Biochemical Analysis Apparatus and Rotary Valve
A first aspect of the present invention relates to the performance of biochemical analysis
of a sample using nanopores, for example sequencing of polynucleotides. Second to fourth
aspects of the present invention relate to a rotary valve for selecting between a large number of
ports.
Regarding the first aspect of the present invention, in recent years there has been
considerable development of biochemical analysis of a sample using nanopores. A nanopore is a
small hole in an electrically insulating layer and may be formed, for example, by protein pores or
channels introduced into an amphophilic membrane. The nanopores may allow a flow of ions to
travel across the amphiphilic membrane, modulated by the nanopore on the basis of an analyte
interaction, thus allowing the nanopore to provide a biochemical analysis. Various types of
nanopore and analysis apparatus for using them have been developed for a range of types of
biochemical analysis. One example of commercial interest is to use nanopores for sequencing of
polynucleotides such as DNA. One example of an analysis apparatus for performing biochemical
analysis of a sample using nanopore is disclosed in WO-2009/077734.
As such nanopores offer the potential of a platform for biochemical analysis on a
commercial scale. However, in such a context it would be desirable to provide efficient handling
of samples in the apparatus in order to maximise throughput and minimise costs of performing
the biochemical analysis.
Regarding the second to fifth aspects of the present invention, it would be advantageous
to provide a valve that is capable of selecting between a large number of ports in a wide range of
applications. One example of such an application is the handling of fluids in an apparatus for
performing a biochemical analysis.
One type of valve that allows selection between ports is a rotary valve comprising a stator
and a rotor rotatably mounted on the stator. In a known type of rotary valve, the stator defines a
plurality of first ports and a second port and the rotor has plural passages that are configured to
connect different first ports to the second port as rotor rotates. This type of rotary valve provides
various advantages particularly, but not exclusively for small volumes of fluid, for example
allowing the valve to provide selection between the first ports with a simple construction and
reliable operation.
Having regard to the third aspect of the invention, simplification of the overall
construction of the rotary valve may be achieved by arranging the first ports of the stator in an
annular surface that extends around the rotational axis of the rotor, facing the rotational axis.
This facilitates the inclusion of relatively high numbers of first ports. However, it can be difficult
to provide adequate sealing of the first ports between the stator and the rotor. This difficulty
increases as the number of ports increases, and hence the overall size of the valve increases.
It would therefore be desirable to provide a valve in which these problems are alleviated.
Having regard to the third and fourth aspects of the invention, as number of first ports
increases, the complexity of the network of passages in the rotor correspondingly increases. This
results in increase of the size of the rotor and hence the overall size of the valve. Such an
increase in size can be disadvantageous in itself in many applications where it is desired to
minimise the size of the apparatus in which the valve is incorporated. Furthermore an increase in
size can make it more difficult to provide sealing between the rotor and the stator.
Having regard to the fifth aspect of the invention, constructing valves for use in fluidics
systems, such as systems designed within a plate, can be difficult in terms of providing a valve of
simple construction that can provide and connect the desired flow paths
According to a first aspect of the present invention, there is provided an analysis
apparatus for performing biochemical analysis of a sample using nanopores, the analysis
apparatus comprising:
a sensor device that is capable of supporting plural nanopores and being operable to
perform biochemical analysis of a sample using the nanopores;
at least one reservoir for holding material for performing the biochemical analysis;
a fluidics system configured to controllably supply material from the at least one
reservoir to the sensor device; and
a plurality of containers for receiving respective samples, the fluidics system being
configured to supply the samples selectively from the containers to the sensor device.
The analysis apparatus has a construction that encapsulates the components and material
necessary to perform the biochemical analysis. In particular, the analysis apparatus incorporates
the sensor device operable to perform biochemical analysis of a sample using the nanopores with
at least one reservoir for holding the necessary material and a fluidics system that may supply the
material to the sensor device, under suitable control. This allows for efficient performance of the
biochemical analysis.
Furthermore, by providing a plurality of containers for receiving respective samples, the
analysis apparatus is configured to handle multiple samples that may be introduced into the
respective containers. As the fluidics system is configured to supply the samples selectively from
the containers to the sensor device, the analysis apparatus provides for sequential processing and
easy manipulation of the multiple samples. This allows the efficient handling of multiple
samples, allowing work flows to be improved. This in turn allows a reduction of the overall cost
of performing the biochemical analysis per sample.
The fluidics system may be configured to supply the samples selectively from the
containers to the sensor device by including a rotary valve according to any of the second to
fourth aspects of the invention as discussed below. In this case, any features of the valves in
accordance with the second to fourth aspects of the invention may be combined with any features
of the first aspect of the invention in any combination.
Advantageously, the analysis apparatus comprises:
a body on which the analysis apparatus, the at least one reservoir and the fluidics system
are mounted, and
a container element that is separate from the body and attachable thereto, the plurality of
containers being formed in the container element.
As the container element is a separate element, the introduction of the samples into the
containers may be performed before attachment to the body of the analysis apparatus. This
facilitates the filling of the containers, improving the efficiency of the filling operation.
Furthermore, as the container element is a separate element, it may be a disposable
element, allowing convenient re-use of the analysis apparatus by filling and attaching a new
container element.
For example, the container element may be a well plate, the containers being wells
formed in the well plate. In this case, the well plate may be filled using existing plate-based
parallel manipulation techniques that are intrinsically efficient.
Optionally, the plurality of containers comprises 24 containers or more. When
processing a large number of samples, such as 24 or over, it becomes less practical to provide an
individual system for processing each sample, and so an integrated fluidics approach becomes
preferable. Typically the plurality of containers is provided as a 96 well plate.
Optionally, the analysis apparatus, further comprises a controller configured to measure a
performance target of the biochemical analysis and control the analysis to meet the performance
target. The controller can be configured to control the analysis to utilise a selection of the
plurality of containers in sequence, the selection of the plurality containers containing the same
sample, until the performance target is met. By providing the analysis apparatus with multiple
versions of the same sample, the analysis can be controlled to use several samples of the same
type, if required, or to ignore wells containing a sample for which an analysis has been
successfully performed. As a result, if a particular analysis becomes unexpectedly lengthy, the
analysis can continue for the required length of time, but without needlessly processing every
sample provided.
Optionally, the analysis apparatus is a cartridge for cooperation with another device. As
such, the analysis apparatus can be disposable, or at least replaceable.
According to a second aspect of the present invention, there is provided a rotary valve
comprising:
a stator defining a plurality of first ports in an annular surface that extends around a
rotational axis, facing the rotational axis, and a second port; and
a rotor mounted on the stator for rotation about the rotational axis inside a liner arranged
between the annular surface of the stator and an annular surface of the rotor that faces the
annular surface of the stator, the liner being made of a material having a greater compliance than
the rotor and than the stator,
the rotor having a passage extending from a first port defined in the annular surface of the
stator and being in communication with the second port of the stator,
the liner having at least one channel extending through the liner between the annular
surface of the stator and the annular surface of the rotor and capable of providing communication
between the first port of the rotor and any one of the plurality of first ports of the stator,
depending on the rotational position of the rotor.
The rotary valve incorporates the first ports of the stator in an annular surface that
extends around the rotational axis of the rotor, facing the rotational axis, thereby simplifying the
overall construction and facilitating the inclusion of relatively high numbers of ports. Sealing is
achieved by providing a liner arranged between the annular surface of the stator and a facing,
annular surface of the rotor. At least one channel extending through the liner between the annular
surface of the stator and the annular surface of the rotor provides communication between the
first port of the rotor and any one of the plurality of first ports of the stator, depending on the
rotational position of the rotor. The liner is made of a material selected to have a greater
compliance than the rotor and than the stator. This makes it easier to provide the required degree
of sealing. Without the liner, the sealing is directly between the facing annular surfaces of the
rotor and stator, in which case there is difficulty in selecting materials that provide sufficient
sealing whilst maintaining the other desired mechanical properties for operation of the valve, for
example sufficient rigidity and sufficiently low resistance. The difficulty in sealing directly
between the facing annular surfaces of the rotor and stator increases as the number of ports
increases, so the present invention facilitates the formation of valves with relatively high
numbers of ports.
The rotary valve may be advantageously applied to the handling of small volumes of
fluid, in which the difficulty in sealing is worse, for example in which the ports of the stator and
the rotor, the passage of the rotor and the at least one channel of the liner have cross-sectional
areas of no more than 10mm2, preferably no more than 1mm2. In one advantageous use of the
rotary valve, the stator is on a body that is arranged to allow attachment of a well plate
comprising a plurality of wells corresponding to the plurality of first ports, the body defining
channels connecting the wells to the corresponding first ports.
In one advantageous construction, the passage extends to a second port defined in the
rotor that is positioned on the rotational axis and is in communication with the second port of the
stator. In this construction, the same passage is always connected to the second port and provides
communication with any one of the first ports selected by the rotational position of the rotor.
Such selective connection of any one of the first ports to the second port is achieved using a very
simple configuration that is relatively compact and is scalable to any number of first ports. The
first ports need merely to be spaced around the rotational axis, so increasing the number of
second ports only increases the size marginally. In approximate terms, the circumference and
hence diameter of the rotary valve scales linearly with the number of first ports. The size is much
reduced as compared to providing the rotor with respective passages for connecting each first
port to the second port.
The passage in the rotor may communicate with a passage in the liner that is in
communication with the second port of the stator. In this configuration, the liner is also used to
seal the connection between the rotor and the second port of the stator.
According to a third aspect of the present invention, there is provided a rotary valve
comprising:
a stator; and
a rotor rotatably mounted on the stator for rotation about a rotational axis;
the stator defining a plurality of first ports arranged around the rotational axis and a
second port,
the rotor defining a first port capable of communication with any one of the first ports of
the stator depending on the rotational position of the rotor, a second port positioned on the
rotational axis and in communication with the second port of the stator, and a passage extending
between the first port and the second port.
This rotary valve achieves selective connection of any one of the first ports to the second
port using a very simple configuration. Selection of the first ports is achieved by rotation of the
rotor, but, as the second port is positioned on the rotational axis, the second port of the stator and
the second port of the rotor remain in communication as the rotor rotates. This provides a simple
configuration for the valve and allows for sealing between the second port of the stator and the
second port of the rotor. This configuration is relatively compact and is scalable to any number
of first ports. The first ports need merely to be spaced around the rotational axis, so increasing
the number of second ports only increases the size marginally. In approximate terms, the
circumference and hence diameter of the rotary valve scales linearly with the number of first
ports. The size is much reduced as compared to providing the rotor with respective passages for
connecting each first port to the second port.
Such a rotary valve may be advantageously applied to the handling of small volumes of
fluid, for example in which the first ports, the passage, the collection chamber and the second
port have cross-sectional areas of no more than 10mm2, preferably no more than 1mm2. In one
advantageous use of the rotary valve, the stator is on a body that is arranged to allow attachment
of a well plate comprising a plurality of wells corresponding to the plurality of first ports, the
body defining channels connecting the wells to the corresponding first ports.According to a
fourth aspect of the present invention, there is provided a rotary valve comprising:
a stator; and
a rotor rotatably mounted on the stator for rotation about a rotational axis;
the stator defining a plurality of first ports arranged around the rotational axis facing the
rotor;
the valve comprising a collection chamber extending in at least part of an annulus around
the axis of rotation of the valve member,
the stator defining a second port in communication with the collection chamber, and
the rotor providing a passage extending between the collection chamber with which the
passage is in communication and a position where the passage is capable of communication with
any one of the plurality of first ports depending on the rotational position of the rotor.
The rotary valve includes a collection chamber that extends in at least part of an annulus
around the axis of rotation, and the first ports are also arranged around the rotational axis. As a
result of this configuration of the collection chamber and the first ports, it is possible for the rotor
to be arranged with a passage that can connect the collection chamber to any selected one of the
plurality of first ports depending on the rotational position of the rotor. As the collection
chamber is in communication with the second port, this results in the selected first port also
being connected to the second port.
Such selective connection of any one of the first ports to the second port is achieved
using a very simple configuration of the collection chamber and the passage in the rotor. This
configuration is relatively compact and is scalable to any number of first ports. The first ports
need merely to be spaced around the rotational axis, so increasing the number of second ports
only increases the size marginally. In approximate terms, the circumference and hence diameter
of the rotary valve scales linearly with the number of first ports. The size is much reduced as
compared to providing the rotor with respective passages for connecting each first port to the
second port.
Such a rotary valve may be advantageously applied to the handling of small volumes of
fluid, for example in which the first ports, the passage, the collection chamber and the second
port have cross-sectional areas of no more than 10mm2 , preferably no more than 1mm2 . In one
advantageous use of the rotary valve, the stator is on a body that is arranged to allow attachment
of a well plate comprising a plurality of wells corresponding to the plurality of first ports, the
body defining channels connecting the wells to the corresponding first ports.
In one advantageous construction, the rotor and the stator have interfacing contact
surfaces that extend transversely, preferably perpendicular, to the rotational axis, the plurality of
first ports and the second port opening in the contact surface of the stator. With this construction,
sealing of the interfacing contact surfaces may be facilitated by constructing the valve to apply a
high load between the rotor and stator along the rotational axis. This makes sealing easier than if
the ports are provided in interfacing contact surfaces extending parallel to the rotational axis.
For example, the loading may be achieved by the valve further comprising a biasing
arrangement arranged to bias the rotor against the stator, for example including a resilient biasing
element engaging the rotor.
According to a fifth aspect of the invention, there is provided a rotary valve comprising: a
stator defining a plurality of first ports, and a second port, a rotor mounted on the stator for
rotation about a rotational axis, the valve comprising a passage being in communication with the
second port of the stator and extending to a position for communicating with any one of the
plurality of first ports of the stator individually, depending upon the rotational position of the
rotor.
According to this arrangement, it is possible for the rotor to simply connect the passage
of the valve to the desired first port of the stator. As a result, the stator can be formed directly in
the plate of a fluidics system, and the rotor can provide the means for connecting the desired
ports formed in the plate.
Optionally, the first ports, the passage, and the second port have cross-sectional areas of
no more than 10mm2, preferably no more than 1mm2.
Optionally the rotary valve can further comprise a liner arranged between the rotor and
the stator, wherein the liner is fixed relative to the rotor. The liner can optionally be a made of a
material more compliant than both the stator or rotor. The provision of the liner in fixed relation
to the rotor allows for an improved seal between the stator and rotor.
In the hereinafter described embodiments, the second and third aspects of the present
invention are implemented in combination in a rotary valve. However, this is not essential. A
rotary valve in accordance with the second aspect of the invention may be implemented in
combination with the fourth aspect of the invention, with the second port of the rotor
communicating with the collection chamber.
Embodiments of the present invention will now be described by way of non-limitative
example with reference to the accompanying drawings, in which:
Fig. 1 is a schematic view of a biochemical analysis instrument;
Fig. 2 is a perspective view of a module of the instrument;
Fig. 3 is a perspective view from above of a cartridge that is replaceable in the module;
Fig. 4 is a cross-sectional view of a part of a sensor device of the cartridge;
Figs. 5 and 6 are top and bottom perspective views of the sensor device mounted on a
PCB;
Fig. 7 is a perspective view of the module;
Fig. 8 is a schematic diagram of the electrical circuit of a module;
Fig. 9 is a schematic diagram of the control unit;
Fig. 10 is a diagram of a detection channel;
Figs. 11 and 1 are perspective views from below of the cartridge, showing a well plate,
respectively, attached and separated;
Fig. 13 is a sectioned perspective view of part of the well plate;
Figs. 14 and 15 are perspective views from above and below respectively of a valve
assembly incorporating a valve of a first construction;
Fig. 16 is a cross-sectional view through the valve assembly of Fig. 14 taken along line
XVI-XVI;
Fig. 17 is a partial plan view from above of a body of the valve assembly around a stator
of the valve of the first construction;
Fig. 18 is a plan view from below of a rotor of the valve of the first construction;
Fig. 19 is a partial cross-sectional view of the body of the valve assembly and a well of
the well plate;
Fig. 20 is a plan view from below of a second plate of the valve assembly;
Fig. 2 1 is a perspective view of the valve assembly including a motor;
Fig. 22 is an exploded perspective view from above of a valve assembly incorporating a
valve of a second construction;
Fig. 23 is perspective view from above of the valve assembly of Fig. 22 in an assembled
state; and
Fig. 24 is a cross-sectional view through the valve assembly of Fig. 22 taken along line
XXIV-XX1V.
There will first be described an instrument for performing biochemical analysis using
nanopores in the form of protein pores supported in an amphiphilic membrane, but this is not
limitative of the invention.
The instrument 1 is formed a plurality of modules 2 that are each connected to a data
network 3. In this example, the network 3 is formed as a conventional local area network by each
module 2 being connected by a cable 4 to a network switch 5. In general, the modules 2 may be
connected to any type of data network, including wireless networks, wide-area networks and the
internet
Attached to the network 3, there may also be a storage device 6 of any type, for example
a NAS, and an external computer 7 that is used to address the modules 2 and may be a
conventional computer having an HTTP browser.
Due to the networked configuration of the instrument 1, any number of modules 2 may
be provided in a given location, depending on the local requirements, for example from a small
number of modules 2 or even a single module 2 in a small-scale research facility to a large bank
of modules 2 in a commercial sequencing centre. Similarly the modules 2 need not be physically
close and so the instrument 1 may be formed from modules 2 that are distributed in different
locations, even different countries.
An individual module 2 will now be described.
As shown in Fig. 2, the module 2 has a cartridge 10 that is replaceable in the housing 11
of the module 2. The cartridge 10 forms an analysis apparatus for performing a biochemical
analysis as will now be described. The cartridge 1 has a construction shown in Fig. 3.
The cartridge 0 comprises a body 37 formed for example of moulded plastic. The body
37 of the cartridge 10 mounts a sensor device 14 that is an apparatus as described in detail in
WO-2009/077734 which is incorporated herein by reference. Without limitation to the generality
of the teaching therein, the sensor device 14 has a construction as shown in cross-section in Fig.
4 comprising a body 20 in which there is formed a plurality of wells 2 1 each being a recess
having a well electrode 22 arranged therein. A large number of wells 2 1 is provided to optimise
the data collection rate. In general, there may be any number of wells 21, although only a few of
the wells 2 1 are shown in Fig. 4. In one example, the number of wells is 256 or 1024, but there
could be one, two or three orders of magnitude more. The body 20 is covered by a cover 23 that
extends over the body 20 and is hollow to define a chamber 24 into which each of the wells 2 1
opens. A common electrode 25 is disposed within the chamber 23.
The sensor device 14 is prepared to form an amphiphilic membrane 26, such as a lipid
bilayer, across each well 21 and to insert nanopores that are protein pores into the amphiphilic
membrane 26. This preparation is achieved using the techniques and materials described in detail
in WO-2009/077734, but may be summarised as follows. Aqueous solution is introduced into the
chamber 24 to form the amphiphilic membrane 26 across each well 21 separating aqueous
solution in the well 2 1 from the remaining volume of aqueous solution in the chamber 24.
Protein pores are provided into the aqueous solution, for example by being introduced into the
aqueous solution before or after that is introduced into the chamber 24 or by being deposited on
an internal surface of the chamber 24. The protein pores spontaneously insert from the aqueous
solution into the amphiphilic membranes 26.
A protein pore is an example of a nanopore and may be used to perform a biochemical
analysis, as follows. In respect of any given well 21, when an amphiphilic membrane 26 has
been formed and a protein pore is inserted therein, the well 1 is capable of being used as a
sensor element to sense interactions between molecular entities and the protein pore that are
stochastic physical events because the output electrical signal across the amphiphilic membrane
26 is dependent on those interactions in that the interactions cause characteristic changes therein.
For example, there will typically be interactions between the protein pore and a particular
molecular entity (analyte) that modulate the flow of ions through the pore, creating a
characteristic change in current flow through the pore. The molecular entity may be a molecule
or part of a molecule, for example a DNA base. Thus the interaction appears as a characteristic
event in the electrical signal across the protein pore in each amphiphilic membrane 26.
The electrical signals may be detected as the signals between the well electrodes 22 and
the common electrode 25, and may subsequently be analysed to produce output data representing
the results of the biochemical analysis. Separate electrical signals are derived from the protein
pores in the amphiphilic membranes 26 in different wells 21, each resulting in a different
channel of the output data.
A wide range of types of biochemical analysis may be performed. One such biochemical
analysis is sequencing of polynucleotides. In this case, the electrical signal is modulated
differently for each different base, allowing discrimination thereof.
The body 37 of the cartridge 10 encapsulates the components and material necessary to
perform the biochemical analysis and is capable of preparing the sensor device 14 automatically.
For this purpose, the cartridge 10 mounts reservoirs 30 containing sufficient volumes the
necessary materials, such as buffer solutions, lipids, protein pores (in solution), pre-treatment (if
required), and sample, such that many 'refreshes' of the analysis apparatus are possible. Thus the
cartridge 10 is fully self-contained in that all reagents and other materials required for the
biochemical analysis are present and may be used for sample preparation. The cartridge 10
mounts a waste reservoir 35 for disposal of waste products from the sensor device 14.
The body 37 of the cartridge 10 also mounts a fluidics system 3 1 for supplying the fluids
from the reservoirs 30 to the sensor device 14. The fluidics system 31 includes supply channels
32 and inlet pumps 33 for pumping fluids from the reservoirs 30 to the sensor device 14. The
fluidics system 31 also includes an output pump 34 for pumping fluids out of the sensor device
14 through an outlet channel 36 connected to the waste reservoir 35 for disposal of the fluids.
The pumps 33 and 34 may be syringe pumps depending on volume and flow rate required (for
example as supplied by Hamilton Company, Via Crusch 8, Bonaduz, GR, Switzerland CH-
7402).
The fluidics system also includes a selector valve 45 disposed in the supply channels 32
between the inlet pumps 33 connected to the reservoirs 30 and the output pump 34. The selector
valve 45 selectively connects the sensor device 14 to the reservoirs 30 or to the waste reservoir
35. The waste reservoir 35 is open to atmosphere.
One of the reservoirs 30 holds the lipid and the fluidics system 3 1 supplies the lipid to the
sensor device 14 in the same manner as the other materials. As an alternative for supplying the
lipid, the supply channels 32 of the fluidics system 3 1 may pass into the sensor device 14
through a lipid assembly holding lipid so that the fluid flowing into the sensor device 14 acquires
lipid and introduces it into the sensor device 14.
The pumps 33 and 34 may thus be operated to control the flow of fluids to prepare the
sensor device 14 to form an amphiphilic membrane 26 across each well 2 1 and to insert
nanopores that are protein pores into the amphiphilic membrane 26, as discussed above.
The cartridge 10 is capable of receiving a plurality of samples as follows. As shown in
Fig. 11, the body 37 of the cartridge 10 is arranged to allow attachment of a well plate 100. In
particular, the body 37 has a pair of clips 101 protruding from its underside and to which a well
plate 100 may by attached by pressing the well plate 100 against the clips 101 in the direction of
the arrows in Fig. 12.
As shown in Fig. 13, the well plate 100 is of standard construction and forms a plurality
of wells 102 opening a flat upper surface 103 of the well plate 100. In this example the well plate
100 has 96 wells 102, but in general may have any number of wells 102. Preferably, the plate has
at least 24 wells 102, more preferably at least 48 wells 102. The plate can have at least 96 wells,
at least 384 wells or even at least 1536 wells 102. The wells 102 are used as containers for
receiving respective samples. In use, the samples are introduced into the respective wells 2
before attachment of the well plate 102 to the cartridge 1 and before loading of the cartridge 10
into the module 2. The well plate 102 may be filled with samples using known plate-based
parallel manipulation techniques that are intrinsically efficient. As the well plate 100 is a
separate element from the body 37 of the cartridge 10 it is easily filled prior to attachment
facilitates the filling of the wells 102. More generally, similar advantages could be achieved by
replacing the well plate 100 by any other type of container element comprising a plurality of
containers that might be wells or closed containers.
After introduction of the samples, the well plate 100 is attached to the cartridge 0 with
the flat upper surface 103 against the body 37, to encapsulate the well plate 100 into the cartridge
10. Subsequently, the cartridge 0 is loaded into the module 2.
The fluidics system 3 1 is configured to supply the samples selectively from the wells 102
to the sensor device 14, using a valve 110 that is a rotary valve. Two possible constructions for
the valve 110 will now be described.
In the first possible construction in accordance with the fourth aspect of the invention, the
valve 110 is formed in a valve assembly 111 illustrated in Figs. 1 to 2 1 that is incorporated into
the body 37 of the cartridge 10.
The valve 110 comprises a stator 112 and a rotor 113. The stator 112 is provided on a
body 120 formed by a first plate 1 , a second plate 122 and a third plate 123 that are fixed
together by interfacing contact surfaces 124 between the first and second plates 1 1 and 122 and
by interfacing contact surfaces 125 between the first and second plates 122 and 123.
The rotor 113 is rotatably mounted on the stator 12 for rotation about a rotational axis R.
A bearing for the rotational mounting is provided by the rotor 1 3 comprising a bearing stub 114
that is mounted in a bearing recess 15 formed in the stator 112. In particular, the bearing stub
114 is has a length chosen to provide a clearance between the end of the bearing stub 115 and the
first sheet 121. Around the bearing recess 115, the second sheet 122 has an annular boss 126 that
protrudes towards the first sheet 121 and the stator 13, the second sheet 3 having a circular
aperture 127 in which the annular boss 126 fits.
In addition the bearing for the rotational mounting is provided by the rotor 113
comprising a disc 116 having a cylindrical outer surface 117 that is mounted in an annular wall
1 8 formed in the stator 112 and protruding therefrom, in particular from the third plate 123
outside the circular aperture 127. Alternatively, there may be a clearance gap between the disc
116 and the annular wall 118.
The stator 112 and rotor 13 have interfacing contact surfaces 130 that are annular and
extend perpendicular to the rotational axis R, being provided as follows. The contact surface 130
of the rotor 113 is formed by a lower surface of the disc 116 that extends perpendicular to the
rotational axis R both overlapping the annular boss 126 of the second plate 122 and overlapping
the third plate 123 outside the aperture 127. Thus the contact surface 130 of the stator 112 is
formed by the adjacent parts of the upper surface of the annular boss 126 of the second plate 122
and the upper surface of the third plate 123, which are flush with each other.
Sealing of the interfacing contact surfaces 130 of the stator 112 and the rotor 113 is
facilitated by applying a load between the stator 112 and the rotor 113 along the rotational axis
R. This is achieved by a biasing arrangement arranged as follows to bias the rotor 113 against the
stator 112. A clamping ring 13 is attached to the stator 113, in particular screwed to the annular
wall 118. A disc spring 132 is disposed between and engages the clamping ring 131 and the rotor
112. The disc spring 132 provides resilient biasing between the stator 1 2 and the rotor 113,
although could be replaced by another type of resilient biasing element.
The contact surface 130 of the stator 112 is arranged as shown in Fig. 17 which is a plan
view of the stator 112 without the clamping ring 131. In particular, a plurality of inlet ports 133
are formed in the contact surface 130 of the stator 112 arranged in a circle around the rotational
axis R. In this example, the valve 110 has 96 inlet ports 233 but in general the valve 110 may
have any number of ports 233. Preferably, the valve has at least 24 inlet ports 233 and more
preferably at least 48 inlet ports 233. . The valve can have at least 96 ports, at least 384 ports or
even at least 1536 ports 233. The inlet ports 133 are evenly spaced, except for a gap at one
position, lowermost in Fig. 17. The inlet ports 3 are formed in particular in the upper surface
of the annular boss 126 of the second plate 122, facing the contact surface 130 of the rotor 113.
Also, a collection chamber 134 is formed in the contact surface 130 of the stator 112. The
collection chamber 134 is formed as a groove in the upper surface of the third plate 122, facing
the contact surface 130 of the rotor 113. The collection chamber 134 extends outside the inlet
ports 133 in a circular annulus around the rotational axis R aligned angularly with the inlet ports
133, that is with a gap aligned angularly around the rotational axis R with the gap in the inlet
ports 133.
The stator 112 further includes an outlet port 135 in communication with the collection
chamber 134 by being formed in the lower surface of the collection chamber 134.
The rotor 113 is provided with a passage 136 formed as a groove in the contact surface
130 of the rotor 113. The passage 136 extends radially from the position of the inlet ports 133 to
the position of the collection chamber 135. Thus, the passage 136 is capable of communication
with any one of the inlet ports 133 depending on the rotational position of the rotor 113. Rotation
of the rotor 113 allows different inlet ports 133 to be selected. As the collection chamber 134 is
aligned angularly with the inlet ports 133, at all rotational positions where the passage 136
communicates with an inlet port 133, the passage 136 also communicates with the collection
chamber 134, thereby connecting the selected inlet port 133 to the outlet port 135. Therefore,
rotation of the rotor 136 selectively connects individual inlet ports 133 to the outlet port 135.
When the rotor 133 is aligned with the gap in the inlet ports 133 and the gap in the
collection chamber 134, the passage 136 is closed against the contact surface 130 of the stator
112, thereby closing the valve 110. However, as an alternative, the inlet ports 133 can be brought
together to omit the gap so that inlet ports are arranged in a complete annulus and the valve 10
cannot be closed.
As an alternative to forming the collection chamber 134 in the contact surface 130 of the
stator 112, a similar operation could be achieved by alternatively forming the collection chamber
134 as a groove in the contact surface 130 of the rotor 113 opening into the passage 136.
To provide positioning of the rotor 112, the contact surface 130 of the stator 112 has a
circular array of pits 137 at the same pitch as the inlet ports 133, and the contact surface 130 of
the rotor 113 has pips 138 that fit into the pits 137. The pips 138 may be pushed out of the pits
137 on rotation of the rotor 112 but are aligned to hold the rotational position of the rotor 2 in
stepped rotational positions that each locate the passage 136 in communication with each a
respective inlet port 133, or in one of the stepped rotational positions to locate the passage 136
over the gap in the inlet ports 133 and the gap in the collection chamber 134.
The size of the valve 10 is minimised by arranging the inlet ports 133 as close together
as possible, but the same operation could be achieved by increasing the size of the gap in the
inlet ports 133 so that the inlet ports 133 extend around a smaller part of the annulus. In this case,
the collection chamber 134 can be correspondingly reduced in length to extend in a shorter part
of the annulus.
The body 120 defines channels connecting the wells 102 of the well plate 100 to the inlet
ports 133 as follows.
The first plate 121 is disposed on the underside of the cartridge 10 at the position where
the well plate 100 is attached and has an array of nozzles 140 protruding outwardly and having
the same spacing as the wells 102 of the well plate 100 to align therewith. As a result, when the
plate 100 is attached to the cartridge 10, each nozzle 140 protrudes into a respective well, as
shown in Fig 19. Each nozzle 140 comprises a through hole 141 that extends through the nozzle
140 and through the first plate 121 to the contact surface 124 of the first plate 121 to form part of
a channel in respect of the well 102.
The nozzles 140 extend into the wells 102 by a sufficient distance that the end of the
nozzle 140 is submerged below the surface of a sample 142 in the well 102. In this manner, the
sample 142 effectively seals the nozzle 140. This avoids the need for a hermetic seal between the
well plate 100 and the first plate 121 .
The contact surface 124 of the second plate 122 is formed with a set of grooves 143 that
form part of the channel in respect of each well 102. Each groove 143 communicates at one end
with the through hole 141 that extends through the nozzle 140 and through the first plate 121. As
shown in Fig. 20, the grooves 143 extend from the nozzles 0 to the stator 112, in particular to
the annular boss 126 on the opposite side of the second plate 122 from the outlet ports 133. The
remainder of the channels are formed by through holes 144 extending through the boss 126 of
the second plate 122 from a respective groove 144 in the contact surface 124 of the second plate
122 to a respective inlet port 133.
The body 1 0 also defines a channel connecting to the outlet port 135 as follows. The
third plate 123 has a through hole 145, shown in dotted outline in Fig. 16, that extends from the
outlet port 135 through the third plate 123 to the contact surface 125 of the third plate 123,
forming part of the channel. The remainder of the channel is formed by a groove 146 in the
contact surface 125 of the third plate 123 extending away from the through hole 145. As shown
in Fig. 16, the groove 146 extends to a dosing pump 147 operable to pump a sample from a well
102 selected by the rotational position of the valve 110 through the valve 110 to the sensor
device 14.
The first, second and third plates 121-123 may be formed from any suitable material that
provides sealing for channels defined between the contact surfaces 24 and 5. Suitable
materials include PMMA (poly(methyl methacrylate)), PC (polycarbonate) or COC (cyclic olefin
co-polymer). The first, second and third plates 121-123 may be sealed by any suitable technique
for example ultrasonic welding, laser welding or bonding. PMMA is particularly effective due to
the ability to use PMMA diffusion bonds. The first, second and third plates 121-123 may be
injection moulded.
Similarly, the rotor 113 may be formed from any suitable material that provides sealing
and sufficiently low friction for rotation. One suitable material is PTFE (polytetrafluoroethylene)
that may be machined with a section made of an elastomer (e.g. silicone) to provide
compression. PTFE can lower the torque required for rotation and has good sealing properties.
The elastomer allows the rotor 112 to be clamped but still rotate. Alternatively the rotor 113 can
be made from a material that can be injection moulded, for example, FEP (fluorinated ethylene
propylene) or UHMWPE (ultra-high-molecular-weight polyethylene).
In the second possible construction in accordance with the second and third aspects of the
invention, the valve 110 is formed in a valve assembly 2 11 illustrated in Figs. 22 to 24 that is
incorporated into the body 37 of the cartridge 10.
The valve 110 comprises a stator 212 and a rotor 213, the stator 212 being mounted into
the body 37 that may have a construction comprising plural plates fixed together with channels
defined therebetween, similar to the construction of the body 120 in the first possible
construction of the valve 110 described above.
The rotor 213 has an outer, annular surface 214 and is mounted on the stator 212 in a
recess 216 in the stator 212 having an inner, annular surface 215 that faces the annular surface
214 of the rotor 213. The rotor 213 is mounted inside a liner 215 also arranged inside the recess
14 between the annular surface 214 of the rotor 213 and the annular surface 215 of the stator
212.
The liner 217 comprises an annular wall 22 1 disposed between the annular surface 214 of
the rotor 213 and the annular surface 215 of the stator 212. The annular wall 221 has a rim 218
that protrudes outwardly and sits in a widened opening 219 of the recess 21 . The rim 2 18 has a
radial protrusion 220 fitting in a notch 222 in the widened opening 219 that prevents rotation of
the liner 217 relative to the stator 212, and the liner 217 is fixed to the stator 212 for example by
adhesive. Thus the liner 2 17 has a fixed position relative to the stator 2 2.
The liner has a base 224 covering an end surface 225 of the rotor 213 that extends
transversely to the rotational axis R. The rotor 213 has a boss 226 formed on its end surface 225
and protruding into a recess 227 formed in the base 224 of the liner 217.
The rotor 213 is capable of rotation about a rotational axis R relative to the liner 217 and
hence also relative the stator 212. The annular surface 214 of the rotor 213 and the annular
surface 215 of the stator 212 are both parallel to the rotational axis R, although either or both of
annular surface 214 of the rotor 213 and the annular surface 215 of the stator 212 could
alternatively extend at an acute angle to the rotational axis R. The rotor 213 also has an annular
lip 223 protruding from the annular surface 214 that engages the liner 217 and retains the rotor
2 13 in the liner 217 along the rotational axis R.
The stator 212 defines a plurality of inlet ports 233 in the annular surface 214 of the
stator 212 around the rotational axis R. In this example the valve 1 0 has 96 inlet ports 233 but
in general the valve 110 may have any number of ports 233. The inlet ports 233 are evenly
spaced, except for a gap at one position. The size of the valve 110 is minimised by arranging the
inlet ports 233 as close together as possible, but the same operation could be achieved by
increasing the size of the gap in the inlet ports 233 so that the inlet ports 233 extend around a
smaller part of the annulus. The inlet ports 233 are formed in the end of channels 234 that extend
through the stator 212 to the outer surface 232 of the stator 212 where the channels 234
communicate with channels formed in the body 37 that connect the wells 102 of the well plate
100 to respective inlet ports 233.The channels formed in the body 37 provide this connection to
the wells 102 through an array of nozzles 140 arranged as shown in Fig. 1 and described above.
The stator 212 further defines an outlet port 235 in the annular surface 214 of the stator
21 separated from the inlet ports 233 along the rotational axis R, level with the base 224 of the
liner 217. The outlet port 135 is formed in the end of a channel 2 1 that extends through the
stator 212 to the outer surface 232 of the stator 212 where the channel 231 communicates with a
channel formed in the body 37.
The rotor 213 defines a passage 236 that extends from a first port 237 to a second port
238. The first port 237 is formed in the annular surface 214 of the rotor 213 and is axially
aligned with the inlet ports 233 of the stator 212. The second port 238 is positioned on the
rotational axis R, being formed in particular in the boss 226. The passage 236 has a radial portion
239 extending from the first port 237 to the rotational axis R and an axial portion 240 extending
along the rotational axis R to the second port 238.
The liner 21 provides communication between the inlet ports 233 of the stator 212 and
the first port 237 of the rotor 213, as follows. The liner 217 has a plurality of inlet channels 241
that extending through the annular wall 221 between the annular surface 214 of the rotor 213 and
the annular surface 215 of the stator 212. Each inlet channel 241 is aligned with, and
communicates with, an inlet port 233 of the stator 212. thus, the inlet ports 233 are evenly
spaced, except for a gap at one position. Depending on the rotational position of the rotor 213,
the first port 237 of the rotor 213 may be aligned with, and communicate with, any one of the
inlet channels 241, or may be aligned with the gap to close the valve 110.
The liner 217 also provides communication between the second port 238 of the rotor 213
and the outlet ports 235 of the stator 212, as follows. The liner 217 defines a passage 242 in its
base 224 that extends from the second port 238 of the rotor 213 to the outlet port 235 of the
stator 212. The passage 242 has an axial portion 244 extending along the rotational axis R from
the second port 238 in the recess 227 and a radial portion 243 extending to the outlet port 238.
As a result, the passage 236 in the rotor 213 is in communication with outlet port 235 of the
stator 212 through the passage 242 in the liner 217.
Thus, the valve 110 is capable of providing communication between any one of the inlet
ports 233 and the outlet port 236 depending on the rotational position of the rotor 113. Rotation
of the rotor 213 allows individual inlet ports 233 to be selectively connected. When the first port
237 of the rotor 213 is aligned with the gap in the inlet ports 233 and the gap in channels 241, the
passage 236 is closed against the annular wall 217 of the liner 215, thereby closing the valve
110. However, as an alternative, the inlet ports 233 can be brought together to omit the gap so
that inlet ports are arranged in a complete annulus and the valve 110 cannot be closed.
As compared to the first construction of the valve 110, the arrangement of the inlet ports
233 of the stator 2 in the annular surface 215 that extends around the rotational axis R, facing
the rotational axis R, simplifies the overall construction with the inclusion of relatively high
numbers of inlet ports 233. This is achieved whilst still providing adequate sealing around the
inlet ports 233 and the first port 237 by means of the liner 217 sealing between the annular
surface 214 of the rotor 213 and the annular surface 215 of the stator 212. The stator 212 and the
rotor 213 may be made from materials having suitable mechanical properties, whilst making the
liner 2 17 of a material selected to have a greater compliance than the stator 212 and the rotor
213, to provide the required degree of sealing between the annular surface 214 of the rotor 213
and the annular surface 215 of the stator 212. Sealing between the passage 236 and the passage
242 is provided between the boss 226 and recess 227, on either or both of the radial or axial
surfaces thereof. In contrast, if the liner 217 was absent, then it would be difficult to select
materials for the stator 212 and the rotor 213 provide the required mechanical properties and the
required sealing between the annular surface 214 of the rotor 213 and the annular surface 215 of
the stator 21 , in particular when providing a large number of ports and handling small volumes.
The rotor 213 may be formed from a variety of materials that provide sufficient rigidity,
and preferably selected to provide a low coefficient of friction against the liner 217. By way of
example, the rotor 213 may be made from any one of ultra-high-molecular- weight polyethylene
(UHMWPE), polytetrafluoroethylene (PTFE), perfluoroalkoxy (PFA) or fluorinated ethylene
propylene (FEP) (or indeed from any combination of such materials).
The stator 212 may be made from a variety of materials that provide sufficient rigidity.
This may be the same material as the rotor 213 or may be a different material, a wider choice of
materials being available because there is no need to provide a low coefficient of friction. By
way of example, the stator 212 may be made from any one of ultra-high-molecular- weight
polyethylene (UHMWPE), polytetrafluoroethylene (PTFE), perfluoroalkoxy (PFA), fluorinated
ethylene propylene (FEP), poly(methyl methacrylate) (PMMA) or cyclic olefin co-polymer
(COC) (or indeed from any combination of such materials).
The liner 217 may be made from a variety of materials that have a greater compliance
than the stator 212 and the rotor 213, and preferably selected to provide a low coefficient of
friction against the rotor 213. By way of example, the liner 217 may be made from any one of
polytetrafluoroethylene (PTFE), perfluoroalkoxy (PFA), fluorinated ethylene propylene (FEP) or
combination of PTFE and an elastomer (e.g. silicone) (or indeed from any combination of such
materials).
In this example, the liner 217 has a fixed position relative to the stator 213, but as an
alternative the liner 217 could have a fixed position relative to the rotor 212, in which case only a
single channel 214 would be required.
The valve 110 having the second possible construction is connected to other components
of the fluidics system 31 and the cartridge 10, and is operated, in the same manner as described
above for the first possible construction for the valve 110. The valve 110 in its first or second
possible construction is not limited to use in the cartridge 10 and can be used in other
applications. The valve 110 may be used for flow in the opposite direction to the inlet ports 133
or 233 from outlet port 135 or 235 so more generally the inlet ports 133 or 233 may be referred
to as first ports and the outlet port 135 or 235 may be referred to as a second port. The valve 110
is particularly suited as a miniature element for handling low volumes of fluid, in which the
fluidics channels, (for example the inlet ports 133, the passage 136, the collection chamber 134
and the outlet port 135 in the first possible construction or the inlet ports 233, the passage 236,
the passage 242 and the outlet port 235 in the second possible construction) have cross-sectional
areas of no more than 10mm2, preferably no more than 1mm2.
The rotor 1 or 2 3 is actuated by a motor 150, as shown in Figs. 1 and 24. As shown
in Fig. 2 The rotor 113 has a coupling element 152 protruding upwardly from the rotor 113 and
into which is fitted a drive shaft 151 that mounts a gear wheel 153. The motor 151 has an output
shaft 154 that mounts a gear profile 155 engaging the gear wheel 153 so that the motor 150
drives rotation of the drive shaft 151 and hence the rotor 113 or 213. The drive shaft 151 also
mounts an encoder wheel 156 whose position is sensed by a sensor 157. The motor 150 is driven
based on the output of the sensor 157, allowing the rotor 113 to be rotated around to select the
desired inlet port 133.
The fluidics system 31 is controlled to perform the biochemical analysis in respect of
successive samples sequentially. The sensor device 14 is prepared and then the fluidics system
31 is controlled to supply the sample from one of the wells 102 to the sensor device 14. After the
biochemical analysis has been performed, the sensor device 14 is emptied and flushed to clear
the sample. Then the sensor device 1 is prepared again and the fluidics system 31 is controlled
to supply the sample from the next well 102 by rotating the rotor 112 or 212 of the valve 110. A
specific example of the method of using the cartridge 10. The materials used are those described
in detail in WO-2009/077734.
First, a pre-treatment coating is applied to modify the surface of the body 20 of the sensor
device 14 surrounding the wells 2 to increase its affinity to the amphiphilic molecules. The
required volume pre-treatment is a hydrophobic fluid, typically an organic substance, in an
organic solvent is drawn from a reservoir 30 and dispensed by an inlet pump 33 by means of the
supply channels 32 to fill the chamber 24 covering the body 20 and the wells 21. The excess
material is expelled into the waste reservoir 35.
The cartridge 10 may be used in various configurations to expel the excess pre-treatment.
One example is to apply a gas flow with an inlet pump 33 through the supply channels 32 and
chamber 24 to move the fluid through the outlet channel 36 into the waste reservoir 35.
Alternatively, the pre-treatment may be dispensed from the inlet pump 33 with gas behind the
required volume and the excess expelled through the chamber 24 into the outlet channel 36 into
the waste reservoir 35 in a single action. The gas flow is continued through the chamber 24 to
flush solvent vapour from the system until the final pre-treatment coating is achieved. In further
modification, this final step may be achieved more rapidly by warming the gas flow or the body
20.
After application of the pre-treatment coating an aqueous solution, containing
amphiphilic molecules, is flowed across the body 20 to cover the wells 2 1. The required volume
of aqueous solution is drawn from the appropriate reservoir 30 and dispensed by an inlet pump
33 by means of the supply channels 32 to fill the chamber 24 covering the body 20 and the wells
21.
Formation of the amphiphilic membrane 26 is formed with the amphiphilic molecules
either directly or improved if a multi-pass technique is applied in which aqueous solution covers
and uncovers the recess wells 1 at least once before covering the wells 1 for a final time. The
aqueous solution containing amphiphilic molecules may be drawn directly from a reservoir 30 or
in the alternative approach mentioned above formed by passing aqueous solution through the
lipid assembly in the flow path of the supply channel 32 to the chamber 24.
In a first example, multiple passes of the solution air interface can be achieve by reversal
of the flow in the chamber 24. The flow to and from the reservoirs 30 is prevented by operation
of the selector valve 45 and operation of the output pump 34 drawing the amphiphilic molecule
containing solution through the supply channels 32 from the chamber 24 and pulling air from the
outlet channel 36 to the waste reservoir 35. The direction of the outlet pump 34 is reversed and
solution returned across the solution filled wells 2 1.
The formation of the amphiphilic membrane 26 may be observed by monitoring of the
resultant electrical signals across the electrodes 22 and 25 when a potential is applied the
formation introducing a resistive barrier and a decreases in the measured current. In the event
that an amphiphilic membrane 26 fails to form, it is a simple matter to perform another pass of
the aqueous solution air interface.
Alternatively, in a second example, multiple passes of solution air interface can be
achieved by flow in a single direction by inclusion of air slugs in the solution supply. In this
second example, the aqueous solution containing amphiphilic molecules is drawn into an inlet
pump 3 from the reservoir 30 and then with operation of non-return valves pumped into the
supply channels 32. An air slug may be formed by stopping the amphiphilic molecule aqueous
solution flow altering the position of the selector valve 45 and required air volume into the
channel behind the solution from the waste reservoir 35 (as it is open to atmosphere) by action of
another inlet pump 33. The selector valve 45 is returned to the previous position and further
amphiphilic molecule aqueous solution pumped forward. As the inlet pump 33 moves the
solution forward through the supply channels 32 to the chamber 24 and through into the outlet
channel 36 into the waste reservoir 35, the aqueous amphiphilic molecule solution stream
including slugs of air are passed over the wells 2 1. The process is repeated to achieve the desired
number of passes.
Excess amphiphilic molecules are removed from the chamber 24 by flushing aqueous
buffer solution from a reservoir 30 by action of an inlet pump 33. Multiple volumes of aqueous
buffer solution passed through the chamber 24 into the outlet channel 36 for supply to the waste
reservoir 5.
Preparation of the sensor device 1 is completed by flow of aqueous solution containing
a membrane protein, for example alpha-hemolysin or a variant thereof, from a reservoir 30 by
action of an inlet pump 33 into the chamber over the layer 26 allowing the membrane protein is
inserted spontaneously into the layer 26 of amphophilic molecules after a period of time.
In an alternative approach, the membrane proteins may be stored dried. In this case, the
aqueous solution may be directed into a second reservoir 30 containing the membrane protein in
dried form from an appropriate reservoir 30 by an inlet pump 33 via the supply channels 32 by
altering the position of the selector valve 45 used to rehydrate the membrane proteins before
using an inlet pump 33 to flow the resulting solution into the chamber 24 over the layer 26.
The insertion process into the layer 26 may be observed by monitoring of the resultant
electrical signals across the electrodes 22 and 25 when a potential is applied insertion resulting in
an increase in ionic conduction and an increases in the measured current.
When the insertion period is complete removed from the supply channels 32 and chamber
24 by flush of aqueous buffer solution from a reservoir 30 by action of an inlet pump 33.
Multiple volumes of aqueous buffer solution passed through the chamber 24 into the outlet
channel 36 for supply to the waste reservoir 35.
Analysis of the samples contained in the well plate 100 may start on completion of
preparation of the sensor device 14. The rotary valve 110 is configured to allow fluid contact
with the first inlet port 133. The selector valve 45 is positioned to stop flow from the fluid
reservoirs 30 and the outlet pump 34 operated to draw the sample material from the sample well
102. The rotary valve 110 is repositioned to direct flow towards the supply channels 32 and fill
the chamber 24 to cover the membrane layers 26 of the sensor system. On completion of the
analysis the selector valve 45 is positioned to allow flow of aqueous buffer from the inlet pump
33 to flush the sample from the supply channels 32, the rotary valve 110 and the chamber 24
with multiple volumes of buffer through the outlet channel 36 into the waste reservoir 3 to
prevent contamination of succeeding samples.
The selector valve 45 is positioned to stop flow from the fluid reservoirs 30 and valve
110 is re-positioned to form fluid connection to the next sample well 102 in the well plate 100.
This process repeated for all samples.
After all the samples have been analysed, either the cartridge 10 may be disposed of.
Alternatively, as the well plate 100 is a separate element, it may be removed, disposed of and
replaced by a new well plate 100 loaded with fresh samples. Such use of the well plate 100 as a
disposable element allows re-use of the cartridge 10.
The sensor device 1 is formed in a chip that is mounted on a printed circuit board (PCB)
38 electrically connected to the PCB 38. Electrical contacts from the PCB 38 are arranged as an
edge connector pad for making electrical connection to the sensor device 14. On insertion of the
cartridge 10 into the module 2, the contacts 39 make electrical connection to the remainder of the
electrical circuit in the module 2 that is described below. Three alternative designs for the sensor
device 14 and PCB 38 are as follows.
In the first possible design shown in Figs. 5 and 6, the sensor device 14 is formed as
disclosed in WO-2009/077734 as an array of electrodes embedded in wells fabricated on silicon
with wells made in a suitable passivation layer on top of the silicon, with the electrical
connections at the base of the silicon substrate using through wafer vias, solder-bump bonded to
the PCB 38. The PCB provides has an equivalent number of connections to two (or in general
any number of) application specific integrated circuits (ASICs) 40 bonded in similar fashion to
the opposite side of the PCB 38. The ASICs 40 include some of the components of the electrical
circuit of the module 2 described below. The ASICs 40 may include components of the
processing circuit for processing the electrical signals from the sensor device 14, for example an
amplifier, a sampling circuit and an analog-to-digital converter (ADC) to provide a digital
output. The digital output is supplied from the contracts 39 to enable the digital output to leave
the sensor device 14 using a suitable interface, for example low-voltage differential signalling
(LVDS). Alternatively, the output signal may be provided in amplified analog form with ADC
provided within the module. The ASICs 40 may also include some components of control
circuits for example accepting power and control commands via the contacts in order to set and
monitor functioning parameters, including for example current measurement sample rate (IHz to
00kHz), integration capacitors, bit resolution, applied bias voltage.
The second possible design is to form the sensor device 14 as a simple electrode array
chip fabricated on silicon, mounted on the PCB 38 and wire-bonded to the contacts 39. This
connection can then interface into the electrical circuit, either as a series of discrete channels, or
using an appropriate ASIC. Such an ASIC may be a conventional electronic readout chip, for
example as supplied by FLIR Systems, (e.g. FLIR ISC 9717) as an arrayed electrode
measurement device.
The third possible design is to fabricate the sensor device 14 and ASIC 40 as one device
that is then mounted on the PCB 38.
The configuration of the module 2 will now be described with reference to Fig. 7 which
shows the module 2 with the housing 1 removed to show the physical layout. The module 2
includes an internal board 50 and an embedded computer 51 connected together by a PCI data
acquisition module 52, which together provide an electrical circuit described below. The internal
board 50 makes contact with the contacts 39 of the cartridge 10 on insertion into the module 2.
The embedded computer 51 may be a conventional computer, including a processing unit
and a storage unit. The embedded computer 5 1 includes a network interface 53 that allows the
module 2 to connect to the network 3, thereby turning the module 2 into a standalone network
device yet also providing 'hooks' to enable many modules 2 to be run, managed and controlled
as a cluster, as described below. For example, the embedded computer 51 may run a slimmed
down operating system (e.g. LINUX) and applications to perform the various functions
described below. Complete development kits for such embedded systems are commercially
available.
The module 2 includes a loading mechanism 54 for automatically loading and ejecting
the cartridge 10 to and from the module 2. The loading mechanism 54 may be for example a
proprietary mechanism driven by a high precision stepper motors.
The module 2 also includes a microcontroller 58 and an FPGA 72 mounted on the
internal board 50 that control various components of the module 2 as described below.
The module 2 also includes fluidics actuation unit 60 that is mounted on the internal
board 50 and controls the fluidics system 31.
The module 2 also comprises a thermal control element 42 arranged to control the
temperature of cartridge 10 and the sensor device 14 in particular. The thermal control element
42 may be for example a Peltier thermal controller, such as a 32 watt Single Stage
Thermoelectric Module (for example as supplied by Ferrotec Corp, 33 Constitution Drive,
Bedford NH 03110 USA - part number 9500/07 1/060B). The thermal control element 42 may be
mounted, for example, underneath the cartridge 0 and so is not visible in Fig. 7. The thermal
control element 42 may be considered as part of the analysis apparatus formed primarily by the
cartridge 10 and could alternatively be mounted on the cartridge 10.
Lastly, the module 2 includes a display 55 for displaying basic operational status
information, a power supply 56 for supplying power to the various components of the module 2,
and a cooler assembly 57 for cooling the module 2.
The electrical circuit provided by the internal board 50 and the embedded computer 51
will now be described with reference to Figs. 8 and 9. The electrical circuit has two main
functions, namely a signal processing function and a control function, so that it acts as both a
signal processing circuit and as a control unit for the module 2.
The signal processing function is distributed between the internal board 50 and embedded
computer 5 1 and is provided as follows.
The sensor device 14 is connected to a switch arrangement 62 formed in an ASIC 40 on
the PCB 38 of the cartridge 0 and controlled by the control interface to the ASIC 40. The switch
arrangement 62 is arranged to selectively connect the well electrodes 22 of the sensor device 1
to a respective contact for supply to a detection channel of the signal processing function, there
being a greater number of wells 1 than detection channels. The switch arrangement 62 is
arranged and operated as described in detail in US Application No. 61/170,729 which is
incorporated herein by reference.
Alternatively the switch arrangement 62 may be provided and controlled separately from
the ASIC 40 as a standalone functional block between the sensor device 4 and the detection
channels 65, the detection channels 65 being provided within a readout chip, for example as
supplied by FLIR Systems, (e.g. FLIR ISC 9717).
The ASIC 40 provides an array of detection channels 65 each arranged as shown in Fig.
10 to amplify the electrical signal from one of the well electrodes 26. The detection channel 65 is
therefore designed to amplify very small currents with sufficient resolution to detect the
characteristic changes caused by the interaction of interest. The detection channel 65 is also
designed with a sufficiently high bandwidth to provide the time resolution needed to detect each
such interaction. These constraints require sensitive and therefore expensive components.
The detection channel 65 includes a charge amplifier 66 that is arranged as an integrating
amplifier by means of a capacitor 67 being connected between an inverting input of the charge
amplifier 66 and the output of the charge amplifier 66. The charge amplifier 66 integrates the
current supplied thereto from the well 2 1 to provide an output representative of the charge
supplied in successive integration periods. As the integration periods are of fixed duration the
output signal is representative of current, that duration being short enough to provide sufficient
resolution for monitoring of events occurring in the well 2 1 connected thereto. The output of the
charge amplifier 66 is supplied through a low pass filter 68 and a programmable gain stage 69 to
a sample-hold stage 70 that is operated to sample the output signal from the charge amplifier 66
and produce a sampled current signal. The output current signal is supplied to an ADC 7 1 to
convert it into a digital signal. The digital signals from each detection channel 65 are output from
the ASIC 40.
The digital signals output from the ASIC 40 are supplied via the contacts 39 from the
PCB 38 of the cartridge 10 to a field programmable gate array (FPGA) 72 provided on the
internal board 50 of the module 2. The FPGA 72 includes a buffer arranged to buffer the digital
signals from each detection channel 65 before supply via the PCI data acquisition module 52 to
the embedded computer 51.
In an alternative arrangement, the digital output from the detection are provided from a
readout chip located on the internal board 50 of the module 2 and supplied to the FPGA 72.
The embedded computer 51 is arranged as follows to process the digital current signals
from each detection channel 65 as follows. A PCI data acquisition module 52 controls the
transfer of the digital current signals from the FPGA 72 to the embedded computer 5 1 where it is
stored as digital data.
Thus the digital data stored in the embedded computer 51 is raw output data representing
the current measured by each well electrode 22 in respect of a nanopore in the amphiphilic
membranes 26 of the corresponding well. The current from each nanopore is a channel of the
measured electrical signal. This raw data is processed by a processing module 73 that includes a
pipeline 74 in respect of each channel. The processing module 73 is implemented by software
executed in the embedded computer 51.
The nature of the signal processing performed in each pipeline 74 of the processing
module 73 is as follows. The pipeline 74 processes the raw data to produce output data
representing the results of the biochemical analysis in respect of the corresponding channel. As
discussed above, interactions between the nanopore and the sample cause characteristic changes
in the electrical current that are recognisable events. For example, an analyte passing through the
nanopore may cause the electrical current to reduce by a characteristic amount. Thus, the
pipeline 74 detects those events and generates output data representing those events. Examples
of such processing are disclosed in WO2008/102120 which is incorporated herein by reference.
The output data may simply represent the fact that the event has occurred.
Additionally, the pipeline may classify the event and the output data may represent the
classification of the event. For example, the nanopore may have an interaction that differs as
between different analytes in the sample causing a different modulation of the electrical signal.
In this case, the pipeline 74 classifies the analyte on the basis of the modulated electrical signal.
An example of this is that a nanopore may have an interaction with bases of a polynucleotide in
which each base modulates the electrical signal differently. For example, a base passing through
the nanopore may cause the electrical current to reduce by an amount that is characteristic of the
base. In this case, the pipeline 74 classifies the event by identifying the base from the modulation
of the electrical signal. In this manner, the biochemical analysis is sequencing of a
polynucleotide in the sample, and the output data includes sequence data representing a sequence
of the polynucleotide. This may be referred to as "base calling".
The pipeline 74 also produces output data that is quality data representative of the quality
of the output data that represents the results of the biochemical analysis. This may represent a
probability of the detection and/or classification of the events being incorrect.
The output data may be represented in any suitable format. In the case of sequencing of a
polynucleotide, the output data and the quality data may be represented in the FASTQ format
which is a conventional text-based format for a nucleotide sequence and its associated quality
scores. The output data is stored in the embedded computer 51 and may also be transferred over
the network 3 and stored on the storage device 6 . The raw data representing the electrical signals
across each nanopore may be stored as well as the final output data, depending on user
requirements.
The processing module 73 may also derive and store quality control metrics representing
parameters of the biochemical analysis itself.
Aspects of the signal processing performed by the pipeline 74 may be performed on the
internal board 50 before data is transferred to the embedded computer 51. This approach is of
particular use for large numbers channels and the FPGA 72 may be particularly suited to this
type of task.
There will now be described the control function that is arranged to control the operation
of the module 2. The control function is distributed between the internal board 50 and embedded
computer 5 1 and is provided as follows.
The control function includes a controller 58, for example a Cortex M3 Microcontroller,
provided on the internal board 50. The controller 58 controls the operation of all the components
of the analysis apparatus 13. The controller 58 is arranged to send, via standard protocols and
through low level device drivers, commands to the pumps 33 and 34 of the fluidics system 3 1
and other pre-requisites for reading data. Status information is stored based on error codes
derived from drivers.
The controller 58 is itself controlled by a control module 80 that is implemented in the
embedded computer 1 by software executed thereon. The control module 80 communicates
with the controller 58 via an RS232 interface 81. The control module 80 controls the controller
58 as follows so that they operate together to constitute a control unit for the module 2.
The controller 58 controls the loading mechanism 54 to load and eject the cartridge 10.
On loading the controller 58 detects that proper electrical contact is made between the contacts
39 and the internal board 50.
The controller 58 controls the fluidics actuation unit 60 to control the fluidics system 31
to prepare the sensor device 14.
During this preparation, the control module 80 may monitor the electrical signals output
from the sensor device 14 to detect that preparation occurs correctly, for example using the
analysis techniques disclosed in WO-2008/102120 which is incorporated herein by reference.
Typically, the control module 80 will determine which of the wells 22 are set-up correctly at the
start of a run. This may include sensing bi-layer quality, electrode quality, occupancy by a pore
and even whether the nanopore is active following the sensing of a sample.
On the basis of this monitoring, the controller 58 also controls the switching controller 63
to cause the switch arrangement 62 connect detection channels 65 to the well electrodes 26 of
wells 22 of the sensor device 14 that have acceptable performance, in the manner disclosed in
detail in US Application No. 61/170729.
In the case of sequencing of polynucleotides, the control module 80 may also sense the
presence and state of any modifications to nanopores that might be required in order to process
and measure DNA, e.g. attachment of exonuclease enzymes, cyclodextrin adaptors.
The controller 58 controls a bias voltage source 59 that supplies a bias voltage to the
common electrode 25. In this way, the controller 58 controls the bias voltage across each
nanopore. The controller 58 controls the thermal control element 42 to vary the temperature of
the analysis apparatus 13. The controller 58 controls the operation of the ASIC 40 to vary the
sampling characteristics, for example the sampling rate, the integration period and reset period of
the capacitor 67, and the resolution of the resultant signal.
The controller 58 may execute the above control functions and other experimental
parameters via the FPGA 72. In particular, control of the ASIC 40 is provided via the FPGA 72.
Once the sensor device 14 has been prepared correctly, then the controller 58 controls the
cartridge 10 to introduce the sample into the sensor device 14 and to perform the biochemical
analysis. The biochemical analysis is then performed with the result that electrical signals are
output from the sensor device 14 and processed by the processing module 73 to produce output
data representative of the analysis.
The control module 80 has local performance targets that are derived on the basis of input
as discussed below. The local performance targets represent the desired performance for the
operation of the module 2. The performance targets can relate to any combination of: the time
within which output data is produced; the quantity of output data that is produced; or the quality
of output data that is produced, depending on the requirements for the biochemical analysis.
During operation, the control module 80 determines measures of performance of the
biochemical analysis, these being of the same nature as the local performance targets, i.e. the
time within which output data is produced; the quantity of output data that is produced; or the
quality of output data that is produced. On the basis of the measures of performance, the control
module 80 controls the controller 58 to control the analysis performed by the module 2 to meet
the performance targets. This is done by starting and stopping operation of the analysis apparatus
and/or varying the experimental parameters.
In one mode of operation, the plurality of wells 102 may not each contain a different
sample 142. That is, a selection of the wells 102 may contain the same sample 142. The
information as to which wells 102 contain the same sample 1 2 could, for example, either be
programmed into the analysis apparatus, or could be provided by a default setting in which
predetermined wells 102 (for example those on the same row or column) are known to be
provided with the same sample 142. In this mode, the control module 80 can be configured to
determine whether a performance target has been met after a sample 142 in a well 102 has been
used up. In the event the performance target has not been met, the control module 80 can control
the analysis to continue using a sample 142 from the selection of wells 102 containing the same
sample 142 as that which has been used up. This procedure can be repeated until it is determined
that the performance target has been met, at which time the control module 80 can control the
apparatus to analyse another sample 2 (of a different type) or bring the analysis to a
conclusion.
As a result, there is no need to process repeats of samples 142 for which a successful
analysis has been performed, but unexpectedly lengthy analyses can be run until completion.
Further, in this mode it may be preferable to supply the samples 142 to the sensor 14
immediately after each other, without an intermediate washing step, especially for example when
the samples 142 are the same. In other modes, an intermediate washing step may be desirable
between analyzing samples 1 2, especially for example when the samples 142 are different to
each other. However, instead of a washing step, another option is to use a first portion of the
next sample 142 to be analysed to displace the previous sample 142, effectively using the sample
142 itself as a washing medium.
This operation of the control module 80 using local performance targets and measures of
performance is described in detail in US Patent Application No. 61/265,488 to which reference is
made and which is incorporated herein by reference.
In the manner described above, each module 2 is a standalone device that can perform a
biochemical analysis independently of the other modules 2. A cluster of modules 2 are operated
as a common instrument 1 to perform a common biochemical analysis. This operation of a
cluster of modules 2 as a common instrument 1 and manner in which the modules 2 connect to
the network 3 and communicate on a peer-to-peer basis are described in detail in US Patent
Application No. 61/265,488 to which reference is made and which is incorporated herein by
reference.
More details on the nature of the biochemical analysis that may be performed are as
follows. The following paragraphs refer to numerous documents that are all incorporated by
reference.
The cartridge 10 described above can perform biochemical analysis using nanopores in
the form of protein pores supported in an amphiphilic membrane 26.
The nature of the amphiphilic membrane 26 is as follows. For amphiphilic systems the
membrane 26 is typically composed of lipid molecules or their analogues and can be either
naturally occurring (e.g. phosphatidylcholine) or synthetic (DPhPC,
diphytanoylphosphatidylcholine), Non-natural lipid analogues may also be used such as 1,2-
dioleoyl-3-trimethylammonium-propane (DOTAP). Amphiphilic membranes may be comprised
of a single species or a mixture of species. Additives such as fatty acids, fatty alcohols,
cholesterol (or similar derivatives) may also be used to modulate membrane behaviour.
Amphiphilic membranes provide a high resistive barrier to the flow of ions across the membrane.
Further details of amphiphilic membranes that are applicable to the present invention are given
in WO-2008/102121, WO-2008/102120, and WO-2009/077734.
In the analysis apparatus 13, the amphiphilic membrane 26 is formed across a well 22,
but the cartridge lOcan be adapted to support an amphiphilic membrane in other manners
including the following. The formation of electrically addressable amphiphilic membranes can
be achieved by a number of known techniques. These can be split into membranes or bilayers
that are incorporated onto one or more electrodes and those that provide a divider between two
or more electrodes. Membranes attached to the electrode may be bilayers or monolayers of
amphiphilic species and may use direct current measurements or impedance analysis, examples
of which are disclosed in (Kohli et al. Biomacromolecules. 2006; 7(12):3327-35; Andersson et
al., Langmuir. 2007 ;23(6):2924-7; and WO-1997/020203. Membranes dividing two or more
electrodes can be formed in a number of ways including but not limited to: folded (e.g. Montal et
al., Proc Natl Acad Sci U S A. 1972, 69(12), 3561-3566); tip-dip (e.g. Coronado et al, Biophys.
J. 1983, 43, 231-236); droplets (Holden et al., J Am Chem Soc. 2007; 129(27):8650-5; and
Heron et al., Mol Biosyst. 2008;4(12): 1191-208); glass supported (e.g. WO-2008/042018); gelsupported
(e.g. WO-2008/102120); gel-encapsulated (e.g. WO 2007/127327); and tethered and
porous-supported (e.g. Schmitt et al., Biophys J. 2006;91(6):2 163-71).
The nanopores are formed by protein pores or channels introduced into the amphiphilic
membranes 26. The protein pores or channels may be proteins that are either natural or synthetic,
examples being disclosed in WO-00/79257; WO-00/78668; US-5368712; WO-1997/20203; and
Holden et al., Nat Chem Biol.;2 (6):3 14-8)]. Natural pores and channels may include structures
where the membrane spanning portion of the protein comprises a beta-barrel, such as alphahemolysin
(e.g. Song et al., Science. 1996;274(5294): 1859-66), OmpG (e.g. Chen et al., Proc
Natl Acad Sci U S A. 2008; 105(1 7):6272-7), OmpF (e.g. Schmitt et al., Biophys J.
2006;91(6):2163-71) or MsPA (e.g. Butler et al., Proc Natl Acad Sci U S A.
2008; 105(52):20647-52). Alternatively, the membrane spanning portion of the protein may
consist of an alpha-helix, such as a potassium channel (e.g. Holden et al., Nat Chem Biol.;2
(6):314-8), (Syeda et al., J Am Chem Soc. 2008 ;130(46): 15543-8)]. The pore or channel may be
a naturally occurring proteins that is modified either chemically or genetically to provide desired
nanopore behaviour. An example of a chemically modified protein pore is given in WO-
01/59453 and an example of a genetically modified protein pore is given in WO-99/05167.
Adapters may also be added to the system to provide greater control and more targeted analyte
detection, examples of which are disclosed in US 6,426,231; US 6,927,070; and
WO2009044170.
The nanopores allow a flow of ions to travel across the amphiphilic membrane 26. The
flow of ions is modulated by pore on the basis of an analyte interaction, thus allowing the
nanopore to provide a biochemical analysis. There are many examples of such modulation being
used to as the basis for biochemical analysis, for example in US- 6,426,23 1; US-6,927,070; US-
6,426,231; US-6,927,070; WO-99/05167; WO-03/095669; WO-2007/057668; WO1997020203;
Clarke et al. NatNanotechnol. 2009;4(4):265-270; and Stoddart et al., Proc Natl Acad Sci S A.
2009;106(19):7702-7707.
The cartridge 10 may use nanopores for sequencing of polynucleotides, including DNA
and RNA, and including naturally occurring and synthetic polynucleotides. It may apply a
variety of techniques that have been proposed for deriving sequence information in a rapid and
cost effective manner, typically utilising measurement of changes in the electrical signal across a
single nanopore as a single strand of DNA passes through the nanopore. Such techniques include
without limitation: nanopore-assisted sequencing by hydridisation; strand sequencing; and
exonuclease-nanopore sequencing (e.g. D.Branton et al, Nature Biotechnology 26(10), pl-8
(2009)). The technique may involve the polynucleotide passing through the nanopore as an intact
polymer (modified or unmodified), or broken into the constituent nucleotide components or
bases (for example using the techniques disclosed in: US-5,795,782; EP-1,956,367; US-
6,015,714; US-7,189,503; US-6,627,067; EP-1, 192,453; WO-89/03432; US-4,962,037; WO-
2007/057668; International Appl. No. PCT/GB09/001690 (corresponding to British Appl. No.
0812693.0 and US Appl. No. 61/078687); and International Appl. No. PCT/GB09/001679
(corresponding to British Appl. No. 0812697.1and US Appl. No. 61/078695).
In general, present invention may be applied to any apparatus providing the measurement
of nanopores by providing two electrodes, one either side of an insulating membrane, into which
a nanopore is inserted. When immersed in an ionic solution, a biased potential between the
electrodes will drive ionic flow through the nanopore that can be measured as current in an
external electrical circuit. This current alters as DNA passes through the nanopore, and with
sufficient resolution, the constituent bases can be recognised from the changes, for example as
disclosed in Clarke et al. Nat Nanotechnol. 2009;4(4):265-270; International Appl. No.
PCT/GB09/001690 (corresponding to British Appl. No. 0812693.0 and US Appl. No.
61/078687); and D. Stoddart, PNAS doi 10.1073/pnas.090 1054 106, April 2009.
Further, the present invention may be applied to any apparatus in which arrays of
nanopores measure the same sample by providing individually addressable electrodes on one
side of each nanopore in the array connected to either a common electrode or an equivalent
number of addressable electrodes in the sample on the other side. External circuitry can then
perform measurements of DNA passing through each and every nanopore in the array without
the synchronisation of base addition to each nanopore in the array, i.e. each nanopore is free to
process a single DNA strand independently of every other, for example as disclosed in US-
2009/0167288; WO-2009/077734; and US Application No. 61/170,729. Having processed one
strand, each nanopore is also then free to begin processing a subsequent strand.
One advantage of nanopore-based analysis is that the quality of measurement does not
change over time for a fully-functioning nanopore, i.e. the accuracy of base identification is the
same at the start of sequencing as at any point in the future, subject to the expect experimental
limitations. This enables each sensor to perform, at constant average quality, multiple analyses in
a sequential fashion on the same sample or on multiple samples over time.
Besides sequencing of polynucleotides, the nanopores may be applied to a diverse range
of other biochemical analysis, including without limitation: diagnostics (e.g. Howorka et al., Nat
Biotechnol. 2001;19(7):636-9); protein detection (e.g. Cheley et al., Chembiochem.
2006;7(12): 1923-7; and Shim et al., J Phys Chem B. 2008;112(28):8354-60); drug molecule
analysis (e.g. Kang et al., J Am Chem Soc. 2006;128(33):10684-5); ion channel screening (e.g.
Syeda et al., J Am Chem Soc. 2008 Nov 19; 130(46): 15543-8), defence (e.g. Wu et al., J Am
Chem Soc. 2008;130(21):6813-9; and Guan et al., Chembiochem. 2005;6(10):1875-81); and
polymers (e.g. Gu et al., Biophys. J. 2000; 79, 1967-1975; Movileanu et al., Biophys. J . 2005;
89, 1030-1045; and Maglia et al., Proc Natl Acad Sci U S A.. USA 2008; 105, 19720-19725).
The present invention may also be applied to an analysis apparatus in which nanopores
are provided in solid state membranes. In this case the nanopore is a physical pore in a
membrane formed from a solid material. Such membranes have many advantages over fluid or
semi-fluid layers, particularly with respect to stability and size. The original concept was
proposed by researchers at the University of Harvard for examining polymers, such as DNA (e.g.
WO- 00/79257; and WO 00/78668). Since then the work has expanded to include the following
techniques that may be applied in the present invention: fabrication methods (e.g. WO-
03/003446; US-7,258,838; WO-2005/000732; WO-2004/077503; WO-2005/035437; WO-
2005/061373); data acquisition and evaluation (e.g. WO-01/59684; WO-03/000920; WO-
2005/017025; and WO-2009/045472), incorporation of nanotubes (e.g. WO-2005/000739; WO-
2005/124888; WO-2007/084163); and the addition of molecular motors ( e.g.WO-2006/028508)
; the use of field effect transistors or similar embedded within nanopore structures (e.g. US6,413,792,
US-7,00 1,792); the detection of fluorescent probes interacting with a nanopore or
nanochannel (e.g. US-6,355,420; WO-98/35012); and the illumination and detection of
fluorescent probes being removed from their target substrates as they translocate a nanopore (e.g.
US-2009-0029477). Even the use of mass spectrometry may be employed in the analysis
apparatus, for example as a polymer of interest passes through a nanopore or channel and whose
monomers are then cleaved and ionised sequentially analysed using mass spectrometry.
The analysis may be a chemical or biological assay, and could be used to carry out
biomarker validation studies, clinical tests and high-throughput screening. These tests may
involve carrying out chromatography (HPLC (high performance liquid chromatography, TLC
(thin layer chromatography), FPLC (fast protein liquid chromatography), flash chromatography,
with detection of analyte in the liquid eluent (by absorbance, fluorescence, radiometric methods,
light scattering, particle analysis, mass spectrometry), or an immunoassay or using direct mass
spectrometry (MALDI (matrix assisted laser desorption ionization), APCI (atmospheric pressure
chemical ionization), ESI (electrospray ionization) ionization with Quadrupole (single and
multiple), time-of-flight, ion trap detection). Immunoassays include an ELISA (enzyme-linked
immunosorbent assay), lateral flow assay, radioimmunoassay, magnetic immunoassay or
immunofluorescence assay.
These tests and assays can be used in the context of: identification of foetal abnormalities
such as Down's Syndrome, genome-wide association studies, pharmacokinetic and
pharmacodynamic investigations on tissues and whole animals, drug testing in sport, testing for
micro-organisms in environmental matrices (sewage, polluted water etc.), testing for hormones
and growth factors in treated water and so on.
The analysis may be applied to biomarker validation studies. The present invention can
allow very high numbers of samples to be analysed quickly and easily. For example, the current
process of biomarker discovery is hampered by the validation step, i.e. once a candidate marker
has been found, large numbers of samples must be examined in order to statistically confirm its
altered levels in the tissues of interest. An assay must therefore be developed for each marker.
The system of the present invention has a single readout for all analytes, for example DNA,
RNA, protein or small molecule, cutting down on the assay development stages.
The analysis may be applied to clinical tests and ELISA substitute. When a sample is
submitted for tests at a hospital or clinic, the testing procedure is very likely to involve either
mass spectrometry or ELISA. Both of these can be supplanted by the system of the present
invention. Development of suitable tests on the system of the invention will give huge increases
in throughput and savings in sample preparation time and handling. This will apply to large
proteins such as growth factors, peptides such as insulin, or small molecules such as drugs of
abuse or prescription drugs.
The analysis may be applied to high-throughput screening. Any quantitative screen can
be carried out on the system of the present invention. Thus, if an assay (for example a protease
assay) that gives a peptide or small molecule as a product is currently used in high-throughput
screening, the present invention can increase the throughput and cut down on sample handling
and preparation time.
Claims
1. An analysis apparatus for performing biochemical analysis of a sample using nanopores,
the analysis apparatus comprising:
a sensor device that is capable of supporting plural nanopores and being operable to
perform biochemical analysis of a sample using the nanopores;
at least one reservoir for holding material for performing the biochemical analysis;
a fluidics system configured to controllably supply material from the at least one
reservoir to the sensor device; and
a plurality of containers for receiving respective samples, the fluidics system being
configured to supply the samples selectively from the containers to the sensor device.
2. An analysis apparatus according to claim 1, wherein the analysis apparatus comprises:
a body on which the analysis apparatus, the at least one reservoir and the fluidics system
are mounted, and
a container element that is separate from the body and attachable thereto, the plurality of
containers being formed in the container element.
3. An analysis apparatus according to claim 2, wherein the container element is a well plate,
the containers being wells formed in the well plate.
4. An analysis apparatus according to claim 3, wherein the fluidics system comprises a
plurality of nozzles arranged to protrude into the wells of the well plate when attached to the
body.
5. An analysis apparatus according to any one of claims 1 to 4, wherein the fluidics system
includes a valve having an outlet port and a plurality of inlet ports corresponding to the plurality
of containers and being configured to selectively connect one of the inlet ports to the output port,
the fluidics system including channels connecting the containers to the corresponding inlet ports
and channels connecting the outlet port to the sensor device.
6. An analysis apparatus according to any one of claims 1to 5, wherein the plurality of
containers comprises 24 containers or more.
7. An analysis apparatus according to any one of claims 1to 6, further comprising a
controller configured to measure a performance target of the biochemical analysis and control
the analysis to meet the performance target.
8. An analysis apparatus according to claim 7, wherein the controller is configured to
control the analysis to utilise a selection of the plurality of containers in sequence, the selection
of the plurality of containers containing the same sample, until the performance target is met.
9. An analysis apparatus according to any one of claims 1 to 8, wherein the analysis
apparatus is a cartridge for cooperation with another device.
10. A rotary valve comprising:
a stator defining a plurality of first ports, and a second port;
a rotor mounted on the stator for rotation about a rotational axis,
the valve comprising a passage being in communication with the second port of the stator
and extending to a position for communicating with any one of the plurality of first ports of the
stator individually, depending upon the rotational position of the rotor.
11. A rotary valve according to claim 10, wherein the first ports, the passage, and the second
port have cross-sectional areas of no more than 10mm2, preferably no more than 1mm2 .
12. A rotary valve according to claim 0 or 11, wherein the plurality of first ports comprises
24 first ports or more.
13. A rotary valve according to any one of claims 10 to 12, further comprising a liner
arranged between the rotor and the stator, wherein the liner is fixed relative to the rotor.
14. A rotary valve according to claim 10, wherein:
tthe plurality of first ports defined by the stator is arranged around the rotational axis, and
the rotor defines a first port capable of communication with any one of the first ports of
the stator depending on the rotational position of the rotor, a second port positioned on the
rotational axis and in communication with the second port of the stator, and the passage
extending between the first port and the second port.
15. A rotary valve according to claim 14, wherein the ports of the stator and the rotor, and the
passage of the rotor have cross-sectional areas of no more than 10mm2, preferably no more than
lmm 2 .
16. A rotary valve according to claim 14 or 15, wherein
the plurality of first ports of the stator are provided in an annular surface that extends
around a rotational axis, facing the rotational axis,
the first port of the rotor is defined in an annular surface of the rotor that faces the
annular surface of the stator,
the rotor is mounted on the stator inside a liner arranged between the annular surface of
the stator and an annular surface of the rotor, the liner being made of a material having a greater
compliance than the rotor and than the stator,
the liner having at least one channel extending through the liner between the annular
surface of the stator and the annular surface of the rotor and capable of providing communication
between the first port of the rotor and any one of the plurality of first ports of the stator,
depending on the rotational position of the rotor.
17. A rotary valve according to claim 16, wherein the liner has a base covering an end
surface of the rotor that extends transversely to the rotational axis, the base having a passage that
extends from the second port of the rotor to the second port of the stator so that the passage in
the rotor is in communication with the second port of the stator through the passage in the liner.
18. A rotary valve according to claim 17, wherein the rotor has a boss formed on the end
surface of the rotor protruding into a recess in the liner, the second port of the rotor being formed
in the boss and the passage extending from the recess, the boss and the recess providing a seal
therebetween.
19. A rotary valve according to claim 17 or 18, wherein the second port of the stator is
defined in the annular surface of the stator.
20. A rotary valve according to claim 16, wherein the passage in the rotor communicates
with a passage in the liner that is in communication with the second port of the stator.
2 1. A rotary valve according to any one of claims 16 to 20 wherein the liner has a fixed
position relative to the stator, and said at least one channel is a plurality of channels each in
communication with one of the plurality of first ports of the stator and capable of communication
with the first port of the rotor, depending on the rotational position of the rotor.
22. A rotary valve according to any one of claims 16 to 21, wherein the rotor has an annular
lip engaging the liner and retaining the rotor in the liner along the rotational axis.
23. A rotary valve according to any one of claims 16 to 22, wherein the annular surface of
the rotor is parallel to the rotational axis.
24. A rotary valve according to any one of claims 16 to 23, wherein the annular surface of
the stator is parallel to the rotational axis.
25. A rotary valve according to any one of the claims 14 to 24, wherein the stator is on a
body that is arranged to allow attachment of a well plate comprising a plurality of wells
corresponding to the plurality of first ports, the body defining channels connecting the wells to
the corresponding first ports.
26. A rotary valve according to claim 10, wherein:
the stator defines the plurality of first ports in an annular surface that extends around the
rotational axis, facing the rotational axis;
the rotor is mounted on the stator for rotation about the rotational axis inside a liner
arranged between the annular surface of the stator and an annular surface of the rotor that faces
the annular surface of the stator, the liner being made of a material having a greater compliance
than the rotor and than the stator,
the rotor comprises the passage, the passage extending from a first port defined in the
annular surface of the rotor and being in communication with the second port of the stator, and
the liner has at least one channel extending through the liner between the annular surface
of the stator and the annular surface of the rotor and capable of providing communication
between the first port of the rotor and any one of the plurality of first ports of the stator,
depending on the rotational position of the rotor.
27. A rotary valve according to claim 26, wherein the ports of the stator and the rotor, the
passage of the rotor and the at least one channel of the liner have cross-sectional areas of no
more than 10mm , preferably no more than 1mm .
28. A rotary valve according to claim 26 or 27, wherein the passage extends to a second port
defined in the rotor that is positioned on the rotational axis and is in communication with the
second port of the stator.
29. A rotary valve according to claim 28, wherein the liner has a base covering an end
surface of the rotor that extends transversely to the rotational axis, the base having a passage that
extends from the second port of the rotor to the second port of the stator so that the passage in
the rotor is in communication with the second port of the stator through the passage in the liner.
30. A rotary valve according to claim 29, wherein the rotor has a boss formed on the end
surface of the rotor protruding into a recess in the liner, the second port of the rotor being formed
in the boss and the passage extending from the recess, the boss and the recess providing a seal
therebetween.
31. A rotary valve according to claim 29 or 30, wherein the second port of the stator is
defined in the annular surface of the stator.
32. A rotary valve according to any one of claims 26 to 28, wherein the passage in the rotor
communicates with a passage in the liner that is in communication with the second port of the
stator.
33. A rotary valve according to any one of claims 26 to 32, wherein the liner has a fixed
position relative to the stator, and said at least one channel is a plurality of channels each in
communication with one of the plurality of first ports of the stator and capable of communication
with the first port of the rotor, depending on the rotational position of the rotor.
34. A rotary valve according to any one of claims 26 to 33, wherein the rotor has an annular
lip engaging the liner and retaining the rotor in the liner along the rotational axis.
35. A rotary valve according to any one of claims 26 to 34, wherein the annular surface of
the rotor is parallel to the rotational axis.
36. A rotary valve according to any one of claims 26 to 35, wherein the annular surface of
the stator is parallel to the rotational axis.
37. A rotary valve according to any one of claims 26 to 36, wherein the stator is on a body
that is arranged to allow attachment of a well plate comprising a plurality of wells corresponding
to the plurality of first ports, the body defining channels connecting the wells to the
corresponding first ports.
38. A rotary valve according to claim 10, wherein:
the plurality of first ports defined by the stator is arranged around the rotational axis
facing the rotor;
the valve comprises a collection chamber extending in at least part of an annulus around
the axis of rotation of the valve member,
the second port of the stator is in communication with the collection chamber, and
the rotor provides the passage, the passage extending between the collection chamber
with which the passage is in communication and a position where the passage is capable of
communication with any one of the plurality of first ports depending on the rotational position of
the rotor.
39. A rotary valve according to claim 38, wherein the first ports, the passage, the collection
chamber and the second port have cross-sectional areas of no more than 10mm , preferably no
more than 1mm2.
40. A rotary valve according to claim 38 or 39, wherein the rotor and the stator have
interfacing contact surfaces that extend transversely to the rotational axis, the plurality of first
ports and the second port opening in the contact surface of the stator.
4 1. A rotary valve according to claim 40, wherein the interfacing contact surfaces extend
perpendicular to the rotational axis.
42. A rotary valve according to claim 40 or 41, wherein the valve further comprises a biasing
arrangement arranged to bias the rotor against the stator.
43. A rotary valve according to claim 42, wherein the biasing arrangement comprises a
resilient biasing element engaging the rotor.
44. A rotary valve according to claim 43, wherein the biasing arrangement further comprises
a clamping ring attached to the stator, the resilient biasing element being arranged between the
clamping ring and the rotor.
WO 2012/042226 "4 - PCT/GB201 1/001432
45. A rotary valve according to any one of claims 34 to 44, wherein the passage is defined by
a groove formed in the contact surface of the rotor.
46. A rotary valve according to any one of claims 34 to 45, wherein the collection chamber is
defined by a groove formed in the contact surface of one of the stator or the rotor.
47. A rotary valve according to claim 46, wherein the collection chamber is defined by a
groove formed in the contact surface of the stator.
48. A rotary valve according to any one of claims 38 to 47, wherein the rotor comprises a
bearing stub and the stator comprises a bearing recess in which the bearing stub is mounted.
49. A rotary valve according to any one of claims 38 to 48, wherein the stator is on a body
that is arranged to allow attachment of a well plate comprising a plurality of wells corresponding
to the plurality of first ports, the body defining channels connecting the wells to the
corresponding first ports.
50. A rotary valve according to claim 49, wherein the body comprises first and second plates
having interfacing contact surfaces, the first plate being arranged to allow attachment of the well
plate, the contact surface of the base being formed in the second plate and each channel being
formed by a through passage extending through the first plate to the contact surface of the first
plate, a groove formed in the contact surface of one of the first plate and the second plate, and a
through passage extending through the second plate from the contact surface of the second plate.
5 1 A rotary valve according to claim 50, wherein the first plate comprises a plurality of
nozzles arranged to protrude into the wells of a well plate when attached to the first plate, the
through passages that extend through the first plate extending through the nozzles.
52. A rotary valve according to claim 50 or 51, wherein the base further comprises a third
plate, the second and third plates having interfacing contact surfaces, the base further comprising
a channel formed by a through passage extending through the third plate to the contact surface of
the third plate, and a groove formed in the contact surface of one of the second plate and the
third plate.
53. An analysis apparatus according claim 5, wherein the valve is a rotary valve according to
any one of claims 10 to 52.