U.S. patent application number 15/577076 was filed with the patent office on 2018-06-07 for sensor device.
This patent application is currently assigned to IMEC VZW. The applicant listed for this patent is IMEC VZW. Invention is credited to Kris Covens, Tim Stakenborg, Willem Van Roy.
Application Number | 20180156788 15/577076 |
Document ID | / |
Family ID | 53502509 |
Filed Date | 2018-06-07 |
United States Patent
Application |
20180156788 |
Kind Code |
A1 |
Van Roy; Willem ; et
al. |
June 7, 2018 |
Sensor Device
Abstract
A device (1) for sensing an analyte, the device (1) comprises at
least a sample inlet (10) for receiving a sample, affinity probes
(111) selected to have a preferential binding to the analyte, a
transducer (11) sensitive to a characteristic of the analyte and/or
a label attached to the analyte, the transducer not being a FET
transducer, and a desalting unit (13) for desalting the received
sample.
Inventors: |
Van Roy; Willem; (Bierbeek,
BE) ; Stakenborg; Tim; (Heverlee, BE) ;
Covens; Kris; (Kessel-Lo, BE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
IMEC VZW |
Lauven |
|
BE |
|
|
Assignee: |
IMEC VZW
Leuven
BE
|
Family ID: |
53502509 |
Appl. No.: |
15/577076 |
Filed: |
June 30, 2016 |
PCT Filed: |
June 30, 2016 |
PCT NO: |
PCT/EP2016/065449 |
371 Date: |
November 27, 2017 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 33/523 20130101;
C12Q 1/6825 20130101; G01N 33/54393 20130101; C12Q 1/002
20130101 |
International
Class: |
G01N 33/543 20060101
G01N033/543; C12Q 1/00 20060101 C12Q001/00; C12Q 1/6825 20060101
C12Q001/6825; G01N 33/52 20060101 G01N033/52 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 30, 2015 |
EP |
15174417.4 |
Claims
1. A biosensor device for sensing an analyte, the device
comprising: at least a sample inlet for receiving a sample;
affinity probes selected so as to have an preferential binding to
the analyte; a transducer sensitive to a characteristic of the
analyte and/or a label attached to the analyte and adapted to
convert an interaction of the analyte with the affinity probes into
a readout signal, the transducer not being a Field Effect
Transistor transducer; and a desalting unit for desalting the
received sample so as to reduce a response time and/or increase the
signal of the transducer.
2. The device according to claim 1, wherein the desalting unit
further comprises a port for receiving buffer fluid for being flown
to the received sample.
3. The device according to claim 1, wherein the desalting unit
comprises a buffer fluid reservoir for containing buffer fluid.
4. The device according to claim 3, wherein the desalting unit
further comprises a mixer for mixing received sample with the
buffer fluid.
5. The device according to claim 1, wherein the transducer is an
optical transducer.
6. The device according to claim 1, wherein the desalting unit is
integrated on or in a same substrate or in a same enclosure as the
transducer.
7. The device according to claim 6, wherein the desalting unit
comprises a port, a buffer fluid reservoir and a mixer (132).
8. The device according to claim 1, wherein the device is an
affinity-based sensing device having affinity probes on the
transducer.
9. A diagnostic device comprising: a biosensor device according to
claim 1 for sensing an analyte and generating a sensing signal, and
an output unit for providing an output of said biosensor
device.
10. A diagnostic device according to claim 9, wherein the output
device is adapted for outputting a signal representative for
presence/absence or concentration of the analyte.
11. A method for measuring a concentration of an analyte in a
biological sample, the method comprising: i) receiving a biological
sample; ii) desalting the sample, thereby obtaining a desalted
sample, iii) measuring at least one signal of the desalted sample
by means of an affinity-based sensing device based on affinity
probes and a transducer, the transducer not being a Field Effect
Transistor transducer; and iv) determining the concentration of the
analyte in the sample using the at least one signal.
12. The method according to claim 11, wherein the step (ii) of
desalting the sample comprises a step of bringing the sample to an
ionic strength ranging from 10 nM to 150 mM.
13. The method according to claim 11, wherein the step (ii) of
desalting the sample and the step (iii) for measuring the at least
one signal of the desalted sample are performed simultaneously or
successively.
14. The method according to claim 11, wherein the method
furthermore comprises a step of comparing the at least one signal
to a reference signal obtained with a standard solution.
15. The method according to claim 11, wherein the step (iii) of
measuring the at least one signal of the desalted sample is
performed over time to obtain a measurement curve.
16. The method according to claim 11, wherein the step (ii) of
desalting the sample comprises a step of diluting the sample.
17. The method according to claim 11, wherein the step (ii) of
desalting the sample comprises a step of performing
electrodialysis.
18. The method according to claim 11, wherein the step (ii) of
desalting the sample comprises a step of bringing the sample to an
ionic strength ranging from 10 mM to 150 mM.
19. The method according to claim 15, further comprising a step
(iiia) of determining a slope of the measurement curve.
20. The method according to claim 11, wherein the affinity-based
sensing device comprises the device of claim 1.
Description
TECHNICAL FIELD OF THE INVENTION
[0001] The present invention relates to a device for sensing an
analyte and to a method for measuring the presence and/or
concentration of an analyte in a sample. In particular embodiments,
the present invention may relate to a biosensor device.
BACKGROUND OF THE INVENTION
[0002] Affinity-based sensors are devices for sensing and detecting
analytes in a sample, for instance in a liquid sample. Such sensors
may operate on the basis of electrical, electrochemical, chemical,
optical, magnetic, electromagnetic, mechanical, and/or acoustic
detection principles. The detection of analytes in the sample is
performed through interaction and reaction between specified
reactants and the analytes in the sample. In particular in an
affinity-based biosensor, the detection is based on the formation
of a complex (hybridisation) between at least two entities, i.e.
the analyte and a receptor or capture probe which may be
immobilized on or in a substrate. The complex formation between the
analyte and the capture probe leads to a signal that is measurable
by a signal measurement unit. In order to make the binding
detectable, in particular embodiments, a label element may be
attached to the analyte. In alternative embodiments, however,
detection may be based on a label-free operation.
[0003] Real time sensing of biomolecules as a particular type of
analytes, is particularly useful in many applications such as
disease diagnosis or food safety, for example. Unfortunately, the
response time of a biosensor device is often slow. This response
time depends on a huge number of parameters such as, among other,
the concentration of the analyte, the diffusion of the analyte, the
kinetics of the hybridisation reaction and the stability of the
obtained complex. For biosensors, the response times can vary from
a few seconds to hours or more. It is generally admitted that in
point-of-care (POC) or point-of-need applications, response time
must be no longer than about 10 minutes. Moreover, the Limits of
Detection (LOD) of existing biosensors can become higher (worse) if
the various incubation times are reduced below their recommended
values.
[0004] There is therefore still a need to dispose of a device
having short response time in the detection of the presence and/or
in the measurement of concentrations of analyte and, preferably,
having low limit of detection values. Furthermore, the methods
implemented at present in devices for sensing an analyte, for
instance in biosensors, need to be improved in order to decrease
the response time.
SUMMARY OF THE INVENTION
[0005] It is an object of embodiments of the present invention to
provide a device for sensing an analyte, for instance a biosensor,
having a rapid response time for the detection of the presence
and/or for the determination of the concentration of the analyte in
a sample. Alternatively or additionally, the device according to
embodiments of the present invention may present an increased
signal of the transducer, thereby allowing to detect the analyte
more rapidly and/or at smaller concentrations.
[0006] It is also an object of the present invention to provide a
method to be implemented in a device for measuring an analyte, for
instance in a biosensor, the method leading to fast response times
for the detection of the presence and/or for the determination of
the concentration of the analyte in a sample.
[0007] In a first aspect, the present invention relates to a device
for sensing an analyte, for instance a biosensor, the device
comprising at least a sample inlet for receiving a sample, in
particular for instance a liquid sample, affinity probes selected
to have a preferential binding to the analyte, and a transducer
sensitive to a characteristic of the analyte and/or a label
attached to the analyte, and adapted to convert an interaction of
the analyte with the affinity probes into a readout signal, the
transducer not being a field-effect transducer, such as a
field-effect transistor (FET), and a desalting unit for desalting
the received sample so as to increase the binding rate between the
affinity probes and the analyte and consequently to reduce the
response time and/or increase the signal of the transducer.
[0008] The inventors have surprisingly found that the presence of a
desalting unit in a device for sensing an analyte, for instance a
biosensor, permits to obtain a faster response from the device. The
measurable signal (the output signal) increases faster as compared
to a similar device without the desalting unit. Moreover, by using
a desalting unit in a device for sensing an analyte, the limit of
detection is decreased (=improved).
[0009] The desalting unit may be any of a dilution means, a
concentration/redispersion means, an electrodialysis means, or any
other suitable means.
[0010] By the term "dilution means", is meant a means suitable to
decrease the ionic strength of the sample containing the analyte by
dilution with a fluid, for instance a buffer fluid. The fluid may
be a solution having a lower ionic strength than the ionic strength
of the provided sample. In particular embodiments, the dilution
means comprises a mixer and/or a fluid reservoir, for instance a
buffer fluid reservoir. The use of a dilution means has the
advantage that it is easy to implement and fast in operation, thus
allowing a short sample-to-answer time, but it has the disadvantage
that not only the ionic strength of the sample is reduced, but that
also the analyte concentration is reduced. Nevertheless, the
overall performance of the sensor device is improved.
[0011] By the term "concentration/redispersion means", an analyte
concentrator coupled to a redispersion means is meant. By using the
analyte concentrator, analyte is brought into a more concentrated
state. The redispersion means is suitable to redisperse the
concentrated analyte in a solution having an ionic strength lower
than the initial state (e.g. lower than physiological ionic
strength if the sample was a physiological sample). In particular
embodiments, the analyte concentrator may be a centrifuge, a filter
(such as a paper filter, a micropillar filter, a bead filter), or a
microsieve. The redispersion means may be selected from the group
consisting of magnetic stirrer, mechanical stirrer, ultrasonic
stirrer, flow-through device, or microfluidic device. An advantage
of using a combined concentration/redispersion means is that it
permits to reduce the ionic strength while the concentration of the
analyte remains unaffected, if the amount of liquid added during
redispersion is equal to the amount of liquid removed during
concentration. The concentration of the analyte can also be
increased or decreased, if desired, by adding a different volume
during redispersion compared to the volume that was removed in the
concentration step.
[0012] By the term "electrodialysis means", is meant a means
comprising at least two ion-selective membranes (also known as ion
exchange membranes) suitable for performing electrodyalisis. By the
term "ion-selective", is meant that the membrane is permeable to
some ions (e.g. in a cation-selective membrane: to cations such as,
among other, Li.sup.+, Na.sup.+, K.sup.+, Ca.sup.2+ and Mg.sup.2+,
preferably Na.sup.+), and not to others (e.g. in a cation-selective
membrane: anions such as, among others, F.sup.-, Cl.sup.-, Br.sup.-
and HCO.sub.3.sup.-, preferably Cl.sup.-), through channels across
the membrane (e.g. pores or holes). The ion-selective membranes are
selected so as to be not permeable to the analyte. The
electrodialysis means comprises electrodes on the side of the
membranes opposite to the one in contact with the sample containing
the analyte to be detected and/or measured. The electrodes may be
actuated so as to attract the cations, for instance Na.sup.+, and
more preferably more cations, through the Na.sup.+-selective or
cation-selective membrane, and to attract the anions, for instance
Cl.sup.-, and more preferably more anions, through the
Cl.sup.--selective or anion-selective membrane. An advantage of an
electrodialysis means is that it permits to reduce the ionic
strength without diluting the analyte concentration in the
sample.
[0013] It is an advantage of embodiments of the present invention
that a biosensor is provided which can be used for sensing
biological samples. Such biological samples may for instance be
received, e.g. taken, from a patient, for instance a blood or
plasma sample, a saliva sample, a urine sample, etc. Embodiments of
the present invention are particularly well-suited for detection of
biological targets. A biosensor device in accordance with
embodiments of the present invention is a sensor, adapted for
sensing the presence/absence and/or the concentration of the
analyte. The medium in which the sample is received is an aqueous
medium, and may contain dissolved salts, e.g. at physiological
conditions (ionic strength .about.150 mM).
[0014] In embodiments of the present invention, the desalting unit
is internal to the sensor device, e.g. integrated on a same
substrate, e.g. semiconductor substrate, or in a same enclosure as
the transducer. In alternative embodiments, the desalting unit is
external to the sensor device, e.g. not integrated on a same
substrate or in a same enclosure as the transducer.
[0015] In embodiments, the desalting unit may furthermore comprise
a port for receiving a buffer fluid for being flown to the received
sample.
[0016] In embodiments of the present invention, the desalting unit
may comprise a buffer fluid reservoir for containing buffer fluid.
The buffer fluid reservoir may be part of the desalting unit, or
may be the desalting unit as such (i.e. the desalting unit consists
of the buffer fluid reservoir). In particular embodiments, the
buffer fluid reservoir may be selected from the group consisting of
an ampoule, a syringe, a blister, a well, a tube connecting two
liquid reservoirs, an Eppendorf tube, a channel, and an on-board
reservoir provided on or in a chip, being for instance a
semiconductor chip or a microfluidics chip. It is advantageous to
use a blister pack, a channel, or an on-chip reservoir, as the
blister pack, the channel, or the on-chip reservoir is easy to be
incorporated.
[0017] In alternative embodiments of the present invention, a
buffer fluid reservoir for containing buffer fluid may be located
outside the desalting unit.
[0018] In embodiments of the present invention, the sample inlet
and the desalting unit are connected to each other by a
transferring means suitable for the transfer of a sample from the
sample inlet to the desalting unit.
[0019] In embodiments of the present invention, the desalting unit
and the transducer are connected to each other by a transferring
means suitable for the transfer of a desalted sample from the
desalting unit to the transducer.
[0020] In embodiments of the present invention, an outlet port may
be provided for evacuating excess sample and/or waste. On top
thereof or alternatively, an internal reservoir may be provided for
storing excess sample and/or waste. The outlet port and/or the
internal reservoir may be connected to other parts of the sensor
device by suitable transferring means.
[0021] In embodiments of the present invention, the transferring
means is or are based on capillary flow. In embodiments of the
present invention, the transferring means is or are based on
capillary flow in an open channel. In alternative embodiments of
the present invention, the transferring means is or are based on
capillary flow in a closed channel. In embodiments of the present
invention, the desalting unit comprises a mixer for mixing the
received sample with buffer fluid. In particular embodiments, the
mixer may be selected from the group consisting of a microfluidic
mixer, a vortex mixer, a shaker, a magnetic mixer, an ultrasonic
mixer, mechanical mixer and rapid-mixing apparatus. The rapid
mixing-apparatus may comprise two syringes, one for the delivery of
a sample through the sample inlet and one for the delivery of a
buffer fluid through a buffer fluid inlet and a mixing chamber. In
particularly advantageous embodiments, the mixer for mixing
received sample with buffer fluid is a microfluidic mixer, the
advantage of the microfluidic mixer being that the mixer has no
moving parts.
[0022] In embodiments of the present invention, the transducer may
be an optical transducer, i.e. a transducer that converts an
optical signal into an electronic signal. The optical signal may be
any suitable type of optical signal, such as for instance a
variation of fluorescence or of refractive index or of colour.
[0023] In embodiments of the present invention, the desalting unit
may be located on a same substrate or in a same enclosure as the
transducer. The desalting unit may comprise the port for receiving
the buffer fluid for being flown to the received sample, the buffer
fluid reservoir and the mixer.
[0024] In a second aspect, the present invention provides a
diagnostic device comprising a biosensor device according to
embodiments of the first aspect of the present invention, for
sensing an analyte and generating a sensing signal, and an output
unit for providing an output of said biosensor device which can be
used, alone or in combination with other factors, for basing a
diagnosis on. The output device may be adapted for outputting a
signal representative for presence/absence or concentration of the
analyte. Such diagnostic device is intended for use in diagnosis of
disease or other conditions, including a determination of the state
of health, in order to cure, mitigate, treat or prevent disease or
its sequelae. Such diagnostic device or parts thereof are intended
for use in the collection, preparation and examination of samples
taken from a human or animal body.
[0025] In a third aspect, embodiments of the present invention
relate to a method for measuring the concentration of an analyte,
typically for instance a biomolecule, a protein, an antibody, an
antigen, a biomarker, a cytokine, a nucleic acid, a small molecule
(a small molecule typically having a molecular weight lower than a
few kiloDaltons, for instance lower than 10 kDa, e.g. lower than 5
kDa, e.g. lower than 2 kDa, such as for instance between 50 Da and
1 kDa), or a metabolite, in a sample, the method comprising: [0026]
i. Obtaining or receiving a sample, e.g. a biological sample,
[0027] ii. desalting the sample, thereby obtaining a desalted
sample, [0028] iii. measuring at least one signal of the desalted
sample by means of an affinity-based sensing device based on
affinity probes and a transducer, the transducer not being a
FET-transducer, [0029] iv. determining the presence and/or
concentration of the analyte in the sample using the at least one
signal.
[0030] By the expression "desalting the sample" is meant obtaining
a decrease of the ionic strength of the sample, for example a
sample in an aqueous medium with physiological salt concentration.
The obtained desalted sample of step ii. has an ionic strength
lower than the ionic strength of the original sample, e.g. lower
than physiological ionic strength in case of a physiological
sample. However, the ionic strength does not necessarily need to be
zero.
[0031] The inventors have surprisingly found that thanks to the
method according to embodiments of the present invention, the
response time may be decreased to only a few minutes (e.g. 20
minutes or less, for instance 10 minutes or less, preferably to 5
minutes or less, more preferably to 1 minute or less) and even to
only a few seconds (e.g. to 30 seconds or less, preferably to 20
seconds or less, more preferably to 10 seconds or less). This is
particularly advantageous for use of a sensor in POC
applications.
[0032] In embodiments of the present invention, the step ii. of
desalting the sample comprises, consists essentially of, or
consists of, a step of bringing the sample to an ionic strength
ranging from 10 nM to 150 mM, preferably from 1 mM to 150 mM, more
preferably from 10 mM to 150 mM.
[0033] In embodiments of the present invention, the step ii. of
desalting the sample and the step iii. for measuring the at least
one signal of the desalted sample may be performed successively. In
particular embodiments the sample is first desalted and then
applied on affinity probes and a transducer. In alternative
embodiments, the sample is desalted on the affinity probes and the
transducer, but before the measurement is started.
[0034] In alternative embodiments of the present invention, the
step ii. of desalting the sample (e.g. biological sample) and the
step iii. for measuring the at least one signal of the desalted
sample may be performed simultaneously. In other words, in this
embodiment the sample is desalted on the affinity probes and the
transducer during the measurement.
[0035] In embodiments, the method according to the invention may
furthermore comprise a step of comparing the at least one signal to
a reference signal obtained with a standard solution. By the
expression "standard solution" is meant a sample in which no
analyte is present, or in which a known concentration of analyte is
present.
[0036] In embodiments of the present invention, the step iii. of
measuring the at least one signal of the desalted sample may be
repeated over time, thus obtaining a measurement curve. In
particular embodiments, the step iii. of measuring the at least one
signal of the desalted sample comprises a step of determining a
slope of the measurement curve. In this embodiment, the measurement
is performed before a stable situation is reached.
[0037] In embodiments of the present invention, the step ii of
desalting the sample comprises, preferably consists essentially of,
more preferably consists of, a step of diluting the sample. The
step of diluting of the sample is a simple and fast step;
nevertheless the diluting leads also to the diluting of the
analyte. In particular embodiments, the solvent used in the step of
diluting may be a water based buffer fluid. In particular
embodiments, the water based buffer fluid may have a pH ranging
from pH 2 to 12, or 5 to 9, or around 7.
[0038] In embodiments of the present invention, the step ii. of
desalting the sample comprises, consist essentially of, or consists
of, a step of performing electrodialysis. The advantage linked to
the use of electrodialysis is that the sample is desalted without
being diluted. Furthermore, the desalting step may be done on the
affinity probes and the transducer, before the measurement is
started, or during measurement.
[0039] In embodiments, the method according to the invention is
such that the analyte is a biomolecule, a protein, an antibody, an
antigen, a biomarker, a cytokine, a nucleic acid, a small molecule
(a small molecule typically having a molecular weight lower than a
few kiloDaltons, for instance lower than 10 kDa, e.g. lower than 5
kDa, e.g. lower than 2 kDa, such as for instance between 50 Da and
1 kDa), or a metabolite.
[0040] Particular and preferred aspects of the invention are set
out in the accompanying independent and dependent claims. Features
from the dependent claims may be combined with features of the
independent claims and with features of other dependent claims as
appropriate and not merely as explicitly set out in the claims.
[0041] Although there has been constant improvement, change and
evolution of devices in this field, the present concepts are
believed to represent substantial new and novel improvements,
including departures from prior practices, resulting in the
provision of faster, more sensitive, more efficient, stable and
reliable devices of this nature.
[0042] The above and other characteristics, features and advantages
of the present invention will become apparent from the following
detailed description, taken in conjunction with the accompanying
drawings, which illustrate, by way of example, the principles of
the invention. This description is given for the sake of example
only, without limiting the scope of the invention. The reference
figures quoted below refer to the attached drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0043] FIG. 1 is a graph of the occupied fraction, at equilibrium,
of the capture probes [PA]/[P.sub.tot] versus the analyte
concentration [A] for different affinity constants, K.sub.a.
[0044] FIG. 2 is a graph of the occupied fraction of the capture
probes [PA]/[P.sub.tot] versus the analyte concentration [A], for
different measurement times.
[0045] FIG. 3 is a graph of the amount of occupied capture probes
[PA] versus the time of an affinity-based sensing device.
[0046] FIG. 4 is a graph of the occupied fraction, at equilibrium,
of the capture probes [PA]/[P.sub.tot] versus the analyte
concentration [A] for different affinity constants, K.sub.a.
[0047] FIG. 5 is a schematic representation of a device for sensing
an analyte according to embodiments of the present invention.
[0048] FIG. 6 is a schematic representation of a device for sensing
an analyte according to alternative embodiments of the present
invention.
[0049] FIG. 7 is a schematic illustration of another embodiment of
a device for sensing an analyte according to the present
invention.
[0050] FIG. 8, FIG. 9, FIG. 10 and FIG. 11 are schematic
illustrations of yet other embodiments of a device for sensing an
analyte according to the present invention.
[0051] FIG. 12 represents a diagrammatic illustration of an
embodiment of the method for measuring the concentration of an
analyte according to the invention.
[0052] In the different figures, the same reference signs refer to
the same or analogous elements.
DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
[0053] The present invention will be described with respect to
particular embodiments and with reference to certain drawings but
the invention is not limited thereto but only by the claims. The
drawings described are only schematic and are non-limiting. In the
drawings, the size of some of the elements may be exaggerated and
not drawn on scale for illustrative purposes. The dimensions and
the relative dimensions do not correspond to actual reductions to
practice of the invention.
[0054] It is to be noticed that the term "comprising", used in the
claims, should not be interpreted as being restricted to the means
listed thereafter; it does not exclude other elements or steps. It
is thus to be interpreted as specifying the presence of the stated
features, integers, steps or components as referred to, but does
not preclude the presence or addition of one or more other
features, integers, steps or components, or groups thereof. Thus,
the scope of the expression "a device comprising means A and B"
should not be limited to devices consisting only of components A
and B. It means that with respect to the present invention, the
only relevant components of the device are A and B.
[0055] Similarly, it is to be noticed that the term "coupled",
should not be interpreted as being restricted to direct connections
only. The terms "coupled" and "connected", along with their
derivatives, may be used. It should be understood that these terms
are not intended as synonyms for each other. Thus, the scope of the
expression "a device A coupled to a device B" should not be limited
to devices or systems wherein an output of device A is directly
connected to an input of device B. It means that there exists a
path between an output of A and an input of B which may be a path
including other devices or means. "Coupled" may mean that two or
more elements are either in direct physical or electrical contact,
or that two or more elements are not in direct contact with each
other but yet still co-operate or interact with each other.
[0056] Reference throughout this specification to "one embodiment"
or "an embodiment" means that a particular feature, structure or
characteristic described in connection with the embodiment is
included in at least one embodiment of the present invention. Thus,
appearances of the phrases "in one embodiment" or "in an
embodiment" in various places throughout this specification are not
necessarily all referring to the same embodiment, but may.
Furthermore, the particular features, structures or characteristics
may be combined in any suitable manner, as would be apparent to one
of ordinary skill in the art from this disclosure, in one or more
embodiments.
[0057] Similarly it should be appreciated that in the description
of exemplary embodiments of the invention, various features of the
invention are sometimes grouped together in a single embodiment,
figure, or description thereof for the purpose of streamlining the
disclosure and aiding in the understanding of one or more of the
various inventive aspects. This method of disclosure, however, is
not to be interpreted as reflecting an intention that the claimed
invention requires more features than are expressly recited in each
claim. Rather, as the following claims reflect, inventive aspects
lie in less than all features of a single foregoing disclosed
embodiment. Thus, the claims following the detailed description are
hereby expressly incorporated into this detailed description, with
each claim standing on its own as a separate embodiment of this
invention.
[0058] Furthermore, while some embodiments described herein include
some but not other features included in other embodiments,
combinations of features of different embodiments are meant to be
within the scope of the invention, and form different embodiments,
as would be understood by those in the art. For example, in the
following claims, any of the claimed embodiments can be used in any
combination.
[0059] Furthermore, some of the embodiments are described herein as
a method or combination of elements of a method that can be
implemented by a processor of a computer system or by other means
of carrying out the function, such as for instance a microfluidics
system. Thus, a processor with the necessary instructions for
carrying out such a method or element of a method, e.g. a
controller that actuates valves, mixers, etc., forms a means for
carrying out the method or element of the method. Alternatively or
on top thereof, a capillary circuit with liquid delay lines to
perform a particular sequence of sample loading, mixing, moving to
the affinity probes and the transducer, etc. also forms a means for
carrying out the method or element of the method. Furthermore, the
means of carrying out the function are not limited to capillary
circuits, and any element described herein of an apparatus
embodiment is an example of a means for carrying out the function
performed by the element for the purpose of carrying out the
invention.
[0060] In the description provided herein, numerous specific
details are set forth. However, it is understood that embodiments
of the invention may be practiced without these specific details.
In other instances, well-known methods, structures and techniques
have not been shown in detail in order not to obscure an
understanding of this description.
[0061] The invention will now be described by a detailed
description of several embodiments of the invention. It is clear
that other embodiments of the invention can be configured according
to the knowledge of persons skilled in the art without departing
from the true spirit or technical teaching of the invention, the
invention being limited only by the terms of the appended
claims.
[0062] As used herein and unless provided otherwise, the term
"analyte", indicated by A in the description, refers to the
substance to be measured, the substance having or not having a
biological origin. By the expression "substance having a biological
origin", we intend to mean a substance that is present or produced
in a living organism. Particularly, the substance may be a
biomolecule. For instance, the analyte may be a protein, an
antibody, an antigen, a biomarker, a cytokine, a polysaccharide, a
lipid, a nucleic acid, a small molecule, or a metabolite, the small
molecules typically having a molecular weight lower than a few
kiloDaltons, for instance lower than 10 kDa, such as lower than 5
kDa, or lower than 2 kDa, e.g. between 50 Da and 1 kDa, such as
primary metabolites, secondary metabolites, and natural
products.
[0063] By the term "biomolecule" is meant any molecule that is
present in living organisms, including large macromolecules such as
proteins, polysaccharides, lipids, and nucleic acids, as well as
small molecules. The term "biomolecule" also encompasses molecules
with similar properties and/or structure and/or composition, but
that have been manufactured artificially rather than in a living
organism.
[0064] As used herein, the term "sample" means the liquid, e.g. an
aqueous solution, also called container liquid, in which it is
desired to detect the presence and/or concentration of an analyte.
This sample can be an original patient sample, like a quantity of
blood, plasma saliva, urine, sperm; the original sample after
desalting, e.g. after diluting; or the original or desalted sample
to which one or more steps have been applied, which are typically
done by a person skilled in the art of assay, e.g. with the
intention to associate a label with an analyte, for instance by
direct labelling of the analyte, by having the analyte compete with
a labelled species, or by quenching a label.
[0065] The term "affinity probe", indicated by P in the
description, refers to the substance having a certain affinity,
e.g. a natural attraction or preferential binding, to the analyte,
the substance having or not having a biological origin. By the
expression "substance having a biological origin", we intend to
mean a substance that is present or produced in a living organism,
or has similar properties and/or structure and/or composition. For
instance, the affinity probe may be an antibody, an antigen, an
enzyme, a receptor, an aptamer, a nucleic acid aptamer, a peptide
aptamer, or a molecularly imprinted polymer (MIP). Although we list
examples of affinity probes in the singular, typically there is
more than one affinity probe, even many more than one affinity
probe present in the system. The affinity probes may be free in the
solution, or they may be immobilized on a surface, or they may be
immobilized in a 3D matrix such as e.g. a gel or a dextran
matrix.
[0066] By the expression "affinity-based sensing device" is meant a
sensor based on a hybridisation reaction between affinity probes
and analyte, for instance an affinity-based biosensor.
[0067] By the expression "response time" is meant the time
necessary for obtaining a signal that is large enough to allow the
determination of the presence and/or the concentration of the
analyte of interest. Actual response time values depend on the
relevant concentration range of the analyte, and on the noise
sources, whereby the noise occurring may depend for instance on the
type of assay performed, on biological noise, on transducer noise,
on data processing noise, on noise due to optical detection,
etc.
[0068] By the expression "physiological conditions", we intend to
mean a pH equal to about 7.4 and an ionic strength equal to about
0.15 M or about 150 mM.
[0069] The term "transducer" in the context of the present
invention refers to a means to convert the interaction of the
analyte with affinity probes into a readout signal. The transducer
may be, but does not need to be, an optical or an electronic
device, and the readout signal may be, but does not need to be, an
optical or electronic signal. In embodiments of the present
invention, affinity probes may be present on or in, or they may
form part of, the transducer. In particular embodiments, the
transducer may be a means, such as an enzymatic reaction, which
converts the interaction of the analyte with affinity probes into a
visually discernible signal, for instance a colour indication of a
particular colour depending on the type of analyte present in the
sample. The intensity of the generated signal is related to, e.g.
proportional to, such as directly or inversely proportional to, the
amount of analyte bound to the affinity probes.
[0070] By the expression "sensing a characteristic of the analyte
and/or a label attached to the analyte" is meant that the
transducer of the device for sensing an analyte, for instance of
the biosensor, detects presence, events or changes in quantities of
analyte bound to affinity probes, and provides a corresponding
output signal, generally as an electrical or optical signal. For
example, measurements of the concentration, presence or absence of
analyte can be obtained. "A characteristic of the analyte and/or a
label attached to the analyte" includes any derived or indirect
characteristic, or any characteristics that are the results of
steps, actions or assays that result in a particular characteristic
being associated with the sample. Sometimes the characteristic
cannot be measured on the analyte itself, and in such cases labels
may be provided, which bind to the analyte, and on which
characteristics can be measured. For instance, in particular cases
the analyte may not be fluorescent in itself, but fluorescent
labels may be used, and the fluorescence of such labels may be
detected.
[0071] In general, a sensor converts bulk concentration of an
analyte to an output signal. If the sensor is an affinity-base
sensor, as in the context of the present invention, the sensor
includes the affinity probes and the transducer.
[0072] In an affinity-based sensing device, the detection of
analytes in the sample may be performed through a hybridisation
reaction between specified reactants and the analytes in the
sample. The hybridisation reaction is based on the formation of a
complex between at least two molecules, e.g. at least two
biomolecules, e.g. the analyte and an affinity probe, which is a
molecule or an entity acting as receptor, also called a capture
probe, which may be immobilized on a substrate, or immobilized in a
3D matrix, or free in solution. The complex formation between the
analyte (A) and the affinity probes (P) leads to a signal that is
detectable, e.g. measurable by a signal measurement unit, or
visually discernable.
[0073] The transducer converts concentration or density of affinity
probes--analyte complexes to an output signal.
[0074] The response of the affinity-based sensing device may be
limited by the rate of the hybridisation reaction. In the case of
an affinity-based sensor based on the complexation reaction between
affinity probes (P) such as for instance antibodies (Ab) acting as
capture probes and an analyte (A) such as for instance an antigen
(Ag), the hybridisation reaction is a binding reaction, e.g., but
not limited thereto, a first order binding reaction based on the
chemical equation (I):
P + A k off k on PA ( I ) ##EQU00001##
where P represents the (empty) affinity probes (e.g., but not
limited thereto, immobilized on the surface) [0075] A represents
the analyte (e.g. in the bulk of the liquid) [0076] PA represents
the complex affinity probe--analyte (e.g., but not limited thereto,
on the surface) [0077] k.sub.on represents the on-rate constant,
also called association (or complexation) rate constant k.sub.a
[0078] k.sub.off represents the off-rate constant, also called
dissociation rate constant k.sub.d; or applied more specifically to
antibody-antigen complexation:
[0078] Ab + Ag k off k on AbAg ##EQU00002##
where Ab represents the (empty) antibodies (e.g., but not limited
thereto, immobilized on the surface) [0079] Ag represents the
antigens (in the bulk of the sample) [0080] AbAg represents the
complex antibody-antigen (e.g., but not limited thereto, on the
surface)
[0081] The affinity (or association constant) of the reaction,
K.sub.a is given by the equation (II):
K a = k on k off ( II ) ##EQU00003##
[0082] At equilibrium, the concentrations of the various species
obey the equation (III):
[ PA ] [ P ] [ A ] = K a = k on k off ( III ) ##EQU00004##
where [x] represents the concentration of x. or applied more
specifically to antibody-antigen complexation:
[ AbAg ] [ Ab ] [ Ag ] = K a = k on k off ##EQU00005##
[0083] In the context of the present invention, "concentration" can
mean either bulk concentration or surface concentration, depending
on whether the reaction is taking place in the bulk of the liquid
(e.g. with affinity probes in the bulk of the liquid), or on a
surface (e.g. with affinity probes immobilized on a surface),
respectively. Surface concentration is sometimes also called
surface density, and both terms are intended to be equivalent.
[0084] After reorganizing some terms in the equation (III), at
equilibrium, the occupied fraction F of the available affinity
probes is given by the equation (IV):
F = [ PA ] [ P tot ] = K a [ A ] 1 + K a [ A ] ( IV )
##EQU00006##
where [P.sub.tot]=[P]+[PA] represents the total affinity probe
concentration (free+occupied); or applied to antibody-antigen
complexation:
F = [ AbAg ] [ Ab tot ] = K a [ Ag ] 1 + K a [ Ag ]
##EQU00007##
where [Ab.sub.tot]=[Ab]+[AbAg] represents the total antibody
concentration
[0085] If the concentration [A] of analyte in the bulk, e.g. the
concentration [Ag] of antigens in the bulk, equals 1/K.sub.a, then
50% of the affinity probes, e.g. the antibodies, will be occupied
at equilibrium. This is illustrated in FIG. 1 which shows the
direct influence of the affinity, K.sub.a, on the limit of
detection of an affinity-based sensor. For a same transducer, the
use of different biological systems, e.g. different couples of
affinity probes and analytes, e.g. antibodies and antigens, with
different K.sub.a values, leads to different limits of detection
(LOD). It can be seen that for higher K.sub.a values a same
occupied fraction of affinity probes is obtained at lower analyte
concentrations; the transducer converts the occupied fraction of
affinity probes into an output signal, hence a same output signal,
e.g. sufficient to exceed the total system noise, may already be
obtained at lower analyte concentrations.
[0086] FIG. 2 illustrates a simulation of the time dependence of
the signal (representative for the amount of captured analyte, e.g.
antigens) as a function of the concentration of the analyte, e.g.
antigen, in the example illustrated for a reaction with an on-rate
constant k.sub.on equal to 10.sup.5 M.sup.-1s.sup.-1 and an
off-rate constant k.sub.off equal to 10.sup.-5s.sup.-1. Despite the
fact that the affinity constant, K.sub.a, used in this example has
been fixed as equal to 10.sup.10 M.sup.-1, and thus the affinity
probes are considered "good" affinity probes, it takes a very long
time to build up the equilibrium response (e.g. in the example
illustrated more than 2 days). This means that if a signal of, for
instance, 0.4 is measured, the corresponding analyte concentration
which can be determined therefrom depends on the time from start of
the complexation reaction. Hence it is desired to have a fast
complexation reaction between affinity probes and analyte, such
that equilibrium is reached after only a short period of time, for
instance after 10 to 15 minutes, such that a measurement signal
obtained after that period of time, results in a measurement value
which is representative for the actual analyte concentration
(endpoint measurement), or such that the slope of the measurement
signal generated by the transducer is steep (slope
measurement).
[0087] The rate at which the hybridisation occurs is limited by
k.sub.on. For a typical macromolecular analyte having a molecular
weight in the range of 10 to a few 100 kDa, k.sub.on is in the
range of 10.sup.5-10.sup.6 M.sup.-1s.sup.-1 when both analyte, e.g.
antigen, and affinity probes, e.g. antibodies, are free molecules,
in other words when the affinity probes are not fixed on a surface.
The limitation of the hybridisation is linked, among other, to the
diffusional encounter between analyte and affinity probes, and in
the majority of cases it is difficult to increase this. For surface
bonded affinity probes, e.g. antibodies (Ab), the diffusional
encounter rate could be even slower and thus the error in
determination of analyte concentration based on a measurement value
could even be higher. For example, the inventors have measured
values in the range of k.sub.on=10.sup.5 to 3.times.10.sup.5
M.sup.-1s.sup.-1.
[0088] The time evolution to reach the equilibrium of the chemical
equation (I) is given by the relation (V):
d [ PA ] dt = k on [ P ] [ A ] - k off [ PA ] ( V )
##EQU00008##
wherein [A]=represents the concentration of analyte, or applied to
antibody-antigen complexation:
d [ AbAg ] dt = k on [ Ab ] [ Ag ] - k off [ AbAg ]
##EQU00009##
wherein [Ag] represents the antigen concentration
[0089] In relation (V), the concentration [A] of analyte, e.g. the
concentration [Ag] of antigen, represents the concentration
directly above or in contact with the affinity probes, e.g. the
antibodies. In the case of a mass transport limited reaction, this
concentration may drop below the bulk concentration (also known as
depletion of the analyte). In this case, the concentration directly
above or in contact with the affinity probes can be related to the
bulk concentration by taking into account both the reaction rate,
as given by relation (V) in the case of a first order affinity
reaction, and the appropriate mass transport laws, e.g. diffusion
equations such as Fick's law in the case of mass transport by
diffusion, convection-diffusion equations in the case of mass
transport by convection, where the liquid flow is treated by the
appropriate fluid dynamics models, such as models based on the
Navier-Stokes equations, as can be done by one of ordinary skill in
the art.
[0090] The time evolution of the hybridisation reaction, e.g. of
the formation of the complex between the analyte, e.g. antigen
(Ag), and the affinity probes, e.g. antibodies (Ab), is given by
the relation (VI):
[ PA ] ( t ) [ P tot ] = K a [ A ] 1 + K a [ A ] ( 1 - e - ( k on [
A ] + k off ) t ) .about. ( 1 - e - t / .tau. ) ( VI ) [ AbAg ] ( t
) [ Ab tot ] = K a [ Ag ] 1 + K a [ Ag ] ( 1 - e - ( k on [ Ag ] +
k off ) t ) .about. ( 1 - e - t / .tau. ) ##EQU00010##
This leads to a time constant, .tau.
.tau. = 1 k on [ A ] + k off ##EQU00011##
or applied to antibody-antigen complexation:
.tau. = 1 k on [ Ag ] + k off ( VII ) ##EQU00012##
[0091] FIG. 4 illustrates a simulation of the occupied fraction
[PA]/[P.sub.tot] at equilibrium, e.g. [AbAg]/[Ab.sub.tot] at
equilibrium as a function of the analyte concentration [A] (e.g.
the antigen concentration) in the sample for different affinity
constants. This is the same as FIG. 1, but with a logarithmic scale
instead of a linear scale on the vertical axis. Area I corresponds
to situation of saturation at equilibrium, where the equilibrium
situation consists of essentially 100% complexation of the affinity
probes P, e.g. antibodies Ab, by the analyte A, e.g. antigen Ag,
has taken place, or in other words [PA].apprxeq.[A.sub.tot]. In
this situation [A]>>1/K.sub.a, e.g. [Ag]>>1/K.sub.a,
leading to a time constant r, that can be approximated by the
relation (VIII):
.tau. = 1 k on [ A ] ( VIII ) ##EQU00013##
or applied to antibody-antigen complexation:
.tau. = 1 k on [ Ag ] ##EQU00014##
Area II corresponds to a situation where at equilibrium less than
50% of the affinity probes P, e.g. antibodies Ab, are complexed by
the analyte A, e.g. antigens Ag. In this situation
[A]<<1/K.sub.a, e.g. [Ag]<<1/K.sub.a, leading to a time
constant r, that can be approximated by the relation (IX):
.tau. = 1 k off ( IX ) ##EQU00015##
Hence it can be seen that area I and area II indicate different
simplified expressions for the time constant.
[0092] From FIG. 2 and the relations given here above, the
inventors have found that analyte concentration measurements
require a long measurement time. For measurements performed at
short measurement times, the signal has had no time to build up,
leading to the determination of erroneous analyte concentration
values, or, if the considerations leading to FIG. 2 are taken into
account, to smaller signals, which results in a lower
signal-to-noise ratio and a lower accuracy of the measurement
result. In short measurement times, the response of the sensor is
thus determined by the complexation rate constant, k.sub.on, of the
hybridisation reaction (I).
[0093] It is therefore a solution provided by embodiments of the
present invention to increase the association kinetics (represented
by k.sub.on) of the hybridisation reaction, to build up the signals
more quickly and to reach lower (=better) limit of detection. The
dissociation times of the complex (e.g. AbAg) typically are in the
range of hours or days, so that the dissociation reaction can be
neglected on the desired time scale of the measurement (e.g. less
than 20 minutes, such as for instance around 10 min and even less)
and mainly the complexation rate constant, k.sub.on, is
important.
[0094] The inventors have surprisingly found that the hybridisation
rate for some affinity probe-analyte (e.g. antibody-antigen)
combinations is not constant, and that the hybridisation may be
highly speeded up by decreasing the ionic strength of the analyte.
The inventors have found that a reduction of the ionic strength by
1 order of magnitude, for example from 100 to 10 mM, resulted in 4
or 5 orders of magnitude increasing of the complexation rate
constant, k.sub.on, and thus to a decreasing of the LOD and/or of
the response time.
[0095] The reduction of the ionic strength, in accordance with
embodiments of the present invention, is performed by using a
device for sensing an analyte comprising a desalting unit.
[0096] FIG. 5 illustrates an embodiment of a device (1) for sensing
an analyte according to embodiments of the present invention. The
device (1) for sensing an analyte, for instance, but not limited
thereto, a biosensor, comprises at least a sample inlet (10) for
receiving a sample, affinity probes (111) selected so as to have a
preferential binding to the analyte, a transducer (11) for sensing
a characteristic of the analyte and/or a label attached to the
analyte, the transducer being not a FET transducer, and a desalting
unit (13). The transducer (11) is sensitive to a characteristic of
the analyte and/or a label attached to the analyte and converts an
interaction of the analyte with the affinity probes (111) into a
measurable signal (12), e.g. the output signal. This output signal
may for instance be an electrical signal, an optical signal, or a
visual signal.
[0097] The desalting unit (13) may be any suitable device for
desalting the analyte. This may for instance be performed by
diluting the analyte, or by extracting salt from the analyte. In
embodiments of the present invention, the desalting unit (13) may
comprise a port (130) and a buffer fluid reservoir (131). The
buffer fluid reservoir (131) may for instance be any of an ampoule,
a syringe, a blister, a well, a tube, an Eppendorf tube, a channel,
or an on-chip reservoir. In particularly advantageous embodiments,
the buffer fluid reservoir is a blister pack, a channel, or an
on-chip reservoir.
[0098] The device (1) may be implemented with discrete components,
or as an integrated chip. In the latter case, desalting may be
performed off-chip, or on-chip.
[0099] The sample received at the sample inlet (10) and a stream of
buffer fluid may be flown together in the desalting unit (13), and
may be led to the affinity probes (111) linked to the transducer
(11) as illustrated in FIG. 5, for instance by capillary forces.
Alternatively, pumps may be provided to pump the sample and the
buffer fluid together towards the affinity probes (111) and the
transducer (11).
[0100] In alternative embodiments, the sample received at sample
inlet (10) may be led through a buffer fluid reservoir (131)
comprising buffer fluid, such that the dilution is performed
automatically, such as for instance illustrated in FIG. 6. Also in
this embodiment, the flow of the sample through the buffer fluid
reservoir (131) may be driven by capillary forces, or by external
driving means such as pumps, for pumping sample towards the fluid
reservoir (131), and for pumping a mix of sample and buffer fluid
towards the affinity probes (111) and the transducer (11).
[0101] In embodiments of the present invention, the desalting may
be performed when or while the analyte is associated with a label.
The type of association with a label is irrelevant for embodiments
of the present invention; it may include for instance direct
labelling of the analyte, having the analyte compete with a
labelled species, or by quenching a label. This can be done by
adding a low-ionic strength buffer or solvent to the labelled
sample mix. Alternatively, this can be done by preparing the label
solution at low ionic strength, and mixing it in a suitable ratio
with the sample. After the desalting step, the low ionic strength
labelled sample may be sent over affinity probes where the
hybridisation reaction relevant for the present invention takes
place.
[0102] In embodiments of the present invention, labelling the
analyte may take place at normal (e.g. physiological) ionic
strength, and then the ionic strength may be reduced before sending
the labelled analyte over the affinity probes. This has the
advantage that the kinetics of premixing in the bulk are somewhat
better than the kinetics of capturing the analyte or analyte
complex on the surface, so slow kinetics for the bulk premixing is
less critical. Furthermore, the off-rate constant k.sub.off is less
affected by the reduced ionic strength, so once the
analyte-affinity probe complex has been formed, it remains stable
also at reduced ionic strength.
[0103] In embodiments of the present invention, labelling the
analyte may take place after the analyte has been sent over the
affinity probes. This can e.g. be done by sending a second solution
containing second affinity probes over the surface with the
captured analytes, the second affinity probes being labelled and
also having an affinity for the analyte. In embodiments of the
present invention, the second solution can be at physiological
ionic strength. The second affinity probes may be provided at high
concentration such that the kinetics is fast. Alternatively, the
second solution can be at low ionic strength, to speed up also this
interaction.
[0104] In particular embodiments, the desalting unit (13) may
comprise a mixer (132) for mixing received sample with buffer
fluid. The mixer may for instance be any of a microfluidic mixer, a
vortex mixer, a shaker, a magnetic mixer, an ultrasonic mixer, a
mechanical mixer or a rapid-mixing apparatus. The rapid
mixing-apparatus may comprise two syringes, one for the delivery of
a sample through the sample inlet and one for the delivery of a
buffer fluid through a buffer fluid inlet and a mixing chamber.
[0105] The transducer (11) may be an optical transducer such as for
instance, the present invention, however, not being limited
thereto, a luminescence transducer, such as a fluorescence
transducer, a total internal reflection fluorescence (TIRE)
transducer, an evanescent field based fluorescence transducer, a
phosphorescence transducer, a chemiluminescence transducer, a
bioluminescence transducer; a refractive index transducer, such as
a Surface Plasmon Resonance (SPR) transducer, a Biolayer
interferometry/reflectance interference spectroscopy (BLI/RIfS)
transducer, a Photonic ring resonator, an Optical interferometer
(MZI, Young); an absorbance transducer (also known as colorimetric
transducer); and Photonic crystals. Alternatively, the transducer
may be of a non-optical type. Examples thereof, without being
limiting for the present invention, are for instance an electrical
transducer other than a FET-transducer, e.g. an amperometric
transducer, a capacitive transducer, an electrical impedance
transducer, an electrochemical transducer, an electrocatalytic
transducer; a mechanical transducer, such as a quartz crystal
microbalance (QCM), a micro-electromechanical system (MEMS), a
nano-electromechanical system (NEMS), a microcantilever, a
suspended microchannel resonator; a magnetic transducer, such as a
magnetometer, a Hall effect transducer, a spin valve, a magnetic
tunnel junction, a transducer based on nitrogen-vacancy (NV)
centers in diamond; or a radioactivity transducer.
[0106] FIG. 7 illustrates a device (1) for sensing an analyte
according to embodiments of the invention, wherein the desalting
unit (13) comprises a mixer (132). A separate buffer fluid
reservoir (131) is provided, and both sample obtained from the
sample inlet (10) and buffer fluid from the reservoir (131) are led
to the mixer (132), for instance by capillary forces or under
influence of pumps or the like. The device (1) for sensing an
analyte, for instance, but not limited thereto, a biosensor,
comprises a transferring means (133) permitting the transfer of the
desalted sample from the mixer (132) to the affinity probes (111)
and the transducer (11). This transfer may take place by capillary
forces or under influence of pumps or the like. FIG. 8 illustrates
an alternative device (1) for sensing an analyte according to
embodiments of the invention, wherein the reservoir (131) and the
mixer (132) are implemented as a single entity, i.e. the mixer is
provided in the reservoir (131). Sample obtained from the sample
inlet (10) is led to the reservoir (131), where it is mixed with
the buffer fluid, after which the mix is led to the affinity probes
(111) and the transducer (11). Transport of sample (from sample
inlet to reservoir/mixer) and sample mixed with buffer fluid (from
reservoir/mixer to transducer) may take place under capillary
forces, or driven by pumps or the like. The embodiment illustrated
in FIG. 8 is similar to the embodiment illustrated in FIG. 5,
except that in the embodiment of FIG. 8 mixing means are provided
in the reservoir (131), which is not the case in the embodiment of
FIG. 5. The mixing means may be active mixing means (comprising a
mechanical actuator such as a magnetic stirrer, a vortex mixer or
any other suitable mixing device) or passive mixing means (not
comprising any moving parts, but modifying the flow to enhance the
mixing efficiency; e.g. by creating turbulent flow, by modifying
laminar flow, by increasing the residence time). FIG. 9 illustrates
another embodiment of a device (1) according to the present
invention, wherein sample obtained from a sample inlet (10), and
buffer fluid obtained from a reservoir (131) are flown together
towards affinity probes (111) and a transducer (11) on top of which
mixing means (132) are provided for better mixing the sample and
the buffer fluid. The transport of fluids through the device (1)
may be provided by capillary forces, or may be driven by pumps or
similar.
[0107] In yet alternative embodiments, as for instance illustrated
in FIG. 10, essentially the same process as illustrated in FIG. 9
takes place, but instead of first flowing together the sample and
the buffer fluid, and flowing these together towards the affinity
probes (111) and the transducer (11), in this embodiment sample and
buffer fluid are each flown separately, under capillary forces or
driven by pumps or similar devices, towards the affinity probes
(111) and the transducer (11), where they are mixed by means of a
mixing means (132).
[0108] In still another embodiments, as illustrated in FIG. 11, a
fluid reservoir (131) is provided on the affinity probes (111) and
the transducer (11), optionally with a mixer (132) being provided
in the fluid reservoir (131), and sample obtained from the sample
inlet (10) is flown towards and into the fluid reservoir (131),
where it is desalted, before or during the binding to the affinity
probes (111) and the measurement by the transducer (11) takes
place.
[0109] FIG. 12 represents diagrammatically an embodiment of a
method for measuring the concentration of an analyte, for instance
an antigen, in a sample of analyte, according to embodiments of the
invention. The method comprising the steps of: [0110] i. obtaining
a sample, for example receiving a sample in the biosensor, for
example a sample taken from a patient, [0111] ii. desalting the
sample, thereby obtaining a desalted sample having a ionic strength
lower than the ionic strength in the original sample, for instance
an ionic strength ranging from 10 nM to 150 mM, more preferably
from 1 mM to 150 mM, more preferably from 10 mM to 150 mM, [0112]
iii. measuring at least one signal of the desalted sample, by means
of an affinity-based sensing device based on affinity probes and a
transducer, the transducer not being a FET-transducer, and [0113]
iv. determining the concentration of the analyte in the sample
using the at least one signal.
[0114] In particular embodiments of the present invention, step ii.
may be a dilution step, wherein the sample is diluted with a
solvent, for instance a buffer fluid. The solvent used for the
dilution may be a water based buffer fluid, for instance at a pH
ranging from pH 2 to 12, or 5 to 9, or around 7.
[0115] In alternative embodiments of the present invention, the
desalting step may be a step wherein the sample is provided in a
reservoir with one or more, preferably at least two, semipermeable
walls. The reservoir is adapted for allowing Na.sup.+ ions and
Cl.sup.- ions to leave the reservoir through the semipermeable wall
thereof, while the remainder of the sample is kept in the
reservoir. Suitably actuated electrodes may be provided for
attracting the ions through the semi-permeable wall. This way, the
sample is desalted, without decreasing the concentration of analyte
molecules in the sample.
[0116] In particular embodiments, the desalting step may be part
of, e.g. integrated with, the sample collection. In alternative
embodiments, the sample is collected first, and is only desalted
thereafter. The desalting may take place prior to the measurement.
Hereto, the desalting may take place before the sample reaches the
affinity probes and the transducer, or the desalting of the sample
may take place on the affinity probes and the transducer.
[0117] In particular embodiments, the desalting may take place in a
separate instrument, which is for instance provided in a different
enclosure, separated from the enclosure where the signal
measurement takes place. Alternatively, desalting and measurement
may take place within a same enclosure.
[0118] By desalting the sample to be analysed, thus reducing its
ionic strength, the association kinetics of the hybridisation
reaction may be increased by a significant factor, up to multiple
orders of magnitude.
[0119] The measurement signal may be followed in real time, and one
can monitor and use the faster and larger signal in real time, and
terminate the measurement more quickly.
[0120] FIG. 3 illustrates a measurement signal in function of time.
The measurement signal, i.e. the signal generated by the affinity
probes (111) and the associated transducer (11), is related to,
e.g. proportional such as directly or inversely proportional to,
the occupied fraction [PA] of affinity probes.
[0121] The inventors have found that a measurement of the slope of
the curve of the signal may be done in order to decrease the error
linked to short measurement times (less than 20 minutes, for
instance less than 10 minutes) as shown on FIG. 3. Thus,
embodiments of the present invention may advantageously increase
the signal of the transducer, and additionally or alternatively
they may reduce the response time thereof. The slope of the curve
depends on the concentration of the analyte, e.g. antigen, just
above the sensor surface: the more analyte is present there, the
faster analyte will bind to corresponding affinity probes, hence
the faster the amount of occupied affinity probes will increase,
and the steeper the slope of the curve will be. Agitation may be
used to avoid depletion of the analyte above the affinity probes
and thus to avoid problems linked to the mass transport of the
analyte, e.g. antigen.
[0122] Advantages of slope measurements are as follows.
Traditionally, a particular level is measured, i.e. how large the
measurement signal is at a certain point in time (called an
endpoint). So a dose-response relationship is assumed. If the
incubation times are long enough to reach (or approach)
equilibrium, then the dose-response curves are those at
equilibrium, similar to the ones shown on FIGS. 1 and 4. In this
case, the exact timepoint at which the measurement is taken, is not
important as the signal no longer changes with time. This is equal
to what is shown far to the right on FIG. 3). However, the
discussion above has shown that the required incubation times are
often unreasonably long, e.g. much longer than what would be
acceptable for a point-of-care (POC) application, such as for
instance more than one hour, more than a couple of hours, even more
than a day. For POC tests where results are desired in less than
about 20 min, often in less than about 10 min, measurements are
most often done when the system has not yet reached equilibrium. In
this case the dose-response curves become those of FIG. 2, i.e. the
response not only depends on the analyte concentration but also on
the incubation time. As a result, any variation in the incubation
time translates into an error on the measurement. And the shorter
the incubation time, the larger the relative error on the time.
This is the main uncertainty that is solved by doing a slope
measurement in accordance with embodiments of the present
invention.
[0123] In the initial stages of the association (complexation)
reaction, the time evolution (described by equation VI) can be
approximated by a linear expression. This means that the slope is
independent of the exact time, as can be seen on the left-hand side
of FIG. 3, and only depends on the analyte concentration (as shown
by equation VI).
[0124] In addition, by continuously following the time evolution of
the signal, the shape of the time-dependent curve can be
reproduced, and a determination can be made as to whether it
matches an expected behavior. So it can be checked whether the
linear approximation of equation (VI) is still valid. If not, the
full exponential dependence can be taken into account, and the full
eq. VI can be used, instead of a linear approximation, to deduce
the concentration.
[0125] Following the functional shape of the signal also allows to
detect (and correct for) other parasitic effects. For instance in a
lateral flow system, when a switch is made from buffer flow to
sample flow, there may be some intermixing at the liquid front
between them. This gives an error on the exact incubation time, and
also an error on the slope (the slope will be smaller during this
transient stage). However, in continuous measurements this can be
seen, and the slope can be calculated after this transient has
settled (i.e. the points affected by the transient can be
discarded). As another example: if there are mass transport
limitations showing up, slope measurement may allow to detect these
and correct for them in the data analysis
[0126] A further advantage of slope measurement is that the slope
calculations can be based on many datapoints, which helps to cancel
out (random) measurement errors or noise.
[0127] Yet a further advantage of slope measurement is that the
measurement can be terminated as soon as a good enough signal is
obtained, e.g. as soon as a required or desired accuracy is
reached. This can be very fast for a sample having a high
concentration of a particular biomarker, and longer for a sample
where the concentration is lower, e.g. closer to the LOD. This is
not possible in endpoint measurements, where the incubation times
are set in advance to cover all possible conditions, hence may be
unnecessarily long for certain samples.
* * * * *