droplets. It is also to be appreciated that the array of wells 1042 may also be vacuumed to minimize air trapping in the array of wells 1042. In a first variation, a pipette having a tip smaller than the opening of each well 1042 is selected and used, since the relatively small dimensions of tip of the pipette allows it to reach the bottom region of the wells 1042 (or placing the tip of the pipette against the internal walls of a well 1042 (to be worked on) to dispense the aqueous phase 201 downwardly into or adjacent to the bottom region of that well 1042, which in the process will help to expel any air trapped in the well 1042, as will be apparent to skilled persons. Under a second variation, the aqueous phase 201 is first to be deposited in the upper region of a well 1042 (which is defined to be adjacent to the opening of the well 1042) and thereafter the aqueous phase 201 is centrifuged down to the bottom region of the well 1042. In a third variation, the internal walls of each well 1042 is configured with hydrophiiic surfaces, and the aqueous phase 201 is loaded into the specific well 1042 using capillary forces generated by means of the hydrophiiic surfaces. In this third variation, the wells 1042 may also be configured with at least one air vent to expel any air trapped in the wells 1042 through the air vent as the aqueous phase 201 is introduced into the wells 1042. It will be appreciated that the air vent is simply an opening preferably positioned adjacent the bottom region of each well 1042, and may be formed by way of arranging the well 1042 to have a porous bottom (e.g. using Teflon filter paper or a polypropylene track etched filter paper), just like afore described in the fifth embodiment above. Alternatively, according to a fourth variation, the wells 1042 are first preloaded with the aqueous buffer, with the biological and/or chemical materials then deposited adjacent to the respective openings of the wells 1042. The deposited biological and/or chemical materials are then moved into the respective wells 1042 by diffusion, body force (e.g. gravity and/or centrifugation force), electrophoretic forces, di- electrophoretic forces, flow induced hydrodynamic forces, electric forces, and magnetic forces. It is also to be highlighted that once the aqueous phase 201 is introduced into the array of wells 1042, as per the first to fourth variations, the protector layer 108 and acrylic embracer 114 are then replaced onto the microfluidic device 100 to cover up the wells 1042, and configuring the open space adjacent to the array of wells 1042 into the headspace channel 1062. This is to be done prior to inverting the microfluidic device 100 as per step 202b in Figure 2b.

Alternatively, in another series of variations, the step 202a of Figure 2a (relating to the introduction of the aqueous phase 201 ) may also be performed using microfluidic loading. But specifically, in all of the fifth to ninth variations to be discussed below, the protector layer 108 and acrylic embracer 114 (i.e. collectively the cover) are now kept in place on the microfluidic device 100 for the headspace channel 1062 to be present. This arrangement is necessary because the headspace channel 1062 is required for loading the aqueous phase 201 into the array of wells 1042 using the microfluidic means to be discussed below. To clarify, the discussions with be with reference to the microfluidic device 100 of Figure 1. In a fifth variation, which is performed using vacuum loading, the wells 1042 are first subjected to vacuum conditions, and thereafter the aqueous phase 201 is introduced into the headspace channel 1062, and consequently into the wells 1042, as will be appreciated. In a sixth variation, which is performed using centrifugal loading, the aqueous phase 201 is moved into the wells 1042 by means of centrifugation force, the specific application of which will be apparent to skilled persons. Based on a seventh variation, which is performed using pressure loading, air pressure is utilised to push the aqueous phase 201 into the wells 1042.

Further, based on an eighth variation, which is performed using capillary loading, the aqueous phase 201 is moved into the wells 1042 by means of capillary force, the application of which will be apparent to skilled persons. It is to be highlighted that in the seventh and eighth variations, the wells 1042 are each preferably arranged with an air vent, similar to the arrangement described in the third variation, and thus not repeated for brevity. But yet optionally, according to a ninth variation, the wells 1042 are first preloaded with the aqueous buffer, which may be carried out using any of the methods discussed in the fifth to the eighth variations (where the aqueous phase 201 is instead replaced with the aqueous buffer), and the biological and/or chemical materials are then deposited adjacent to the respective openings of the wells 1042. Subsequently, the deposited biological and/or chemical materials are moved into the respective wells 1042 by diffusion, body force (e.g. gravity and/or centrifugation force), electrophoretic forces, di-electrophoretic forces, flow induced hydrodynamic forces, electric forces, and magnetic forces.

According to a tenth variation, it is to be appreciated that at step 202a of Figure 2a, an optional step may be performed (prior to step 202a) to lyse nucleic acid- containing biological particles including cells, virus, protozoa, tissues, plants and etc. to release nucleic acids from the said biological particles, only if the aqueous phase 201 includes those types of biological particles. Hence, it is to be noted that for aqueous phases 201 that contain only nucleic acids, with no cells in the sample, this step may however be omitted. If the aqueous phase 201 contains nucleic acid-containing biological particles, there are two options for adding in a lysis buffer. The first option is to mix the lysis buffer into the aqueous phase 201 before loading into the wells 1042. Loading of the aqueous phase 201 is then to be completed in short time after adding the lysis buffer, so that the biological particles are not lysed before being introduced into, the wells 1042, which is important for single cell analysis where the nucleic acids from each biological particle are all contained inside one well 1042. The second option is to pre-deposit the lysis buffer into the wells 1042 (in wet or dry form) before loading the aqueous phase 201. In any event, lysing of the nucleic acid- containing biological particles needs to be performed before the organic phase 203 is introduced into the microfluidic device 100. Besides adding the lysis buffer, it is to be appreciated that other physical means such as ultrasound, electric current, thermal stress via freeze thawing process cycles or rapid heating and cooling cycles, or solid beads grinding under agitation such as vortex and ultrasound may also be used in place of the lysis buffer.

According to an eleventh variation, it is further to be appreciated that at step 202b of Figure 2b, the organic phase 203 (after being introduced into the headspace channel 1062) may not be set in a circulating motion within the headspace channel 1062. In other words, the organic phase 203 may be arranged to be kept stationary over the wells 1042 for a defined period, and may be agitated to flow/move only within the headspace channel 1062, when necessary. Based on this variation, if for a case where the well size is of a sufficiently small dimension (e.g. such as a few hundreds of micrometre or smaller) and the organic phase 203 is moving/flowing within the headspace channel 1062, impurities in the aqueous phase 201 may be partitioned to the organic phase 203 through diffusion thereto over an acceptable time period. For a case where the well size is instead of a larger dimension (e.g. such as greater than a few hundreds of micrometre), the organic phase 203 may also be maintained stationary over the wells 1042 (for diffusion of the impurities to the organic phase 203 to take place), if a user can wait for a longer period of time.

Based on a twelfth variation, the inversion of/angularly arranging the microfluidic device 100 is optional, and needs to be performed only if the organic phase 203 is denser than the aqueous phase 201. Furthermore, if the wells 1042 are arranged to be of a small size (i.e. between a range of 0.05 mm to 1 mm), then inversion/angular arrangement of the microfluidic device 100 may also be unnecessary, since surface tension from the well surface of the wells 1042 is able to retain the aqueous phase 201 within the wells 1042. Of course, whether sufficient surface tension is generated to retain the aqueous phase 2.01 i the wells 104 depends on the type of aqueous phase 201 , type of well surface, type of the organic phase 203, and other relevant factors (as will be appreciated by skilled persons). Based on a thirteenth variation, the proposed method 200 of Figure 2 may further involve repeating the steps 202b to 202d by sequentially introducing and removing different constituents of the organic phase 203 into the headspace channel 1062, after the organic phase 203 is removed at step 202d of Figure 2d. For example, this may involve by first introducing phenol, followed by chloroform, and then finally isoamyl alcohol. Alternatively, it may comprise introducing into the headspace channel 1062 any desired combination of those mentioned constituents of the organic phase 203 in sequence. It is importantly to be appreciated that this presently discussed variation is to be performed prior to the steps 202e to 202h.

In a fourteen variation, the organic phase 203 may alternatively be a mixture of phenol, and/or chloroform and/or isoamyl alcohol, and/or other chemicals that may facilitate the partitioning of the unwanted impurities from the aqueous sample 201 to the organic phase 203.

In a fifteen variation, it is to be appreciated that after drying the nucleic acids as shown in Figure 2f, an aqueous buffer such as a TE buffer may also be introduced into the wells 1042 to re-suspend the dried nucleic acids and thereafter removing the re-suspended nucleic acids from the wells 1042 for further analyses. Means for removing the re-suspended nucleic acids from the wells 1042 may include by pipetting (after removing the cover above the wells 1042), centrifugation into the headspace channel 1062 or into the matching well- plate or tubes, etc. The recovered aqueous sample containing the purified nucleic acids may then be used for further analyses such as nucleic acid amplifications, hybridizations and DNA sequencing. Figure 21 shows an alternative embodiment of the wells 1042 of the microfluidic device 100 of Figure 1. In particular, the wells 2102a, 2102b are arranged with both a top and a bottom opening. Both the top and bottom openings of each well 2102a, 2102b respectively lead to headspace channels 1062, 1062". . In other words, besides the original headspace channel 1062 as per the first embodiment in Figure 1 , there is now a second headspace channel 1062" which is configured in opposition to the original headspace channel 1062. Specifically, the second headspace channel 1062" is located/positioned at an opposite side of the wells 2102a, 2102b, in relation to the original headspace channel 1062. Thus, in a configuration where the top opening of each well 2102a, 2102b leads to the original headspace channel 1062, then the bottom opening of each well 2102a, 2102b leads will lead to the second headspace channel 1062", and vice versa. Also, the inner surfaces of the wells 2102a, 2102b are made to be sufficiently hydrophilic and the surfaces outside the wells 2102a, 2102b are made to be sufficiently hydrophobic. Combined with a suitable well size, such proposed wells 2102a, 2102b may hold the aqueous sample inside the wells 2102a, 2102b by way of surface tension. Furthermore, the headspace channels 1062, 1062" are configured for introducing the organic phase 203 to contact the aqueous phase 201 over the top and the bottom well openings for partitioning of the impurities from the aqueous phase 201 into the organic phase 203. The rest of the steps of method 200 are the same as those shown in Figure 2. The advantage of this embodiment is that the aqueous phase 201 may more easily be removed from the wells 2102a, 2102b due to absence of corners over the bottom of the wells 2102a, 2102b.

It is also to be appreciated that, not limited to the above described, any part of the microfluidic device 100 of Figure 1 , the microfluidic device 1700 of Figure 17 or the microfluidic device 1900 of Figure 19 may be formed of other suitable materials substantially inert to the biological/chemical materials, samples or fluids which the microfluidic device 100 may come into contact with, and the materials include (for example) PDMS, Teflon, Polypropylene, plastics, glass, metal, ceramics and the like. It is to be appreciated that for the afore described embodiments, in particular where there is usage/application of droplets, the droplets may optionally be generated by suitable microfluidic means for generating uniformly sized droplets, which include a microfluidic droplet generation chip with a T-junction and/or a cross-junction, and/or electrical field, vibration means such as piezo-excitation, and/or droplets generated by a chip with a gradient confinement (i.e. as set out in Remi Panola, S. Capri Kayi, and Charles N. Baroud, ''Droplet microfluidics driven by gradients of confinement , 2013, Vol. 1 10, No. 3, pp.853-858). Furthermore, if droplet uniformity may not be essential, then other appropriate means such as vortex can be used to generate the droplets, as will be appreciated by skilled persons.

While the invention has been illustrated and described in detail in the drawings and foregoing description, such illustration and description are to be considered illustrative or exemplary, and not restrictive; the invention is not limited to the disclosed embodiments. Other variations to the disclosed embodiments can be understood and effected by those skilled in the art in practising the claimed invention.

Claims

1. A method of isolating nucleic acids in an aqueous sample using a microfluidic device, the device having at least one well in fluid communication

5 with an adjacent space external to the opening of the at least one well, wherein the aqueous sample comprises biological and/or chemical materials and/or impurities, and is held in the at least one well, the method comprising:

introducing a first fluid substantially immiscible with the aqueous sample into the adjacent space to partition impurities to the first fluid; and

Ί0 removing the first fluid with the impurities leaving the nucleic acid in the aqueous sample in the at least one well.

2. The method of claim 1 , further comprising moving the first fluid in the adjacent space for a defined period, after the first fluid is introduced into the

15 adjacent space.

3. The method of claim 1 , further comprising angularly arranging the microfluidic device for a defined period to partition the impurities to the first fluid, if the first fluid is denser than the aqueous sample, and/or if the size of the at

20 least one well is able to hold the aqueous sample therewithin by surface tension.

4. The method of claim 3, wherein angularly arranging the microfluidic device includes inverting the microfluidic device.

25 5. The method of claim 1 , further comprising drying the at least one well having the nucleic acids, after the first fluid is removed.

6. The method of claim 1 , further comprising sequentially introducing and removing different constituents of the first fluid into the adjacent space, after the

30 first fluid is removed.

7. The method of claim 1 , wherein the biological and/or chemical materials are selected from the group consisting of: primers, short nucleotides, and adaptors for nucleic acid amplification, reverse transcription, and next

35 generation sequencing applications, cells, cell debris, tissues, plants, viruses, antibodies, proteins, enzymes, molecules, peptides, nucleic acids, polynucleotides, oligonucleotides, short fragments of genes or probes, reaction constituents, lysis buffer constituents, bacteria, protozoa, pathogens, fluorescent chemicals or molecules, crystals, liquid droplets, metal ions, and solid particles.

. . ^{' '} , . · . ·^■ .. .

8. The method of claim 7, wherein the biological and/or chemical materials comprises solid, dried, partially dried, or liquid forms.

9. The method of claim 7, wherein the solid particles include fluorescent particles, fluorescent dye chemicals, nanoparticles, glass beads, or magnetic beads.

10. The method of any one of the preceding claims, wherein the nucleic acids include DNA, RNA, mRNA, microRNA, or cDNA.

1 1. The method of any one of the preceding claims, wherein the first fluid includes a composition of phenol, and/or chloroform and/or isoamyl alcohol, and/or other chemicals to facilitate the partitioning of the impurities from the aqueous sample to the first fluid.

12. The method of claim 1 1 , wherein the composition of phenol, chloroform and isoamyl alcohol is in a volume ratio of approximately 25:24:1 , if the nucleic acids to be isolated are DNAs.

13. The method of claim 12, wherein a pH value of the first fluid is approximately 8.0.

14. The method of claim 11 , wherein the composition of phenol, chloroform and isoamyl alcohol is in a volume ratio of approximately 125:24:1 , if the nucleic acids to be isolated are RNAs.

15. The method of claim 14, wherein a pH value of the first fluid is approximately 4.6.

16. The method of claim 1 , wherein the impurities include proteins, DNA, RNA, unwanted chemicals, metal ions and salt.

17. The method of any one of the preceding claims, further comprising .5 providing a cover to the at least one well to configure the adjacent space into a fluid channel, prior to introducing the first fluid into the adjacent space.

18. The method of any one of the preceding claims, wherein drying the at least one well includes using vacuum evaporation and/or heat drying and/or

10 freeze drying.

19. The method of any one of the preceding claims, wherein the defined period is approximately 15 minutes.

15 20. The method of any one of the preceding claims, wherein circulating the first fluid comprises arranging the first fluid to circulate with forward and reverse flows for a plurality of cycles, wherein each cycle lasts approximately 5 seconds.

21. The method of claim 20, wherein the first fluid is circulated at a flow rate -20 of 0.25 ml/min, 0.45 ml/min, or 0.65 ml/min for one cycle.

22. The method of any one of the preceding claims, wherein if the device is inverted, the device is restored back to the position with the adjacent space above the at least one well after removing the first fluid.

25

23. The method of any one of the preceding claims, further comprising introducing the aqueous sample into the at least one well, prior to introducing the first fluid into the adjacent space.

30 24. The method of claim 23, wherein introducing the aqueous sample into the at least one well comprises introducing biological and/or chemical substances together with an aqueous fluid.

25. The method of claim 23, wherein introducing the aqueous sample into the at least one well comprises introducing an aqueous fluid into the at least one well, which is pre-loaded with biological and/or chemical substances.

26. The method of claim 24 or 25, further comprises introducing into the at least one well lysis buffers and/or physical means such as ultrasound, electric current, thermal stress via freeze thawing process cycles or rapid heating and cooling cycles, or solid beads grinding under agitation such as vortex and ultrasound to lyse the biological substances to release nucleic acids therefrom.

27. The method of claim 24, further comprising depositing the aqueous fluid into the at least one well; and introducing the biological and/or chemical materials into the at least one well using diffusion, body forces, electrophoretic forces, dielectrophoretic forces, flow-induced hydrodynamic forces, electric forces or magnetic forces,

wherein the aqueous liquid and the biological and/or chemical materials together form the aqueous sample.

28. The method of claim 27, wherein the body forces include gravity and centrifugal forces.

29. The method of claim 27 or 28, wherein the bottom of the at least one well is arranged to be substantially porous to enable at least some particulate impurities to pass through, and wherein a pore size of the porous bottom is configured to be smaller than the size of the nucleic acids.

30. The method of claim 29, wherein the bottom of the at least one well includes being formed using a Telfon filter paper or a Polypropylene track- etched filter paper.

31. The method of claim 23, wherein introducing the aqueous sample into the at least one well includes using manual or robotic pipette loading.

32. The method of claim 31 , further comprising vacuuming the at least one well to remove air trapped therein.

33. The method of claim 31 , further comprising depositing the aqueous sample adjacent to the bottom of the at least one well using a pipette.

34. The method of claim 31 , further comprising depositing the . aqueous sample substantially adjacent to the top of the at least one well; and centrifuging the microfluidic device to cause the aqueous sample to move to the bottom of the least one well.

35. The method of claim 31 , wherein the at least one well comprises walls with hydrophilic surfaces and a porous base, further comprising depositing the aqueous sample into the at least one well through capillary forces between the aqueous sample and the hydrophilic surfaces, wherein air and/or particulate impurities in the at least one well are expelled through the porous base while the aqueous sample moves into and is retained in the at least one well.

36. The method of claim 23, wherein introducing the aqueous sample into the at least one well includes using microfluidic loading, wherein the adjacent space is configured as a fluid channel.

37. The method of claim 36, further includes depositing the aqueous sample into the at least one well using vacuum loading, centrifugal loading, pressure loading, or capillary loading.

38. The method of claim 5, further comprising washing the at least one well with a cleaning fluid after the at least one well is dried to substantially remove residual first fluid.

39. The method of any one of the preceding claims, wherein the cleaning fluid comprises ethanol of a concentration between 40% to 100%.

40. The method of claim 39, wherein the concentration of the ethanol is approximately 70%.

41. The method of any one of claims 38 to 40, further comprising removing the cleaning fluid from the adjacent space after the at least one well is washed.

42. The method of any one of claims 38 to 40, further comprising drying the at least one well to substantially remove residual cleaning fluid after trie at least one well is washed.

43. The method of claim 42, further comprising vacuum evaporating the at least one well for approximately 5 minutes.

44. The method of any one of the preceding claims, wherein the at least one well or the cover is formed of a material selecting from the group consisting of: Polydimethylsiloxane (PDMS), Teflon, Polypropylene, glass and ceramics.

45. The method of any one of the preceding claims, wherein the at least one well has an edge length of approximately between 0.05 pm to 10000 pm.

46. The method of claim 42 or 43, further comprises introducing a reaction mixture suitable for polymerase chain reaction (PCR), q-PCR, q-RT-PCR, isothermal amplification, reverse transcription, DNA amplification used for DNA sequencing, into the at least one well having the nucleic acids.

47. The method of claim 46, further comprises introducing a layer of mineral oil into the adjacent space to seal the at least one well filled with the reaction mixture to enable PCR, RT-PCR, q-PCR, q-RT-PCR, isothermal amplification, reverse transcription, or DNA amplification used for DNA sequencing assays to be subsequently performed on the nucleic acids.

48. The method of claim 1 or 47, further comprising removing the aqueous sample in the at least one well for storage and/or further analysis.

49. The method of claim 48, wherein removing the nucleic acids includes using pipette aspiration, centrifugation of the nucleic acids into a desired collection device, or collecting the nucleic acids through an aperture configured at the bottom of the at least one well.

50. A method of isolating nucleic acids from an aqueous sample comprising: introducing the aqueous sample into at least one internal well at the base of a tube, introducing a first fluid substantially immiscible with the aqueous sample in the space of the tube external to the opening of the well to partition impurities into the first fluid, optionally agitating the first fluid to improve partitioning; removing the first fluid from tube leaving the nucleic acids in the internal well of the tube; and drying the tube.

51. The method of claim 50, further comprises disposing materials into the at least one internal well to enable biological assays being one of nucleic acid amplification, cell assay and assays involving a plurality of biological particles and chemical agents.

52. The method of claim 51 , wherein the materials disposed in the at least one well include primers and/or probes for nucleic acid amplification, or same or different primers and/or probes.

53. The method of claim 50, further comprising removing the nucleic acids from the internal well for storage and/or further analysis.

54. The method of claim 50, further comprising removing the nucleic acids from the internal well for further analysis, wherein the nucleic acids are partially or fully amplified in the internal well.

55. A method of isolating nucleic acids in an aqueous sample using a microfluidic device, the device having at least one well in fluid communication with an adjacent space external to the opening of the at least one well, the method comprising:

introducing a first fluid substantially immiscible with the aqueous sample into the adjacent space and the at least one well to partition impurities to the first fluid;

introducing the aqueous sample as at least one droplet into the first fluid which subsequently settle into the at least one well, wherein the aqueous sample comprises biological and/or chemical materials and/or impurities; and removing the first fluid with the impurities leaving the nucleic acid in the aqueous sample in the at least one well.

56. The method of claim 55, further comprising angularly arranging the m^'icrofluidic device for a defined period to partition the impurities to the first fluid, if the first fluid is denser than the aqueous sample, and/or if the size of the at least one well is able to hold the aqueous sample therewithin by surface tension.

57. The method of claim 56, wherein angularly arranging the microfluidic device includes inverting the microfluidic device.

58. The method of claim 55, further comprising drying the at least one well having the nucleic acids, after the first fluid is removed.

59. The method of claim 55, wherein the size of the at least one drop is arranged to be substantially equally to the size of an opening of the at least one well.

60. The method of claim 55, wherein the at least one droplet includes a plurality of droplets, and the at least one well includes a plurality of wells, and wherein a number of the droplets generated is less than a number of the wells.

61. The method of claim 60, wherein each droplet includes different biological and/or chemical materials from the other droplets.

62. The method of claim 61 , further comprises pre-loading each of the wells with a biological and/or chemical material to enable interaction with the different materials held in the droplets.

63. A method of isolating nucleic acids in an aqueous sample using a microfluidic device, the device having at least one well in fluid communication with an adjacent space external to the opening of the at least one well, the method comprising:

introducing an aqueous fluid into the adjacent space to fill the at least one well; introducing biological and/or chemical materials into the aqueous fluid which subsequently settle into the at least one well, wherein the biological and/or chemical materials include the nucleic acids;

removing the aqueous fluid from the adjacent space;

introducing a first fluid substantially immiscible with the aqueous fluid into the adjacent space to partition impurities to the first fluid; and

removing the first fluid with the impurities leaving the nucleic acid in the aqueous sample in the at least one well..

64. The method of claim 63, further comprising angularly arranging the microfluidic device for a defined period to partition the impurities to the first fluid, if the first fluid is denser than the aqueous sample, and/or if the size of the at least one well is able to hold the aqueous sample therewithin by surface tension.

65. The method of claim 64, wherein angularly arranging the microfluidic device includes inverting the microfluidic device.

66. The method of claim 63, further comprising drying the at least one well having the nucleic acids, after the first fluid is removed.

67. The method of claim 63, wherein the size of the biological and/or chemical materials is arranged to be substantially equally to the size of an opening of the at least one well.

68. The method of claim 63, wherein the at least one well includes a plurality of wells, and wherein a number of the biological and/or chemical materials introduced is less than a number of the wells.

69. A method of isolating nucleic acids in an aqueous sample using a microfluidic device, the device having at least one well in fluid communication with an adjacent space external to the opening of the at least one well, the method comprising:

introducing an aqueous fluid pre-mixed with biological and/or chemical materials into the adjacent space to fill the at least one well, wherein the biological and/or chemical materials include the nucleic acids; removing the aqueous fluid from the adjacent space after the biological and/or chemical materials have settled into the at least one well;

removing the first fluid with the impurities from the adjacent space leaving the nucleic acid in the aqueous sample in the at least one well.

70. The method of claim 42 or 43, further comprises introducing an aqueous buffer into the at least one well having the nucleic acids to re-suspend the dried nucleic acids; and removing the re-suspended dried nucleic acids for further analyses.

71. A method of isolating nucleic acids in an aqueous sample using a microfluidic device, the device having at least one well in fluid communication with an adjacent space external to the opening of the at least one well, the method comprising:

forming a mixture of the aqueous sample as at least one droplet with a first fluid substantially immiscible with the aqueous sample;

introducing the said mixture into the adjacent space and the at least one well to partition impurities to the first fluid, allowing at least one droplet to settle into the at least one well, wherein the aqueous sample comprises biological and/or chemical materials and/or impurities; and