Classifications

• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J19/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01F—MIXING, e.g. DISSOLVING, EMULSIFYING OR DISPERSING
  • B01F33/00—Other mixers; Mixing plants; Combinations of mixers
  • B01F33/30—Micromixers
  • B01F33/3032—Micromixers using magneto-hydrodynamic [MHD] phenomena to mix or move the fluids
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
  • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
  • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
  • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
  • B01L3/50273—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by the means or forces applied to move the fluids
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01F—MIXING, e.g. DISSOLVING, EMULSIFYING OR DISPERSING
  • B01F25/00—Flow mixers; Mixers for falling materials, e.g. solid particles
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01F—MIXING, e.g. DISSOLVING, EMULSIFYING OR DISPERSING
  • B01F33/00—Other mixers; Mixing plants; Combinations of mixers
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01F—MIXING, e.g. DISSOLVING, EMULSIFYING OR DISPERSING
  • B01F33/00—Other mixers; Mixing plants; Combinations of mixers
  • B01F33/30—Micromixers
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01F—MIXING, e.g. DISSOLVING, EMULSIFYING OR DISPERSING
  • B01F33/00—Other mixers; Mixing plants; Combinations of mixers
  • B01F33/45—Magnetic mixers; Mixers with magnetically driven stirrers
  • B01F33/452—Magnetic mixers; Mixers with magnetically driven stirrers using independent floating stirring elements
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
  • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
  • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
  • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
  • B01L3/502746—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by the means for controlling flow resistance, e.g. flow controllers, baffles
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
  • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
  • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
  • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
  • B01L3/502761—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads, for physically stretching molecules
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
  • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
  • G01N27/416—Systems
  • G01N27/447—Systems using electrophoresis
  • G01N27/44756—Apparatus specially adapted therefor
  • G01N27/44791—Microapparatus
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
  • B01L2200/06—Fluid handling related problems
  • B01L2200/0647—Handling flowable solids, e.g. microscopic beads, cells, particles
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2300/00—Additional constructional details
  • B01L2300/06—Auxiliary integrated devices, integrated components
  • B01L2300/0627—Sensor or part of a sensor is integrated
  • B01L2300/0645—Electrodes
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2300/00—Additional constructional details
  • B01L2300/08—Geometry, shape and general structure
  • B01L2300/0809—Geometry, shape and general structure rectangular shaped
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2300/00—Additional constructional details
  • B01L2300/08—Geometry, shape and general structure
  • B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
  • B01L2300/087—Multiple sequential chambers
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2400/00—Moving or stopping fluids
  • B01L2400/04—Moving fluids with specific forces or mechanical means
  • B01L2400/0403—Moving fluids with specific forces or mechanical means specific forces
  • B01L2400/043—Moving fluids with specific forces or mechanical means specific forces magnetic forces
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
  • Y10T436/00—Chemistry: analytical and immunological testing
  • Y10T436/25—Chemistry: analytical and immunological testing including sample preparation

Definitions

• the current invention relates to a microfluidic device and to methods of its use for isolating and detecting an analyte from a biological sample .
• MEMS Micro-Electro-Mechanical Systems
• ⁇ -TAS Micro Total Analysis Systems
• LOC Lab-on-a-Chip
• ⁇ -TAS The main advantages of ⁇ -TAS over traditional devices lie in lower fabrication costs, improvement of analytical performance regarding quality and operation time, small size, disposability, precise detection, minimal human interference and lower power consumption. Moreover, the problem of rare chemical and samples which restrain the application of genetic typing and other molecular analyses has been resolved by employment of ⁇ -TAS.
• Suzuki, H., et al J. microelectromechanical systems, 2004, vol 13, no.5 779-790 disclose a magnetic force driven chaotic mixer in which physical obstacles in the microchannel are used in conjunction with microconductors embedded in the base of the channel, which act to manipulate magnetic beads back and forth, to facilitate mixing of the sample and the beads .
• EP 1462174 Al discloses a device for controlled transport of magnetic beads between a position X and a position Y, wherein the beads are transported by applying successively a series of local magnetic fields generated by triangular current carrying structures in which the current density is non constant, resulting in the beads accumulating at the tips of the current carrying structures in the region having the highest charge density.
• WO 2006004558 discloses a biochip for sorting and lysing biological samples which makes use of dielectrophoretic forces to retain and recover desired cells from a sample.
• a microfluidic device comprising;
• a first layer comprising at least first and second current carrying structures, wherein the at least first and second current carrying structures each comprise a plurality of teeth, and wherein the teeth of the first and second current carrying structures are optionally offset such that the teeth of the first current carrying structure are positioned between the teeth of the second current carrying structure;
• a second layer comprising a first microfluidic chamber in fluid communication with the inlet and positioned above the at least first and second current carrying structures of the first layer;
• a third layer comprising at least third and fourth current carrying structures wherein the at least third and fourth current carrying structures each comprise a plurality of teeth, and wherein the teeth of the third and fourth current carrying structures are optionally offset such that the teeth of the third current carrying structure are positioned between the teeth of the fourth current carrying structure;
• the at least third and fourth current carrying structures are positioned in the third layer so as to be above the first microfluidic chamber and such that the teeth of the third current carrying structure are positioned substantially vertically above or offset from the teeth of the first current carrying structure and the teeth of the fourth current carrying structure are positioned substantially vertically above, or offset from the teeth of the second current carrying structure; wherein the teeth have a stem having substantially- elliptical tip.
• a microfluidic device comprising;
• a first layer comprising at least a first current carrying structure comprising a plurality of teeth
• a second layer comprising a first microfluidic chamber in fluid communication with the inlet and positioned above the at least first and second current carrying structures of the first layer;
• a third layer comprising at least a second current carrying structure comprising a plurality of teeth
• the second current carrying structure is positioned in the third layer so as to be above the first microfluidic chamber and such that the teeth of the second current carrying structure are positioned substantially vertically above or offset from the teeth of the first current carrying structure;
• teeth have a stem having substantially elliptical tip.
• first and third layers of the device each comprise a current carrying structure, rather than first and second, and third and fourth current carrying structures respectively. This however does not preclude the possible inclusion of further current carrying structures in the first and third layers .
• the current carrying structure of either the first or the third layer may be orientated to include turns or changes in direction such that individual teeth of the structure may be orientated such that they are opposite one another.
• the individual teeth may also be offset from one another.
• the term offset encompasses a range of possible spacings for the teeth of the first and second current carrying structures.
• the teeth may for example be spaced regularly and with the same spatial interval between teeth in the first and the second current carrying structure, although this need not be the case.
• the teeth of the first current carrying structure may for example be offset such that they are present halfway between the teeth of the second current carrying structure, or alternatively at another fraction of the distance between the teeth.
• the term offset also encompasses irregular spacing between the teeth of the current carrying structures and between the current carrying structures themselves. Teeth will be understood to refer to projections along the path of the current carrying structure.
• the shape of each tooth may therefore comprise further shapes and structure, for example the stem portion of the projection may terminate in an elliptical tip.
• the current carrying structures may be of the kind described as "key-type” or “multiple turn key-type”.
• the spatial layouts of examples of such configurations are illustrated in figures 18 to 20.
• elliptical refers to a tip having an ovoid or circular conformation. In a preferred embodiment, the tip is circular.
• the inventors have found that the elliptical configuration of the teeth of the device result in a magnetic field which is more evenly distributed about the tooth, as compared to other shapes of tooth, such as triangular, where the magnetic field is only stronger at the tip.
• the current carrying structures are embedded in the first and third layers. More preferably, the current carrying structures are between 0. l ⁇ m to lO ⁇ m below the surface of the first and third layers. Even more preferably, between 0. l ⁇ m and 5 ⁇ m. Most preferably, between 0. l ⁇ m and 2 ⁇ m .
• the device may also include a high permeable (e.g permalloy) layer located within or adjacent the first and/or third layers distal to the microchannel to increase the magnetic field generated by the device .
• a high permeable (e.g permalloy) layer located within or adjacent the first and/or third layers distal to the microchannel to increase the magnetic field generated by the device .
• the first microfluidic chamber is a substantially straight channel.
• the substantially straight channel has a region having increased dimensions forming a chamber proximal to the inlet .
• this region acts to increase the rate at which a sample liquid can be mixed.
• the sample is a liquid which is liable to thicken or coagulate, for example whole blood.
• the use of blood as the sample is of particular interest in devices which are designed as home use or point of care use, because the sample can be easily obtained by a simple needle prick.
• the inlet opens directly into the region having increased dimensions and the current carrying devices extend into this region such that chaotic mixing of the sample begins immediately the sample enters the device .
• the first and/or third layers further comprises a fifth current carrying structure. More preferably, the fifth current carrying structure is located so as to be distal to the inlet.
• the first microfluidic chamber forms a lysis and extraction unit.
• the device is useful for the analysis of whole blood.
• the microfluidic device further comprises a second microfluidic chamber in fluid communication with the first microfluidic chamber, wherein the second microfluidic chamber is an amplification chamber. More preferably, the amplification chamber is a PCR chamber.
• the microfluidic device comprises a third microfluidic chamber in fluid communication with the second microfluidic chamber, said third microfluidic chamber comprising a sensor for detecting the presence of an analyte.
• the senor comprises a mutual inductance device .
• the microfluidic device comprises at least one integrated pump for effecting movement of a fluid from chamber to chamber.
• the integrated pumps are magnetic pumps .
• the microfluidic device further comprises means for applying a voltage to each of the current carrying structures independently in a predetermined order and for a predetermined period.
• the period is in the range of 1-10 seconds, more preferably, less than 5 seconds.
• the microfluidic device further comprises at least a first fluid reservoir.
• the at least a first reservoir is in fluid communication with the first microfluidic chamber.
• the at least first reservoir is integrated into the device .
• the first microfluidic chamber forms the first fluid reservoir.
• the fluid comprises superparamagnetic beads.
• the fluid also comprises lysis buffer.
• the microfluidic device further comprising at least a second fluid reservoir.
• the fluid may comprise other constituents, for example, it may optionally comprise an anticoagulant .
• a lab-on-chip system for preparing a sample comprising a biological molecule, the system comprising; a) the device according to the first aspect;
• a lab-on-chip system for preparing a sample comprising a biological molecule, the system comprising;
• the first, second, third and fourth current carrying structures of the device have a voltage applied thereto in a predetermined sequence .
• a fifth current carrying structure acts to retain the superparamagnetic particles in the first microfluidic chamber.
• the superparamagnetic particles may have any suitable diameter, preferably they have an average diameter from 50nm to lO ⁇ m. For example an average diameter of 3 ⁇ m is contemplated. Other diameters are possible .
• the superparamagnetic particles are functionalised so as to bind to an analyte of interest.
• the analyte is a nucleic acid.
• system further comprises a second reservoir containing a wash buffer in fluid communication with the first microfluidic chamber.
• system further comprises a third reservoir containing an elution buffer in fluid communication with the first microfluidic chamber.
• the sample may be any suitable biological material .
• the sample comprises at least one cell. More preferably, the sample comprises a whole blood sample.
• the fluid further comprises a lysis buffer.
• the fluid further comprises an anticoagulant.
• a third aspect of the current invention there is provided a method for the isolation of an analyte comprising a biological molecule from a sample, said method comprising the steps of : -
• introducing the sample into the inlet of the device according to the first aspect: ii) introducing a fluid comprising superparamagnetic particles into the first microfluidic chamber of the device; iii) applying a voltage to the first, second, third and fourth current carrying structures of the device in a predetermined sequential order so as to cause electric currents to pass therethrough;
• step i) can be performed prior to, concomitantly with or subsequently to step ii) ; and wherein, said superparamagnetic particles are functionalised so as to bind to the analyte of interest;
• step iii) is performed concomitantly with or immediately after step i) ;
• introducing the sample into the inlet of the device according to the variation of the first aspect: ii) introducing a fluid comprising superparamagnetic particles into the first microfluidic chamber of the device; iii) applying a voltage to the current carrying structures of the device in a predetermined sequential order so as to cause electric currents to pass therethrough;
• step i) can be performed prior to, concomitantly with or subsequently to step ii) ; and wherein, said superparamagnetic particles are functionalised so as to bind to the analyte of interest;
• step iii) is performed concomitantly with or immediately after step i) ;
• the elliptical configuration of the teeth of the device result in a magnetic field which is more evenly distributed about the tooth, as opposed to other shapes of tooth, such as triangular, where the magnetic field is stronger only at the tip. This results in greater mixing due to chaotic movement of the beads.
• the device further comprises a fifth current carrying structure, the fifth current carrying structure having a voltage applied thereto subsequently to step iii) wherein the superparamagnetic particles are attracted to and retained on the fifth current carrying structure through magnetic interactions.
• the current passing through each current carrying structure is in the range of 100mA to 1OA. More preferably, 100mA to 75OmA. Most preferably, less than 50OmA
• the method comprises the further step of introducing a wash solution into the first microfluidic chamber of the device, preferably, once the superparamagnetic particles have been retained on the fifth current carrying structure .
• the method optionally comprises the further step of introducing an elution solution into the first microfluidic chamber of the device .
• the voltage is applied to each of the first, second, third and fourth current carrying devices for sufficiently long so as to allow the beads to move to a predetermined location in the first microfluidic chamber.
• the current carrying structures have the voltage applied in the order one, four, three, two.
• the voltage can be supplied to the current carrying structures in any desired order so as to obtain optimum mixing of the fluid comprising the superparamagnetic particles and the sample.
• the sample comprises at least one cell. More preferably, the sample is a blood sample .
• the fluid further comprises lysis buffer and mixing of the sample with the buffer causes the cell to lyse.
• the analyte is a nucleic acid. More preferably, DNA.
• the method of the third aspect preferably comprises the further step of detecting the presence of the analyte.
• the velocity of flow of the sample through the first microfluidic chamber is in the range 20 - 100 ⁇ m/s.
• a device for detecting the presence of an analyte in a sample comprising; i) a mutual inductor ii) an insulating layer having a first surface adjacent the spiral mutual inductor and an opposed second surface, ii) a sample contacting layer having a first surface having at least one probe immobilised thereon and a second surface opposed to the first surface and positioned so as to be adjacent the second surface of the insulating layer,
• the mutual inductor comprises a first coil and a second coil.
• the mutual inductor comprises a circular coil spiral, a square shaped spiral coil, serpentine stacked-spiral coils, or a castellated stacked -type conductor.
• first and second coils are positioned such that the first coil is positioned vertically above the second coil.
• the first and second coils are interwound. It will be understood by the skilled person that the presence of the analyte is detected by passing an alternating current through the first coil and monitoring the second coil for changes in induced voltage.
• the probe is a nucleic acid. More preferably, the probe is DNA.
• the device further comprises a suitable high permeability material layer, such as permalloy, located adjacent the spiral mutual inductor distal to the insulating layer.
• a suitable high permeability material layer such as permalloy
• the insulating layer comprises silicon dioxide
• the immobilisation layer may comprise any suitable material, for example, gold, agarose or Si 3 N 4 .
• the immobilisation layer comprises gold.
• a method of detecting an analyte in a liquid sample comprising the steps of;
• an increase in the mutual inductance indicates the presence of the analyte in the sample.
• the analyte is a nucleic acid.
• the probe is a nucleic acid.
• the magnetic beads may for example be paramagnetic beads.
• Figure 1 is an exploded view of a microfluidic device according to the first aspect.
• Figure 2 shows a diagrammatic representation of the configuration of the current carrying structures forming one mixing unit in one layer of the device.
• Figure 3 shows one tooth of a current carrying structure showing the variation in magnetic field intensity.
• Figure 4a shows a diagrammatic representation of a lab-on- chip device comprising the microfluidic device according to the first aspect
• Figure 4b shows a diagrammatic representation of an embodiment of the device according to the first aspect.
• Figure 5 shows a representation of Sprott • s method for calculating the Lyapunov component .
• Figures 6a and 6b show advection of cells within three and a half mixing units, a) without perturbation of cells and b) with magnetic perturbation.
• Figure 7 shows simulated chaotic advection of four particles.
• Figure 8 shows the initial positions of individual particles for calculating the Lyapunov Exponent.
• Figure 11 shows a diagrammatic representation of the detector device according to the current invention showing hybridised DNA tagged with magnetic beads .
• Figure 12 shows a diagrammatic representation of the sensor model used in design simulations a) top view of coil, b) lateral cross section.
• Figure 13 shows an electrical model of the sensor.
• Figure 14 shows the percentage change in coil inductance against outer coil diameter for different bead permeabilities .
• Figure 15a is a graph showing the optimal outer coil diameter at which output signal is maximised against bead permeability for different conductor thickness values.
• Figure 15b is a graph showing the corresponding maximised inductance percentage change for the inductors of Fig 15a.
• Figure 16a is a graph showing the optimal outer coil diameter at which output signal is maximised against bead permeability for different frequencies.
• Figure 16b is a graph showing the corresponding maximised sensor voltage for the frequencies of Fig 16a
• Figure 17 shows a DNA extraction chip according to the present invention in exploded view.
• Figure 18 shows a 3 dimensional view of a key type electrode arrangement
• Figure 19 shows the dimensions of the key type electrode arrangement
• Figure 20 shows a multiple-turn key-type electrode arrangement (dimensions: same as Fig. 19, except the width of each turn is 100 micrometers, inter-spacing between turns is 50 micrometers and thickness ⁇ 100 micrometers)
• Figure 21 shows a photograph of the proof-of concept chip.
• Figure 22 shows the results of PCR performed on samples prepared using the proof of concept chip as shown in Figure 21.
• Figure 23 shows an electrical model of a coupled inductor showing resistance and inductance of primary and secondary- windings .
• Figure 24 shows Common types of planar coupled inductors [Fig 24 (a)&(b) stacked-type windings, Fig 24(c)&(d) inter- wound windings]
• Figure 25 shows square shaped stacked-spiral coils suitable for use as planar coupled inductors in the detecting device of the invention
• Figure 26 shows serpentine stacked-spiral coils suitable for use as planar coupled inductors in the detecting device of the invention
• Figure 27 shows castellated stacked-type conductors suitable for use as planar coupled inductors in the detecting device of the invention
• the micromixer 10, as shown in Fig. 1 comprises a base layer 12 formed from glass having three serpentine conductors 14, 16, 18 embedded therein.
• a central layer 20 formed from PDMS comprising a straight channel 22 which is located above the serpentine conductors 14, 16, 18 and a upper layer 24 formed from glass having two further serpentine conductors 26, 28 embedded therein, two inlet ports 30, 32 and an outlet port 36.
• Each mixing unit comprises two adjacent teeth from each conductor.
• Channel 22 is 150 ⁇ m wide and 50 ⁇ m deep.
• Conductors 14, 16 are in the shape of teeth 38 having circular tips 40 and are 35 ⁇ m high and 35 ⁇ m wide in the section and distances between centres of circular tips 40 of the conductors are 100 ⁇ m and 65 ⁇ m in x and y directions, respectively.
• Each row of upper and lower conductors 14, 16 is connected to the power supply alternately.
• the mixing operation cycle consists of two phases. In the first half- cycle, one of the conductor arrays in switched on while the other one is off.
• Each mixing unit consists of two adjacent teeth 38 from opposite conductor arrays and the mixer is composed of a series of such mixing units which are connected together. In 3-D configuration, the switching between conductors will occur every 0.25 of a cycle.
• Fig. 3 shows one tooth 38 with the magnetic field generated near the circular tip 40 of the conductor when a current of 750 mA is injected into one conductor array and is turned off in the opposite array during a half cycle of activation.
• the greyscale map represents variations in the magnetic field intensity at 10 ⁇ m above the surface of the conductor where the maximum magnitude of the field is about 6000 A/m at the centre of the circular tip (point P) .
• the maximum force (5.5 pN) is applied on particles near the conductor and inside the circle of its tip where the intensity of magnetic field is at its maximum value.
• the magnetic field is maximum at the centre point P, the force on particles is relatively small at this point. This is due to the fact that the magnetic force is proportional to the gradient of the field which is almost constant in the neighbourhood of the point P. In moving away from the conductor, the force drops significantly due to a dramatic decrease in the magnetic field which in turn affects the magnetic moment.
• the microfluidic device as shown in Figs 1 and 2 may be integrated into "lab-on-chip" devices such as those shown diagrammatically in Figs 4a and b.
• the device comprises a sample preparation device 10, as shown in Fig 1, linked in series to an amplification chamber 50 and a sample analysis unit 60 comprising a detector.
• Figure 4b shows the sample preparation device 10 in greater detail.
• the device comprises an inlet to a micropump, linked to a mixing region and a separation region distal to the inlet.
• the first two steps are performed in the chaotic mixer followed by downstream processes in separator.
• human blood and particle laden lysis buffer are introduced to the device, e.g. into the microchannel, through two inlet ports, for example by direct injection, under gravity, by negative pressure applied downstream, or using external pumps or integrated micropumps .
• Mixing Qf the particles is performed by applying local and time-dependent magnetic field generated by micro-conductors to produce chaotic advection in the motion of the particles through magnetophoretic forces.
• the embedded high aspect-ratio conductors allow a relatively large current to generate strong magnetic fields to move magnetic particles.
• Conductors on both top and bottom glass wafers are required to perform an efficient spatial mixing. Using a proper concentration of particles in lysis buffer, chaotic advection of the particles can be transferred to the fluids pattern, therefore, mixing the lysis buffer and blood.
• released DNA molecules are adsorbed onto the particles' surface.
• the whole solution is then flowed downstream and the intact DNA/particles are separated from other contaminants by using another serpentine conductor fabricated at the bottom of the channel.
• the bottom coil or coils
• This conductor is activated by a constant DC current and due to the generated magnetic field; particles are gathered at the bottom surface of the channel while other contaminants are washed out with flow.
• washing buffer is introduced into the channel, which washes and removes remaining contaminants.
• conductors are switched off and resuspension buffer is pumped into the system and the purified DNA/particles are resuspended in it.
• the sample can now be used directly for PCR as the DNA is released upon heating the DNA/particle complex above 65°C as required by a standard PCR protocol.
• Functionalized nano and microparticlees or beads offer a large specific surface for chemical binding and may be advantageously used as a "mobile substrate” for bioassays and in vivo applications (Gijs 2004) .
• magnetite Fe 3 O 4
• ⁇ - FE 2 O 3 oxidized form maghemite
• magnetic particles are magnetized in an external magnetic field.
• Such external field generated by a permanent magnet or an electromagnet, may be used to manipulate these particles through magnetophoretic forces and therefore result in migration of particles in liquids.
• Conductors are utilized to produce magnetophoretic (hereafter, magnetic) forces and, therefore, chaotic pattern in the motion of particles and intensify the labelling of bio-cells.
• Two flows; target cells suspension and particle laden buffer, are introduced into the channel and manipulated by pressure-driven flow (see Fig. 2) . While the cells follow the mainstream in upper half section of the channel (transported by convection of the suspending bio- fluid) , the motion of magnetic particles is affected by both surrounding flow field and localized time-dependent magnetic field generated by periodical activation of two serpentine conductor arrays. Particles from various positions in the upstream and downstream sides are attracted towards the centre of the nearest activated tip where the maximum magnetic field exists. Chaotic patterns are produced in the motion of particles through utilizing a proper structural geometry and periodical current injection in conductors, thereby enhancing the spread of particles in the channel.
• the magnetic force on particles is a function of the external magnetic field gradient and the magnetization of the particle.
• the magnetic force exerted on the particle in the linear area is described by: where: d is the diameter of the spherical particle
• Magnetic force is applied along the gradient of the external field and the particles are attracted towards higher magnetic field regions .
• Relative permeability and diameter of the reference particle used in this study (M-280, Dynabeads, Dynal, Oslo, Norway) are 2.83 ⁇ m and 1.76, respectively .
• the magnetic force is three- dimensional and the z-component of the force is downward, which together with gravity, pull the particles towards the bottom of the channel and restrict their motion to a two- dimensional pattern. In fact, this component has no contribution to the chaotic motion of the particles and is assumed not to be influential on the process of mixing. Therefore, in this study planar forces close to the surface of the channel's bottom are of interest and simulation procedure is conducted on a two-dimensional basis.
• Total velocity of the particle at each moment would be the sum of the velocity due to fluid field (V f ) and the velocity due to magnetic field (V n ,) .
• a two-dimensional numerical simulation is carried out assuming that there are no magnetic or hydrodynamic interactions between particles (one-way coupling) and motion of the particles is treated as if they are moving individually. This assumption is valid for small particles at low concentration in suspension, namely less than 10 15 particles/m 3 (C. Mikkelsen and H. Bruus, "Microfluidic capturing-dynamics of paramagnetic bead suspensions," Lab Chip, vol.
• Newtonian fluid (water) field and time-dependent magnetic field are computed using commercial multiphysics finite element package Comsol (COMSOL, UK) and velocities of the particles due to these fields are extracted. Then trajectories of the particles are evaluated by integrating the sum of velocities using Euler integration method in Matlab:
• St £ ⁇ (4)
• f is the frequency
• L is the characteristic length (here, distance between two adjacent teeth)
• the size of biological entities may vary from a few nano-meters (proteins) to several micrometers (cells) . In this study, cells are considered to be spheres of 1 ⁇ m diameter.
• the bulk velocity of flow is in the order of 10 ⁇ m/s, which yields a Reynolds number of the order of 10 ⁇ 3 , indicating that the flow is laminar.
• largest Lyapunov exponent was used to quantify the chaotic advection of magnetic particles as a common definition of the mixing quality.
• Sprott's method J. C. Sprott, Chaos and Time-Series Analysis, Oxford University Press, Oxford, 2003
• ⁇ i the largest Lyapunov exponent
• the new distance, d(t) between real and virtual particles and also the value of ln ⁇ d(t)/d(0) I are calculated.
• the virtual particle is then placed at distance d(0) along its connecting line to the real particle.
• ⁇ i will be converged and is evaluated by: where ⁇ t is the duration of one time-step and n is the number of steps. Examination of ⁇ i for various particles reveals that generally after a period of 20s, ⁇ i approaches its converged value. Therefore, both indices of LE and ⁇ i are calculated for a period of 20s of mixing. Fig.
• FIG. 6a illustrates the position of the particles and cells while advecting within three and half mixing units.
• Bio- cells red dots, upper part of the diagram
• magnetic particles blue dots, lower part of the diagram
• A-A first mixing unit
• both cells and particles remain in their initial section and simply follow the streamlines of the parabolic velocity profile in Poiseuille flow. In this situation, tagging might occur only in the central region of the channel along the interface between two halves. All dimensions are normalized to the characteristic length.
• first array (conductor I) is on and second array (conductor II) is off.
• Particle I feels a strong magnetic force in y direction and tends to move in this direction while it is advected by the mainstream in x direction. Note that depending on its location in the channel which determines both drag force in the Poiseuille flow and magnetic force, particle I can have a positive or negative velocity in x direction.
• Particle 2 is farther from the conductor I and does not find any chance to be attracted upwards completely during the first half cycle. Therefore, two initially nearby particles diverge inducing the mechanism of stretching which is marked with a rectangle. In this phase particle I is exposed to the target cells across different streamlines and captures them in case on any collision.
• Particles 3 and 4 which are too far from the conductor I to be attracted, are dragged downstream by the fluid and gradually move towards the upper half of the channel. After passing a few mixing units, almost all particles penetrate to cells' region and fluctuate in a chaotic regime confied to the tips of two conductors.
• Devices according to the present invention can be fabricated for example using basic building blocks in MEMS technology.
• MEMS technology has the ability to deposit thin films of materials on substrate, to apply a patterned mask on top of the films by photolithographic imaging, and to etch the films selectively to the mask. It is a structured sequence of these operations to form actual device .
• the MEMS process starts with a rigid substrate material such as PMMA/ Glass/Silicon/Polystyrene.
• a high permeability layer e.g. permalloy/ Nickel
• An insulating layer of SiO2/PMMA/PDMS/Polystyrene may then be deposited on top of the permeable layer.
• the current carrying structure also known as a coil structure
• a thin layer of PDMS/PMMA/Polystyrene may then be spin coated on top of the coils to form a planar surface.
• a microfluidic channel/chamber may for example be constructed using a pre-prepared PDMA/PMMA/Polystyrene cast of the desired thickness, for example of 150 microns and it is punched out of this sheet. This latter structure is sandwiched between two identical rigid substrate construction containing the coil electrodes and bonded using plasma bonding. The input and output ports may for example be punched or drilled through the structure.
• a central thin plane of an appropriately biocompatible material e.g. PDMS
• PDMS e.g. PDMS
• a central hole formed through it preferably of rectangular shape.
• the length and width of this hole are calculated to give an appropriate final chamber volume, say 20 microlitres.
• This component formed the central part of the main lysis/mixing chamber and is closed by being sandwiched between two layers of similar or compatible material 10 to 100 micrometres in thickness.
• cover-plates carry holes to allow inlet and outlet port-ways to the chamber thus formed.
• Such current carrying structures when fed with appropriately switched currents, will cause a magnetic field to form and collapse normally to the principal plane of the cavity.
• the magnetic field strength if further amplified by the introduction of a backing of an appropriately permeable magnetic material, such as a Permalloy alloy, nickel, mu- metal or similar.
• an insulating layer ⁇ 100 micrometres in thickness is introduced.
• Micron-sized magnetic beads have also been widely used as labels in DNA detection (J. Fritz, et al, "Electronic detection of DNA by its intrinsic molecular charge,” Proc . Nat. Acad. Sci., vol. 99, no. 22, pp. 14 142- 6, 2002) (L,Moreno-Hagelsieb, et al , "Sensitive DNA electrical detection based on interdigitated A1/A12O3 microelectrodes, " Sens. Actuators B, Chem., vol. 98, pp.
• This example relates to a DNA hybridization detection sensor that uses magnetic beads attached to DNA strands as detectable particles. Increased concentration of magnetic beads due to DNA hybridization is detected in the form of inductance variations. The response of a planar spiral coil sensor to different types of magnetic beads is investigated and the effects of coil geometry as well as frequency on the performance of the sensor are numerically evaluated. Results and mathematical analysis provided for one coil can be extrapolated to multiple coils.
• the sensor 100 of the current invention for DNA hybridization detection is illustrated in Fig. 11.
• the sensor 100 comprises a core 102 which is a planar spiral inductor -which is sandwiched between an insulating layer 104 on the top and a layer of permalloy 106 in the bottom.
• the insulating layer 104 is covered with a permeable layer 108 to which probe DNAs 110 can attach and be immobilized.
• This layer could be any of standard surface treatments on gold coating or SiO 2 -Si 3 N 4 .
• Magnetic beads functionalized with target DNAs 112 are applied to this surface. Specific Hybridization of target and probe DNA will result in formation of a layer of magnetic beads 112 above this surface 108.
• This layer is of high magnetic permeability and acts as one half of the magnetic core for the inductor.
• the underlying permalloy layer 106 acts as the other half of the magnetic core and completes the magnetic circuit. Formation of this magnetic circuit allows the magnetic flux to pass through easily and leads to an increase in the coil inductance. This property is used for detection of hybridization process.
• the inductance of the spiral coil is a function of various geometrical as well as physical parameters.
• the important geometrical parameters as depicted in Fig. 12 are defined as follows : d ml : Coil outer diameter d m : Coil inner diameter t *c. : Conductor thickness t p : Thickness of permalloy layer
• the effect of interwinding distance S and the conductors thichness w are expressed in terms of fill factor (FF) .
• FF fill factor
• the electrical model of the sensor is shown in Fig. 13.
• the coil is driven by an AC current source and the coil voltage is measured as the sensor output. After formation of the bead layer, the coil inductance is increased and the sensor output, V s , will be changed. This amplitude of this voltage is used in order to detect the hybridization.
• the amplitude of K 5 can be expressed as follows:
• V s ⁇ R c 2 +( ⁇ L c ) 2 I 5 (1)
• the voltage V s is measured and its normalized variation is calculated to indicate the presence of the bead layer due to occurrence of hybridization.
• the frequency of the current source may be chosen in a range where R c is constant. This means that for a particular sensor and source frequency, the voltage v s is merely dependent on the inductance L c and hence, the normalized variations of V s may be calculated as follows:
• a three dimensional model of the sensor was simulated using the finite element package COMSOL FEMLAB Multiphysics v.3.2. Details of the model used in the simulation are shown in Fig. 12. The model was simulated for a layer of magnetic beads with effective thickness of 2 ⁇ m and different relative permeabilities.
• the graphs of Fig. 14 show how ⁇ L changes with respect to the outer diameter d oul for different values of ⁇ rB .
• the values adopted for the other parameters are shown in Tablel .
• the sensor output is maximum at a specific value of d oul which may be denoted as £> max . It should be noted that the value of D max is increasing with respect to ⁇ rB as shown by the dashed curve in Fig . 14.
• ⁇ v is computed for different bead permeabilities.
• the parameter values are as in Table 1 and the simulation results are shown in Fig. 16.
• the sensor output is maximum at a specific value of d out which is again denoted as £> max .
• the graphs of Fig. 16a show how these values are related to frequency.
• a preferred embodiment used in the sensor utilizes a transformer arrangement.
• Figure 23 shows a simplified model of a transformer.
• the series resistances of Rp and Rs are ohmic resistance of the conductors in the primary and secondary windings, respectively.
• Eqn. (1) shows the relationship between different parameters of the model.
• the output voltage is directly- proportional to the primary (or secondary) reactance as well as the coupling factor km. Based on this result and through computer simulation, the output voltage is calculated for coils of different diameters and conductor thicknesses and optimum performance of the sensor has been obtained for magnetic beads of different permeabilities.

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