Field of the invention
[0001] The present invention relates to a fluidic device for analysis of a fluid, said device
having a plurality of analytical units, a method of use of said device, an instrument
for analyzing fluids using said device and a system including said device and said
instrument. The field of application of the fluidic device according to the invention
is mainly in analytics of fluid, for instance in health care, for the analysis of
nucleic acids. Analyses performed using this device are considerably improved, as
it is possible to increase the throughput of samples to be analyzed in automated manner.
Background of the invention
[0002] Particularly in analytical laboratories there is a great interest in conducting analyses
in a convenient, safe and reliable way. Particular problems are the contamination
of reagents, samples and devices for performing an analysis sample and the contamination
of the environment by reagents or samples. Therefore devices have been proposed for
the analysis of a sample and/or reagents that minimize the contamination of the environment.
[0003] In
EP 318 256 there is shown a device comprising a chamber through which the fluid is forced. This
device cannot perform more than one analysis.
[0004] In
WO 93/22058 there is disclosed a device having a few chambers each having different temperatures.
Again, this device does not allow more than one analysis. Furthermore, the fluid flow
in this device is complicated.
[0005] It was an object of the present invention to provide a device with improved properties
over the devices according to the prior art, particularly a device allowing multiple
analyses of fluids to be performed, preferably analyses for different analytes.
Summary of the invention
[0006] A first subject of the invention is a fluidic device comprising a body comprising
a fluidic unit comprising
- a) a first chamber having an outlet portion,
- b) a first channel exiting said outlet portion,
characterized in that said body comprises two or more of said fluidic units.
[0007] A second subject of the invention is an analytical instrument comprising
- a fitting for holding a device according to the invention, and
- a head comprising two or more liquid handling units.
[0008] Another subject of the invention is a system for analysis of a fluid in a device,
comprising
- a device according to the invention, and
- an instrument according to the invention.
[0009] Another subject of the invention is the use of a device according to the invention
for the analysis of a fluid.
[0010] Still another subject of the invention is a method of analysis of components of more
than one fluid comprising
- providing a device according to the invention or a system according to the invention,
and
- in each fluidic unit
- introducing a fluid into said first chamber,
- releasing said component of said fluid from other components of said fluid this component
is associated with in said first chamber,
- transferring the resulting fluid through said outlet portion and said first channel
into a second chamber, said second chamber containing a solid phase for immobilization
of said component to be analyzed, thereby binding said component to said solid phase,
- introducing a second fluid into said second chamber directly through said first channel.
Brief description of the drawings
[0011] In FIG. 1a a first, simple embodiment of the device (1) according to the present
invention is shown comprising eight fluidic units (2a, 2b, 2c, ...) each containing
a first, sample lysis chamber (3a, ...), a first channel (5a, ...) leading from said
lysis chamber to a second, nucleic acid purification, chamber (6a, ...).
[0012] FIG 1b shows the device of FIG 1b in a view cut through the first chamber (3a) and
the channel (5a).
[0013] In FIG. 2a a second, more sophisticated embodiment according of the invention is
shown. This device contains eight analytical units, each in addition containing a
second channel, leading to the second chamber via a junction to said first channel.
[0014] FIG. 2b shows the device of FIG. 2a in exploded view further detailing the body and
the sealing foil.
Detailed description of the invention
[0015] The device of the present invention is useful for several fluid actions commonly
performed or desirable, during treatment of fluids, such as physical treatment and
chemical treatment of fluids, particularly in the field of analytics. Due to the present
invention, even complex fluidic methods are made possible. However, even for simple
steps the present invention provides advantages. More than one fluid can be treated
in parallel.
[0016] A fluid that can be treated according to the present invention can be any fluid that
is of interest to be subjected to a particular treatment. Preferably, the fluid is
a liquid. More preferable, the liquid is an aqueous solution. In the preferred use
of the device according to the invention, components of the liquid or compounds derived
there from are intended to be analyzed. In a diagnostic device, the liquid contains
components to be determined in an analysis, e.g. nucleic acids or antigens. Such liquids
may be selected from the group of liquids from the environment, like water from a
river or liquids extracted from soil, food fluids, like a juice or an extract from
a plant or fruit, or a fluid received from a human or animal body, like blood, urine,
cerebrospinal fluid or lymphatic fluid, or liquid derived there from, like serum or
plasma, or liquids containing components isolated from the before mentioned liquids,
like liquids containing purified antibodies or nucleic acids. The liquid may further
contain additional components useful for the analysis of components of the liquid
or reagents for chemical reactions to be performed within the device. Those reagents
can comprise labelled binding partners, for instance labelled oligonucleotide probes
or dyes. Such reagents are generally known to those skilled in the art.
[0017] A fluidic unit is a part of the device that has a fluidic behavior which is independent
from the individual fluidic behavior of other fluidic units in the device. The function
of fluidic units of the same device may be the same or different. However, preferably
the function is generally the same, but the reagents used for the treatment may differ
from one fluidic unit to another. Preferably, the geometry of the fluidic units on
the same device is the same, e.g. each unit has the same number and arrangement of
chambers and channels as any other fluidic unit on the same device. Preferably, the
fluidic units allow the analysis of different fluids in parallel. For example, two
or more samples from different sources, e.g. from different human beings, but of the
same kind, e.g. serum, can be analyzed in parallel. In another mode, aliquots of one
specimen from the same source, e.g. the same human being, is analyzed for the presence
or the amount of different analytes, e.g. the presence of different viruses, such
as HBV and HIV, in parallel. The measures to be taken to analyze fluids according
to those embodiments of the invention will be described below.
[0018] A device according to the invention preferably is a composite of at least one body
and at least one sealing wall.
[0019] A body is a part of the overall device that mainly provides stiffness or rigidity
to the device. Therefore, the body preferably is rigid. Preferably, the body is formed
from a thermoplastic material, more preferred from a material selected from thermoplastic
organic polymers. Most preferred the thermoplastic organic polymer is selected from
the group consisting of polypropylene, polyethylene, polystyrene, polycarbonate and
polymethylmethacrylate. Further preferred, the material is, at least at parts needed
for analysis, light transparent. The body may have a length of between 20 and 150
mm, a breadth of between 5 and 20 mm and a height of between 40 and 150 mm, dependent
upon the amount and kind of treatment steps to be performed in the device. Usually,
the more fluid(s) is (are) to be analyzed, the larger the volume of the body.
[0020] Preferably, the body has an area that is generally flat over an area of between 100
and 10000 mm
2, more preferred between 160 and 2250 mm
2 or more preferably between 4500 and 8500 mm
2. This area is in the following called the sealing area. The term flat means that
the body towards the outside of the device is as geometrically homogenous to allow
a sealing unit to approach and thermally contact the body such that sufficient heat
can be applied to the material of the body to melt a part of the body in contact with
the sealing unit. In other parts, the body may contain areas that rise from the flat
surface, e.g. in the vicinity of chambers formed in the body.
[0021] Most preferable, any cavities in the body are closed by a sealing wall attached to
the body.
[0022] A sealing wall preferably is a generally flat piece of material. It may be made from
one material or may be a composite. Preferably it has the form of a foil which is
less rigid than the body. The present invention has found that it is very advantageous,
if the sealing wall is a composite of the same thermoplastic material as the body
- this part being called the thermoplastic part - and a carrier part made of a material
having a melting temperature which is higher than the melting temperature of the thermoplastic
part. Preferably, the carrier part is selected to provide tear strength to the sealing
wall. Said tear strength is important for the reliability of the sealing process.
The preferred tear strength is preferably between 3 and 50 N/mm
2, more preferably between 4 and 40 N/mm
2. Preferred materials for the carrier part are selected from the group of metal foils;
more preferred the material comprises aluminum. The thickness of the foil is preferably
between 40 and 400 µm.
[0023] Preferably, the sealing wall is a heat-transfer wall. A heat-transfer wall preferably
comprises a heat-transfer material, i.e. a material having good heat conductivity.
The overall heat transfer rate of the heat transferring or sealing wall used according
to the invention is typically greater than 200 W/m
2/K, more preferred greater than 2000 W/m
2/K. Preferred heat-transfer materials are selected from the group of aluminum and
copper, more preferred is aluminum. Preferably, the heat-transfer wall comprises 2
layers, preferably, wherein one of said layers is a metal layer and a second layer
is a thermoplastic layer, and said layers are welded together.
[0024] In order to insure proper, particularly liquid tight, sealing of the sealing wall
to the body in the area surrounding the cavity, the sealing wall preferably is substantially
planar. Substantially planar means that it is flat over more than 80%, preferably
more than 90% and most preferably 100%, of its surface. The part of the body intended
for sealing to the sealing wall should be substantially planar to a similar extend
in the areas surrounding the cavity, but excluding the grooves that are intended to
form channels or chambers in the device after sealing.
[0025] The sealing wall preferably is between 20 and 1000 µm thick, more preferably between
50 and 250 µm. Preferably, there is one sealing wall per body of the device, covering
all grooves to be sealed in the body.
[0026] Between those components at least one cavity is formed in said fluidic unit. A cavity
comprises at least one chamber and at least one channel. A fluidic unit according
to the invention thus requires at least one chamber, called the first chamber, and
at least one channel, called the first channel. This chamber and channel can be located
at any position of the fluidic unit, for instance at the beginning of a fluid path,
or in between a fluid path or at the end of a fluid path.
[0027] A channel according to the invention is a cavity in the device which has a longitudinal
dimension that is larger than its width and height. The channel is preferably confined
by walls defining the width and height of the channel. In preferred embodiments, a
wall of the channel is defined by the surface of a groove formed in the body of the
device and a surface of a wall closely sealed to the edges of the grooves of the body.
Channels formed within the device preferably have a cross section of less than 10
mm
2, preferably of between 0.01 and 2 mm
2. Channels for transporting fluids through the device will preferably have smaller
dimensions than chambers for keeping the fluids or/and performing a process, preferably
a chemical reaction.
[0028] The use of a channel can be various, e.g.
- delivery of fluid between two locations within the device (e.g. chambers),
- delivery of fluid in or out of the device,
- measuring fluid, or/and
- processing a fluid or processing matter being solved or suspended in the fluid.
[0029] A chamber according to the invention is another kind of cavity in the device. The
dimensions of said cavity will vary upon the intended use of said chamber. The use
of a chamber can be various, e.g.
- storing, receiving, or / and delivering fluid,
- processing a fluid, e.g. for analysis of matter in the fluid, or / and
- measuring a physical of chemical property of a fluid (e.g. for performing optical
absorption or fluorescence measurement).
[0030] In a first use, the chamber will be used to receive a sample having a large volume,
e.g. for performing a lysis reaction in the original sample, adding a certain volume
of reagent fluid. The volume of a chamber may be less than 1 L, preferably between
50 µl and 10 ml. A preferred embodiment of such chamber is a chamber for chemical
sample preparation, such as the lysis of cellular components of a fluid containing
cells to release constituents of said cells, e.g. nucleic acids or other substances
to be analyzed. Such chamber may be called a lysis chamber. A lysis chamber does not
need to be a flat chamber, but preferably will have an at least partially tube like
form, having an upper opening for introducing a sample and reagents for lysis, and
a lower opening as an outlet to a channel. Conditions under which chemical sample
preparation is performed are well known and can be applied to the present invention
easily.
[0031] In other uses than performing a lysis reaction, it is preferred to use a substantially
flat chamber. Preferably the length is in the same magnitude as the maximum width
or the maximum height, while either the height or the width is considerably smaller
that the maximum height or maximum width. The height of a preferred substantially
flat chamber will be between 10 µm and 49 mm, preferably between 10 µm and 20 mm,
more preferred between 0.01 mm and 2 mm. Most preferably, the height is between 50
µm and 1 mm. The length and width of the chamber may be between 10 µm and 295 mm,
but preferably is between 20 µm and 145 mm. In particular preferred, the chamber is
a flat chamber of a thickness of less than 2000 µm, preferably between 50 µm and 5
mm. For example, the length of such chamber may be between 1 mm and 100 mm, preferred
between 5 mm and 20 mm. Preferred the width of the chamber varies over the length
of said chamber. The maximum width may be between 1 mm and 30 mm, preferred between
3 mm and 20 mm, and the maximum height may be between 0.01 mm and 5 mm.
[0032] A first use of a flat chamber is the detection of a property of a fluid, the amplification
of nucleic acids or / and the purification of a nucleic acid.
[0033] For detecting a property of a fluid, such as a signal, a chamber will preferably
allow entering energy to excite a component of the fluid to create an analyte specific
signal, preferably an electromagnetic signal, and allow the signal to escape to a
detector. Thus, preferably, a detection chamber will have an optical window, e.g.
an optically transparent part, such as a wall. Preferred, a chamber for detection
has a detection area of a length of between 3 and 30 mm, preferably between 6 and
20 mm, and a width of between 3 and 20 mm, preferably between 6 and 12 mm, making
up an area of between 9 and 600 mm
2, preferably between 90 and 400 mm
2, which area of said chamber does not contain no fluid, if the chamber is filled with
fluid.
[0034] A chamber for amplifying nucleic acids, e.g. performing the polymerase chain reaction,
may have a volume of between 0.1 µl and 500 µl. If combined amplification and detection
is intended to be made in the chamber, the chamber will also preferably have a volume
of between 0.1 µl and 500 µl.
[0035] The chamber may comprise protrusions or / and solid materials, such as porous inserts.
A protrusion is a deformation of any of the walls to reduce the width of the chamber.
Such protrusion may improve bubble free filling of the chamber.
[0036] Preferably, the solid material is porous. A porous insert is a substance through
which a fluid can flow, thus filling the pores of said insert temporarily or permanently.
Preferred porous materials are fleece, e.g. made from fibers. Preferred materials
making up a porous insert are selected according to the intended use of the insert.
Porous inserts can be used to specifically or nonspecifically immobilize components
of the fluid. Specific immobilization is preferably performed by contacting the fluid
to a solid material that specifically recognizes and binds said component. Examples
of specific recognition are the recognition of antibodies by antigens or haptens or
the hybridization of nucleic acids having substantial complementarity. Non-specific
immobilization may occur, if more than one component or a group of components are
recognized and bound. An example of such recognition is the recognition of the group
of nucleic acids by glass surfaces under binding conditions. For purifying nucleic
acids on a solid phase the use of a glass fleece is preferred. For example, a chamber
for separating nucleic acids from a fluid will preferably have a volume of between
5 µl and 100 µl. Conditions for binding nucleic acids are well known to those skilled
in the art and may comprise the use of chaotropic salts, such as guanidinium isocyanate.
[0037] In order to perform the basic steps of thermally treating the fluid and monitoring
a property of said fluid, the fluidic unit just needs to have a chamber to contain
the fluid to be treated. Thus, taken a preferred size of the chamber of between 0.01
µl and 1 ml, the size of the device may be as small as 6 ml or less. Preferably, the
device has a substantially flat design, i.e. in its main part it may have a thickness
of less than 50 mm, preferably of between 0.2 and 10 mm, and a length and width of
less than 300 mm, preferably of between 2 and 150 mm.
[0038] A fluidic unit may contain even more channels and / or chambers, e.g. for providing
fluids to the interior of the device or receiving fluids from chambers or channels
in the device.
[0039] In a preferred embodiment, useful for the determination of nucleic acid analytes
in fluids, each fluidic unit contains a first chamber, i.e. a lysis chamber and a
first channel leading from the outlet portion of said first chamber to a second, preferably
flat, chamber, preferably via an inlet portion, said second chamber comprising a fleece
capable of reversibly binding nucleic acids. A second channel leads from an outlet
portion of said second chamber to a third chamber, preferably via an inlet portion
of said third chamber, and a third channel leads from an outlet portion of said third
chamber to an outlet port of said device. Any of those chambers can be a chamber according
to the invention. Preferably, the chamber as defined according to the invention is
a lysis chamber and is the first chamber according to the invention.
[0040] In another preferred embodiment of the invention, a fluidic unit according to the
invention contains a first chamber having an outlet portion, a first channel leading
from the outlet portion of said first chamber to the second chamber, and an additional
channel leading from a second opening of the device to the first channel or to said
second chamber. The additional channel is called inlet channel in the following. It
is preferably designed to allow introducing one or more fluids into the device without
passing the first chamber. In this embodiment the second channel preferably leads
to said first channel in a portion of the fluidic unit located between the outlet
portion of said first and the inlet portion of said second chamber, so that the first
channel extends from the junction of the first and the second channel towards said
first chamber by at least 0.1 mm, preferably between 1 and 100 mm, more preferred
between 2 and 20 mm. The junction between those channels further preferably is located
in a distance of at least 0.1 mm, preferably between 1 and 100 mm, more preferred
between 2 and 20 mm from the inlet portion of said second chamber. The second chamber
preferably is a nucleic acid purification chamber.
[0041] The inlet channel preferably is leading from an inlet port into the device. An inlet
port in the device according to the invention is an opening of the device designed
to allow the fluid to enter a fluidic unit while avoiding the escape of fluid during
treatment. Therefore, preferably such port is sealed, for instance by a stopper. The
inlet port can be closed or opened at any time, or it may be closed or opened at convenience
or according to the needs of the method to be performed in the device. Preferably,
the inlet port can be pierced by a hollow needle.
[0042] In the preferred embodiment, wherein the device contains an inlet channel, it is
preferred that the channel leading from the first chamber to the second chamber comprises
a valve to reversibly or irreversibly close the channel. This prevents fluids from
unintended entering from the first chamber into the second chamber and thus spilling
of the following fluid path. Such valve, more preferred a sealable valve, is preferably
located in the vicinity of the junction of said first and second channel. The closer
the valve is to the junction, the lower is the risk of retaining fluid in the remainder
of said channel downstream of said flow path of the fluid. Retained fluids may lead
to false results of the overall procedure performed, e.g. by contamination. Preferably,
said vicinity is at a distance of between 1 and 20 mm, more preferred at a distance
of between 2 and 10 mm, from said end. Even more preferred the volume between said
valve and said end is between 0.1 and 4 µl, more preferred between 0.2 and 2 µl.
[0043] More preferred each fluidic unit further comprises a second channel leading from
said second chamber to a third chamber for irradiation and detection.
[0044] The last channel, in the above embodiments the third channel, in the fluid path is
leading out of the device through an outlet port. An outlet port in the device according
to the invention, more preferred of the fluidic unit, is an opening of the device
designed to allow the fluid to exit the device in a controlled manner, while avoiding
unintended escaping of fluid during treatment. Thus, preferably the opening is sealed,
for instance by a stopper, which can be pierced by a hollow needle.
[0045] In a preferred embodiment of a device being made of a body and a sealing wall covering
adjacent chambers, heat transfer between chambers of adjacently located fluidic units
is reduced or discontinued by construction. Any means useful for reducing the stream
of heat from a chamber of one unit to a chamber of another unit, preferably to a chamber
having the same or similar function, are useful. This may include reducing the thickness
of the material between the two chambers, i.e. using a thin bridge between the chambers
or even interrupting the material positioned directly between the chambers. Preferably,
when the device comprises a body and a sealing wall comprising a metallic heat transfer
layer, the metal layer is interrupted between the chambers. The interruption may have
any suitable form, e.g. a trench or a gap; preferably the gaps are arranged in parallel.
Examples of such interruptions can be seen in FIGs. 1 and 2. The interruption may
also be larger than the direct connection between two adjacent chambers. For example,
the interruption may extend between two pairs of adjacent chambers, such as shown
in FIGs. 1 and 2.
[0046] The device according to the invention may comprise as many fluidic units as meaningful.
A too large number may be disadvantageous in view of then more difficult handling
of the device. For instance this may require too many actuators in an instrument to
be fluidically accommodated. It has proven to be advantageous to use from 2 to 16
fluidic units in one device, more preferred from 4 to 8 units.
[0047] In order to handle the device conveniently, the fluidic units are preferably arranged
in a parallel mode. This means that the positions of the chambers and channels of
different fluidic units geometrically parallel each other. Any inlet and outlet ports
will then be located at the same side of the device, preferably each kind of port,
e.g. the inlet ports, along an edge of the device, the other kind, e.g. the outlet
ports, being located along another edge. If there are two different kinds of inlet
ports, they may be arranged at the same side or edge of the device.
[0048] In an alternative mode, any channels from the chambers to the outlet can be combined
to have a common outlet port. In a preferred mode of this embodiment the device contains
one or more unidirectional restrictor valves, in order to secure that the pressure
can be directed to a particular analytical unit.
[0049] The form and size of the overall device according to the invention is mainly determined
by the function to be served by the device. Furthermore, the kind and amount of the
fluid in said device and the kind and number of steps to be performed is further determining
the geometric and functional characteristics of the device.
[0050] In a preferable embodiment, the devices according to the here used understanding
have one or more channels with a cross section of more than 0.1 µm
2, more preferable between 10 µm
2 to 10 mm
2. The devices may furthermore or alternatively comprise one or more chambers having
a larger cross section larger than the channels. A chamber of a device may have a
volume of between 10 nl and 10 ml, more preferable between 1 µl and 0.5 ml dependent
on the function and the purpose of the device.
[0051] The device according to the invention can comprise additional elements, such as recesses
and protrusions for interacting with an instrument for receiving or / and treating
said device. Preferably, said device contains grooves to engage with a gripper to
grip the device and transport it to a position in the instrument and secure it at
a predefined opposition.
[0052] A first preferred embodiment of a device according to the invention is shown in FIG.
1a (assembled view with sealing wall being shown as transparent) and FIG. 1b (cut
through the first fluidic unit (2a)).
[0053] A device having 8 fluidic units (2) is shown, three of them being named 2a, 2b and
2c, the other five not being named, but shown. Each contains a chamber (3), exemplified
as 3a, 3b and 3c. Each of them has an outlet portion (4), only the outlet portion
of first one being named (4a). Similarly, for the first unit there is shown a channel
(5), for the first unit named 5a, and a second chamber (6), for the first unit named
6a.
[0054] In FIG. 1b there is shown chamber 3a, outlet portion 4a, channel 5a, second chamber
6a, second channel 7a, third chamber 8a, third channel 9a, and outlet port 10a. Further
shown is an interruption 12 and the sealing wall 11.
[0055] In FIG. 2a a second embodiment of the invention is shown, wherein each fluidic unit
contains an additional channel 16 (exemplified as 16a for the first fluidic unit)
leading from an inlet port 13 (exemplified as 13a) to the junction 15 (exemplified
as 15a) with the first channel 5 (exemplified as 15a) leading from the first chamber
3 (exemplified as 3a) to the second chamber 6 (exemplified as 6a). Further shown in
FIG. 2a is an inlet port 14 (exemplified as 14b for the second fluidic unit 2a).
[0056] In FIG. 2b the device of FIG. 2a is shown in exploded view, making visible the sealing
wall 11 and the interruption 12 (exemplified as 12a) therein.
Another subject of the invention is
[0057] The two parts - body and sealing wall- can be joined by known methods. In the preferred
embodiment, wherein the sealing wall is a thin wall comprising a thermoplastic polymer
and the rigid body is made of polymer, e.g. polystyrene or polypropylene, the two
parts can be combined and then sealed by welding, for example Laser welding, ultrasound
welding, thermo sealing or gluing. The two parts can also only be clamped or stick
together.
[0058] The joining method, the material of body and the material of the sealing wall have
to be selected to fit together. For example, if the joining method is Laser welding,
then the bulk material of the body and the sealing wall are of the same material (e.g.
polypropylene) but one of the two materials is stained to have absorption for the
laser energy. If the joining method is ultrasound welding both materials are typically
the same. If the joining method is thermo sealing the sealing wall is a thermo sealable
wall adapted to thermally seal to the body.
[0059] In the above method for manufacturing, further assembly steps can be added, particularly,
if the device contains additional elements.
[0060] Another subject of the invention is an instrument comprising
- a fitting for holding a device according to the invention or its preferred embodiments,
and
- a head comprising two or more liquid handling units.
[0061] In order to reliably hold and apply instrumentation to the device, the instrument
comprises a fitting for holding the device. This fitting also allows holding the device
in a position wherein the fluids can be introduced into the fluidic units of said
device at the time as wanted. The fitting may be adapted to the outer form of said
device as much as needed to keep the device. The fitting may include a snap-in means
that have a form fit to respective parts of the device. Such form fit may be provided
by protrusions in said fitting that can be inserted into recesses in the device, or
vice versa.
[0062] Furthermore, the instrument according to the invention comprises a head containing
two or more liquid handling units. Such handling units are generally known to those
in the art and may be selected from the group comprising fluid dispensing devices
and fluid aspirating devices. Examples of such devices are pipettes, pipette tips
fixed to sockets for receiving the tips, and hollow needles. Preferably pipette tips
having an outlet opening having an outer conical shape are used according to the invention.
Those devices may be connected to pumps. While it is possible to introduce fluid into
the fluidic units of the device according to the invention consecutively, one by one,
it is highly preferred for high throughput reasons, to use liquid handling units being
in register with the inlet ports of the fluidic units, such that two or more of the
fluidic units can be filled in parallel with fluid without removing the head from
the device after each fluid handling step. Therefore, the distances of liquid handling
units resemble distances between inlet ports. The same holds true for the outlet ports.
[0063] The function of dispense or deliver and remove or receive fluids to and from the
device is to be considered both as active and passive handling. For example, receiving
a fluid from a first fluid handling unit can be made by either applying the fluid
under pressure to the device to press the fluid into the device or by applying negative
pressure to the cavity so as to suck fluid into the device and removing or delivering
fluid from the device to the outside can be achieved by either applying pressure to
the cavity, e.g. by pumping a fluid, such as a liquid or a gas through a first inlet
port, or applying negative pressure to the cavity so as to suck the fluid through
an inlet port. Appropriate means include syringe pumps. The liquid handling units
are situated in the instrument such that they can act on any input and output location
when the device is put into a defined position on the instrument. The position of
the head relatively to the inlet or outlet port of the device may be controlled by
a control unit.
[0064] Preferably, this instrument is an analytical instrument. Instruments for analysis
of a fluid or any components thereof are generally known. They include units as generally
known for analyses. Preferred units are means for determining properties, for instance
optical properties, or changes in properties of the fluid contained in the device,
mechanics to move the fluid from a first position to one or more other positions,
and liquid handling units for dispensing or/and aspirating fluids from tubes, vessels
or reagent containers into the device. As pointed out above, the instrument comprises
a head, which is used to dispense fluids into the fluidic units of the device according
to the invention or/and remove liquids from the device.
[0065] The instrument further preferably contains a heater, preferably a heating or/and
cooling element. This element is positioned such that it contacts or can contact the
device at the outside of the sealed heat transfer wall, preferably when the fluid
is contained in a chamber within the device, such that a heat transfer to and from
the heater or/and cooler to and from the chamber is possible, preferably through said
heat-transfer wall. An example of an instrument comprising a heating or/and cooling
element is a thermocycler. Thermocyclers are generally known to apply a profile of
different temperatures in repeated manner to a fluid. An exemplary thermocycler is
described in
EP 0 236 069. Preferred heating or/and cooling elements are selected from the group consisting
of a Peltier element, a resistance heating element and a passive cooling element,
such as a metal block equipped with a fan.
[0066] In the present invention, preferably for each fluidic unit there is at least one
thermal cycler unit, each being located in the instrument in a position that be moved
relative to the device to contact said sealing wall close to the chamber containing
the fluid to be heated. Preferably, this is the third chamber as pointed out above.
More preferably, each thermal cycler can be regulated independently, i.e. each thermal
cycler can be applied with a different thermal profile. A thermal profile is defined
by the temperature to be reached in the chamber and the length of time to keep this
temperature. The different profiles can be achieved by computer control. The interruptions
provided on the device facilitate the possibility to use different thermal profiles
at adjacent fluidic units.
[0067] In order to perform monitoring of properties or change of properties of the liquid
during processes performed in the device, the instrument further comprises a property
monitor unit optically connected to transparent walls of a chamber in said fluidic
units, e.g. a detection module. Appropriate detection modules are generally known
and depend upon the kind of property or property change performed during the presence
of the fluids in the device. For example, if the property is a change in an optical
signal, for example a fluorescent signal, the detection module will comprise a light
source positioned in the instrument such that the fluidic units of the device, preferably
a detection chamber in that device, such as the third chamber, can be irradiated,
and an irradiation receiving unit, preferably a light sensitive cell for receiving
irradiation from the fluids contained in the device and transmitting an electrical
signal to an evaluation unit. The detection module is located in the instrument where
it can detect light emanating from the fluids contained in the chambers. Preferably,
if there is also an irradiation module located to impinge light into the chamber;
this light preferably has characteristics to either excite a component in the fluid,
either to be absorbed or to be altered.
[0068] If the process to be performed in the device requires connectivity of components
of the device, such as electrodes or heating walls in the device to an electric circuit
of the instrument, such connectors are preferably provided on the instruments on positions
that are located such that the connectors on the instrument are connected to their
counterparts on the device, when the device is inserted into the instrument.
[0069] Furthermore, the instrument preferably contains units to close any valves at a predetermined
point in time during said method. This can be done reversibly or irreversibly. Reversible
sealing is for example by a three way valve. Preferred is irreversible sealing.
[0070] Irreversibly sealing valves means that the valves cannot be reopened to return to
their previous form and function to let fluid pass through the channels. Such irreversible
closing can be done by heating the material around the channels to be closed. In order
to be reliably and irreversibly sealable, the melting temperatures of the material
of said rigid body part and the material of said sealing wall part are identical or
differ by no more than 50 °C, more preferred by no more than 20 °C. Preferably, the
material is the same.
[0071] To achieve this, the instrument according to the invention preferably comprises a
thermal sealing unit. A unit for thermal sealing of the valve is a device for heat
the material of the valve to at least partially melt. The melting allows deformation
of the valve, such that the channel is closed and there is no more an open cross section
in said channel in said valve. Said unit for thermal sealing is preferably moveably
mounted on said instrument. This allows for moving the sealing unit towards said valve
when said device is held in the fitting or/and when said sealing is done. Furthermore,
this allows for applying pressure to the valve during the sealing process. This may
improve the reliability of the sealing process by brining the material of said body
part in contact with the material of said sealing wall part. Thereby, the materials
will be very efficiently connected without leaving any channels between them at at
least one location in said valves. Thus, no fluid can pass said valves any more; the
valves are sealed and closed. The unit therefore preferably comprises a plunger having
the size of any of said the valves, or as many plungers as valves to be closed, and
being heatable to the melting temperature of the material or materials. The preferred
material for the part of the plunger is a metal, e.g. steel. The unit to heat up the
plunger(s) can comprise any means to raise the temperature, such as a resistance heater.
[0072] In order to just close the valves, but not destroy the intended functions of the
device, the instrument preferably further comprises a control unit for initiating
thermal sealing. This will preferably include a means to control the amount of heat
to be introduced to the valves to get sealed, and the time of contact of the sealing
unit(s) with the valves. Those variables can be determined easily by a few experiments
at given time and temperature conditions. Those variables will then be stored in a
memory and can then be used by the control unit to start and heat the valves. Another
important task for the control unit is to initiate the thermal sealing process at
the time, when no more fluid should pass the valves, but not before. Again, this time
variable will be predetermined and kept in a memory of said control unit for use and
control. Another subject of the present invention is a system for analysis of a fluid
in a device, comprising
- a device according to the invention in its general or preferred embodiment, and
- an instrument according to the invention in its general or preferred embodiment.
[0073] Preferably, the system according to the invention comprises in addition a fluid container
(e.g. for waste collection) or/and one or more reagent containers.
[0074] A further subject of the invention is the use of a device according to the invention
in its general and preferred embodiment in a method for analysis of a sample.
[0075] Therefore, another subject of the invention is a method of analysis of components
of more than one fluid comprising
- providing a device according to the invention or its preferred embodiments or a system
according to the invention or its preferred embodiments, and
- in each fluidic unit
- introducing a fluid into said first chamber,
- releasing said component of said fluid from other components of said fluid this component
is associated with in said first chamber,
- transferring the resulting fluid through said outlet portion and said first channel
into a second chamber, said second chamber containing a solid phase for immobilization
of said component to be analyzed, thereby binding said component to said solid phase,
- introducing a second fluid into said second chamber through said first channel.
[0076] The fluids, preferably samples to be analyzed or/and reagents, can be introduced
into the device according to known methods, e.g. by pipetting the fluids into openings
in the fluidic units. Preferably, they are introduced into the fluidic units by a
head as outlined above for the instrument, e.g. such as a head carrying pipette tips,
through said inlet ports into the first chambers. In these chambers the samples are
treated to set the components of the samples to be analyzed free from any cellular
compartments the components may be associated with in the samples. For the analysis
of nucleic acids, this may include disrupting cells by a combination of chemical treatment
with chaotropic salts and a protease to digest cell walls with a physical treatment
with heat, e.g. by warming up the lysis mixture to between 37 °C and 72 °C. The exact
conditions may depend upon the particular type of sample. Some samples may need more
harsh conditions than others. In order to achieve lysis, the samples must be brought
into contact with reagents for the treatment, e.g. for the lysis. This is preferably
done by pipetting aliquots of each of the samples and the reagents into the chambers.
The mixture is moved by pressure and vacuum through all chambers and channels to the
waste, during this process the analytes are immobilized in the second chamber.
[0077] Then the valves are closed by thermally treating said valves in said instrument.
This is done by at least partially melting the material of the body part, preferably
by heating the body part to a temperature above the melting temperature of said material.
Such temperatures may be selected between 200 and 400 °C, more preferably between
260 °C and 333 °C, dependent upon the material. At those temperatures, the material
confining the valves in the channel will deform and the channels will contract towards
the material of the sealing wall and finally close the valves in the channel. In order
to reliably perform the closing step, preferably, said sealing comprises moving the
device, the fitting(s) or/and the unit(s) for thermal sealing towards each other.
This will direct the melted material towards the sealing wall. Most preferred, the
method comprises either
- actively pressing said device towards said sealing unit, or
- actively pressing said sealing unit(s) towards said device,
said pressure being applied during said thermal sealing. The pressure needed to seal
the materials together will depend upon the melting degree of the material, materials
with higher fluidity needing less pressure than materials that are still more solid.
The pressure may be selected from between 2 and 40 kp mm-2, preferably between 8 and 20 kp mm-2.
[0078] If no more than the lysis is intended to be performed in the device, the process
may be completed by removing the pretreated samples from said chambers, for instance
by removing the mixtures through the first channels. However, other steps may be added
in said device that may or ay not include further embodiments of the invention.
[0079] If the method according to the invention shall be performed including the analysis
within the sample, the method according to the invention after treatment in a first
chamber, e.g. after sample lysis, should include transport of the result of the step,
e.g. the pretreated sample, into the second chambers for further treatment. This is
preferably done by subjecting the fluids to positive and/or negative pressure to leave
the first chambers through the outlet portions into the first channels. In a preferred
embodiment, the fluids are transferred for purification purposes of components of
the samples into second chambers. Any components to be immobilized are bound to porous
material contained therein.
[0080] After said fluids have passed the valves, the valves in the first channels will be
closed by thermal sealing as described above. Any further fluid transport is then
conducted through channels other than the channel containing the sealed valve, e.g.
through the inlet channel.
[0081] A particularly preferred embodiment of the invention comprises introducing second
fluids into said second chambers after having sealed said valves. Preferably, said
second fluids are selected from the group consisting of a washing fluid and an elution
buffer and master mix. A washing buffer is a fluid that is designed to remove any
free components of the fluids from the component(s) immobilized to said porous material.
Such buffers are well known in the art and preferred include salt concentrations lower
than the fluid used for immobilization. After drying the porous material at elevated
temperature the elution is done by adding an elution buffer which preferably contains
reagents for the detection of a component of said fluid or a component derived there
from. The heating of the porous material is performed also through the heat transfer
wall. A mixture of an elution buffer and a master mix further contains the reagents
for amplification and detection of nucleic acids, such as primers, probes, enzyme
and reagents.
[0082] For conducting an assay, the method preferably comprises first washing the components
immobilized on the porous material and then eluting them from the material. The liquids
needed for that are conveniently introduced into said second chambers through said
inlet channels of the preferred embodiment (see also FIG 2a).
[0083] Then eluates are preferably led to the third chambers for detection. This can be
done by the pipetting device, preferably through said inlet ports. This will force
the fluids through the second channels to third chambers.
[0084] The chambers each preferably further contain at their ends opposite to the inlet
portions of the third channels outlet portions for a fourth channel, said fourth channel
leading to another fluid port, the output port.
[0085] In a preferred embodiment, there is in each fluidic unit at least one chamber, more
preferred the third chamber as outlined above, designed to allow a step for physical
or chemical treating said fluids. Preferably, physical treatment is a treatment selected
from the group of heating and cooling (thermal treatment), mixing and irradiating
and any combinations thereof. Any thermal treatment may be performed through any wall
of the chamber of said device. Preferably, the heating is done through the heat transfer
wall.
[0086] In a first preferred embodiment, physical treatment is thermocycling as used in the
Polymerase Chain Reaction (PCR,
EP 0 201 184).
[0087] In another preferred embodiment, said second chamber or the third chamber in each
fluidic unit preferably is a detection chamber, and most preferred an amplification
/ detection chamber. In this chamber, preferably a property representative of said
component to be analyzed or of a component derived there from is determined as a measure
of the presence or absence or the quantity of the component of said original liquid.
[0088] Detection may be a two step process, including irradiation and monitoring. After
irradiating the fluid in said chamber a property of the contents of the chamber, i.e.
the fluid, is monitored. Said monitoring a property of the fluid may be performed
through a wall of the body. The requirements of the monitoring process determine the
characteristics of the walls confining the chambers. For instance, determining light
emanating from the fluids using a detector unit located outside the device in an instrument
requires transparency of the walls for light emanating from the chambers. In this
case, the material of the walls will be a material transparent for this light. If
said monitoring in addition requires impinging light onto the fluids contained in
said chambers through said walls, the material of the walls should be transparent
for the impinging light.
[0089] Detection can be made by irradiating the liquids in the cavities with light of a
wavelength at which one of the components or reagents in the fluids has a measurable
absorption. Determination of light leaving the cavities, for example by fluorescence,
can be used to determine the absorbance of the liquids or any changes in absorbance
of the liquids over time or compared to a standard liquid.
[0090] Chemical treatment is the performance of a chemical reaction. Preferred, in the third
chambers the performance of chemical reactions is detected. Preferred chemical reactions
are reactions modifying the chemical constitution of any components of the fluids
or any derivates thereof. More preferred, chemical reactions are selected from the
group consisting of primer extension, hybridization, denaturation and lysis. Most
preferred, the chemical reaction is the PCR as referred to above, or its improvements
such as homogenous PCR, sometimes also called Real-Time-PCR, as described in
EP 0 543 942. In Real-Time-PCR, a signal is determined not necessarily at the end of the amplification
reaction, but at least once between the first and the last thermal cycle. Other amplification
and detecting method can also be applied like linear amplifications and other methods
known to those skilled in the art.
[0091] In order to perform combined amplification / detection including the PCR, the contents
of the chambers are heated and cooled in cyclic manner. In order to achieve efficient
thermocycling the heat transfer wall covering the third chambers contain a metallic
part facilitating heat transfer from the thermocycler into the chamber, a heat transfer
foil. Homogenous PCR allows detection nearly from the start of the thermocycles through
a transparent window in said body.
[0092] In a very preferred embodiment of this method of analysis, the components of the
liquids to be analyzed are nucleic acids suspected to be contained in the fluids,
for example parts of the genome of hepatitis B virus. The reagents for analysis, preferably
the elution buffers, will then contain reagents, e.g. primers, for the amplification
of a particular fragment of said nucleic acids and probes for binding to the amplified
fragments. A very preferred embodiment of such reaction is disclosed in
EP 0 543 942. In order to apply thermal cycles to the fluids contained in the chambers, the instrument
used contains a combined heating/cooling block to bring the contents of the chambers
to the temperatures in a profile as needed to amplify the nucleic acids. The change
in absorbance or fluorescence in the fluids is then used as a measure of the nucleic
acids to be determined in the fluids.
[0093] The reagents used for treatment in the different fluidic units of one device may
be the same or may be different. For example, if in the first fluidic unit HBV is
to be detected and in the second unit HIV is to be detected, the same procedures and
reagents for sample lysis and purification may be used for the two aliquots of the
sample in different units, but different reagents for amplification and detection
(elution buffer and master mix), reflecting the different sequences to be amplified,
should be used. Suitable reagents for sequence specific amplification and detection
are known to the man skilled in the art and can be applied analogously.
[0094] Preferred embodiments are detailed above in the description of the instrument according
to the invention.
[0095] An advantage of the device according to the present invention is that it is possible
to conduct several analyses in parallel, even if the analyses differ, e.g. in that
different analytes are determined or in that the chemical reactions performed are
different or the thermal treatment are different.
Reference numerals:
[0096]
- 1
- Device according to the invention
- 2
- Fluidic unit (2a, 2b, 2c, 2d, 2e, ...)
- 3
- First Chamber (3a, 3b, 3c, ...)
- 4
- Outlet portion (4a, ...)
- 5
- First Channel (5a, ...)
- 6
- Second chamber (6a, ...)
- 7
- Second channel (7a, ...)
- 8
- Third chamber (8a, ...)
- 9
- Third channel (9a, ...)
- 10
- Outlet port (10a, ...)
- 11
- Sealing wall
- 12
- Interruption
- 13
- Inlet port (13a, ...)
- 14
- Opening (14a, 14b, ...)
- 15
- Junction (15a, ...)
- 16
- Inlet channel (16a, ...)
- 17
- Recess for engagement of gripper
[0097] While the foregoing invention has been described in some detail for purposes of clarity
and understanding, it will be clear to one skilled in the art from a reading of this
disclosure that various changes in form and detail can be made without departing from
the true scope of the invention. For example, all the techniques and apparatus described
above can be used in various combinations. All publications, patents, patent applications,
and/or other documents cited in this application are incorporated by reference in
their entirety for all purposes to the same extent as if each individual publication,
patent, patent application, and/or other document were individually indicated to be
incorporated by reference for all purposes.
Examples
Example 1
Manufacture of a device according to the invention
[0098]
- a) A device as shown in FIG. 1a is prepared as follows:
A two-part mould reflecting the outer form of the body of the device is made from
polypropylene according to FIG. 1 and as described in "Handbuch Spritzgießen", Hanser-Verlag 2004, page 77, or "Werkstoff-Führer Kunststoffe", Hanser-Verlag 2001, 8. Aufl., pages 83-89. After solidification, the porous material is inserted. Then, a foil of polypropylene
(30 µm) and aluminium (110 µm, e.g., available from Alcan Packing Rorschach AG/Switzerland)
is welded by thermo welding at 300 °C to the polypropylene body. The outlet openings
are closed by silicon stoppers.
- b) A device as shown in FIG. 2a is prepared analogously, using a mould that in addition
has an additional groove to reflect the additional inlet channel.
Example 2
Performance of a process including sample preparation and PCR and detection in one
device of Example 1a
[0099] In the first step, the device manufactured as in Example 1a is loaded into the process
station of the instrument. This is done using grippers engaging into recesses on the
upper part of the device (see FIG. 1a, reference numerals 17). Then a quantitation
standard solution is added to each of the first chambers (see 3a, ...) using a head
bearing 8 pipette tips. The tips are discarded. Then a lysis solution containing proteinase
K is added to the chambers, again using parallel pipetting. Then, using fresh pipette
tips, an aliquot of 8 samples. The tips are usedfor mixing the reagents and the samples
by sipping and spitting the mixtures within each chamber to mix thoroughly. The mixtures
are then incubated for 10 min to lyse. Then binding solution is added. Binding solutions
are well known to the man skilled in the art.
[0100] Then pressure is applied on top of the solution, while vacuum is applied to the outlet
to transport the mixture through the outlet portion (see 4a, ...) of the first chamber
into the second chamber (see 5a, ...), filled with glass fleece. Any nucleic acids
get bound to the glass fleece surface, while the liquid is removed through the outlet
port (see 10a, ...). A hollow steel needle is docked onto the outlet port to withdraw
the liquid.
[0101] After removal of the pipette tip, washing liquid (800 µl) is pipetted into each first
chamber using a new set of new pipette tips, and was sucked through the second chamber,
thus removing impurities from the nucleic acids bound. This can be repeated several
times. After drying the glass fibre fleece with air and applying heat through the
heat transfer wall to the fleece aliquots of an elution buffer (containing all materials
for the PCR reaction) (50 µl) are added to the first channel and pipetted through
the fluidic units by the multipipetts while the vacuum on the outlet is switched off,
so that the eluted liquids remain in the third chambers.
[0102] The liquids in the third chambers are subjected to the following thermal cycles:
1st cycle:
50°C 120 sec UNG-Step
5 cycles:
+4°C/sec 95°C 15 sec Denaturation
-4°C/sec.59°C.50sec Anealing & fluoresecence measurement after 35 sec
45 cycles:
+4°C/sec 91°C 15 sec Denaturation
-4 °C/sec 52°C 50sec Anealing & fluoresecence measurement after 35 sec
[0103] Light of the wavelengths to impinge the probes is directed (dependent upon the absorption
of the fluorescent dye used) into each third chamber and fluorescence is measured
in the third chambers during irradiation during the annealing phase in each cycle.
Using the quantitation standard, the amount of nucleic acids in each sample is determined
according to standard calculations.
Example 3
Performance of a process including sample preparation and PCR and detection in one
device of Example 1b
[0104] In the first step, the device manufactured as in Example 1b is loaded into the process
station of the instrument. This is done using grippers engaging into recesses on the
upper part of the device (see FIG. 2a, reference numerals 17). Then a quantitation
standard solution is added to each of the first chambers (see 3a, ...) using a head
bearing 8 pipette tips. The tips are discarded. Then aliquots of a lysis solution
containing proteinase K are added to the first chambers, again using parallel pipetting.
Then, using fresh pipette tips, aliquots of 7 samples and one negative control is
added to the first chambers. The tips are used for mixing the solution by sipping
and spitting the mixtures within each chamber to mix thoroughly. The mixtures are
then incubated for 10 min at the temperature optimum of the enzyme, the heat for this
process is also transferred through the heat transfer wall to lyse, while the tips
remain in the first chambers. Now the binding buffer is added.
[0105] Then hydrostatic pressure is put on the solution to transport the mixtures through
the outlet portions (see 4a, ...) of the first chambers into the second chambers (see
5a, ...), filled with glass fleece. Any nucleic acids get bound to the glass surface,
while the liquids are removed through the outlet ports (see 10a, ...). Hollow steel
needles are docked onto the outlet ports to withdraw the liquid. The pipette tips
remain in the first chambers to close the openings.
[0106] Then a set of 8 point heaters is moved towards the device. The metal tops of the
heaters are pressed against the device in parts between the junction of the first
and the inlet channel and the first chamber, until the material surrounding the channels
have liquefied and molten to close the channels at that very location. Then the head
is removed from the device.
[0107] Aliquots of a washing liquid (400 µl) are pipetted into each forth channel through
the inlet port stoppers, using a set of hollow steel needles piercing the stoppers,
and was sucked through the second chambers, thus removing impurities from the nucleic
acids bound. Aliquots of an elution buffer (54 µl) are added through the inlet channel
by the pipetting device to the second chambers and so that the eluted liquids remain
in the third chambers.
[0108] The liquids in the third chambers are subjected to the following thermal cycles:
1st cycle:
50°C 120 sec UNG-Step
5 cycles:
+4°C/sec 95°C 15 sec Denaturation
-4°C/sec.59°C. 50 sec Anealing & fluoresecence measurement after 35 sec
45 cycles:
+4°C/sec 91°C 15 sec Denaturation
-4 °C/sec 52°C 50 sec Anealing & fluoresecence measurement after 35 sec
[0109] Light of the wavelengths to impinge the probes is directed (depending upon the absorption
of the fluorescent dye used) into each third chamber and fluorescence is measured
in the third chambers during irradiation during the annealing phase in each cycle.
Using the quantitation standard, the amount of nucleic acids in each sample is determined
according to standard calculations. The 8
th sample is used as a negative control.