ASSAY DEVICE AND READER
Field of the Invention
The present invention relates to a microfluidic based assay system, comprising
a disposable assay cartridge and associated reading device, as well as the
individual components themselves. The present invention also relates to
methods of conducting assays, using the cartridge and device of the invention,
as well as kits for conducting assays.
Background to the Invention
The in vitro diagnostics (IVD) market is highly competitive and there is a
constant need within the IVD market to develop fast, low volume, precise and
cheap IVD tests. This is coupled with the fact there is a strong market desire to
develop capillary finger stick blood tests with reduced user complexity to allow
total market penetration (e.g. point of care, doctors surgery, home etc). This
capillary finger stick IVD testing model has proved hugely successful for
diabetes testing developing into a $3.5 billion market (ref: Medical Device
Today). The desire and ability to evolve immunoassay IVD towards capillary
finger stick blood testing has been hampered by technology developments,
however this remains a golden aim of many diagnostic companies as it allows
reduced complexity and greater placement of products in existing or untapped
market.
It is amongst the objects of the present invention to provide a cheap and reliable
assay system for carrying out IVD tests.
It is amongst the objects of the present invention to provide an assay cartridge
design platform and reader which may be easily and cheaply fabricated, as well
as being able to be configured to carry out a specified assay or assays.
It is amongst the objects of the present invention to provide an assay cartridge
which may easily be adapted to carry out a variety of different specified assays.
It is amongst the objects of the present invention to provide an assay system
comprising a reader which may preferably be used or easily adapted to perform
a variety of different assays.
Summary of the invention
In a first aspect the present invention provides a microfluidic assay cartridge for
use in detecting an analyte in a sample of fluid, the cartridge comprising:
a substrate comprising one or more microfluidic channels disposed therein and
comprising a binding agent disposed within said channel(s) for binding any of
said analyte within the sample;
a sample port for introducing said fluid sample into the cartridge;
at least one fluid input port for allowing one or more fluids to be introduced to
the cartridge from an associated reader device and transported through the
microfluidic channel(s); and
a fluid outlet sink for removing fluid from said channel(s).
The cartridge may further comprise a detection area where any bound analyte
may be detected. The detection area maybe contained within the sample
channel, which is directly adjacent or downstream from the sample port.
The cartridge design of the present invention may easily be adapted to carry out
a number of different assays and hence can be considered as an assay
platform for a variety of assays. The cartridge and channel(s) disposed therein
may be formed in any manner of ways known to the skilled addresses, which
may include photolithography, wet chemical etching, laser ablation, injection
moulding, embossing and printing techniques. However, in a preferred
embodiment, the cartridge and the channels and other features disposed
therein, are formed by a sandwich of three separate substrates - a top, middle
and bottom substrate.
The cartridge can be formed of any suitable material, such as polycarbonate,
polyester, polystyrene, PM A, etc. and the/each substrate may be formed of a
single or plurality of material(s). In the embodiment comprising three substrates,
the middle substrate comprises a pattern cut through the substrate,
corresponding to certain features of the cartridge, such as the channel(s), fluid
reservoir/reservoirs port, sink area and the like. By applying and sandwiching
(such as by heat sealing, gluing, stapling and the like) appropriately cut top and
bottom substrates, to sandwich the middle substrate between the top and
bottom substrates, a cartridge can be provided in which channels and other
features are disposed. Openings or features in the top and/or bottom substrate
may be designed to co-locate with features in a reader device (as will be
discussed hereinafter), which may facilitate with correct location of the cartridge
in the reader and also importantly allow for a fluid, such as a wash buffer, to be
introduced from a fluid reservoir/reservoirs in the reader to the cartridge or
sample to be applied or air to be vented from the cartridge. The fluid/wash
buffer or gas can be introduced into the cartridge by way of suitable means
such as a pump/pumps means in the reader and the fluid transport means can
therefore control fluid transport within the cartridge itself. Thus once a sample
has been introduced into the cartridge such as by way of capillary action, further
fluid transport within and throughout the cartridge is controlled/facilitated by way
of means provided in the reader device. It will be appreciated that the fluid
introduced into the cartridge by way of the fluid input port may be a liquid and/or
a gas, such as air.
As identified, in use, the sample is applied to the cartridge through a sample
introduction port such as by way of capillary action or other means. In a
preferred embodiment the sample introduction port is an aperture in a side or
face of the cartridge. Desirably the cartridge is in the form of a generally thin
planar device comprising top and bottom faces and four edges. In this
arrangement, the sample introduction port may be formed in one of the edges of
the cartridge, so that a user need only contact the sample with the aperture
formed in the edge, in order to enable sample uptake into the cartridge. In use
the user contacts the fluid sample with the port/aperture and, in certain
embodiments, due to the dimensions of said channel(s) within the cartridge,
fluid is drawn into the cartridge by capillary action. The dimensions of the
sample port/aperture may be smaller than the dimensions of the channel(s).
When fluid is being transported through the cartridge, fluid is not expelled
through the sample port as there are no surfaces to wet. However, because the
sink offers a large void area which can be wetted the preferential fluidic path is
into the sink.
Said fluid input port(s) of the cartridge is/are adapted to co-locate with a feature
in the reader, so that a fluid, such as a wash buffer or gas, such as air,
contained in a reservoir/reservoirs within the reader, can be introduced into the
cartridge. Typically the inlet port is simply an aperture or hole within the top
surface of the cartridge. The cartridge may include more than one input port, so
that a fluid, or fluids, may be added at different time points and/or locations
within the cartridge. It is to be understood that a fluid-tight seal is generally
formed between each said input port of the cartridge and a feature, such as a
valve or tubing within the reader, which may be connected to the reservoir fluid.
Desirably, said channel(s) in the cartridge also comprise one or more fluid stop
features, which are designed to prevent the sample and/or other fluids from
passing through the stop feature, by virtue of capillary action alone. That is, the
sample or any other fluid may be actively forced past said stop feature(s) by a
force, such as that applied by a pump/pumps provided by the reader. A
preferred stop feature is a hydrophobic material (e.g. printable conductive or
non conductive inks) or a process or material that changes the surface
properties of a channel surface therefore creating a hydrophilic/hydrophobic
differential (e.g. by way of laser ablation, surface scoring, surface material
removal, evaporated metallic materials etc), which is designed to abut/be a wall
feature or is coated on a wall of the channel. In the embodiment where the
channels are formed by virtue of three substrates being sandwiched together
thereby forming the channels, the hydrophobic material may be applied to the
top and/or bottom substrates, such that when the three substrates are
sandwiched together, the hydrophobic stop material forms a feature on the top
and/or bottom surface of said channel.
It is also preferred that a stop feature be located upstream of the sink feature, in
order that the sample, upon initial application, does not flow into the sink
feature. Only when a force, such as provided by way of a pump/pumps, within
the reader is applied, can fluid pass the stop feature upstream of the sink
feature and hence allow fluid to pass into the sink. The fluid outlet sink is
designed to be a void area of the cartridge into which spent fluid or fluid which is
not required or deemed undesirable, may be evacuated. For example, whole
blood contains many proteins and other agents which can interfere with assay
reactions and/or detection of captured analyte, by way of fluorescence
detection, for example. The present invention allows the initial binding of any
analyte to be carried out within the sample of whole blood, but all or
substantially all of the unbound material can subsequently be evacuated to the
sink feature, enabling further reactions and/or detection to be carried out in a
defined media or buffer.
As well as the microfluidic channel(s), the cartridge of the present invention may
comprise one or more electrode features which contact with the channel and
hence the sample once introduced into the cartridge. The electrodes are
designed to contact electrical contacts within the reader, enabling a variety of
readings to be taken, where appropriate. For example, one or more electrodes
in the cartridge may be designed to detect correct loading of the cartridge and
the reader may signal to the user whether or not the cartridge has a) been
correctly inserted into the reader and/or the sample loaded into the cartridge
correctly. The electrode(s) may also carry out one or more electrical
measurements on the sample itself. For example, when the sample is a sample
of whole blood, the electrode(s) may conduct a hematocrit measurement of the
sample, which may be important in determining an accurate concentration of the
analyte to be detected. Conductivity and/or impedance measurements may be
determined depending on the sample being studied. Thus, the cartridges of the
present invention may not only detect whether or not an analyte is present in a
sample by way of binding any analyte, but electrical measurements on the
sample may also be conducted.
The sample to be applied to the cartridge may be any suitable fluid sample. It
may for example be a sample of fluid obtained from a subject, such as a whole
blood, plasma, saliva, semen, sweat, serum, menses, amniotic fluid, tears, a
tissue swab, urine, cerebrospinal fluid, mucous and the like. It is to be
appreciated that the assay systems of the present invention may be applied in
the human health area, including large and growing IVD markets (e.g. cancer,
cardiology, and infectious disease). The assays may also be used to test drugs
and drug action. However, the system may also be applied in environmental
settings where it is desirable to detect, for example toxic agents or infectious
agents such as bacteria or viruses. Thus, samples from rivers or lakes or swabs
from solid surfaces may be taken in order to obtain a fluid sample for providing
to the cartridge. The assay systems may also be utilised for veterinary
applications. Essentially any assay in which a sample can be provided in a fluid
form may be utilised in the present invention.
The sample may, for instance, include materials obtained directly from a source,
such as a sample of whole blood, as well as materials pretreated using
techniques, such as filtration, precipitation, dilution, distillation, mixing,
concentration, inactivation of interfering agents, etc. These steps may be carried
out prior to the sample being introduced to the cartridge or may be carried out
by the cartridge itself.
The sample may be introduced prior to the cartridge being inserted into the
reader or after the cartridge has been inserted into the reader. The cartridge
may be so designed that the sample is introduced by way of capillary action, or
by virtue of a seal being formed between an input port of the cartridge and the
reader, the sample may be actively drawn into the cartridge by way of air being
drawn through the microfluidic channel(s) by a pump/pumps in the reader, such
as a pump/pumps.
The analyte to be detected can be any desired analyte and may include
proteins, peptides, antibodies, nucleic acid, microorganisms (such as bacteria
and viruses), chemical agents, toxins, pharmaceuticals, metabolites, cellular
moieties and the like. For example, the present system may be adapted to
detect any type of analyte that can bind a suitable binding agent. The binding
agent may be any suitable agent which is able to bind specifically to the analyte
to be detected. For example, if the analyte is a protein or peptide, the binding
agent may be a receptor or antibody which is capable of specifically binding to
the protein/peptide. Conversely an antibody may be bound by a protein/peptide
which the antibody is designed to specifically bind to. Nucleic acids may be
bound by other nucleic acids which are capable of specifically hybridising to the
analyte nucleic acid. Microorganisms may be bound by antibodies which
specifically bind to proteins on the surface of the microorganism. Chemical
agents, toxins, pharmaceuticals, metabolites may be bound by chemical
moieties which are capable or reacting or binding to the aforementioned
chemical analytes via appropriate bonding reactions, or affinities. Many types of
binding techniques are well known to those of skill in the art.
Moreover, the binding agent may be an enzyme or an enzyme substrate. For
example analytes such as glucose through well described enzymatic
methodologies may be deleted, for example the reaction product formed
following the enzyme reacting with the glucose may be detected by using
electrochemical, or optical detection techniques known to the skilled addressee.
Such measurements can be made as standalone measurements or in
combination with other analytes to be detected in the sample.
The binding agent may itself by attached directly to a wall or surface of said
channel within the cartridge, by suitable bonding to the wall or surface for
example, by way of physical adsorption, covalent chemical coupling, non
covalent chemical bonding (e.g. biotin-avidin) or a combination of any of the
above. In a preferred embodiment the binding agent is in the form of a
magnetic or paramagnetic particle, comprising a binding moiety and the binding
moiety is bound by non covalent chemical bonding (e.g. biotin-avidin) to the
surface of the particle. Additional embodiments could also include physical
adsorption, covalent chemical coupling, non covalent chemical bonding (e.g.
biotin-avidin) or any combination of these to the surface of a magnetic agent,
such as a magnetic particle. The magnetic agents/particles which are
functionalised to comprise the binding agent bound thereto, may simply be
deposited within a channel of the cartridge, such that upon the sample being
applied to the cartridge and being drawn into the channel(s), the functionalised
magnetic agents/particles are resuspended by the fluid sample and hence come
into contact with any analyte in the sample. The area of deposition may be
specifically defined using hydrophobic stop features, through the techniques
described previously in order to separate this area from the detection area in
order to ensure that high background readings are not obtained due to reagent
components (e.g. fluorescent latex) being dried down in the measurement /
detection area.
As mentioned above as well as the binding agents, the cartridge may comprise
one or more further reagents deposited within said microfluidic channels(s),
which reagents may facilitate detection of the captured analyte. For example
said one or more reagents may include a label which has been adapted to
specifically bind to the captured analyte, thus facilitating its detection.
Bound analyte may be detected directly providing the bound analyte is capable
of generating a detectable signal, or upon binding of the analyte a reaction may
place, so as to generate a reaction product and the reaction product may be
detected. However, in a preferred embodiment, bound analyte is contacted with
a label which is able to bind the bound analyte and a label/binding agent/analyte
complex is subsequently detected. The label may itself be bound to a further
binding moiety which is also capable of specifically binding to the binding
agent/analyte complex. Typically the label is able to bind to a different portion
of the analyte to which the first binding agent binds, or is capable of binding to a
region of the binding agent/analyte complex which is formed only on generation
of such a complex.
Bound analyte may be transported to the label within a region of the cartridge
by way of the transport means in the reader causing the bound analyte to be
moved. Alternatively a detection agent or label is brought into contact with the
bound analyte by virtue of an amount of fluid being introduced into the cartridge
from a fluid reservoir/reservoirs in the reader.
Desirably the binding agent and any detection agent/label are in a dry state
when deposited in the channel(s) of the cartridge.
In one embodiment, the detection agent/label which is designed to facilitate
detection of the analyte, is initially located upstream (in terms of the direction
the sample flows into the cartridge following introduction) from such a stop
feature. In this manner said detection agent does not initially come into contact
with the sample upon initial sample application to the cartridge. Only when a
fluid such as a buffer is provided to the cartridge through the fluid input port, is
the detection agent constructed with the bound analyte. When a fluid is
introduced into the cartridge from the reader, the detection agent may be
carried by the fluid into contact with the bound analyte resuspended and carried
by the fluid, passing by the stop feature and into contact with the captured
analyte.
In another embodiment, after the initial binding phase between the sample and
the binding agent and optional wash, the magnetic particle-analyte complex
within a buffer media could be transferred to an upstream region of the channel,
where the label is located, in dry form within the channel. The .magnetic particleanalyte
complex within the buffer media would resuspend/rehydrate the label
and allow binding of the label to the analyte. This transfer event is possible due
to the ability of the reader to effectively and accurately remove air from the
channel (which is a sealed system). This method may allow greater control of
rehydration of deposited reagents and homogeneity of reagent dispersion.
In another embodiment, the binding agent and the label are deposited in the
sample channel. The sample rehydrates these reagents allowing the binding
reaction to occur. In this embodiment all the reagents can contact the sample,
the reader then accumulates the magnetic particle-analyte-label complexes to a
region within the sample channel via the application of a magnet/electromagnet.
The reader then expels the unbound label/sample into the sink using an air/fluid
wash. The reader can use a disposable fluid/air reservoir/reservoirs or likewise
a reusable fluid reservoir/reservoirs. The magnetic particle-analyte-label
complex is then quantified in a fluid or air environment.
Each cartridge may be designed to carry out single analyte detection or multiple
analyte detection. Moreover, each cartridge comprises more than one
microfluidic channel system, so that more than one assay may be carried out
using a single cartridge.
Desirably the cartridges may easily be mass produced. The cartridge may
provide in a strip, where a number of cartridges are initially connected for
example, be initially together, such as by way of a perforated seal. In this
manner, the user can easily remove a cartridge from the strip, prior to use.
Once the cartridge has been loaded with a sample, any captured analyte may
be detected by way of a suitable reader. The present invention provides such a
reader and an important aspect of the present invention is the separate
provision of a fluid/buffer reservoir/reservoirs within the reader. One advantage
of this is that the cartridges themselves may be initially "dry", that is contain little
or no fluid within the cartridge prior to sample application. This not only
simplifies manufacturing of the cartridges themselves, but also improves shelflife
and allows many of the cartridges of the present invention to be stored at
room temperature, with little degradation of the chemical or biological
components within the cartridge prior to use.
In a further aspect there is provided a method of conducting an assay on a
sample, the method comprising:
Introducing a sample into a microfluidic cartridge of the present invention such
that any analyte present in the sample is capable of being bound by a binding
agent;
washing any unbound material away from the bound analyte using a suitable
fluid or gas introduced to the cartridge by way of the input port and;
detecting any labelled bound analyte present in the cartridge.
In a further aspect there is provided an assay system for conducting an assay
on a fluid sample, the assay system comprising;
a) a microfluidic cartridge according to the first aspect (or preferred
embodiments thereof) and;
b) a reader device, the reader device comprising:
i) a receiving port for introducing the cartridge into the reader;
ii) an internal reservoir/reservoirs for storing a fluid or a gas;
iii) means for delivering the fluid or gas to the input port(s) of the
cartridge once inserted within the reader, so that fluid or gas may be
transported through the microfluidic channel(s) of the cartridge and;
iv) detection means for enabling detection of any bound analyte or a
reaction product formed as a result of the analyte binding the binding
agent within the cartridge.
The reader includes a receiving port into which the cartridge is to be inserted.
The reader may be adapted so as to ensure correct insertion of the cartridge
and this could take a variety of forms. For example, the cartridge may be initially
located on a carrier mechanism which enters the reader, such as may be found
in computers for loading CDs and the like. Alternatively the receiving port may
be sized to allow the cartridge to be received and an internal stop member may
be found within the reader which the cartridge abuts once inserted correctly.
Additionally, or alternatively, features found on or cut into the surface of the
cartridge may be designed to co-locate with features found within the reader
and only once the cartridge is correctly located in the reader, will the cartridge
be able to be read.
The fluid reservoir/reservoirs is preferably sized such that more than one
sample cartridge may be analysed and read before fluid in the
reservoir/reservoirs needs replacing. Desirably many assays may be carried out
before fluid in the reservoir/reservoirs may need to be replaced. Alternatively, in
the case where the internal reservoir/reservoirs is filled with air, the
reservoir/reservoirs will not require to be replaced as when the
reservoir/reservoirs was completely expelled, it could retract to its starting
position, drawing in air from the atmosphere. In the case of a fluid
reservoir/reservoirs, the fluid may be introduced into the reservoir/reservoirs
manually from another source. Preferably the reservoir/reservoirs takes the form
of a replaceable cartridge, which may be introduced into the reader when
required. For example, a user may have, or be provided with a reader which is
able to be configured to carry out a variety of different types of assay, but the
user is provided with a kit comprising assay cartridges and a fluid
reservoir/reservoirs cartridge which are suitable for a particular analyte or
analytes to be detected. In this manner, prior to use, the user inserts the fluid
reservoir/reservoirs cartridge into the reader. The reservoir/reservoirs cartridge
itself may have a unique identifier feature, such as a bar-code or chip device,
which is recognised by the reader to be associated with a particular assay which
is appropriate for the sample cartridges and reservoir/reservoirs cartridge, or the
user may configure the reader to conduct a particular assay which is associated
with the particular sample cartridge and optionally the reservoir/reservoirs
cartridge. For some assays although differently manufactured sample cartridges
may be required, a single fluid reservoir/reservoirs cartridge may be used to
conduct a variety of different assays. Desirably a single fluid reservoir/reservoirs
cartridge may contain enough fluid to be able to carry out many assays, such as
greater than 25 or 50 assays, before the reservoir/reservoirs cartridge requires
to be replaced. The fluid may be a washing agent such as water, which may
include a buffer, such as PBS, HEPES and the like. Other fluids may also be
suitable.
In the embodiment where the binding agent is bound to the surface of magnetic
agents, such as magnetic beads, it is understood that the reader will comprise a
permanent magnet or electromagnet which is designed to apply a magnetic field
or be brought into close proximity or a magnetic field applied, in order to
concentrate and hold the magnetic particles in a particular area of said
microfluidic channel of the cartridge. This area may be the detection area.
Concentrating the magnetic particles into a particular area may serve to
facilitate detection of any captured analyte and/or increase sensitivity of
detection. Moreover, by holding the particles by way of the magnetic field it also
allows unwanted fluid surrounding the bound analyte to be washed away,
thereby leaving the captured analyte free of potentially interfering
agents/contaminants which may be present in the initial sample. The permanent
or electromagnetic field may be reduced or increased, such as by moving a
permanent magnet closer to, or further away from the cartridge, or by increasing
or decreasing the intensity of the applied field. This may serve to allow the
magnetic particles to "relax" or become less concentrated in a particular
location, whilst still being held to a certain extent by the magnetic field or not.
This may facilitate further reactions to be carried out on the particles, which may
be conducted more efficiently compared to if the magnetic particles where
tightly concentrated. It may also be preferred in certain applications that the
detection is carried out when the particles are less "concentrated" or relaxed.
In use the magnet may be used to hold any bound agent once the magnetic
field has been applied to the sample. Fluid from the fluid input port may be
introduced into the cartridge and the fluid may wash any non-bound
components of the sample away and/or allow other reagents such as a
detection agent to be brought into contact with the captured analyte.
The reader of the present invention further comprises detection means for
detecting any captured analyte within the sample cartridge. The detection
means may be any suitable means depending on the particular assay. For
example, the detection means may be a fluorimeter, which may be used to
detect a fluorescent signal, once appropriately excited, from the labelled or
unlabelled bound analyte or reaction product. The bound analyte/reaction
product may naturally fluoresce once light of an appropriate wavelength has
been used to excite the analyte/product, or a further label may be used to
separately bind the bound analyte and the label detected by fluorescent means.
Other labels which may be employed and hence the detection means adapted
accordingly, include radiolabels, phosphorescent labels, colloidal metal
particles, bioluminescent labels, colourimetric labels, electrochemical labels and
the like. Moreover, as mentioned above the bound analyte or radiation product
itself may be directly detected using techniques such as Raman spectroscopy
and the like.
The detectable labels may be used alone, or n conjunction with a microparticle
or bead, such as a metal oxide, polysaccharide or latex particle. Many types of
latex and other particles are know in the art
The reader comprises suitable means for transporting fluid from the fluid
reservoir/reservoirs into and throughout the cartridge. The reader may also be
configured to enable air, such as filtered air, to be transported into the said
microfluidic channel(s) of the cartridge. The reader comprises appropriate
tubing, valves and/or seals, as necessary, to enable fluid in the
reservoir/reservoirs and/or air to be introduced into the cartridge. The means
may be a pump/pumps and the pump may pump fluid / gas in one-direction, or
may be able to pump fluid / gas back and forth. A preferred pump is a stepper
motor linear actuator, piezoelectric pump, osmosis pump, peristaltic pump or
piston pump. Fluid / gas delivery to the sample cartridge may be controlled by a
microfluidic control assembly, which may control delivery or one or more fluids /
gases to the sample cartridge, to one or more input apertures in the cartridge
and at suitable time points.
The reader may include other features, such as a heating device to allow
assays to be conducted at a particular temperature, as well as appropriate
electrical circuitry and software to allow the reader to be programmed to carry
out one or more different assays.
The platform system of the present invention, comprising cartridge and reader
provides a number of distinct advantages:
1. Reduced Sample Volume: capillary introduction of a fluid, such as a
finger stick blood sample, reduces the complexity for the user and allows
the tests to be performed in any environment (e.g. ambulance, point of
care, doctor's surgery, battle field etc), and similar to glucose testing,
allowing products to be placed anywhere.
2. Performance, Sensitivity and Precision: The ability to perform multiple
step assays will increase sensitivity, precision and reproducibility of
assays, a major requirement of any IVD test. This will become more and
more important as the FDA continues its reduction of the allowable total
error for product launches of new IVD tests (entry into existing and new
product markets will become harder).
3. Room Temperature Stability: Many existing IVD tests require
refrigerated storage and shipping, this requirement adds significant cost
to the product and also restricts the usage and distribution of the
product. The initial "dry" nature of the sample cartridges aids in their
stability and shelf-life.
4. Low material costs and a simple manufacturing process allow for low
costs of goods (COGs), allowing substantial and increased profits to be
generated by the sales of IVD strips. This is especially needed in the
immunoassay and molecular IVD market where the conventional tests
tend to be of high complexity driving both the strip material costs and
overall assay cost higher.
Detailed description of the invention
The present invention will now be further described by way of example and with
reference to the figures which show:
Figure 1 shows a schematic representation of a sample cartridge in accordance
with the present invention;
Figure 2 is a schematic representation of how a cartridge of the present
invention may be formed;
Figure 3 is a photograph of a portion of a cartridge according to the present
invention showing various features;
Figures 4 and 5 show blood entering and filling the portion of the cartridge
shown in figure 3;
Figures 6, 7 and 8 are a photographs of a detailed portion of a cartridge of the
present invention showing magnetic particles being captured by a magnet and
being retained following washing away of a blood sample;
Figure 9 is a photograph of a detailed portion of a cartridge of the present
invention showing magnetic particles being held more diffusely following partial
removal of a magnet;
Figures 10 and 11 are schematic representations of further embodiments of a
sample cartridge in accordance with the present invention;
Figure 12 is a schematic of a reader device in accordance with the present
invention;
Figure 13 is a schematic of the internal mechanisms associated with a reader
device in accordance with the present invention
Figure 1 is a schematic representation of a fluid management system found
within a reader device of the present invention;
Figure 15 shows a schematic representation of a fluid reservoir/reservoirs
system and how this may be used within a reader of the present invention.
Figure 16 shows graphed experimental results of a total PSA washed wet assay
with the assay cartridges measured in the MST Pro Meter V 1 in accordance
with the current invention;
Figure 1 shows graphed experimental results showing the correlation between
the Total PSA washed wet assays measured in strips in the MST Pro meter and
the Victor V reference instrument;.
Figure 18 shows graphed experimental results showing the correlation between
the Total PSA washed wet assays measured in strips in the MST Pro meter and
the Victor V reference instrument;.
Figure 19 shows graphed experimental results showing the total PSA wet assay
performed in the MST Pro Meter and Strip, the meter using a air wash step to
expel unbound label from the channel;
Figure 20 shows graphed experimental results showing the total PSA wet assay
performed in the MST Pro Meter and Strip, does not use a wash step and
measures the fluorescence intensity of the fluorophore after the magnetic
particle-PSA-fluorescent latex complex are accumulated by the magnet;
Figure 2 1 shows graphed experimental results showing the total PSA washed
wet assay performed with the MST Pro Meter V 1 and Strip. For the data, the
MST Pro Meter V 1 uses a fluid wash to expel the sample (containing unbound
label) from the strip channels into the sink;
Figure 22 shows graphed experimental results of a total PSA dry assay
performed with reagents dried in the MST Pro Strip V 1 and the assay performed
on MST Pro Meter V 1 in accordance with the current invention;!
Figure 23 shows graphed experimental results of a total PSA half dried 2 step
assay performed with reagents dried in the MST Pro Strip V 1 and the assay
performed on the MST Pro Meter V 1. In this case the fluorescent latex was
deposited in the test cartridge in dry format.
Figure 24 shows graphed experimental results of a total PSA half dried 2 step
assay performed with reagents dried in the MST Pro Strip V 1 and the assay
performed on the MST Pro Meter V 1. In this case the magnetic particles were
deposited in the test cartridge in dry format;
Figure 25 shows graphed experimental results of a scan across a test sample
channel in the strip MST Pro Strip V 1 using the instrument MST Pro Meter V1;
and
Figure 26 shows a schematic representation of further embodiments of a
sample cartridge (MST pro strip V1, as used in the experimental section) in
accordance with the present invention.
A sample cartridge (10) in accordance with an embodiment of the present
invention is shown in figure 1. A fluid such as blood is applied to the sample
introduction port (12) (via, for example, finger or venous blood). In this
particular embodiment two channels (14,16) span from this one sample
introduction port (12), the channels (14,16) are separate and are not joined,
although to the user who is applying the blood the channel may appear as one.
Although not to be construed as limiting, the further description will relate to the
sample being a sample of whole blood.
The total sample application may be smaller than 1 I depending on the number
of channels to fill therefore when the user applies a sample, such as a drop of
blood, both channels (14,16) will fill under capillary force. This process is very
fast and more in tune with blood glucose strip filling as opposed to the lengthy
blood separation filling of some immunoassay platforms. Deposited in the two
channels (14, 16) are magnetic particles functionalised with antibody (18). As
will be described in more detail, the blood fills each channel (14, 16) to the
fluidic stop features (20, 22), one stop (22) downstream of a sink void (28) and
the other stop (20) in the main sample channel. Fluidic stop features may be
created by applying a printable hydrophobic ink to a surface of the channel.
When the cartridge (10) is formed from three substrates (50, 52, and 54) as
shown in Figure 2b, the hydrophobic ink may be applied to top (50) and bottom
(54) substrates, so as to form a stop feature on the top and bottom surfaces of a
channel. The fluidic stop features (20, 22) in the main sample channel may also
act as fill detect electrodes if made of a suitable hydrophobic electrically
conductive material. As the cartridge (10) is inserted into the reader, a cartridge
heating mechanism may be initiated, heating the cartridge to a predefined
constant temperature for the duration of .the test. This allows many benefits
which are commented on hereinafter.
At the end of each of the 2 sample channels (14, 16) on the cartridge there may
be an electrode (23), see Figure 2. There may also be an electrode (23)
present near the overflow sink (28) (which could also be used as
electrochemical measurement zones). Through the reader, checking the
electrical continuity between the electrodes, the reader will be able to confirm
that the channels (14, 16) have been successfully filled with sample. This can
be performed through a simple conductance measurement. For a specific
channel, if the electrodes (23) have been successfully wetted with blood
(meaning that both channels have been filled completely with sample) then an
electrical current can conduct from one electrode to the other through the blood
sample. Otherwise if the blood sample is not present, or has only partially filled
the channel, then one of the electrodes will not be wetted, meaning the
electrical current cannot flow from one electrode to another.
In the present cartridge/assay system, it shall be possible to measure the
hematocrit of the blood sample. The design of the cartridge means that the
measurement can be performed without any interference from the reagents that
are used for the primary assay functionality.
Figure 3 shows a portion of the cartridge (1) in more detail and in particular the
fluid stop features (20, 22). An additional feature (60) is shown adjacent to the
sample application point (12). This feature (60) is designed to prevent any
sample from wetting the outer surface of the cartridge upon sample application
The hydrophobic stop features (20, 22) are present on both inner surfaces
removing any hydrophilic path resulting in the fluid stopping at this feature. In
one embodiment two hydrophilic surfaces are utilised however alternative
combinations of hydrophilic/hydrophobic surfaces could be used to fill the strip
by capillary action. In an extreme example of this two hydrophobic inner
surfaces could be utilised and by providing a "sucking" action by way of a pump
in the reader the cartridge may be filled with the sample.
As the blood fills the sample channels (14, 16) (see Figures 4 and 5) the
antibody functionalised magnetic particles (18) (which are pre deposited in the
channel as dry reagents) are resuspended by the blood, thereby allowing
binding any analyte/s present. The blood fills to the stop features (20, 22), see
Figure 5. Once the particles (18) are resuspended, incubation with the blood
sample would be allowed to occur for a defined period of time (incubation time)
and controlled by appropriate software and programming of the reader.
Magnetic particles may be chosen as the capture phase due to their high
mobility and functionality (size dependent i.e. diffusion coefficients etc) to
reduce diffusion distances and ultimately incubation time. This type of reaction
will be very efficient and reproducible at binding analyte from blood samples.
During the magnetic particle binding of analyte, a hematocrit measurement may
performed by hematocrit electrodes (24). The hematocrit value can be used by
the reader to calculate the final concentration of the analtye as the reference
value will be a plasma measurement made by a clinical analyser. A hematocrit
measurement may be required to correct for the concentration difference
associated with analyte present in a given volume of sample due to differing
ratios of red blood cells to plasma. Therefore a whole blood measurement may
be corrected for this difference by means of a hematocrit measurement so that
results are consistent with those associated with a plasma sample.
After the antibody functionalized magnetic particles (18) have bound any
analyte in the blood a permanent magnet (80) or electromagnetic field is used to
hold the analyte-antibody magnetic particle complex in place (see figure 6). A
wash buffer or gas is then delivered from an inlet port (26). The wash medium is
provided from a buffer reservoir/reservoirs present in the reader (a particular
buffer reservoir/reservoirs and hence buffer may be inserted into the reader
depending on the particular assay and hence analyte being detected). A defined
volume of buffer (e.g. 1-2mI ) is expelled from the reservoir/reservoirs of the
reader via a pump system into the sample channels (14,16) pushing the blood
past the fluid stop feature (22) into the sink void (28), leaving the magnetic
particles in buffer. (See Figures 7 and 8). The magnetic particles (18) can be
visualised as a discreet band (82) still held within the channel (14, 16).
After this step a series of further wash steps, as above, may be performed (all
using the magnetic particle holding step by an applied magnetic field) and
where other deposited dry reagents (30, 32) may be resuspended in buffer (for
example, the same buffer as the wash buffer) which is then pumped into the
sample channels (14, 16), to allow binding events to occur in a very controlled
manner. Or as previously described, the washed magnetic particle - analyte
complex which is contained within the clean buffer matrix may be transported
upstream, past stop feature (20) to the location in the strip where the label is
dried down in the disposable test cartridge. At this point the magnetic particle -
analyte complex can bind to the label followed by an additional wash step and
measurement of the label. (In both of these examples only the magnetic beadanalyte
binding reaction occurs in the blood, all the other reaction and/or
binding steps occur in a very controlled buffer environment).
However, the magnetic field may also be "relaxed" (see Figure 9) by moving the
magnet away from the cartridge and thereby reducing the magnetic attraction in
this manner the magnetic particles (18) may still be held by the magnetic field,
albeit less strongly and a more diffuse band (84) of particles may form. Moving
the magnet (80) back towards the cartridge again will serve to concentrate the
particles (18) once more.
In summary, this means that any reagents and/or labels never contact the "dirty"
blood matrix, and all reactions/binding (other than the initial analyte capture
step) is very controlled in a buffered, optionally heated environment to maximise
detection efficiency and prevent/minimise non specific binding and interfering
products to be removed (maximising the repeatability/precision of the
measurement). This allows the present system to use reagents that would not
have normally chosen because they were "problematic" in plasma/blood. In
addition it also means all the detection measurements, such as fluorescent
measurements also occur in a "clean" buffer environment meaning sample
quenching/interference (as expected in blood or plasma) is reduced/removed
allowing very sensitive reproducible measurements to be made. This allows a
much greater choice of detection labels e.g. flurophores, because quenching of
excitation or emission light is minimised.
It should be appreciated that the foregoing description, with reference to figure
1, has been made in relation to a two channel cartridge, but the present
invention also relates to single channel as well as multi-channel e.g. 6, 7, 8 etc
cartridges. Each channel may carry out the same reaction for
reproducibility/accuracy purposes, or may be designed to carry out different
assays - in this way each cartridge may be capable of carrying out a "multi¬
plex" reaction.
Figure 10 shows a similar cartridge (10) to that shown in Figure 1, but
additionally shows fill electrodes (23) which may detect correct filling of the
sample by the cartridge (10). Further electrodes (24) are provided to enable a
hematocrit value to be obtained from the blood sample.
A further embodiment of a cartridge ( 1) in accordance with the present
invention, is shown in Figure 11. In this preferred embodiment 6 channels are
fed by a single sample inlet port instead of the two channels being fed by the
single sample inlet port. The 6 channel strip design is an expanded version of
the 2 channel strip shown in Figure 1 whereby additional channels have been
added which all share the same sink (90). This allows a more effective use of
strip footprint and allows increased multiplexing capacity.
Ultimately a measurement is made (e.g. fluorescent) by a reader using optical
or other detection means, suitable for the label to be detected. For example, if
the label is a fluorescent label the detection means may be able to perform and
detect the excitation and emission of the chosen fluophores: a schematic view
of a hand held reader in accordance with the present invention is shown in
Figures 12 - 15. This embodiment of the reader (MST Pro Meter V 1) is the
specific embodiment that was used to perform the experiments as described in
the Experimental section. In addition all experimental results were obtained
using the 6 channel strip design (as shown in figure 26). The reader (100)
comprises a platform (106) for receiving and holding a cartridge (10) of the
present invention and a sealing head (105) the actuation of which can be
controlled by solenoids (108) for the purpose of producing a sealed system
whereby the instrument can pump either a gas, such as air or a fluid such as a
wash buffer into the strip in a controlled manner. Additionally the reader
comprises a fluid reservoir/reservoirs cartridge ( 111) for holding fluid or gas for
subsequent delivery to the cartridge (10). The reservoir/reservoirs cartridge
may contain a separate chamber for each test channel contained on the strip
such that the fluid / air actuation and control for each test channel is driven
directly from what is effectively a separate pump source. Alternatively the
reservoir/reservoirs cartridge may comprise of one chamber which is then split
into multiple outlets such that the fluid / air actuation and control for each test
channel is driven from a common pump source. The fluid or gas is delivered by
way of an actuator ( 13) acting on the reservoir/reservoirs cartridge. There is
also provided suitable optical detection means (107) and electrical circuitry(1 12)
and an associated computer chip or chip(s) and software for controlling the
reader and conducting the assay. In addition because the described system
has the flexibility to perform many wash and reagent delivery steps many assay
formats can be configured using the current system.
A magnet holder (103) and associated magnet which may be orientated at 45
degrees, (104) can be controlled through the use of a motor ( 1 10) in order to
bring the magnet in contact or close proximity to the test strip, for the purpose of
influencing magnetic or paramagnetic particles contained within the test strip. In
order to perform the assay measurement (e.g. fluorescent) the optical reading
head (107) can be moved along the measurement or detection zones (222) of
each of the multiple test channels in the test cartridge controlled by a motor
(109). Thus the optical reading head can be utilized to perform multiple
measurements across one disposable test cartridge. The plot shown in figure 25
shows the results of an example read of the optical reading head across a test
channel in the test cartridge (using the reader design MST Pro Meter V1). From
the results it can be seen that the instrument can make multiple measurements
across the width of the test channel allowing the peak fluorescence signal to be
identified and transformed into a result through the use of an algorithm and
displayed to the user through the LCD (101). In addition the instrument could
interpret the shape associated with the measurements taken across the test
channel and use this as an on board control, for example if the read response
gives the shape of a steady decay or steady increase instead of a parabolic
response then it could be used to determine an erroneous or non uniform result.
It will be appreciated that the reader is required to very accurately control the
fluid / gas delivery of for example, the buffer wash to the disposable cartridge.
The cartridge may involve a number of separate sample channels, and a
number of wash steps may be required for each channel, with each wash step
having to be conducted at a precise flow rate and with a precise volume. Each
channel may have a plurality of wash interface ports ((26) of Figure 1) over
which a seal is made in order to ensure the correct delivery of the buffer / gas
wash from the reservoir/reservoirs in the reader. A suitable fluidic management
system may be made up of 3 main components (as shown in figure 14), a fluidic
pump/pumps ( 13), a buffer reservoir/reservoirs cartridge 1 1 (and figure 15)
and a reader/cartridge fluidic interface or seal (105).
The fluidic pump/pumps that is used to transport the fluid from the buffer
reservoir/reservoirs cartridge to and throughout the cartridge, may be a stepper
motor linear actuator (for example E21H4U-5-900 Haydon Kerk Motion
Solutions ), the features associated with a stepper motor linear actuator that
make it a desirable solution include the fact that it locks in position when
stopped (so that the fluid cannot push back against the buffer
reservoir/reservoirs cartridge and linear actuator) and that stepper motors have
movement of very fine resolution (e.g. 0.0015mm / motor step).
The reader/cartridge fluidic interface may be achieved by using a soft rubber
coated sealing head in the reader that co-locates with said wash inlet ports (26)
of the cartridge. Alternatively a rubber gasket could be located on the
disposable test cartridge, or there may be a rubber gasket present on both the
sealing head and the disposable test cartridge. The sealing head will have an
outlet (e.g. a hole in the rubber membrane containing the outlet from the fluidic
management system) that lines up with each of the cartridge fluid input ports.
The reader may preferably include a buffer wash reservoir/reservoirs. The buffer
wash reservoir/reservoirs may contain the buffer wash fluid for carrying out
assays on a number of cartridges. Alternatively, in the case where the
reservoir/reservoirs is filled with air, it could be a permanent feature of the
reader design as it would not need to be replaced as has been explained
previously.
In order to make the reader suitable for use by the user, such that replacing the
buffer reservoir/reservoirs cartridge does not result in the fluid spilling out and
over the reader or user, the buffer / gas wash reservoir/reservoirs cartridge and
the reader may be designed to have self sealing interfaces, such that when the
reservoir/reservoirs cartridge is removed from the reader any fluid is sealed
within the confines of the reservoir/reservoirs and the instrument. The seal may
be designed such that it only opens when the reservoir/reservoirs is inserted
correctly into the appropriate interface point in the instrument, a feature may be
incorporated to penetrate the self sealing buffer reservoir/reservoirs cartridge.
To ensure that the buffer fluid inside the buffer wash reservoir/reservoirs
cartridge does not evaporate, which would lead to the formation of air voids
within the cartridge, the buffer reservoir/reservoirs cartridge may have foil seals
at both ends, over a syringe plunger end, and over a self sealing seal end.
These foil seals will be broken by the syringe pump driver and the meter
cartridge interface feature respectively. An example of such an embodiment is
shown in Figure 15, where the reader/sample cartridge fluidic interface is
achieved by using a soft rubber coated sealing head in the reader that
interfaces with the sample cartridge. The sealing head will have an outlet (i.e. a
hole in the rubber membrane containing the outlet from the fluidic management
system) that lines up with each of the strip inlet points.
The buffer / gas reservoir/reservoirs cartridge may have a singular chamber
which drives the various wash steps associated with each test channel from a
single source which is then split into multiple exit port points through various
valves and tubing. Preferably the buffer / gas reservoir/reservoirs cartridge may
have a separate chamber for each test channel such that the wash steps
associated with each test channel are driven from individual sources. This type
of design is also shown in figure 15, and does not require any valves in the
reader or test cartridge. Alternatively the reservoir/reservoirs cartridge may have
a number of chambers which are associated with common sub set groups of
test channels contained in the test cartridge.
As discussed above in order to keep the capture phase and bound analyte etc
from getting washed away during the buffer wash and reagent wash steps, the
magnetic beads in the disposable test strip require to be held by the reader.
This function will be fulfilled through utilising either a permanent magnet or an
electromagnet. It is possible to manipulate the magnetic beads in certain ways
that could help improve the accuracy, sensitivity and range of the measurement.
For example for lower assay fluorescent signals, it may be beneficial for the
magnet to gather all of the magnetic beads into a tightly bound clump,
increasing the density of the fluorophores present and so the intensity of the
light emitted towards the detector. In contrast in cases where there are higher
signals, and the light sensor and reader electronics are close to saturation it
may be beneficial to remove or move the magnet so as to relax or spread the
magnetic beads over a certain area, thus reducing the intensity of the light
emitted towards the sensor. This could be seen as a novel way of using the
magnet to influence both the sensitivity and the range of the assay or affecting
the binding kinetics of the multi-step assays. Typically for a magnet, the points
where the flux density is highest (and so where the magnetic beads will tend to
gravitate toward are along the edge of the magnet as this is where the magnetic
flux lines have the shortest travel path from the north to the south pole.
It is a physical feature of assay development that the ambient temperature can
influence the magnitude of response. In the present invention, this temperature
effect will primarily be driven through the effect of temperature on diffusion,
whereby an increase in temperature can result in increased binding efficiency
between the magnetic beads and the target analyte, and the subsequent
binding to delivered reagents. The present system may be used, for example in
a doctors office and home use, and the range of temperatures the system may
be exposed to will be broad, from perhaps as low as 10 Celsius to as high as
35 Celsius. One method of removing this temperature effect is in the heating of
the test strip to a pre determined temperature, for example 40 degrees, this
would remove any variation associated with the assay due to temperature
effects. Thus, the reader may also comprise temperature control means, such
as a heater.
The temperature control of the cartridge can be implemented by utilising the top
surface of the optical block (which will be in contact with the strip and may be
made from a heat conducting metal, such as aluminium, which has good
thermal conductivity properties) as a heater in order to maintain the temperature
of the sample cartridge. Alternatively the support platform on which the
disposable test cartridge rests within the reader could be utilized as the heated
surface that contacts the strip. The heating of the heated surface can be
performed using a high wattage, low value (e.g. 1 ohm) resistor, or though using
a MOSFET with the heatsink tab of the MOSFET attached to the optical block
top surface. The temperature may be controlled by placing a temperature
sensor on the heating block surface and using the output of this temperature
sensor to modify the current flow through the high wattage resistor / MOSFET.
One additional advantage of this implementation is that the MPPC silicon
photodiode gain (The MPPC silicon photodiode is the detector used to measure
the intensity of light emitted by the target fluorophores in the test strip) is
sensitive to ambient temperature, therefore implementing the optical block top
surface as a heater would also ensure that the ambient temperature in proximity
to the MPPC silicon photodiode is controlled.
An alternative method of heating the sample cartridge would be to create a very
thin heating element. The fact that the heating element would be very thin (100-
250 microns) would also mean that a magnet could be positioned underneath
the element and still be in close proximity to the cartridge so that the magnetic
beads can be gathered. The thin heating element could take the form similar to
that of a flexible PCB, with copper tracks sandwiched between two polymer
layers. One side of the layer could then be coated with a reflective material, or a
have a reflective layer adhered onto it to make the mirrored surface if the
heating element were required to reflect light emitted from the target
fluorophores back towards the MPPC silicon photodiode. Alternatively, in the
event that background fluorescence becomes an issue in the system, one side
of the element could have a matt black finish as opposed to a reflective surface.
The Optical block of the reader of the present invention may be capable of
providing the light sources for multiple fluorophore excitation wavelengths and
measuring the subsequent emitted light from the fluorophores. It is the intensity
of this light emitted from the fluorophores in the disposable test cartridge that
will provide the assay measurement. The Optical Measurement Block is
responsible for measuring the amount of target analyte present in the cartridge
through the associated bound labels, such as fluorophores. The Optical
measurement block may comprise a multi pixel photon counter (MPPC) silicon
photodiode (for example Hamamatsu S10362-1 1-100C) and a high power
wideband LED which emits a broad spectrum of wavelengths (for example
HP803WW Roithner LaserTechnik GmbH).
A MPPC silicon photodiode may be preferred as it has a very high internal gain
(in the region of 1 million) compared to a standard photodiode (gain =1) or an
avalanche photodiode (gain = in the region of 100). One convenient feature of
the MPPC silicone photodiode is that its internal gain varies in relation to the
reverse bias voltage that is applied to it (for example a bias voltage of 70V
results in a gain of approx 1 million while a bias voltage of 65V results in a gain
of approx 100,000). This relationship can be used by the reader to manipulate
the dynamic range of the measurement system, i.e. for higher analyte
concentrations the photodiode bias voltage can be reduced to ensure the
photodiode output does not saturate the instrument electronics. It should be
noted that in alternative embodiments a standard photodiode or an avalanche
photodiode could be implemented in place of a MPPC silicon photodiode.
A high power wideband LED is convenient so that a single LED can be used to
generate multiple excitation wavelengths (i.e. the light that is incident on the
target analyte fluorophores) through having a filter slide that can be moved to
place different filters in front of the LED to generate different wavelengths. The
filter slide also contains filters associated with the silicon photodiode in order to
block out the excitation light so that the silicon photodiode measures only the
light emitted by the fluorophores.
With reference to the 2 channel cartridge as shown in figure 1, the Optical
measurement block may be arranged such that there are 2 sets of LED and
silicon photodiodes, one for each channel in the strip. The location of the
photodiodes and LEDs are fixed. The filter slide can be moved such that
different filters can be placed between the LEDs and the MPPC silicon
photodiodes for different measurements. Alternatively, instead of having 2 fixed
sets of LED and silicon photodiodes, there could be a single optical head
implemented that can move between the different test channels.
Alternative embodiments of the stated configuration of the optical block are as
follows:
1. In one embodiment, both the excitation source and the emission
detector are located on the same face of the cartridge. An alternative to
this is that the cartridge is sandwiched between the emission source and
the emission detector.
A reason that the source and detector may be located on the same
surface is to allow space for the integration of the cartridge with the
fluidic management system, cartridge connector interface, heating block
and magnetic particle control.
2. In another embodiment, there is the implementation of a movable filter
slide to allow selectivity in the excitation and detection of a number of
fluorophores. In a simpler configuration where only a single fluorophore
per channel is required to be detected, the filter slide would not be
required and could be replaced with a single, fixed excitation filter and a
single emission filter for each channel.
In a further embodiment, the light source for the excitation of the
fluorophores comes from a broad band emitter. Alternative
implementations where there is not the need to excite at multiple
wavelengths could include wavelength specific light sources such as
narrow band LED's.
Another alternative to a wideband LED would be a xenon flash lamp, the
intensity of a xenon flash lamp is much greater than that of an LED. In
addition a xenon flash lamp emits over a larger range of wavelengths.
Where broad band 'white' or 'warm white' LEDS can emit down to a
wavelength of around 400nm, xenon flash lamps can emit down to
around 200nm meaning that xenon flash lamps can be used for
excitation wavelengths in the UV range.
In another embodiment, the optic block configuration could comprise of
directing the excitation light at a dichroic mirror (or beamsplitter)
mounted at an angle of 45 degrees to the normal which reflects the
excitation light through 90 degrees towards the sample in the disposable
test cartridge. The dichroic mirror is chosen such that the excitation light
generated by the fluorophore is at a wavelength that travels through the
dichroic mirror (i.e. is not reflected back towards the emission source)
where the silicon photodiode is situated for detection of the emitted light.
Additional optical filters may also be placed in front of the light source
and optical detector in this configuration in order to narrow the pass
band of light wavelengths produced by the emitter and accepted by the
detector.
Due to the variation in processes associated with the manufacturing of
disposable assay test cartridges it is normally required for each batch of
cartridges to be characterised and for specific calibration values to be entered
into the reader so that the assay response generated by the test cartridge can
be normalised by the reader internally before the final assay result is reported.
In the present invention, there exists the opportunity to utilise the buffer
cartridge as a tool for transporting particular data of interest, for example assay
calibration parameters about its associated batch of cartridges. One
implementation could include the attachment of an EEPROM memory chip to
the reservoir/reservoirs cartridge, whereby the data in the EEPROM memory
chip can be read by the reader.
In one particular embodiment, the sample cartridge and associated reader are
designed for carrying out an immunoassay, where the analyte to be detected is
an antigen and the binding agent is an antibody. Paramagnetic particles may be
functionalised by attachment of antibodies against either free or free and
complexed antigen.
Due to the present cartridge design, importantly the only binding reaction that
occurs initially is the antibody binding to any antigen in blood, this results in a
number of advantages. Primarily it means multi-step assays can be performed,
meaning the chosen label molecule/particle never contacts ("sees") the blood.
This is a significant benefit over existing POC immunoassay technologies
whereby both capture phase and label are in contact with the sample. The
typical POC immunoassays binding schemes generally consist of either planar
or magnetic capture particles and a label which could be bound to a particle,
conjugate or polymer etc. However, both capture and label phase contact the
sample (blood, plasma etc). This can lead to a number of problems. Within
immunoassays there are many species that interfere with the immunoassay
binding steps. Prime candidates are human anti - animal antibodies such as
Human Anti-Mouse Antibodies (HAMA), animal anti - animal antibodies (in the
case of veterinary applications), rheumatoid factor, anti-BSA antibodies,
fibrinogen etc. Specific non-specific binding (HAMA, rheumatoid factor) and
non-specific binding can result in highly inaccurate results resulting in poor
performance and ultimately inaccurate diagnosis of patient samples. This is
especially true as POC immunoassays are generally referenced to clinical
analyser performance which incorporate multi-step assays, very effective wash
steps and label detection in a clean matrix. Both specific non specific and non
specific binding results in label bound to the capture phase in a manner not
consistent with concentration of analyte (can be higher or lower than the
expected result) resulting in an inaccurate result. Fluorescent latex particles
may be employed which are functionalised by attachment of antibodies against
free and complexed antigen. Thus a sandwich may be formed between the
magnetic particles, antigen to be detected and fluorescent latex particles.
In one embodiment of the present system however, the detection label, such as
a fluorescent moiety never contacts the blood, as the blood is washed away
prior to any label being brought into contact with the bound analyte. Therefore
any of these matrix events that could facilitate the non specific binding cannot
occur because the magnetic beads (18) and detection label are not present in
the blood at the same time, just like some clinical analyser systems. This is very
important when considering the Allowable Total Error (ATE) of an
immunoassay. There is a drive within regulatory authorities, such as the FDA to
tighten the ATE on any new assay products to increase the accuracy of tests
with respect to the reference systems.
For a given population of clinical samples, even if the majority of the
bloods/samples recover accurately the ATE can be heavily affected by a few
inaccurate responses. It is therefore very important that any new platform
technology is designed to minimise these effects. The present invention aims to
do that appropriate design of the cartridge and associated reader, thereby
reducing sample to sample bias (e.g. elimination of specific non specific binding
and non specific binding).
Another advantage of only performing the magnetic particle capture step in the
untreated sample, such as blood is that blood measurements can become very
accurate. Most immunoassays are highly sensitive with regards to reagent
concentrations (i.e. concentration of the capture phase and label phase).
Sufficient reagent concentration is required to drive slope whilst too high a
reagent concentration results in increased assay intercepts. In a whole blood
assay this problem can be further exaggerated as even if you deposit exactly
the same reagent volume and concentration, the reagents will have different
concentrations in different hematocrit bloods (as the reagents will resuspend in
different volumes of plasma). For example, the same deposited reagents in a
60% hematocrit blood will have .87 times less volume than a 25% hematocrit
blood. As a result the reagents will be more concentrated in the 60% hematocrit
blood than in the 25% hematocrit blood. This alone could cause blood to blood
bias problems as both the capture and label phase will vary in concentrations,
therefore the overall capture efficiency (number of capture phase-analyte-label
phase interactions made out of the total number of capture phase-analyte-label
phase interactions possible = capture efficiency) will vary between bloods due
to hematocrit effects alone. This further coupled with the varying viscosities of
different bloods (and plasma's) affects the diffusion coefficients of any mobile
reagent which can result in poor ATE.
Having only the magnetic particles binding analyte in the blood helps to
significantly reduce this problem as the highly mobile and functional magnetic
particles will be highly efficient with regards to binding the analyte. Therefore
the magnetic particle concentration difference in high or low hematocrit bloods
is less important as nearly all the available analyte is bound in this step. The
blood to blood viscosities effects are further minimised by the applied cartridge
heating, as there will be no temperature effect as the cartridge will always have
a constant temperature as previously described.
For an immunoassay once unbound agents in the blood have been actively
washed out of the sample channel out into the sink, the reader then delivers a
small amount of buffer into the sample cartridge through a buffer inlet point on
the strip containing either a dry deposition of antibody functionalised fluorescent
latex bead (or other secondary binding reagents). The antibody labelled
fluorescent latex bead is resuspended by the buffer and then pumped into the
channel containing the magnetic particle-analyte complexes. The permanent
magnet or electromagnet housed in the reader is still applying a magnetic field
at this point and "holding" the magnetic particle-analyte complexes in place in
the channel. Once the antibody functionalised fluorescent latex has been
transported by the reader into the channel containing the magnetic particle -
analyte complexes, the magnetic field is removed allowing a second binding
reaction to occur. (Though it would also be possible to allow the binding phase
while the magnet is in place and the magnetic beads are held in place).
Alternatively the magnetic particle - analyte complex could be transported
upstream in the sample cartridge, to contact the label. Alternatively as
previously described all the binding reagents (magnetic particle capture phase
and label) could be deposited in the sample channel and the binding reaction
would occur in the sample channel. The magnetic accumulation and air/fluid
wash by the reader ensures the magnetic particle-analyte-label complexes are
quantified in an air/fluid environment.
The flexibility of the present system allows the potential for a very flexible and
sensitive measurement. The whole system at this point is set up for high
capture efficiency resulting in very sensitive assays as the magnetic particles
are effectively preloaded with analyte, therefore collisions with .label .should
result in successful binding events. For clarity a binding scheme will be
described, however due to the flexibility of the present platform design nearly
any assay architecture conceived can be formatted.
A wash step is generally performed to remove any unbound label Subsequent
optical measurement is then made in a 'clean' environment. In the case of
fluorescent detection, this allows the use of fluorophores which would not be
usable in some matrices (for example blood) where significant quenching of the
specific signal would (without removal of this matrix) otherwise occur.
Further amplification of signal if needed could be delivered if required by
resuspending and pumping a tertiary binding reagent into the channel. For
example an additional labelled particle (same or different label) with antibodies
against a component of the primary labelled particle could be used (e.g. an
immunogen coupled to the labelled particle to which good antibodies exist).
Once again a magnetic holding and wash step would be performed to remove
any unbound secondary label coated labelled particles. The reader would then
measure the label in the same manner (if the same label was used) or in a
different manner if a different label was used. This additional measurement
could be used to increase the range and sensitivity of the measurement.
Many current POC immunoassays that use fluorescent labels measure the
labels in a blood or plasma matrix. As previously described there are many
components in blood and plasma (human serum albumin, bilirubin,
haemoglobin etc) that interfere with either the excitation or emission wavelength
used to measure the concentration of the captured fluorophore. As a result the
precision within and between bloods of other POC immunoassays can be
affected purely by the measurement of the fluorescent label alone (in addition to
all the matrix binding problems). As a result fluorophores are used that try to
minimise these effects, these are therefore not necessarily the best fluorescent
label to use but a necessity of making the fluorescent measurement in a "dirty
matrix". The present platform will however, allow an extended choice of
fluorescent labels allowing the labels that result in the best assay performance
to be chosen. This alone is an advantage; it also means multiplexing can more
easily be achieved as discussed.
The present invention makes the possibility of multiplexing (i.e. the detection of
more than one analyte using a single sample) within a single channel using
magnetic particles as a capture phase possible. For example, if it is not possible
to use multiple fluorescent labels due to the optical properties of blood and
plasma then multiplexing would not be possible within a channel because there
is no way to provide specificity of measurement as the magnetic particles
cannot be spatially distributed into magnetic particles specific for analyte 1, 2
etc. This is why planar capture is used as capture phase in POC platforms that
can perform multiplexing. For example, some panel tests use the same
fluorescent label (due to the blood and plasma limitations) for the labels with the
capture antibodies for each analyte spatially distributed on the strip to allow
multiplexing capability. As the magnetic particles are mobile and susceptible to
the applied magnetic field, specificity due to spatial distribution of the magnetic
particles (specific against different analytes) is difficult to achieve due to the
fluorescent label optical limitations in plasma or blood. However, by providing a
wash step as in the present invention, multiplexing within a single channel using
magnetic particles' and fluorescent labels become a reality. In addition the 6
channel strip design allows multiplexing to be achieved with one fluorophore
and thus increase the overall multiplexing capability when within channel
multiplexing (multiple flurophores) is taken into account.
Due to the flexibility offered by the ability to perform one step, multi-step assays
and multiple wash steps there is a great opportunity to extend analyte
measurement ranges or linearise the dose response curve of an assay. For
example, typical immunoassay dose response curves are sigmoidal. This is
driven by either reagent saturation (insufficient reagent to maintain linear
binding) or saturation of the label/detection method (i.e. the detection methods
becomes saturated and can no longer measure the label in a linear fashion).
The present platform will however, allow a full linear response across the
measurement range, and this can be achieved by several methods. For
example, the reader can measure the concentration of the fluorescent particles
at each stage of the multi step assay. Therefore after the initial fluorescent
particle binding step has occurred, the reader could measure the fluorescence
intensity. This could be a range measurement, for example, so if the intensity is
over a threshold value (set during calibration) it uses the fluorescence intensity
to calculate the analyte concentration (calibration 1) and the test stops at this
point. If the fluorescence intensity is however below the threshold value this
would indicate a low analyte concentration and the previously described
additional amplification steps would occur (calibration 2) and the subsequent
fluorescence intensity used to calculate the analyte concentration. This could
also be achieved by using the two channels whereby one channel is tuned to
make very sensitive measurements whilst the other is tuned (reagent
concentration and/or binding time) to make linear measurements across the
remaining portion of the analyte measurement range. Optical saturation
resulting in non linearity can also be combated by reader. The present platform
allows many different ways of achieving linear responses across the
measurable ranges, which will allow more accurate calibration resulting in better
within and between blood precision resulting in better ATE. It is envisaged that
magnetic particle distribution could be used to affect both the measurement of
the fluorescent label and the binding reactions. For example, the magnetic
particles could be measured as a homogenous/distributed distribution
throughout the channel allowing range measurements whilst the same magnetic
particles could then be accumulated to increase the fluorescence intensity to
drive the sensitivity of the measurement. Likewise the same principle could be
used to affect the binding reactions and tune the assays accordingly.
In the cartridge design shown in figure 1, two identical channels are shown;
however, measurements do not require this, therefore one channel in the
cartridge may be employed, and this could allow a sub 1mI_ immunoassay
measurement.
In comparison to the 2 channel strip design, the channels within the 6 channel
strip design (shown in Figure 11) are joined. In the 2 channel strip the two
channels are separate entities that are not joined. The 6 channel strip design
can have joined channels (as can the 2 channel strip design) because the
system is a sealed system. In figure , electrodes are shown that could be
used for electrochemical measurements (219) that could be situated in one or
more of the sample channels. Electrodes are also shown that could be used for
electrochemical and / or detection of sample fluid as well as acting as fluidic
stop features (220, 221 ). Figure 11 also shows 2 alternative strip design
features that would lend the strip to be filled with the test sample being
presented to either the end of the strip (217) or the top of the strip (218). Each
test channel within the test cartridge can have one or more wash ports (206,
208, 200, 202).
The strip wash ports (200, 202) are closed (sealed by the reader). After a
certain amount of time, (e.g. 5 minutes), the reader can position a magnet in
contact with the test strip, or in close proximity to it, which will result in the
magnetic beads being accumulated into a tight band which will be held in
position during the primary wash step. The syringe pump cartridge can then be
actuated in order to perform a wash step using liquid or gas, either one channel
at a time, for example using valves to control the opening and closing off of
each channel (e.g., the first port (206) in isolation). Or all channels at the same
time whereby each port (206 and 202) is open to the syringe pump cartridge
through the absence of a valve system, or a valve system where all associated
valves are open. The preferred embodiment is the case where no valves are
present, neither in the reader nor the disposable test cartridge..When the blood
is washed out by buffer or gas (e.g. air) being introduced through the first port
(206) the blood is pushed over a fluidic stop feature (221) into the sink (210).
The previously described magnetic particle accumulation, relaxation processes
by permanent or electromagnets etc remains exactly the same. In the case
where the syringe pump cartridge has washed the test cartridge test channels
with gas for a one step assay, at this point the final fluorescent measurement for
each channel can be made in the detection / measurement area (222). Note
that in this case the measurement is that of an accumulated magnetic bead
band - fluorophore complex in air. In the case of a multiple step assay, the
reader is required to perform additional tasks as stated below.
The blood is not pushed into the other channels (212, 213, 214, 215, 216) as
the ports (200, 202) are either sealed or under positive pressure / force due to
the syringe pump cartridge acting on these ports at the same time. (In this case
no valves are required neither in the reader nor the disposable test cartridge).
Therefore the only place for the displaced blood to go is into the sink (210).
Each channel has at least one fluid input port(s)s, therefore as the first port is
open (e.g. 206) and being used as a port to dispense fluid into the cartridge,
additional ports (e.g. 208) located up channel can be closed preventing any
fluid being dispensed up the channel. The blood is therefore washed from the
channel and into the sink.
A secondary delivery of label can be performed or alternatively the magnetic
particle analyte complex can be transferred up the channel to the zone where
the label is deposited. .(21 1,212,213,214,215,216). Note that printed features
on the strip design can be used to contain these reagents in a pre defined area.
This concept could also be applied to the primary reagents deposited in the test
sample channel (223). This secondary delivery of label process can again be
performed individually in a channel by channel process, or be performed on all
channels at once. When multiple ports are used for a channel, this process
relies on the ports (200, 202, 206, 208) being opened and closed (only one port
is open per channel at a time), In the case of a single port associated with each
channel, this is not necessary. In the case of the delivery of the label the first
strip wash port (206) used for the wash step would be closed, and the second
port located up channel would then be opened and used to dispense fluid to
rehydrate and deliver the label to the magnetic particle-analyte complex or
remove air (resulting in a transferral event). The transferral event is possible
because the strip is a sealed system, and instead of pumping fluid into the strip,
air is sucked from the strip by the "pump" resulting in the magnetic particleanalyte
complex in buffer being sucked along the channel to the deposited
label. This process would then be repeated for the remaining channels (212,
213, 214, 215, 216).
In the case where there is only one port associated with each test channel
(Figure 26, 224), the air volume present in the area (225) between the test
sample and the port can be used to displace the sample from the sample
channel area (filled from the sample entry port (226)). Thus in the case of the
syringe pump cartridge containing fluid, only one port is required to perform a 2
step assay, as the primary wash is performed using air that is already present in
the test cartridge test channel, allowing the subsequent fluid pumped from the
cartridge to rehydrate the secondary reagents (deposited in region identified by
point 227) and present them to the magnetic particle complex present in the
sample channel area of the test channel. It is possible to perform a 1 step assay
with an air wash with only 1 port (224) associated with each channel, this is the
cartridge design ( ST Pro Strip V 1, Figure 26) associated with the experimental
section.
As discussed previously the present platform allows multiple analytes to be
measured within each channel. Multiplexing can be performed easily in one
channel by depositing magnetic particles with different binding agents against
the analytes to be measured. This could be achieved by making individual
preparations of antibody functionalised magnetic particles and then combining
them and depositing them into the sample channel. In comparison a single
preparation could be made whereby a mixed antibody population (against all
the analytes that are going to be measured) are coupled to a magnetic particle
population resulting in magnetic particles having a number of different
antibodies coupled thereto. In both embodiments, fluorescent latex
particles/fluropohores preparations could be made conferring the required
antibody specificity against the analytes to be measured allowing a classical
immunoassay "sandwich" complex to be made (immunoassays employing
fluorescent measurements in blood and plasma could not perform these
measurements as magnetic particles specific to different analytes could not be
spatially distributed as previously described). Further amplification steps could
still be performed if needed; and the reagents could be further tuned to allow
this. There is an extensive range of flurophores available with different
excitation and emission wavelengths and it is envisaged that up to 5 different
analytes could be measured within one channel. In addition to the
immunoassay measurements, electrochemical measurements could also be
incorporated into the strip measurement. For example, an electrochemical
measurement of glucose concentration and a glycosylated haemoglobin
measurement could be performed using the present platform. For example, in
the case of an electrochemical glucose measurement, an additional set of
electrodes would be incorporated mirroring the position of the hematocrit
electrodes in the other channel. All the necessary reagents would be deposited
on the electrodes. As the blood fills the channels and all the previously
described immunoassay events occur, the electrochemical measurement of the
blood glucose occurs, the reader interprets the glucose concentration and the
blood is washed out into the sink. The present platform will therefore be able to
incorporate a very diverse range of measurements upon one cartridge.
Many of the current POC immunoassays platforms require refrigerated storage
of the strips. The present invention may avoid such problems by employing
features that make room temperature stability possible to obtain. For example
other POC immunoassay systems have buffer pouches that contain an
enzymatic substrate (a wet reagent), these substrates have limited room
temperature stability, and as a result the product has a refrigerated stability
profile. In comparison the only "wet" reagent" of the present invention is the
buffer reservoir/reservoirs cartridge. This is not contained within the sample
cartridge and as buffer does not generally go off at room temperature, the
present invention avoids this problem. Likewise none of the reagents are wet
reagents, as they will all be deposited in the cartridge and resuspended by the
buffer when delivered to the sample channel or resuspended by the blood.
Deposited dry reagents will thus avoid any wet reagent instability; likewise
enzyme labels (i.e. enzyme-antibody labels) have been avoided (due to their
poor stability profiles) and stabilisation formulations can be optimised for a
single reagent (e.g. magnetic particle) without impacting the stability profile of
other reagents (e.g. the label).
Experimental Section
Materials:
Maleimide-PEG2-biotin:
Thermo Scientific, Cat 2 1901 (EZ-link maleimide-PEG2-biotin).
Fluorescent amine latex particles:
Invitrogen, Cat F8765 (1 m yellow-green fluospheres with amine surface
functionalisation)
Fluorescent neutravidin latex particles:
Invitrogen, Cat F8776 ( 1pm yellow-green fluospheres with neutravidin surface
functionalisation)
Paramagnetic particles :
Ademtech, Cat 03223 (200nm Strep+ paramagnetic particles)
Antibody 1H12:
Hytest, Cat 4P33 Ab 1H 2 (Anti-PSA, human)
Antibody 5A6:
Hytest, Cat 4P33 MAb 5A6 (Anti-PSA, human)
PBS:
Thermo Scientific, Cat 28372 (BupH phosphate buffered saline packs)
BSA:
Sigma, Cat A4503-50G (Albumin, from bovine serum)
Water:
Sigma, Cat W4502 (water for molecular biology)
2MEA:
Thermo Scientific, Cat 20408 (2-mercaptoethanolamine hydrochloride)
SPDP:
Pierce, Cat 2 1857 (N -Succinimidyl 3-(2-pyridyldithio)-propionate)
PSA:
Hytest, Cat 8P78 (prostate specific antigen)
Biotin quantification kit:
Thermo Scientific, Cat 28005 (Pierce biotin quantification kit)
Size exclusion columns:
Thermo Scientific, Cat 89882 (Zeba spin desalting columns)
Size exclusion columns:
GE Healthcare, Cat 17-0851 -01 (PD1 0 columns)
EDTA:
Sigma, Cat EDS-1 00G (ethylenediamine tetracetic acid, anhydrous)
Tween:
Sigma P7949-100ML (Tween-20)
D SO:
Thermo Scientific, Cat 20684 (dimethylsulfoxide)
Reagent preparation
Preparation of paramagnetic particles and latex particles using
streptavidin-biotin and neutravidin-biotin interactions respectively.
Antibody.
Antibody disulphide bond reduction for biotinylation
Use undiluted antibody (1H12 and 5A6) stock at a concentration between 2 and
7mg/ml. An appropriate volume of antibody stock is removed to give 1mg
antibody. An appropriate volume of 14.28mM EDTA in PBS, pH7.2 is added to
1mg antibody to give an EDTA concentration of 1mM.
6mg of 2MEA is dissolved in 100ul 1mM EDTA in PBS, pH7.2. 1ul of this 2 EA
solution is added per 10ul of antibody solution. This solution is mixed and
incubated in a waterbath at 37deg for 90min.
This solution is then passed through a PD10 column (pre-equilibrated with 1mM
EDTA in PBS, pH7.2) and 500m I fractions collected. A sample from each
fraction is taken and measured on UV spectrophotometer, with the absorbance
at 280nm used to quantify the protein found in each fraction. The fractions
containing significant concentrations of protein are chosen and combined and
remeasured on the UV spectrophotometer. This measurement is used to
determine the antibody concentration using an extinction coefficient of the
antibody of 1mg/ml = 1.4 absorbance units at 280nm.
Binding of maleimide-PEG2-biotin to antibody
Maleimide -PEG2-biotin is dissolved in 1mM EDTA in PBS, pH7.2 to give a
20mM solution. An appropriate volume of this is added to the reduced antibody
to give a 40 times molar excess of maleimide-PEG2-biotin over reduced
antibody. This is then mixed and incubated for 3hours at room temperature.
This is then passed through another PD10 column which has been preequilibrated
with 1mM EDTA in PBS, pH7.2. 500m I fractions are collected and
measured using the UV spectrophotometer at 280nm. The fractions containing
significant protein levels are chosen and combined. A sample of this solution is
measured again at 280nm by absorbance, and the concentration of antibody
determined using the extinction coefficient of the antibody of 1mg/ml = 1.4
absorbance units at 280nm. The number of biotins bound per antibody are then
determined using the Pierce biotin quantification kit, according to the
manufacturer's instructions.
Latex.
Biotinylated antibody 5A6 binding to latex
1 m neutravidin coated latex is washed in 0.1% tween-20 in PBS, pH 7.2 (using
centrifugation at 16100xg for 3.5min, 4degC) and resuspended in the same at a
concentration of 0.5% solids. Biotinylated antibody 5A6 is diluted to a
concentration of 200pg/ml in 0.1% tween-20 in PBS, pH 7.2. An equal volume
of 200pg/ml b5A6 is then added to 0.5% latex. This solution is mixed well and
incubated for 2hours at room temperature with shaking on a rotary mixer
(30rpm) in the dark.
The particles are then washed 4 times (using centrifugation at 16100xg, 3.5min
at 4degC) with an equal volume of PBS, pH7.2 to remove any unbound
biotinylated antibody and resuspended in PBS, pH7.2 to give a latex
concentration of 0.25% solids.
This will be referred to in the text as functionalized latex 1.
Paramagnetic particles
Binding of antibody to particle
200nm streptavidin coated paramagnetic particles are washed (using a
magnetic separator) in 0.1% tween in PBS, pH7.2 and resuspended in the
same to give a concentration of 0.5% solids. Biotinylated antibody 1H12 is
diluted in 0.1% tween in PBS, pH7.2 to give 50ug/ml. An equal volume of 0.5%
paramagnetic particles and 50ug/ml biotinylated antibody are combined, mixed
and allowed to incubate for 70min at room temperature, with shaking using a
rotary shaker at 30rpm.
The paramagnetic particles were then washed 4 times (using a magnetic
separator) in an equal volume of 0.1% tween in PBS, pH7.2 and resuspended
in the same to give a concentration of paramagnetic particles of 0.5% solids.
This will be referred to in the text as functionalized paramagnetic particles.
Preparation of latex particles using amine-SPDP interactions.
Antibody.
Antibody disulphide bond reduction for binding to SPDP
Use undiluted antibody (5A6) stock at a concentration between 2 and 7mg/ml.
An appropriate volume of antibody stock is removed to give 100 g antibody. An
appropriate volume of 14.28mM EDTA in PBS, pH7.2 is added to 100 g
antibody to give an EDTA concentration of 1mM.
6mg of 2MEA is dissolved in 625m I 1mM EDTA in PBS, pH7.2. 1ul of this 2MEA
solution is added per 0ul of antibody solution. This solution is mixed and
incubated in a waterbath at 37deg for 90min.
This solution is then passed through a Zeba spin desalting column (preequilibrated
with 1mM EDTA in PBS, pH7.2) and the flow through collected. A
sample of flow through is taken and measured on UV spectrophotometer, with
the absorbance at 280nm recorded. This measurement is used to determine the
antibody concentration using an extinction coefficient of the antibody of 1mg/ml
= 1.4 absorbance units at 280nm.
Latex.
Amine functionalized fluorescent latex is washed in 1m EDTA in PBS, pH 7.2
(using centrifugation at 16100xg for 3.5min, 4degC) and resuspended in the
same at a concentration of 0.5% solids.
SPDP is dissolved in an appropriate volume of DMSO to give 20mM
concentration SPDP. SPDP in DMSO is then added to the 0.5% latex to give
1mM SPDP. This is mixed and incubated in the dark for 70min with gentle
shaking (30rpm on rotary mixer). The latex is then washed 3 times with 2x
reaction volume of 1mM EDTA in PBS, pH 7.2 (using centrifugation at 16100xg
for 3.5min, 4degC). The latex is then resuspended in the same in the
appropriate volume to give a latex concentration of 0.5% solids. This gives latex
that is bound to SPDP, ready for attachment of reduced antibody.
Binding of reduced antibody latex with bound SPDP.
Reduced antibody 5A6 is diluted to 1mg/ml in 1mM EDTA in PBS, pH 7.2.
1mg/ml antibody is then mixed with 0.5% latex bound to SPDP in a 1:1 ratio.
This gives a binding mixture of 0.25% latex with 500pg/ml antibody in 1mM
EDTA in PBS, pH 7.2.
This binding reaction is incubated at room temperature in the dark for 19.5hours
and then washed 4 times with 1x reaction volume of PBS, pH 7.2 (using
centrifugation at 16100xg for 3.5min, 4degC). The latex was then resuspended
in PBS, pH 7.2 in the appropriate volume to give 0.25% solids.
This will be referred to in the text as functionalized latex 2.
Assay procedures
Assay 1: 1 step wet assay with manual wash
7m I 0.5% functionalized paramagnetic particles (with bound b 1H12) is added to
an eppendorf and placed on a magnetic separator. The supernatant is removed
and the particles resuspended in 49m I 30mg/ml BSA in 0.05% tween-20 in PBS,
pH 7.2. To this, 7m I 0.25% functionalized latex 1(with biotinylated 5A6 bound) is
added and the solution mixed.
8m I of this mixture is removed and added to 2m I PSA protein in 60mg/ml BSA in
PBS, pH 7.2 (N.B. PSA is at 5x required final concentration). This is mixed and
incubated for 5min at room temperature.
The eppendorf is then added to a magnetic separator and the supernatant
carefully removed. 20m I 0.05% tween-20 in PBS, pH 7.2 is then added to the
pellet, whilst remaining on the magnetic separator. The supernatant is removed
and a fresh 20m I 0.05% tween-20 in PBS, pH 7.2 used to resuspend the particle
complexes, once removed from the magnetic separator.
This is repeated with several different concentrations of PSA, producing washed
wet assay complexes which have been diluted 2x from the original reaction.
These diluted, washed complexes are measured in 3 different ways as follows:
Washed wet assay measurement 1:
2m I washed wet assay complexes are added to 38m I PBS in a 384 well black
Optiplate for fluorescent measurement. The plate is then measured using a
Perkin Elmer Victor3 V. The fluorescent signal in the well is measured using the
inbuilt program 'Fluorescein (485nm/535nm, 0.1s)', adapted for use in a 384
well format. This program uses excitation at 485nm and emission at 535nm with
a 0.1s measurement time.
Washed wet assay measurement 2:
8m I washed wet assay complexes are used to fill a 'MST pro strip V1' cartridge
(as shown in figure 26). The washed wet assay reagents were applied to the
cartridge via sample inlet port (226). This allowed the sample (in this case
washed wet assay reagents) to fill the 6 channels up to fluidic stop features
(229, 228, 239, 237) (which can also act as fill detect electrodes via electrical
connection to the reader through connector (230). This cartridge is inserted into
a 'MST pro meter V1' reader (as shown in figure 12) and the signal measured
by the optics in the reader which scans each channel using a linear movement
of the optical head at position (222, 242) In this method, the washed wet assay
complexes are spread homogeneously throughout the channel and hence
detection area (i.e. they are not concentrated into a band by use of a magnetic
field). (It should be noted that measurements are not made in channel (243) in
these experiments as this has been set up as an hematocrit correction channel
with hematocrit electrodes (237) for whole blood measurements which are not
used here. It should also be noted that the hematocrit electrodes, have in this
case been made from a hydrophobic material to prevent filling of the entire
channel and hence waste sample. For functioning hematocrit electrodes an less
hydrophobic or hydrophilic material would be used.)
Washed wet assay measurement 3:
The cartridges (MST pro strip V ) filled with washed wet assay complexes, as
described in washed wet assay measurement 2 above were then remeasured
using the Victor3 V. This was carried out by attaching the cartridge to a 384 well
black Optiplate and aligning the cartridge channel to be measured (at position
222, 241) over a specific well. The fluorescent signal of the channel was then
measured by measuring the signal of the appropriate corresponding well using
the inbuilt program 'Fluorescein (485nm/535nm, 0.1s)', adapted for use in a 384
well format. This program uses excitation at 485nm and emission at 535nm with
a 0.1s measurement time. This was repeated for each channel to be measured.
Results for Assay 1:
Results for comparisons between the different measurement methods of the
washed wet assay complexes are shown in figures 16, 17 and 18.
Figure 6 shows the total PSA washed wet assays measured in a MST pro strip
V 1 in the MST Pro Meter V 1 (washed wet assay measurement 2). Total PSA
wet assays were performed as per the experimental methodology described
above. The total PSA washed wet assay was measured in the MST Pro Meter
V 1 as shown in figure 12. The results clearly show a systematic assay response
with an initial linear phase followed by a non linear phase. The clinical cut off for
Total PSA assays used in the screening of prostate cancer is 4 ng/ml. The
assay is clearly sensitive enough to make accurate measurements above the
below the cut off threshold value in a quantitative manor. In this data set the
paramagnetic particle-PSA-latex bound complexes are homogenously
distributed throughout the strip channels during the optical measurement phase.
The MST Pro Meter has only been utilised to measure Total PSA washed wets
assays in a strip.
Figure 17 shows the Correlation between the Total PSA washed wet assays
measured in strips in the MST Pro meter and the Victor3 V. The same strips
(containing the washed Total PSA washed wet assays) that were measured in
the MST Pro Meter (see figure 16, wet washed assay measurement 2) were
then measured in the Victor3 V (washed wet assay measurement 3), with the
results are summarised in figure 17. A clear correlation between the two
measurement methods (MST Pro Meter V 1 and the Victor3 V) is observed
especially considering the Victor3 V is a conventionally plate reader and is not
intended to measure fluorescent signals in laminate strip. This probably explains
the greater error associated with the Victor V measurements. The data
demonstrates the optical measurement performed by the MST Pro Meter V 1 is
a highly sensitive accurate measurement especially when compared to a very
expensive "gold standard" fluorescent plate reader technology (Victor3 V).
Figure 18 shows the correlation between the Total PSA washed wet assays
measured in strips in the MST Pro meter and the Victor V. The Total PSA
washed wet assays were also measured in a conventional microliter plate assay
using the Victor3 V (washed wet assay measurement 1). These results were
then compared to the Total PSA washed wet assays measured in a strip using
the MST Pro Meter V 1 (washed wet assay measurement 2). The correlation
between the two measurement methodologies is shown in figure 18. An
excellent correlation is observed between the two methodologies demonstrating
the ability to make highly sensitive and accurate measurements using the MST
Pro Meter and Strip (Platform) in very small sample volumes. The sample
channel is 1 mI however the volume measured in the MST pro strip V 1 by the
optical block is only approximately 0.2 mI_.
Assay 2: 1 step wet assay with wash and measurement carried out in 'MST pro
meter V1' reader
Assay reagents are combined in the following volumes and concentrations into
an eppendorf tube:
0.5% functionalized paramagnetic particles (with bound b1H12): m I
30mg/ml BSA, 0.05% tween all in PBS, pH7.2: 6m I
0.25% functionalized latex 1 (with biotinylated 5A6 bound): 1m I
PSA (diluted in 60mg/ml BSA in PBS, pH 7.2): 2m I
All reagents are mixed and then used to fill the channels of a 'MST pro strip V1'
cartridge (see figure 26 for description of cartridge used) via sample inlet port
(226) where it fills the channels up to the fluidic stop features (229, 228, 237).
This cartridge is inserted into a 'MST pro meter V reader (see figure 12 for a
description of the reader used) via connector (230), where a 5min incubation
occurs. The reader then brings a permanent magnet to the cartridge where it
acts to collect the paramagnetic particles and anything bound to them into the
detection area (222, 241). The optical reader head then carries out a
measurement of the fluorescent signal by scanning across the detection areas
of each channel (222, 241 ) . This measurement includes the fluorescent latex
specifically bound to the paramagnetic particles via PSA and also any unbound
fluorescent latex which is found within the detection area (see figure 20 for
description of these results). The sealing head of the reader makes a fluid tight
seal with input ports of channels 1,2,3,4,5,6 (224, 231 , 232, 233, 234, 235
respectively). Whilst the paramagnetic complexes are maintained in place by
the magnet, the reader carries out a wash step by expelling either a) wash
buffer (0.1% tween-20 in PBS, pH 7.2) or b) air from the syringe pump cartridge
(as shown in figure 15, bottom image of 6 chamber syringe cartridge) via the
input ports (231, 232, 233, 234, 235, 224) to displace the sample fluid from the
channel, and hence remove the unbound latex from the detection area, where it
is displaced into the sink (236)..The optical reader head then carries out a
measurement of the remaining fluorescent signal from the binding complexes
within the matrix of the wash substance (fluid or air) by scanning across the
detection areas of each channel (222, 241) (see figure 19 for a description of
these results using the air wash and figure 2 1 for results using the wash buffer
wash).
Results for Assay 2:
Figure 20 shows a Total PSA wet assay performed in the MST Pro Meter and
Strip, does not use a wash step and measures the fluorescence intensity of the
fluorophore after the paramagnetic particle-PSA-fluorescent latex complex are
accumulated by the magnet. A highly effective integrated onboard control is
summarised in figure 20. Before an air or fluid wash is performed by the meter
the paramagnetic particle-PSA-fluorescent latex particle complexes are
accumulated by the magnet. At this point the meter can perform an optical read
of the channels and quantify the concentration of fluorescent latex label as
shown in figure 20. Interestingly the fluorescent response ADC counts is related
to PSA concentration, i.e. a dose response without a wash step, which could be
used as a independent homogenous assay. The meter then performs the wash
step and measures the concentration of the paramagnetic particle-PSAfluorescent
latex complexes. The assay fluorescent signal should always be
lower after the wash step (air or fluid) as the unbound fluorescent label is
removed from the channel.
Figure 19 shows Total PSA wet assay performed in the ST Pro Meter and
Strip, the meter using an air wash step to expel unbound label from the channel.
Total PSA washed wet assays were performed with the MST Pro Meter V 1 and
Strip. The MST Pro Meter V 1 uses an air wash to expel the sample (containing
unbound label) from the strip channels in the sink. The results are summarised
in figure 19, the assay dose response curve clearly demonstrates a rapid,
sensitive, accurate and quantitative assay response for Total PSA. The 5
minute test time would be a significant improvement on current rapid POC PSA
tests, as would the improvements in sensitivity/accuracy and the quantitative
nature of the assay demonstrated. Interestingly the data clearly demonstrates a
"air wash" is a very effective method to remove unbound fluorescent label from
the channel and that accurate sensitive measurements of the fluorescent labels
bound to the paramagnetic particles via analyte (PSA) can be performed in a
non liquid environment. This allows simple but highly functional assays to be
formatted on the MST Pro Platform.
Figure 2 1 shows an example data dose response curve for Total PSA wet
assay performed in the MST Pro Meter and Strip, where the meter uses a fluid
wash step to expel unbound label from the channel. The fluid wash is highly
effective demonstrating a sensitive, accurate rapid PSA test. In comparison to
the air wash methodology the paramagnetic particle-PSA-fluorescent latex label
complexes are optically measured in the fluid environment. The fluid buffer
wash could contain components that could enhance the fluorescent signal
further (for example, chelating agents, fluorescent dye release agents etc).
Assay 3: 1 step dry assay with wash and measurement carried out in 'MST pro
meter V reader
Reagents are deposited and dried within a 'MST pro strip V1' cartridge as
follows:
8m I 0.5% functionalized paramagnetic particles (with bound biotinylated 1H12)
8m I 25mg/ml trehalose in PBS, pH 7.2
8m I 300mg/ml BSA in PBS, pH 7.2
8m I 0.1% tween-20 in PBS, pH 7.2
8m I 0.25% functionalized latex 2 (with 5A6 bound via SPDP)
The above reagents are combined and 1m I of this 1 step deposition mix added
per channel of a 'MST pro strip V1' cartridge (as shown in figure 26). Reagents
are deposited at position (223, 242) in each channel and not allowed to enter
the detection area (222, 241 ) (this is achieved by use of a hydrophobic pen line
to define the reagent deposition area on a single surface of the laminate
cartridge, which is sufficient to prevent reagent spread but not strong enough to
prevent sample filling of the fully assembled cartridge by capillary force). For
deposition, the cartridge is half assembled, with only the bottom and middle
layer of cartridge bonded together. The reagents are pipetted into the reagent
deposition zone of the half assembled test sample channel (see figure 26, with
reagent deposition zone indicated on the cartridge as point 223, 242). These
are dried in an oven at 33deg C for 10min. The top layer of the cartridge is then
bonded to the half assembled cartridge to produce a fully assembled three layer
cartridge (see figure 2 for an example of how the three layers come together to
form an assembled cartridge, for a different cartridge design) with dried
reagents. Figure 26 (240) indicates the shape of the double sided adhesive
material which is cut away to form channels and sink structures when bonded
between two layers of laminate material. The cartridge is then stored in a sealed
foil pouch containing desiccant until use.
PSA protein is diluted to the required concentration in 60mg/ml BSA in PBS, pH
7.2. A cartridge containing dried reagents is inserted into the 'MST pro meter
V1' reader and 8m I PSA is then added to the cartridge to fill the test sample
channels. The sample is applied to the cartridge via sample inlet port (226) and
fills the channels by capillary force up to the hydrophobic fluidic stop features
(229, 228, 239, 237). 5min binding incubation occurs before the reader brings a
permanent magnet to the cartridge where it acts to collect the paramagnetic
particles and anything bound to them into the detection area (222, 241 ). The
sealing head of the reader makes a fluid tight seal with input ports of channels
1,2,3,4,5,6 (224, 231 , 232, 233, 234, 235 respectively). Whilst the paramagnetic
complexes are maintained in place by the magnet, the reader carries out a
wash step by expelling air from the syringe pump cartridge (as shown in figure
15, bottom image of 6 chamber syringe cartridge) to displace the sample fluid
from the channel, and hence remove the unbound latex from the detection area
and displace it into the sink (236). The optical reader head then carries out a
measurement of the remaining fluorescent signal (from the specific sandwich
binding complexes of paramagnetic particles-PSA-fluorescent particles) which
remain in air by scanning across the detection areas of each channel (222, 241 )
(see figure 22 for a description of these results).
Results for Assay 3:
Figure 22 shows results for a Total PSA Dry assay performed in the MST Pro
Meter and Strip, the meter uses air wash step to expel unbound label from the
channel. All the reagents were deposited in the strip. The dynamic range of the
Total PSA assay was increased significantly by using a different fluorescent
latex label preparation and by reducing the input voltage to the optical detector.
The summary results are shown in figure 22 where the assay is a full dry assay
with all reagents deposited within the strip. The assay range has been extended
significantly, the linear response has been increased by 10 times (10 to 100
ng/ml), and this is of great value as some assays require assays with a low limit
of detection but a large dynamic range. With such assays linearity is not
maintained across the large dynamic range, resulting in reduced assay
performance at higher concentrations. The MST Pro platform can overcome this
limitation in several ways. For example, non linearity due to optical detector
saturation can be overcome by reducing the sensitivity of the optical detector by
reducing the input voltage. Therefore if binding is linear, the reduced sensitivity
of the optical detector will allow the dynamic range of the assay to be increased
further (the meter would contain a PSA calibration curve for the high and low
optical detector setting). In comparison, if the non linearity is due to a reagent
limitation, two channels of the MST Pro strip could be used to maximize assay
performance across the assay range. Reagents developed to make very
sensitive measurements that have a limited dynamic range could be deposited
in one channel whilst reagents that are less sensitive but allow the dynamic
range of the assay to be increased significantly could be deposited in another
channel. Each set of reagents/channel would have its own calibration curve
therefore allowing improving assay performance over whole range of the assay.
Assay 4: 2 step half dry assay (dried latex particles) with wash and
measurement carried out in 'MST pro meter V 1' reader
Reagents are deposited and dried within a 'MST pro strip V1' cartridge as
follows:
10m I 0.25% functionalized latex 1 (with biotinylated 5A6 bound)
10m I 25mg/ml trehalose in PBS, pH 7.2
20m I 300mg/ml BSA in PBS, pH 7.2
10m I 0.1% tween-20 in PBS, pH 7.2
The above reagents are combined and 1m I of this deposition mix added per
channel of a 'MST pro strip v cartridge (as shown in figure 26). Reagents are
deposited at position (223, 242) in each channel and not allowed to enter the
detection area (222, 241) (this is achieved by use of a hydrophobic pen line to
define the reagent deposition area on a single surface of the laminate cartridge,
which is sufficient to prevent reagent spread but not strong enough to prevent
sample filling of the fully assembled cartridge). For deposition, the cartridge is
half assembled, with only the bottom and middle layer of cartridge bonded
together. The reagents are pipetted into the reagent deposition zone of the half
assembled test sample channel (see figure 26, with reagent deposition zone
indicated on the cartridge as point 223, 242). These are dried in an oven at
33deg C for 10min. The top layer of the cartridge is then bonded to the half
assembled cartridge to produce a fully assembled three layer cartridge (see
figure 2 for an example of how the three layers come together to form an
assembled cartridge, for a different cartridge design) with dried reagents. Figure
26 (240) indicates the shape of the double sided adhesive material which is cut
away to form channels and sink structures when bonded between two layers of
laminate material. The cartridge is then stored in a sealed foil pouch containing
desiccant until use.
In this 2 step assay, the first binding step (functionalized paramagnetic particles
with PSA) is carried out in a wet format, before the second binding step
(functionalized paramagnetic particles-PSA with functionalized latex 1) occurs
using dried functionalized latex 1 within the 'MST pro strip V1' cartridge as
follows:
Stepl
The following reagents are combined:
2m I 0.5% functionalized paramagnetic particles (with bound biotinylated 1H12)
6m I 30mg/ml BSA in PBS, pH 7.
2m I PSA (diluted in 60mg/ml BSA in PBS, pH 7.2)
This first step binding reaction is incubated for 5min at room temperature before
8m I is added to the cartridge containing dried functionalized latex to fill the test
sample channels as shown in Figure 26. The sampel is applied to sample inlet
port (226) where it fills the channels up to the fluidic stop features (228, 239,
229) (which are made of hydrophobic ink). 5min binding incubation occurs
before the reader brings a permanent magnet to the cartridge where it acts to
collect the paramagnetic particles and anything bound to them into the detection
area. The sealing head of the reader then makes a fluid tight seal with input
ports of channels 1,2,3,4,5,6 (224, 231 , 232, 233, 234, 235 respectively). Whilst
the paramagnetic complexes are maintained in place by the magnet, the reader
carries out a wash step by expelling air from the syringe pump cartridge to
displace the sample fluid from the channel, and hence remove the unbound
latex from the detection area (222, 241) and displace it into the sink (236). The
optical reader head then carries out a measurement of the remaining
fluorescent signal (from the specific sandwich binding complexes of
paramagnetic particles-PSA-fluorescent particles) which remain in air by
scanning across the detection areas of each channel (see figure 23 for a
description of these results).
Results for Assay 4:
Figure 23 shows a Total PSA two step assay performed in the MST Pro Meter
and Strip. The meter uses an air wash step to expel unbound label from the
channel. Fluorescent latex label was deposited in the strip. The summary
results are shown in figure 23. The fluorescent latex label was deposited in the
strip and the sample containing PSA and paramagnetic particles was added to
the strip. The meter performed an air wash and optically measured the
concentration of captured fluorescent latex label. Systematic dose response
curves are observed and demonstrate that two or three step assays can be
formatted on the MST Pro Platform and that fluorescent latex label could be
deposited in one strip and used to perform two step assays.
Assay 5: 2 step half dry assay (dried paramagnetic particles) with wash and
measurement carried out in 'MST pro meter V1' reader
Reagents are deposited and dried within a 'MST pro strip VV cartridge as
follows:
10m I 0.5% functionalized paramagnetic particles (with biotinylated 1H12 bound)
10m I PBS, pH 7.2
0m I 25mg/ml trehalose in PBS, pH 7.2
10mI 300mg/ml BSA in PBS, pH 7.2
10mI 0.1% tween-20 in PBS, pH 7.2
The above reagents are combined and 1m I of this deposition mix added per
channel of a 'MST pro strip V V cartridge (as shown in figure 26). Reagents are
deposited at position (223, 242) in each channel (however it is not vital that
these reagents are kept distinct from the detection area as no label (fluorescent
latex) is being deposited). For deposition, the cartridge is half assembled, with
only the bottom and middle layer of cartridge bonded together. The reagents
are pipetted into the reagent deposition zone of the half assembled test sample
channel (see figure 26, with reagent deposition zone indicated on the cartridge
as point 223, 242). These are dried in an oven at 33deg C for 10min. The top
layer of the cartridge is then bonded to the half assembled cartridge to produce
a fully assembled three layer cartridge (see figure 2 for an example of how the
three layers come together to form an assembled cartridge, for a different
cartridge design) with dried reagents. Figure 26 (240) indicates the shape of the
double sided adhesive material which is cut away to form channels and sink
structures when bonded between two layers of laminate material. The cartridge
is then stored in a sealed foil pouch containing desiccant until use.
In this 2 step assay, the first binding step (functionalized latex 1 with PSA) is
carried out in a wet format, before the second binding step (functionalized latex
1-PSA with functionalized paramagnetic particles) occurs using dried
functionalized paramagnetic particles within the 'MST pro strip V1' cartridge as
follows:
Stepl
The following reagents are combined:
2m I 0.25% functionalized latex 1 (with bound biotinylated 5A6)
6m I 30mg/ml BSA in PBS, pH 7.2
2m I PSA (diluted in 60mg/ml BSA in PBS, pH 7.2)
This first step binding reaction is incubated for 5min at room temperature before
8m I is added to the cartridge (as shown in figure 26) containing dried
functionalized paramagnetic particles to fill the test sample channels. The 8m I
sample is applied to sample inlet port (226) and it fills the channels up to the
fluidic stop features (229, 228, 239) 5min binding incubation occurs before the
reader brings a permanent magnet to the cartridge where it acts to collect the
paramagnetic particles and anything bound to them into the detection area
(222, 241). The sealing head of the reader then makes a fluid tight seal with
input ports of channels 1,2,3,4,5,6 (224, 231 , 232, 233, 234, 235 respectively).
Whilst the paramagnetic complexes are maintained in place by the magnet, the
reader carries out a wash step by expelling air from the syringe pump cartridge
to displace the sample fluid from the channel, and hence remove the unbound
latex from the detection area (222, 241) and displace it into the sink (236). The
optical reader head then carries out a measurement of the remaining
fluorescent signal (from the specific sandwich binding complexes of
paramagnetic particles-PSA-fluorescent particles) which remain in air by
scanning across the detection areas of each channel (222, 241) (see figure 24
for a description of these results).
Results for Assay 5:
Figure 24 shows a Total PSA two step assay performed in the MST Pro Meter
and Strip, the meter using air wash step to expel unbound label from the
channel. Paramagnetic particle capture phase was deposited in the strip. The
meter uses an air wash step to expel unbound label from the channel. The
paramagnetic particles were deposited in the strip and PSA sample/latex label
was added to the strip. The meter performed an air wash and optically
measured the concentration of captured fluorescent latex label. A systematic
dose response curve is shown for the two step assays and demonstrate that
two or three step assays can be formatted on the MST Pro Platform and that
paramagnetic particles could be deposited in one strip and used to perform two
step assays. These results, together with those of Assay 4 (figure 23) show how
it would be possible to dry latex and paramagnetic particles within the same
cartridge and perform a fully dry 2 step assay.
Claims
1. An assay system for conducting an assay on a fluid sample, the assay
system comprising:
a) a microfluidic cartridge comprising one or more microfluidic channels
disposed therein and comprising a binding agent disposed within said
channel(s) for binding any of said analyte within the sample;
a sample port for introducing said fluid sample into the cartridge;
at least one input port for allowing one or more fluids to be introduced to the
cartridge from an associated reader device and transported through the
microfluidic channel(s); and
a fluid outlet sink for removing fluid from said channel(s); and
b) a reader device, the reader device comprising:
i) a receiving port for introducing the cartridge into the reader;
ii) a reservoir for storing a fluid;
iii) means for delivering the fluid to the fluid input port of the cartridge
once inserted within the reader, so that fluid can transported through
the microfluidic channel(s) of the cartridge and;
iv) detection means for enabling detection of the analyte or a reaction
product formed as a result of the analyte binding the binding agent
within the cartridge.
2. The assay system according to claim 1 wherein the reader device comprises
one or more sealing means which are adapted to co-locate with each of said
input ports of the cartridge in order to form a fluid-tight seal with the cartridge
once correctly inserted into the reader.
3. The assay system according to either of claims 1 or 2, wherein the reader
comprises one or more pumps for delivering the fluid into and throughout the
cartridge.
4. The assay system according to claim 3 wherein the pump is abl to drive the
fluid in either direction such that the fluid once delivered to the cartridge may be
moved in a reciprocal motion within the cartridge.
5. The assay device according to any preceding claim wherein the reader
comprises a fluid management system which is capable of controlling fluid
delivery to the cartridge.
6. The assay system according to claim 5, wherein the fluid management
system is capable of delivering fluid to each of said at least one input ports of
the cartridge, independently of the other input ports.
7. The assay system according to any preceding claim which is capable of
conducting a plurality of assays on a single sample.
8. The assay system according to claim 7 wherein the plurality of assays are the
same and/or different assays.
9. The assay system according to any preceding claim wherein the reader
comprises a magnet or means for generating a magnetic field and the binding
agent within the cartridge comprises magnetic properties, such that upon
application of the magnetic field or moving the magnet of the reader into close
proximity with the binding agent with the cartridge, the binding agent is held in
place by the magnetic field or magnet.
10. The assay system according to claim 9, wherein the magnet or magnetic
field can be controlled, such that the magnetic force applied to the binding agent
can be varied, as desired, during the assay.
11. The assay system according to claims 9 or 10, wherein the binding agent is
in the form of magnetic or paramagnetic particles which comprise a binding
moiety on their surface, designed to specifically bind a desired analyte.
12. The assay system according to claim 1 , wherein the binding moiety is an
antibody, protein, peptide or oligonucleotide
13. The assay system according to any preceding claim, wherein the detection
means is a fluorescent measurement.
14. The assay system according to any preceding claim, wherein the wash fluid
(fluid introduced into test cartridge from reader) is air.
15. The assay system according to claim 13, wherein the fluorescent
measurement is carried out in air.
16. A microfluidic assay cartridge for use in the assay system according to any
preceding claim, the cartridge comprising:
a substrate comprising one or more microfluidic channels disposed therein and
comprising a binding agent disposed within said channel(s) for binding any of
said analyte within the sample;
a sample port for introducing said fluid sample into the cartridge;
a fluid outlet sink for removing fluid from said channel(s); and
at least one fluid input port for allowing one or more fluids to be introduced to
the cartridge from the associated reader device and transported through the
microfluidic channel(s).
17. The microfluidic assay cartridge according to claim 16 further comprising a
detection area where any bound analyte may be detected, wherein the
detection area is preferably immediately downstream of the sample port, and
within the test sample channel.
18. The microfluidic assay cartridge according to any of claims 16 or 17,
wherein the cartridge and the channels and other features disposed therein, are
formed by a sandwich of three separate substrates - a top, middle and bottom
substrate.
19. The microfluidic assay cartridge according to claim 18, wherein the top and
bottom substrates is no thicker than 200pm, and preferably 50 m.
20. The microfluidic assay cartridge according to any of claims 16 - 19 wherein
the cartridge comprises top and bottom surfaces and at least one wall disposed
between said top and bottom faces.
2 1. The microfluidic cartridge according to claim 20, wherein the top face
comprises said at least one fluid input port for introducing fluid from the reader
into said microfluidic channel(s).
22. The microfluidic cartridge according to claims 20 or 2 1, wherein the sample
port is formed in said wall disposed between said top and bottom surfaces.
23. The microfluidic assay cartridge according to any of claims 16 - 22 wherein
said input port(s) of the cartridge is/are adapted to co-locate with sealing means
in the reader, so that a fluid, can be introduced into the cartridge, from the
reader.
24. The microfluidic assay cartridge according to any of claims 16 - 23 further
comprising one or more fluid stop features, which are designed to prevent the
sample and/or other fluids from passing through the stop feature, by virtue of
capillary action alone.
25. The microfluidic assay cartridge according to any of claims 16 - 24 further
comprising one or more electrode features which contact with the channel, for
conducting an electrical measurement on the fluid sample.
26. The microfluidic assay cartridge according to any of claims 16 - 25 wherein
the binding agent is attached directly to a wall or surface of said channel within
the cartridge, or to the surface of a magnetic agent, such as a magnetic or
paramagnetic particle disposed within said channel.
27. The microfluidic assay cartridge according to any of claims 16 - 26 further
comprising one or more reagents, such a label deposited within said microfluidic
channels(s), which reagents may facilitate detection of the captured analyte.
28. The microfluidic assay cartridge according to claim 27, wherein said one or
more reagents are deposited within said microfluidic channel(s), such that said
one or more reagents do not come into contact with the sample, upon initial
sample loading of the cartridge.
29. The microfluidic assay cartridge according to any of claims 16 - 28, wherein
the cartridge material forming the detection area is conducive to fluorescent
measurement.
30. A reader device for use in the assay system according to any of claims 1-15,
the reader device comprising:
i) a receiving port for introducing the cartridge into the reader;
ii) a reservoir for storing a fluid;
means for delivering the fluid to the fluid input port of the cartridge
once inserted within the reader, so that fluid can transported through
the microfluidic channel(s) of the cartridge and;
detection means for enabling detection of the analyte or a reaction
product formed as a result of the analyte binding the binding agent
within the cartridge
31. The reader according to claim 30, wherein the means for delivering the fluid
to the fluid input port of the cartridge, is a pump.
32. The reader according to either of claims 30, or 3 1, further comprising a
magnet or magnetic field generating means, for applying a magnetic force to the
cartridge once located within the reader.
33. The reader according to claim 32, wherein the magnetic force which can be
applied to the cartridge can be varied.
34. The reader according to any of claims 30 -33, further comprising fluid
management means for controlling the delivery of fluid to the cartridge.
35. The reader according to any of claims 30 - 34, further comprising one or
more sealing means for co-locating with said one or more respective fluid input
ports of the cartridge, so as to form a fluid seal with said one or more respective
fluid input ports of the cartridge.
36. A method of conducting an assay on a sample, the method comprising:
introducing, by way of capillary action, a fluid sample into a microfluidic cartridge
according to any of claims 16 -29 such that the fluid is drawn through the
cartridge and comes into contact with said binding agent, whereby at least a
portion of said analyte present in the sample is bound by the binding agent;
washing any unbound material away from the bound analyte using a fluid or
fluids introduced to the cartridge by way of the fluid input port and;
detecting any bound analyte present in the cartridge.
37. The method according to claim 36, wherein the binding agent is magnetic or
paramagnetic and bound analyte is held in place using a magnet or magnetic
field, during the washing step.
38. The method according to claims 36 or 37, wherein any unbound analyte is
removed by washing with a fluid introduced into the cartridge through the fluid
input port.
39. The method according to claims 36 - 38, wherein more than one washing
step is carried out.
40. The method according to claims 36 - 39, wherein a label or other reagent
may be brought into contact with the bound analyte.
4 1. The method according to any of claims 36 - 40, wherein detection is
conducted on any bound analyte, which is substantially free of unbound
components of the sample.
42. The method according to any of claims 36 -41, wherein the fluid sample is a
sample of whole blood.
43. The method according to any of claims 36 -42, wherein the fluid sample
introduced into the microfluidic cartridge, is less than 1 microlitre.
44. The method according to any of claims 36 - 43, wherein the detection is a
fluorescent measurement.